Caroline Dellero

Florence Mauve

Nathalie Guérard

Linda Glab

Pauline Debont

Keywords: Photorespiration, Phosphorylation, SHMT1, HPR1, Métabolisme, Stress abiotique

Duminil. I thank them for their accompanying during my PhD. All of you were always behind me and gave me unlimited help. I can't imagine how I

that he gave me excellent guidance on the research work but also for his time and effort on the correction and inspiring advices to my thesis. Thanks to him for finally letting me being a real scientist.

I would like to acknowledge both the invitation from Dr. Mathieu Jossier and the financial support from China Scholarship Council (CSC) for studying in France, without them, I won't have the opportunity to be a member of this great team and get my PhD degree in France.

I would like to thank the jury members: Dr. Nathalie Leonhardt, Dr. Eric Ruelland, Dr.

Arnould Savouré and Dr. Graham Noctor. Your attendence to my PhD defense is a great honor for me. I also would like to thank Marie Boudsocq and Sophie Filleur for being the invited members of my thesis committee.

I sincerely want to thank Michael Hodges, the leader of the group, from the discussion with him, which showing his unique humor, I get a lot of knowledge and useful suggestions both on my experiments and on my project I also greatly appreciate that he spent a lot of time and effort on correcting my manuscript.

I owe a special debt of gratitude to Nathalie Glab and Céline Oury, they gave me a lot of guidance on experimental operations; they also helped me not only to order experimental reagents but also provided me useful assistance. I sincerely thank Axel de Zélicourt and Bertrand Gakière for their kind help all the time. I also want to thank the other team members, Younes Dellero, Caroline Mauve, Florence Guérard, Linda Debont, Pauline Duminil, Françoise Gilard, Marlene Lamothe-Sibold, Valérie Cantonny and Sophie Blanchet for their kindness and availability. I would like to thank Dr. Severine Domenichini for her kindly help in using the microscope; thank Dr. Kevin Magne for providing me the yeast strain; thank Maël Jeuffard for fixing my computer. I also would like to thank the members in I2BC, Julie Hodin and Besse Laetitia, for their instrumental and experimental supports. Xylulose-5-phosphate paramètres cinétiques et entraîne une activité réduite et une modification de l'activité selon la nature du cofacteur (NADH ou NADPH). Cette faible activité explique pourquoi HPR1-T33D était incapable de totalement complémenter le phénotype photorespiratoire de croissance dans l'air du mutant hpr1. La modification du ratio NADH/NADPH de l'activité de HPR1 indique que la phosphorylation de T335 doit modifier les propriétés des paramètres cinétiques pour les rapprocher de ceux de l'enzyme cytosolique HPR2.

List of abbreviations

La phosphorylation de la Ser31 N-terminale a été étudiée par complémentation du mutant d'Arabidopsis shm1-1 par une forme sauvage de SHMT1 ainsi que des versions mimant (S31D) ou non (S31A) une phosphorylation. Les résultats ont montré que toutes les formes de SHMT1 ne pouvaient pas totalement complémenter le phénotype de croissance de shm1-1 puisque chaque lignée transgénique n'avait que 50% d'activité SHMT1 comparée aux plantes contrôles. En réponse à un stress dû au sel ou à la sécheresse, les lignées Compl-S31D ont montré un déficit de croissance plus accentué que les autres lignées transgéniques. Cette sensibilité accrue au sel reflète une diminution de l'activité de SHMT1-S31D dépendante du stress et impactant le métabolisme des feuilles, entraînant à son tour une sous-accumulation de proline et une sur-accumulation de polyamines. Les différences de croissance entre les lignées shm1-1 complémentées et la plante contrôle sont directement liées à la photorespiration puisqu'elles ne sont plus observables lorsque les plantes sont cultivées en atmosphère riche en CO2. La lignée SHMT1-S31D présente aussi une réduction de la fermeture stomatique induite par le sel et l'ABA. Ainsi, nos résultats soulignent l'importance du maintien de l'activité du SHMT1 photorespiratoire dans des conditions de stress salin et sécheresse et indiquent que la phosphorylation de SHMT1 S31 pourrait être impliquée dans la modulation de la stabilité de la protéine SHMT1.

Phosphoregulation of photorespiratory enzymes in Arabidopsis thaliana

Keywords: Photorespiration, Phosphorylation, SHMT1, HPR1, Metabolism, Abiotic stress

Photorespiration is an essential process in oxygenic photosynthetic organisms, that is triggered by the oxygenase activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) leading to the production of 3-phosphoglycerate (3PGA) and 2-phosphoglycolate (2PG). In plant, 3PGA is a useful three carbon molecule that can be metabolized to either regenerate RuBP or to make complex sugars, however 2PG is a toxic two carbon molecule that can be detoxified and recycled the carbon to Calvin-Cycle by the photorespiratory cycle which involves eight core enzymes and takes place in four cellular compartments including chloroplasts, peroxisomes, mitochondria and the cytosol. Photorespiration leads to a reduction of photosynthetic CO2 assimilation and the photorespiratory cycle has a high energetic cost, so for many years, it was been considered as a wasteful process. However, the severe growth phenotypes of photorespiratory enzyme mutants (such as small, chlorotic plants that are often non-viable in air) which are not observed under high CO2 condition reflect the importance of this process in air-grown conditions. It is known that the photorespiratory cycle also interacts with many primary metabolic processes in plants such as photosynthetic carbon assimilation, nitrogen assimilation, amino acid metabolism, the TCA cycle and C1-metabolism. Therefore, the regulation of this cycle is very important for plant growth and development. Current data indicate that seven of the eight core photorespiratory enzymes are associated with phosphopeptides in the PhosPhAt database, thus suggesting that protein phosphorylation is a good candidate to be a key regulatory component of the photorespiratory cycle. In order to understand the regulation of the photorespiratory cycle, we explored the role of mitochondrial serine hydroxymethyltransferase 1 (SHMT1) and peroxisomal hydroxypyruvate reductase 1 (HPR1) phosphorylation on enzymatic activities, plant metabolism and abiotic stress resistance. This was achieved by producing phospho-mimetic (serine/threonine mutated to aspartate) or non-phophorylatable (serine/threonine mutated to alanine) recombinant enzymes either in E. coli or in relevant Arabidopsis thaliana mutants (shm1-1 and hpr1).

Two HPR1 phosphorylation sites (S229 and T335) have been studied but only recombinant phospho-mimetic HPR1-T335D exhibited the main modification of the enzymatic kinetic parameters, a reduced activity assays and altered NADH/NADPH-dependent activity ratio.

This low activity explains why HPR1-T33D was unable to fully complement the photorespiratory phenotype of hpr1 in air condition. The modification of the NADH/NADPHdependent activity ratio indicated the T335 phosphorylation might modify HPR1 kinetic properties towards those of cytosolic HPR2

The phosphorylation of the N-terminal Ser31 of SHMT1 was studied by complementing Arabidopsis shm1-1 with SHMT1 wild-type, phospho-mimetic (S31D) or nonphophorylatable (S31A) forms. It was found that all SHMT1 forms could not fully complement the photorespiratory growth phenotype of shm1-1 since each transgenic line only had 50% of normal rosette SHMT activity. In response to either a salt or drought stress, Compl-S31D lines exhibited a more severe growth deficiency compared to other transgenic lines. This increased sensitivity reflected a salt-dependent reduction of SHMT1-S31D protein and activity levels that impacted leaf metabolism leading to proline under-accumulation and

INTRODUCTION INTRODUCTION 6

I. Photosynthesis

In the light, C3 plants can directly fix atmospheric CO 2 via the carboxylase activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) to produce 3phosphoglycerate (3PGA), a phosphate acid which will be subsequently converted to glyceraldehyde-3-phosphate (G3P), a sugar with three carbons. These two molecules will serve as building blocks for the biosynthesis of all organic molecules: organic and amino acids, simple sugars (such as glucose and fructose) and more complex carbohydrates (such as sucrose, starch, and plant wall constituents), ribonucleic acids, proteins, lipids etc. The process, operated by plants and some other organisms, of converting light energy into chemical energy that is required for the fixation of CO 2 is called photosynthesis which is also accompanied by the release of O 2 . Photosynthesis is composed of two major processes. The light-dependent reactions capture the energy of light and use it to make the energy-storage molecule, ATP and NADPH. The so-called "dark" reactions use the ATP and NADPH generated by the light phase to regenerate ribulose-1,5-bisphosphate (RuBP) via the Calvin-Benson cycle. RuBP is consumed by the RuBisCO to fix CO 2 .

1) Light-dependent reactions

The conversion of light energy into chemical energy is achieved through the photosynthetic electron transfer chain (ETC), located in the thylakoid membranes of chloroplasts. This linear electron transport chain leads to the production of oxygen (from water oxidation), ATP and NADPH. This production of photosynthetic oxygen has played a decisive role in the development of life on Earth. This chain consists of several complexes (Figure 1): photosystems I and II (PSI and PSII), the light-harvesting complexes of PS II and PS I (LHC I and LHC II)), the oxygen-evolving complex (OEC) which is associated with PSII, the cytochrome-b6f complex (cyt-b6f) and the ATP-synthase (ATPase) (Figure 1) (for a detailed review of the composition of PSI and PSII, see [START_REF] Caffarri | A Comparison Between Plant Photosystem I and Photosystem II Architecture and Functioning[END_REF]). The energy of sunlight can be absorbed by plant chlorophyll pigments of the light-harvesting complexes, which will then enter a relatively unstable, excited state. They can return to their fundamental energetic state by transferring energy excitation to neighboring pigments by energy radiation or by emitting fluorescence or heat. They can also change their chemical structure (conversion of energy) and keep a high energy state (singlet excited Chl going to a triplet excited state). As a result, energy is transferred to the reaction center Chls P680 and P700. When energy arrives at PSII, electrons are transferred to Q A and subsequently to PQ bound at the Q B pocket. PQ is a soluble electron carrier in the lipid phase of the thylakoid membrane, and when PQ becomes reduced to plastohydroquinone (PQH2) by the uptake of two protons from the stromal phase.

On the lumen side of the membrane, the PQH2 molecules transfer electrons to the cytochrome-b6f complex and it becomes oxidized with the release of protons to the thylakoid lumen. An electron is then transferred to plastocyanin (PC), a soluble redox mediator that reduces the P700 of PSI which has been oxidized by light energy transfer. Within the PSI complex, electrons are transferred to Fd and FNR that reduces NADP + to form NADPH in the stroma.

Thus, photosynthetic electron transfer leads to the movement of protons across the thylakoid membrane from the stroma to lumen [START_REF] Steffen | Time-resolved spectroscopic investigations of photosystem II[END_REF]. These protons together with protons released by the oxidation of water lead to the acidification of the lumen (pH of about 4.5-4.8)

when compared to the pH of the stroma (of about pH 7.5-7.8) [START_REF] Rumberg | pH changes in the inner phase of the thylakoids during photosynthesis[END_REF].

This pH-difference is used by the ATPase to drive ATP synthesis (see Figure 1).

PSII is a multi-enzymatic chlorophyll protein complex in the thylakoid membrane. PSII is associated with several light-harvesting antenna proteins: The light harvesting II (LHCII) composed of different Chl a and Chl b containing proteins( Lhcb1, Lhcb2, Lhcb3) as well as some other peripheral proteins (Lhcb4, Lhcb5 and Lhcb6); CP43 and CP47 composed of Chl a, as well as carotenoids (neoxanthin, violaxanthin, lutein, β-carotene). The center of the PSII complex consists of a heterodimer composed of a D1 and a D2 protein, that bind two dimers of Chla (P680, absorbing light at 680 nm), two "special" Chls (pheophytin (Pheo)) and ChlA0), two β-carotenes, two quinones (Q A and PQ), cytochrome b559 (cytb559) and the metallic catalytic center (4Mn-1Ca) of the OEC. When the P680 is excited, it will give an electron to the primary acceptor Pheo of the D1 protein (producing a charge separation; P680 + Pheo -). At this stage, the reaction center is "closed" and it can no longer accept excitation energy from the different antenna proteins. The electron of Pheo -is transferred to a secondary quinone acceptor Q A , which then gives its electron to the "mobile" plastoquinone PQ, previously fixed at the Q B site via an iron atom. The P680 + is then reduced by an electron donated by tyrosine (Tyr-z) of the D1 protein. Tyr-z had previously been reduced by the catalytic center (4Mn + -1Ca) of the OEC. Then P680 becomes "Open" and can be "excited" again. For two molecules of H 2 O oxidized by the OEC, four electrons are accepted by the catalytic center (4Mn + -1Ca) that is required in the transfer of four electrons from P680 to PQ located at the QB site.

Light energy can also excite the antenna of PSI (Lhca1,Lhca2,Lhca3,Lhca4). PSI is also associated with other peripheral proteins (PsaG, PsaH and PsaK). The PSI reaction center consists of a heterodimer of the subunits PsaA and PsaB containing two Chl a (P700; light absorbing at 700 nm) dimers, two Pheo, two quinones (PhQA), a Fe-S (Fx) cluster and the PsaC protein which is the PSI terminal acceptor containing the Fe-S cluster (F A F B ). The F A F B cluster can then reduce Fd, a soluble Fe-S protein. So LHCI transfers excitation energy by resonance to P700 to form P700 + which reduces the F A F B cluster via ChlA 0 , PhQA and the Fx cluster. The PC serves to reduce the P700 + in order to "reopen" the PSI reaction center. At this stage, the reduced Fd can be used as an electron donor for various chloroplastic enzymes including FNR, nitrite reductase (NiR), Fd-glutamate synthase (Fd-GOGAT) and also FTR.

The FNR reduces NADP + to NADPH which can be used for all NADPH-dependent reactions in the chloroplast, including some stages of the Calvin-Benson cycle. Reducing power can also be moved to the cytosol via the "malate" shuttle [START_REF] Heineke | Redox Transfer across the Inner Chloroplast Envelope Membrane[END_REF].

As already mentioned above, during photosynthetic electron transfer, protons are transferred from the stroma to the lumen. For two oxidized water molecules, the OEC generates four protons while the PQ / PQH2 cycle and the cytochrome b6f transport eight protons from the stroma to the lumen. This proton gradient across the thylakoid membrane is used by the ATP synthase. This membrane enzyme consists of a stator and a rotor made up of twelve subunits.

The stator rotates the rotor and produces three ATP molecules for each turn (using twelve protons) from three molecules of ADP and inorganic phosphates (Pi). So, the photochemical phase of photosynthesis produces two molecules of NADPH, three molecules of ATP and a molecule of O 2 from two molecules of water and eight photons collected by the antenna complexes. ATP and NADPH will be subsequently used by the Calvin-Benson cycle during the "dark" phase for the reduction of 3PGA to G3P as well as for the regeneration of RuBP, the substrate of RuBisCO. Finally, cyclic electron transfer around PSI also generates an electrochemical potential (pH gradient) used for ATP synthesis that is independent of NADPH production (For a review, see Johnson, 2011).

In the absence of cyclic electron transfer and other Fd-linked processes, the net-reaction of the light-dependent reactions in oxygenic photosynthesis is:

2H 2 O + 2NADP + + 3ADP + 3P i → O 2 + 2NADPH + 3ATP + 2H +
The light reactions are also involved in the regulation of photosynthetic CO 2 assimilation via the Fd/thioredoxin system. Here, Fd reduced by the photosynthetic electron transfer chain is used to activate / inactivate enzymes via thioredoxins, which are small oxidoreductase proteins containing two cysteines close together in an "active site" that can form a disulfide bridge by the oxidation of their thiol groups. Five different forms of thioredoxin are present in the chloroplast: f, m, x, y and z. Thioredoxins f, m, x, and y are reduced by the FTR, and then the Trxs reduce specific target proteins including enzymes (for a review see [START_REF] Lemaire | Thioredoxins in chloroplasts[END_REF]). Several enzymes of the Calvin-Benson cycle are activated in the light by thioredoxins f and/or m: fructose-1,6-biphosphatase (F1,6bPase) [START_REF] Hodges | Purification and characterization of pea thioredoxin f expressed in Escherichia coli[END_REF],

sedoheptulose-1,7-biphosphatase (S1,7bPase) [START_REF] Schürmann | Plant thioredoxin systems revisited[END_REF], phosphoribulokinase (PRK) [START_REF] Brandes | The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f[END_REF], and G3P dehydrogenase [START_REF] Sparla | Regulation of Photosynthetic GAPDH Dissected by Mutants[END_REF]. It is possible that all enzymes in the Calvin cycle are regulated by thioredoxins (see [START_REF] Lemaire | Thioredoxins in chloroplasts[END_REF], RuBisCO activase [START_REF] Zhang | Mechanism of light regulation of Rubisco: A specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f[END_REF], NADP-dependent malate dehydrogenase [START_REF] Hodges | Purification and characterization of pea thioredoxin f expressed in Escherichia coli[END_REF] and ATP synthase [START_REF] Schürmann | Plant thioredoxin systems revisited[END_REF] are also "activated" by plastidial thioredoxins.

2) Light-independent reactions

During the "dark" phase, NADPH and ATP produced in the light by the photosynthetic electron transfer chain will be used by the Calvin-Benson cycle in the chloroplast stroma. This cycle will allow the fixation of inorganic CO 2 into matter organism. It consists of three phases:

1. The first step is the fixation of CO 2 , carried out by the RuBisCO carboxylase activity which will produce 6 molecules of 3PGA from 3 molecules of CO 2 and 3 molecules of RuBP.

2. The second step is the reduction of 6 molecules of 3PGA to 1,3-biphosphoglycerate

(1,3bPGA) by 3PGA kinase with the consumption of 6 molecules of ATP. Then the 6 molecules of 1,3bPGA are reduced to 6 molecules of glyceraldehyde-3-phosphate (G3P) by G3P-dehydrogenase with the consumption of 6 molecules of NADPH.

3. The third step consists in the regeneration of the three RuBP molecules initially used by RuBisCO from 5 G3P molecules. This makes it possible to preserve a molecule of G3P (carbon gain) for the synthesis of numerous organic molecules. Regeneration of RuBP involves 10 enzymatic steps:

-2 molecules of G3P are isomerized into 2 molecules dihydroxyacetone phosphate (DHAP) by triose phosphate isomerase.

-1 molecule of fructose-1,6-bisphosphate (F1,6bP) is synthesized from 2 molecules of DHAP and a molecule of G3P by F1.6bP aldolase -1 molecule of F1,6bP is dephosphorylated in fructose-6-phosphate (F6P) by the F1,6bP

phosphatase-1 molecule of F6P and 1 molecule of G3P are transformed into 1 molecule of erythrosis-4-phosphate (E4P) and 1 molecule of xylulose-5-phosphate (X5P) by glycolaldehyde transferase.

-1 molecule of sedoheptulose-1,7-biphosphate (S1,7bP) is synthesized from 1 molecule of E4P and 1 molecule of DHAP by sedoheptulose-bisphosphate aldolase.

-1 molecule S1,7bP is dephosphorylated in sedoheptulose-7-phosphate (S7P) by the sedoheptulose-1,7-bisphosphatase -1 molecule of S7P and 1 molecule of G3P are transformed into 1 molecule of R5P and 1 molecule of X5P by glycolaldehyde transferase -2 molecules of X5P are transformed into 2 molecules of Ru5P by the Ru5P-3-epimerase.

-1 molecule of R5P is isomerized into 1 molecule of Ru5P by Ru5P isomerase.

-3 molecules of Ru5P are phosphorylated by phosphoribulokinase with consumption of 3 molecules of ATP to form 3 molecules of RuBP.

During a cycle, the consumption of 9 ATP, 6 NADPH and 3 molecules of CO 2 makes it possible to produce 1 molecule of G3P (not used for the regeneration of RuBP). This molecule can therefore be used in various pathways of biosynthesis by the plant. It will serve in particular to starch production in chloroplasts, or it will be exported to the cytosol for serve for the synthesis of sucrose.

II. Photorespiration 1) General description

The photorespiratory cycle or C2 cycle is an essential metabolic pathway not only in higher plants but also in cyanobacteria and algae when grown in oxygen-rich environments (Eisenhut et al., 2008;Hagemann et al., 2016 andTimme 2016,). It is initiated by the oxygenase activity of RuBisCO which substitutes for carboxylase activity and leads to the production of one molecule of 2-phosphoglycolate (2PG) and one molecule of 3PGA. Because 2PG is toxic for plant by inhibiting several essential enzymes, such as triosephosphate isomerase and phosphofructokinase and sedoheptulose 1,7-bisphosphate phosphatase (see below, Anderson, 1971; Kelly and Latzko, 1976;Flügel et al., 2017), it must be removed and the carbon recycled. This process takes place in several subcellular compartments: chloroplast, peroxisome, mitochondrion and the cytosol and requires the cooperation of eight core photorespiratory enzymes (each enzyme will be detailed later) and several transporters [START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF]Bauwe et al. 2010;[START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF]Hodges et al., 2013;[START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF].

In this way, the 2PG and other potentially toxic metabolic intermediates (glyoxylate and glycolate, see below, [START_REF] Cook | Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate[END_REF]Chastain and Ogren, 1989;[START_REF] Servaites | Chemical inhibition of the glycolate pathway in soybean leaf cells[END_REF][START_REF] Gonzalez-Moro | Glycolate accumulation causes a decrease of photosynthesis by inhibiting RUBISCO activity in maize[END_REF] produced within the photorespiratory cycle are metabolized to produce useful 3PGA. However, this C2-cycle also leads to the release of CO 2 and NH 4 + while it requires ATP and NADH.

As mentioned above, the photorespiratory cycle is initiated by the RuBisCO's oxygenase activity thus producing 2PG and 3PGA. Since each enzyme will be detailed later, we will only briefly outline here the reactions taking place in each compartment. The 2PG is transformed in the chloroplast by PGLP1 and the resulting glycolate is transported out of the chloroplast by two transporters named PLGG1 and BASS6. The glycolate is then taken up into the peroxisomes where it is converted into glyoxylate (with production of a molecule of H 2 O 2 ) by the photorespiratory glycolate oxidase(s) (GOX). Then, the glyoxylate is converted into glycine via a glutamate:glyxoxylate aminotransferase (GGT1) and the glycine is transported into mitochondrion to be transformed into serine (with the production of CO 2 , NH 4 + and NADH) via the glycine decarboxylase (GDC) complex and the serine hydroxyl methyl transferase (SHMT1). The serine is transported into the peroxisome to be successively transformed into hydroxypyruvate (via a second aminotransferase, serine:glyoxylate aminotransferase or SGAT1) then into glycerate (by the hydroxypyruvate reductase, HPR1)

with consumption of NADH. Finally, the glycerate is transported into chloroplast by PGGL1 transporter, then the cycle ends in the chloroplast with the phosphorylation of glycerate to 3PGA (carried out by the glycerate kinase, GLYK) with the consumption of ATP (see reviews by Bauwe et al., 2010;Hodges et al., 2013, Hodges et al.,2016) (Figure 2).

The photorespiratory cycle represents a metabolic recycling pathway: (i) by recovering a molecule of 3PGA (with three carbon atoms) from two molecules of 2PG (four carbon atoms), 3PGA can re-enter the Calvin-Benson cycle to make other sugar phosphates or other organic molecules (see the review in [START_REF] Bauwe | Photorespiration has a dual origin and manifold links to central metabolism[END_REF]; (ii) by detoxifying the inhibitory metabolites produced in the course of the recycling process itself including 2PG, glyoxylate and glycolate. 2PG inhibits triose-phosphate isomerase (Anderson, 1971) and sedoheptulose 1,7-bisphosphate phosphatase and thus has a negative effect on the Calvin-Benson cycle (Flügel et al., 2017)) and it also inhibits the glycolytic enzyme phosphofructokinase (Kelly and Latzko, 1976). Glyoxylate has been shown to inhibit RuBisCO in vitro, perhaps by altering its activation state (Chastain and Ogren, 1989) while Cook et al 1985 concluded that glyoxylate inhibits activated RuBisCO [START_REF] Cook | Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate[END_REF]. It has been reported that the inhibition of glycolate pathway in soybean leaf cells led to the reduction of the intermediates of the Calvin-Benson cycle as well as the decrease of the photosynthesis, and glycolate accumulation in Maize is also reported to inhibit the activity of RuBisCO leading to decrease of photosynthesis [START_REF] Servaites | Chemical inhibition of the glycolate pathway in soybean leaf cells[END_REF][START_REF] Gonzalez-Moro | Glycolate accumulation causes a decrease of photosynthesis by inhibiting RUBISCO activity in maize[END_REF]. In addition, photorespiration also protects from photoinhibition by stably consuming reducing equivalents to reduce acceptor limitation and reactive oxygen species (ROS) generation under highlight conditions as well as by helping to avoid the inhibition of the synthesis of the D1 protein of Photosystem II (Takahashi et al., 2007;[START_REF] Takahashi | Photoprotection in plants: A new light on photosystem II damage[END_REF]. Photorespiration also has a positive effect on plant defense reactions against pathogen attack by producing H 2 O 2 (Rojas et al., 2012;[START_REF] Takahashi | Photoprotection in plants: A new light on photosystem II damage[END_REF]Rojas and Mysore, 2012;[START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF] and is involved in the control of stomatal conductance with respect to CO 2 availability (Eisenhut et al., 2017). However, photorespiration also leads to (i) the generation of two H 2 O 2 molecules which need to be detoxified by a peroxisomal catalase (CAT2) [START_REF] Queval | Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel[END_REF]; (ii) the liberation of assimilated carbon and nitrogen as CO 2 and NH 4 + ; (iii) the consumption of energy molecules (ATP and NADH). A part of the photorespiratory CO 2 produced by the activity of glycine decarboxylase (GDC) is probably re-assimilated by RuBisCO, and a part of the ammonium produced by the same enzyme will be re-assimilated by the activity of chloroplastic glutamine synthetase (GS2, which will be detailed later [START_REF] Wallsgrove | Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis[END_REF]). In addition, the photorespiration is the most important process in terms of carbon flow except the photosynthetic pathway (Bauwe et al., 2010;[START_REF] Peterhansel | Photorespiration Redesigned[END_REF], and about half of the photorespiratory CO 2 is not reassimilated but rather lost to the environment, leading to a significant different between the gross and net photosynthesis [START_REF] Timm | The regulatory interplay between photorespiration and photosynthesis[END_REF]. Therefore, the photorespiration has become a prime target for crop improvement over decades [START_REF] Evans | Improving Photosynthesis[END_REF][START_REF] Long | Meeting the global food demand of the future by engineering crop photosynthesis and yield potential[END_REF][START_REF] Orta | Redesigning photosynthesis to sustainably meet global food and bioenergy demand[END_REF].

Initial approaches to improve crop productivity by manipulating photorespiration are main focus on identifying or producing mutants with reduced or impaired photorespiratory enzyme expression. Since the first identification of Arabidopsis thaliana photorespiratory mutants phosphoglycolate phosphatase (PGLP) by Somerville and co-workers [START_REF] Somerville | A phosphoglycolate phosphatasedeficient mutant of Arabidopsis[END_REF], all key photorespiratory enzymes have been identified (Figure 2), for instance, serine:glyoxylate aminotransferase (SGAT), serine hydroxymethyl transferase (SHMT) and glycine decarboxylase (GDC) complex protein P (GLDP) (Somerville and Ogren, 1980;1981;1982). Up to now, by analyzing each mutant of the key photorespiratory enzymes, from the growth phenotypes of photorespiratory enzyme mutants and the development of air-grown plants, it can be demonstrated that, in general, blocking the photorespiratory cycle by the absence or sever reduction of photorespiratory enzyme does not improve but rather reduce the photosynthetic activity. Indeed, the absence or sever reduction of photorespiratory enzyme leads to various phenotypes including reduce growth, chlorosis or even lethality in air, while these phenotypes disappeared when grown in high CO 2 conditions which limit photorespiration by reducing the oxygenase activity of RuBisCO (Timm and Bauwe, 2013).

The variety of photorespiratory phenotypes will be detailed later.

Photorespiration is an essential high flux metabolic pathway in all oxygen-producing photosynthetic organisms. This pathway comprises eight core enzymes and expands to four cellular compartments, the chloroplast, the peroxisome, the mitochondrion and the cytosol.

Then, the photorespiratory pathway itself is a complex network. In addition to the complex of photorespiratory cycle itself and interaction with photosynthesis, it is also known to interact with and impact several plant metabolic pathways and functions including nitrate assimilation, the TCA cycle (Krebs cycle), C1 and amino acid metabolisms (Bauwe et al., 2010;Hodges et al., 2013, Hodges et al., 2016) (Figure 2). These interactions will also be detailed later. In fact, the variety of the photorespiratory enzyme mutant phenotype, to some extent indicates the importance of certain photorespiratory enzyme interaction with other metabolic pathway or suggests. However, right now, how photorespiratory cycle alone is regulated and how it interacts with and impacts on other plant metabolic pathways are two mechanism both little understood [START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF]. In C4 plants, photorespiration is much lower compared to C3 plant, since the CO 2 concentrating mechanism involving the PEPCase (phosphoenolpyruvate carboxylase) and two different cell types (bundle sheaths and mesophyll cells) leads to high CO 2 levels in the vicinity of the RuBisCO that reduces its oxygenase activity [START_REF] Yamori | Temperature response of photosynthesis in C3, C4, and CAM plants: Temperature acclimation and temperature adaptation[END_REF]. Although photorespiration is lower in C4 plants, it still appears to be essential for normal plant growth and development in ambient air. For example, a maize glycolate oxidase (GOX) mutant (Zmgo1) identified in maize exhibited a photorespiratory phenotype when grown in air (Zelitch et al., 2009). And in addition, using recombinant GOX proteins and isotope labeling it was revealed that GOX enzymes from a C3 (Arabidopsis) and a C4 (maize) plant showed similar kinetic properties and reaction mechanism of the photorespiratory [START_REF] Dellero | Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth[END_REF].

2) Enzymes and transporters of the photorespiratory cycle in

Arabidopsis thaliana

As stated earlier, the photorespiratory cycle in higher plants takes place in four subcellular compartments: chloroplast, peroxisome, mitochondrion and cytosol. It includes eight core enzymes: phosphoglycolate phosphatase (PGLP), glycolate oxidase (GOX), glutamate:glyoxylate aminotransferase (GGT), the glycine decarboxylase complex (GDC complex, composed of P, T, L, and H protein subunits), serine hydroxymethyl transferase (SHMT), serine:glyoxylate aminotransferase (SGAT), hydroxypyruvate reductase (HPR) and glycerate 3-kinase (GLYK) (Figure 2). The cycle also requires metabolite transporters to shuttle photorespiratory metabolites to the different subcellular compartments. Unlike the core enzymes, to date, only a few of the necessary transporters have been identified: two plastidial transporters both involving glycolate transport the glycolate/glycerate transporter (PLGG1) and a glycolate transporter (BASS6); plastidial 2OG/malate transporter 1 (DiT1) and plastidial glutamate-malate transporter 1 (DiT2-1): chloroplastic decarboxylate transporter system involved in ammonium reassimilation by the chloroplastic GS2/FD-GOGAT cycle; a mitochondrial transporter A BOUT DE SOUFFLE (BOU) which is believed to be required for GDC activity (Figure 2). Since the first established experimental setup for screening the photorespiratory mutants by Somerville and co-workers [START_REF] Somerville | An early Arabidopsis demonstration resolving a few issues concerning photorespiration[END_REF], many more mutants became available, which are not just restricted to the core photorespiratory cycle but also covers associated proteins, such as transporters or cofactor synthesis (Timm and Bauwe, 2013). Then, research on photorespiration has greatly benefited from the increasing availability of defective mutants in Arabidopsis thaliana. A summary of all Arabidopsis thaliana photorespiratory genes listed in this section can be found in Table 1. In the chloroplast, the oxygenase activity of the RuBisCO produces 1 molecule of 3PGA and 1 molecule of 2PG. The first step of the photorespiratory cycle is to transform the 2PG into glycolate, this is carried out by a phosphoglycolate phosphatase (PGLP). The Arabidopsis thaliana genome, contains thirteen putative PGLP-encoding genes. Two Arabidopsis genes, At5g36700 (AtPGLP1) and At5g47760 (AtPGLP2), both produce proteins exhibiting PGLP activity as detected using recombinant proteins [START_REF] Schwarte | Identification of the Photorespiratory 2-Phosphoglycolate Phosphatase, PGLP1, in Arabidopsis[END_REF]. Only a plastidial PGLP1 T-DNA knockout mutant (pglp1) has an altered leaf PGLP activity (40 times that of control, wild-type Col-0 rosettes) and a strong Class II (the variety of photorespiratory phenotype will be detailed later) photorespiratory phenotype restored by an extremely elevated CO 2 concentration of 10000 ppm [START_REF] Schwarte | Identification of the Photorespiratory 2-Phosphoglycolate Phosphatase, PGLP1, in Arabidopsis[END_REF]. PGLP2 is cytosolic and T-DNA knockout mutant did not show any developmental differences compared to wildtype plants [START_REF] Schwarte | Identification of the Photorespiratory 2-Phosphoglycolate Phosphatase, PGLP1, in Arabidopsis[END_REF]. Thus, PGLP1 is believed to be the photorespiratory PGLP enzyme of A. thaliana. Recently, in order to study the potential role of 2PG in the photosynthetic carbon metabolism, the antisense and overexpression lines showed fine adjustment of PGLP activity were generated and analyzed for the plants growth (Flügel et al., 2017). All the antisense lines (A1, A2 A8 lines) grew well in high CO 2 condition, while when grew in ambient air condition, all the lines showed slower growth phenotype. Among them, the A2 line which had a slightly reduction of PGLP activity also showed a slight growth reduction and could produce fertile seeds, while the A1 and A8, particularly A1 in which only had little PGLP activity left, showed more severe impairment without viable seeds production.

On the contrary to the antisense lines, the overexpression lines showed an improved plant growth indicated by the increased fresh and dry weight and also increase rosette diameter and leaf number (Flügel et al., 2017).

Glycolate oxidase (GOX)

Glycolate oxidase is a peroxisome enzyme containing a flavin mononucleotide (FMN) cofactor that catalyzes the transformation of glycolate to glyoxylate with the production of hydrogen peroxide. This enzyme evolves from a bacterial lactate oxidase and is a member of the α-hydroxy-acid oxidase superfamily [START_REF] Esser | Plant and animal glycolate oxidases have a common eukaryotic ancestor and convergently duplicated to evolve long-chain 2-hydroxy acid oxidases[END_REF]. In Arabidopsis, there are five GOX-related genes: At3g14420, At3g14415, At4g18360, At3g14130 and At3g14150

(encoding GOX1, GOX2, GOX3, HAOX1 and HAOX2, respectively). According to transcriptomic analysis, At3g14415 (AtGOX2) and At3g14420 (AtGOX1) are highly expressed in leaves and are the major GOX isoforms in Arabidopsis thaliana [START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF]Dellero et al., 2016a). GOX3, mainly expresses in roots and senescing leaves, can also function as a lactate oxidase and plays an important role in lactate metabolism [START_REF] Engqvist | GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis[END_REF].

HAOX1 and HAOX2 are mainly expressed in seeds and prefer medium-and long-chain hydroxyl acids as substrates [START_REF] Esser | Plant and animal glycolate oxidases have a common eukaryotic ancestor and convergently duplicated to evolve long-chain 2-hydroxy acid oxidases[END_REF]. Knockout mutant of each Arabidopsis GOX gene presented a residual 30% GOX activity and did not show any photorespiratory phenotype and they were more sensitive to Pseudomonas syringae (Rojas et al., 2012). Later Dellero et al. (2016a) showed that single GOX1 and GOX2 mutants exhibited 35% and 60% residual leaf GOX activity, respectively, and in order to identity the potential role of GOX genes involved in the photorespiratory cycle, an artificial miRNA GOX line was produced that knocked-down both leaf GOX1 and GOX2 genes of Arabidopsis (Dellero et al., 2016a).

The amiRgox1/2 line only had 5-10 % leaf GOX activity and showed a Class II photorespiratory phenotype in air that was reversed by elevated CO 2 (3000 ppm). The transfer of amiRgox1/2 plants from high CO 2 to ambient air lead to a large accumulation of glycolate (700-fold) and a reduced carbon allocation to sugars, organic acids, amino acids and also led to the early senescence of older leaves. Therefore, GOX1 and GOX2 appear to have redundant photorespiratory functions (Dellero et al., 2016a). However, by crossing gox1-1 and gox2-1 single mutant with cat2-2 mutant (mutant lacking the peroxisomal CATALASE 2 (cat2-2) displaying a photorespiratory phenotype), Kerchev and coworkers (2016) showed that cat2-2×gox2-1 plant exhibited weaker photorespiratory phenotype compared with the cat2-2 mutant while it was not the case for cat2-2×gox2-1. These observations indicated that GOX1 is the isoform predominantly involved in glycolate and H 2 O 2 production under conditions promoting photorespiration [START_REF] Kerchev | Lack of GLYCOLATE OXIDASE1, but Not GLYCOLATE OXIDASE2, Attenuates the Photorespiratory Phenotype of CATALASE2-Deficient Arabidopsis[END_REF].

Glutamate:glyoxylate aminotransferase (GGT)

Glutamate:glyoxylate aminotransferase, a peroxisomal enzyme, can transfer the amino group from glutamate to glyoxylate to form glycine and 2-oxoglutarate. In the photorespiratory cycle, this reaction is linked to the chloroplastic GS/GOGAT that produces glutamate and requires 2OG and involves two chloroplastic transporters, DiT1 and DiT2.1 (see below, the section "Transporter in photorespiratory cycle"). In Arabidopsis thaliana, two genes encode GGT, AtGGT1 (At1g23310) and AtGGT2 (At1g70580) [START_REF] Igarashi | Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[END_REF][START_REF] Liepman | Alanine Aminotransferase Homologs Catalyze the Glutamate:Glyoxylate Aminotransferase Reaction in Peroxisomes of Arabidopsis[END_REF].

AtGGT1 shows a 50-fold higher expression compared to AtGGT2 in rosette leaves [START_REF] Igarashi | Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[END_REF]. Both GGT recombinant proteins show an alanine:2-oxoglutarate aminotransferase and glutamate:pyruvate aminotransferase activities [START_REF] Liepman | Alanine Aminotransferase Homologs Catalyze the Glutamate:Glyoxylate Aminotransferase Reaction in Peroxisomes of Arabidopsis[END_REF]. The ggt1 knockout mutant exhibits a Class III photorespiratory phenotype, retarded growth and development in ambient air that is not seen when grown in high CO 2 [START_REF] Igarashi | Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[END_REF]Dellero et al., 2015a). The photorespiratory phenotype was also absent when the mutant was grown at a low light intensity or when seedlings were grown on in vitro plates supplemented with 3% sucrose [START_REF] Igarashi | Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[END_REF]. More recently, metabolic and physiological analyses were carried out on Arabidopsis ggt1 mutants to elucidate the mechanisms involved in the air-grown growth phenotype. It was found that air-grown ggt1 mutants exhibited reduced RuBisCO amounts and low photosynthetic activities. When, the mutants were transferred from high CO 2 to air, the increase in RuBisCO content observed in WT plants was not seen in the ggt1 mutants.

Glycine decarboxylase complex (GDC complex, composed of P, T, L, and H proteins)

The glycine produced in the peroxisomes is transported to the mitochondrion via unknown carriers. In the mitochondrion, two molecules of glycine are required to synthetize one molecule of serine by the combined actions of the glycine decarboxylase complex (GDC) and the serine hydroxymethyltransferase (SHMT). In fact, the GDC complex also known as the glycine cleavage multienzyme system is comprised of four proteins, three enzymes: P-protein (glycine decarboxylase); T-protein (aminomethyltransferase) and L-protein (dihydrolipoyl dehydrogenase) and a protein with no enzymatic activity called the H-protein (hydrogen carrier protein) [START_REF] Douce | The glycine decarboxylase system: A fascinating complex[END_REF]. This latter protein contains the lipoic acid (a shared substrate successively interacting with the P-, T-, and L-proteins). The whole reaction cycle requires a molecule of glycine, THF (tetrahydrofolate) and NAD + to produce one molecule of 5,10-CH 2 -THF, one molecule of CO 2 , a molecule of ammonium and one molecule of NADH.

The CO 2 and NH 4 + are released by the P-and T-proteins, respectively while NADH is produced by the L-protein (Figure 3). In Arabidopsis thaliana genome, there are two Pprotein genes (At2g26080 and At4g33010), two L-protein genes (At1g48030 and At3g17240), three H-protein genes (At1g32470, At2g35120 and At2g35370) and a single T-proteinencoding gene (At1g11860). GDC, which is the site of CO 2 and NH 4 + release, is not only linked to photosynthesis, N-assimilation and amino acid biosynthesis but it is also associated with one-carbon metabolism (Bauwe and Kolukisaoglu, 2003;[START_REF] Douce | The glycine decarboxylase system: A fascinating complex[END_REF].

The P-protein is a glycine decarboxylase containing a pyridoxal phosphate cofactor. It catalyzes the decarboxylation of glycine and leads to the liberation of CO 2 , along with the production of an amino-methylene moiety (Bauwe and Kolukisaoglu, 2003). The study of Pprotein knockout plants showed that mutants lacking both P-protein genes were not viable, even when grown in high CO 2 conditions. The most likely explanation of this lethality is that the P-protein functions not only within the photorespiration cycle but it could also important for one carbon metabolism. Both of them are essential for plant growth and development [START_REF] Engel | Deletion of Glycine Decarboxylase in Arabidopsis Is Lethal under Nonphotorespiratory Conditions[END_REF].

The T-protein is an aminomethyltransferase that transfers the amino-methylene to THF to generate 5,10-methylene-THF, along with the production of NH 4 + . Recently, it was revealed that the absence of T-protein led to an unconditional lethal phenotype similar to the double Pprotein mutant. However, an Arabidopsis knockdown mutant of the T-protein, retaining only 5% of leaf T-protein levels, still exhibited ~30% GDC activity. It was able to grow and propagate in ambient air and exhibited only slight symptoms of metabolic stress, including a higher glycine, a slightly lower photosynthetic performance and a slightly slower growth.

Furthermore, the overexpression of the T-protein in Arabidopsis did not bring about the expected stimulation of the photorespiratory cycle and so there was no improvement in photosynthetic performance and plant growth. Such results suggest that the GDC-T-protein has little/no control over the photorespiratory cycle [START_REF] Timm | T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis[END_REF].

The H-protein is a small hydrogen carrier protein that does not exhibit enzymatic activity.

Instead, it contains a lipoamide cofactor and works as a carrier to transfer electrons and intermediates between neighboring GDC complex protein, from P-via the T-to the L-protein, interacting with all three enzymes [START_REF] Douce | The glycine decarboxylase system: A fascinating complex[END_REF][START_REF] López-Calcagno | Overexpressing the H-protein of the glycine cleavage system increases biomass yield in glasshouse and field-grown transgenic tobacco plants[END_REF].

The L-protein is a FAD-containing flavoprotein with a dihydrolipoyl dehydrogenase that catalyzes the re-oxidation of the reduced lipoamide, which in turn is re-oxidized again by reducing NAD + to NADH (Bauwe and Kolukisaoglu, 2003). In contrast to the overexpression of the T-protein, the overexpression of either the H-or L-proteins in Arabidopsis both facilitate photorespiratory carbon flux and leads to an improvement of photosynthesis and plant biomass [START_REF] Timm | Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana[END_REF][START_REF] Simkin | Simultaneous stimulation of sedoheptulose 1,7-bisphosphatase, fructose 1,6-bisphophate aldolase and the photorespiratory glycine decarboxylase-H protein increases CO2 assimilation, vegetative biomass and seed yield in Arabidopsis[END_REF].

Serine hydroxymethyltransferase (SHMT)

In photorespiratory pathway, SHMT catalyzes the transfer of a 5,10-methylene-THF hydroxymethyl group to a glycine molecule to form a serine molecule and THF. Since this enzyme is a major part of my research during my PhD, it will be described in detail in the section "V. SHMT, Serine hydroxymethyltransferase".

Serine:glyoxylate aminotransferase (SGAT)

In the peroxisome, SGAT transfers an amide group from serine to glyoxylate to form a molecule of glycine and a molecule of hydroxypyruvate. In Arabidopsis thaliana, a single gene (SGAT1, At2g13360) encodes this enzyme. Knockout mutants for this gene have been identified and they show a photorespiratory phenotype (Somerville and Ogren, 1980a). Earlier results demonstrated that the SGAT recombinant protein could perform in vitro three types of aminotransferase activities (alanine:glyoxylate, serine:glyoxylate and serine:pyruvate), with 5-fold more serine:glyoxylate aminotransferase activity compared to the others [START_REF] Liepman | Peroxisomal alanine: Glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana[END_REF]. Other results show that SGAT can use also asparagine (Asn) as a substrate, and that it is the most efficient amide group donor for each SGAT activity [START_REF] Zhang | Characterization of Arabidopsis serine:glyoxylate aminotransferase, AGT1, as an asparagine aminotransferase[END_REF]. In addition, earlier work by Somerville and Ogren showed that leaves of the SGAT mutants (CS51, CS108 and CS117) assimilated 3-fold less CO 2 compared to control (wild-type) plants.

Each of these three mutants accumulated Gly and Ser while the amounts of starch and sucrose decreased compared to wild type plants (Somerville and Ogren, 1980b). Surprisingly, overexpression of SGAT1 in Arabidopsis led to a reduced photosynthetic CO 2 assimilation as well as to fewer leaves and reduced leaf expansion. The overexpressing lines had lower Asn content during the day but not at night, suggesting that the excess SGAT1 activity allowed more Asn to enter the peroxisome to increase photorespiration. This increased import of Asn nitrogen into the photorespiratory cycle was proposed to compete with leaf nitrogen export.

Therefore, it appears that SGAT activity must be well controlled to ensure adequate photorespiratory carbon flux and, at the same time, avoid negative effects on whole-plant nitrogen balance [START_REF] Modde | High serine:glyoxylate aminotransferase activity lowers leaf daytime serine levels, inducing the phosphoserine pathway in Arabidopsis[END_REF].

Hydroxypyruvate reductase (HPR)

Hydroxypyruvate reductase (HPR) catalyzes the NADH/NADPH-dependent reduction of hydroxypyruvate to glycerate. Since this enzyme is also a part of my thesis research work it will be described in detail in the section "VI. HPR, hydroxypyruvate reductase".

Glycerate kinase (GLYK)

Glycerate kinase (GLYK) is the final enzyme of the C2-cycle that produces 3PGA, thus connecting the photorespiratory cycle to several metabolic pathways including the Calvin mRNA can undergo a phytochrome-regulated alternative splicing that leads to a lightdependent change in subcellular localization. In the light, a longer GLYK1 mRNA is present that encodes the chloroplastic-localized GLYK1 protein, while in the darkness a shorter GLY1 mRNA produces a cytosolic GLYK1. The cytoplasmic GLYK1 accumulates in shade conditions and constitutes a photorespiratory bypass to alleviate fluctuating light-induced photoinhibition [START_REF] Ushijima | Light Controls Protein Localization through Phytochrome-Mediated Alternative Promoter Selection[END_REF].

b) Enzymes associated with the photorespiratory cycle

Ribulose-1,5-bisphosphate carboxylse/oxygenase (RuBisCO)

RuBisCO has two activities (carboxylase and oxygenase) thus allowing the fixation of CO 2 and O 2 . The carboxylase activity needs a molecule of RuBP and a molecule of CO 2 to produce 2 molecules of 3PGA that will be converted into C3-sugar phosphates (e.g.glyceraldehyde-3phosphate) that will be the starting point for the biosynthesis of all organic molecules of the plant. The oxygenase activity will use RuBP and O 2 as a substrate instead of CO 2 to produce one molecule of 3PGA and one molecule of 2PG [START_REF] Parry | Rubisco regulation: A role for inhibitors[END_REF]. This reaction is the starting point of the photorespiratory cycle. RuBisCO is composed of 8 small subunits (RBCS) of about 15 kDa and 8 large subunits (RBCL) of about 55 kDa. In A. thaliana, there are 4 nuclear genes encoding RBCS (RBCS1A, At1g67090; RBCS1B, At5g38430; RBCS2B, At5g38420; RBCS3B, At5g38410) and a single chloroplastic gene encoding the large subunit (RBCL, AtCG00490) [START_REF] Parry | Rubisco regulation: A role for inhibitors[END_REF]. The large subunits contain all of the structural information necessary for catalysis, while the function of the small subunits is still not clear.

RuBisCO has a low catalytic turnover (3 to 10 s -1 ) but it is the most abundant protein in leaves, so its abundance makes it possible to compensate for its low catalytic turnover to sustain sufficient rates for photosynthetic activity [START_REF] Parry | Rubisco regulation: A role for inhibitors[END_REF].

The carboxylase and oxygenase activities of RuBisCO are modulated by the amounts of CO 2 and O 2 in the vicinity of this enzyme [START_REF] Servaites | Chemical inhibition of the glycolate pathway in soybean leaf cells[END_REF]. Indeed, the global activity of RuBisCO is dependent on its activation state. RuBisCO must be activated by a fixation of a CO 2 to a lysine on the catalytic site (carbamylation) which is stabilized by simultaneously binding of a molecule of a molecule of divalent cation (Mg 2+ or Mn 2+ ). Then active site of the enzyme becomes "open", forming an active ternary complex [START_REF] Cleland | Mechanism of Rubisco: The Carbamate as General Base[END_REF][START_REF] Bloom | a Photorespiratory Pathway That Increases the Energy Efficiency of Photosynthesis[END_REF] and the "activated" residue will act as a base for transferring several protons from RuBP during the enzymatic reaction [START_REF] Cleland | Mechanism of Rubisco: The Carbamate as General Base[END_REF]. Subsequently, the fixation of CO 2 or O 2 on the activated enzyme containing RuBP (enol-RuBP formed) will lead to the formation of 2-carboxy-3-ketoarabinitol-1,5-bisphosphate or 2-peroxy-3-ketoarabinitol-1,5bisphosphate, respectively. These metabolites are unstable and in the presence of protons and water, they transform into 3PGA and 2PG [START_REF] Cleland | Mechanism of Rubisco: The Carbamate as General Base[END_REF]. In addition, RuBisCO can be inhibited by some inhibitors by binding to the catalytic site before or after carbamylation leading to the closure of the catalytic site. So these inhibitors, that must be removed, are sugar phosphates and they can be produced in the light, but they ate also present in darkness [START_REF] Parry | Rubisco regulation: A role for inhibitors[END_REF]. For this purpose, RuBisCO activase is required to allow the remove of the inhibitors from the active site of the enzyme to rapid format the carbamate at the active site of RuBisCO, by consuming ATP [START_REF] Spreitzer | RUBISCO: Structure, Regulatory Interactions, and Possibilities for a Better Enzyme[END_REF]. Moreover, the RuBisCO activase can be positively regulated by thioredoxin-f which makes the ATP/ADP ratio much less inhibitory [START_REF] Zhang | Mechanism of light regulation of Rubisco: A specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f[END_REF] and by electron transport through PSI [START_REF] Campbell | Electron transport through photosystem I stimulates light activation of ribulose bisphosphate carboxylase / oxygenase (rubisco) by rubisco activase[END_REF], and by illumination [START_REF] Lan | Light-dependent changes in ribulose bisphosphate carboxylase activase activity in leaves[END_REF].

Catalase (CAT)

Catalase is located in peroxisome where it converts GOX-generated H 2 O 2 to H 2 O and O 2 . In Arabidopsis, there are 3 catalase encoding genes: CAT1, At1g20630; CAT2, At4g35090; CAT3, At1g20620. Among them, CAT2 and CAT3 are highly expressed genes in leaves with CAT1

gene expression being less abundant. [START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF]. Among three genes, the cat2 mutants with 10-15% residual catalase activity in Arabidopsis leaves has been identified [START_REF] Queval | Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel[END_REF]. They showed growth retardation in the air (in Class III phenotype) that was absent in high CO 2 (4.5 mL.L -1 ). In addition, it was interesting that when growing under ambient air with moderate light intensity, cat2 mutants exhibited a photoperiod-dependent leaf lesion phenotype that was exacerbated by increasing the photoperiod from 8h to 12h to 18h of light [START_REF] Queval | Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel[END_REF]. Moreover, these conditions led to a further accumulation of oxidized glutathione and perturbed intracellular redox state [START_REF] Queval | Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel[END_REF]. These data therefore suggest that CAT2 plays a major role in scavenging the photorespiratory H 2 O 2 to regulate oxidative signaling and environmental conditions enhancing the photorespiratory flux can trigger peroxisomal H 2 O 2 production and ultimately lead to cell death in cat2 mutants [START_REF] Queval | Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel[END_REF]. Moreover, transcriptomic analyzes and salicylic acid assays showed that the appearance of cell death in long days in these mutants was due to a photoperiod-dependent activation of genes in the salicylic acid biosynthetic pathway, causing its accumulation in the leaves [START_REF] Mhamdi | Catalase function in plants: A focus on Arabidopsis mutants as stress-mimic models[END_REF]. These data are in favor of a major role for the CAT2 gene in the regulation of oxidative stress in A. thaliana and the activation of the salicylic acid pathway by H 2 O 2 signaling.

Glutamine synthetase 2 (GS2)

GS2 plays a major role in the re-fixation of ammonia released by the mitochondrial GDC during the photorespiratory cycle. It combines glutamate and ammonium to generate glutamine along with the consumption of ATP [START_REF] Wallsgrove | Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis[END_REF]. In Arabidopsis thaliana, there are six genes encoding GS proteins; five GS1 genes (GS1-1, At5g37600; GS1-2, At1g66200; GS1-3, At3g17820; GS1-4, At5g16570; GS1-5, At1g48470) and one GS2 gene (At5g35630) [START_REF] Wilkinson | Differential expression of glutamine synthetase genes during the senescence of Arabidopsis thaliana rosette leaves[END_REF]. GS1 genes are expressed in the cytosol while the GS2 is expressed in the chloroplast [START_REF] Moison | Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis[END_REF]. The expression of GS2 in Arabidopsis rosette leaves is 10-fold higher than GS1 and this is further increased under high nitrogen conditions [START_REF] Lothier | The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting[END_REF]. Though GS2 mutants have not yet been reported in Arabidopsis thaliana at present, mutants for the corresponding gene in other species have been characterized. EMS mutant lines in Lotus japonicus displayed normal growth and development in elevated CO 2 conditions, but they had stress symptoms when transferred to ambient CO 2 . The mutants containing 30% of GS2 activity in leaves accumulated 50-fold more ammonia than the control after one day transfer [START_REF] Orea | Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase[END_REF]. The results were consistent with those of the barley GS2 mutant that grew normally under high CO 2 but could not grow in ambient air (in Class II phenotype). After a 30 min transfer from high CO 2 to ambient air, the concentration of ammonia increased 50-fold and the rate of CO 2 fixation was decreased to 40% when compared to wild-type plants [START_REF] Wallsgrove | Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis[END_REF]. Such results demonstrate that GS2 plays a major role in photorespiratory metabolism.

Ferredoxin-dependent glutamate synthase1 (Fd-GOGAT1)

The Ferredoxin-dependent glutamate synthase 1 (Fd-GOGAT1) is required in photorespiration and plays an important role in GS/GOGAT cycle for the generation of Glutamate used by the photorespiratory enzyme GGT [START_REF] Coschigano | Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primar y Nitrogen Assimilation[END_REF]. It catalyzes the transfer of an amide group from the glutamine on 2OG to form two molecules of glutamate, along with the cost of power reducer in the form of ferredoxin (Fd-GOGAT) or in the form of NADH (NADH-GOGAT). Then one molecule of glutamate re-enters the GS/GOGAT cycle, and the other molecule of glutamate can be used for other processes including photorespiration, amino acid biosynthesis, and proteins synthesis [START_REF] Renné | The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2[END_REF]. Arabidopsis thaliana contains two genes (Fd-GOGAT1, At5g04140; Fd-GOGAT2, At2g41220) encoding two Fd-GOGAT proteins localized in chloroplasts. Fd-GOGAT1 is the major foliar enzyme and Fd-GOGAT2 is rather in the roots [START_REF] Coschigano | Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primar y Nitrogen Assimilation[END_REF]. The first Fd-GOGAT EMS mutants (CS30, CS103 and CS113) containing 5% glutamate synthase activity in the leaves compared to the WT showed a Class II photorespiratory phenotype which was absent under high-CO 2 growth conditions (Somerville and Ogren, 1980b).

Subsequently, the mutation mapping revealed that the deficient in the Fd-GOGAT1 gene indicating that Fd-GOGAT1 was essential for photorespiration (Coshigano et al.1998).

In addition, these mutants (CS30, CS103 and CS113) had a net 3-fold lower CO 2 uptake compared to control, as well as a decrease of glutamate amount and a high accumulation of glutamine and ammonium (Somerville and Ogren, 1980b) and these data suggested that Fd-GOGAT1 was the predominant isoform involved in the production of glutamate for the photorespiratory cycle and for re-assimilation of photorespiratory ammonium via the GS-GOGAT cycle.

A point mutation line glu1-201 for the Fd-GOGAT1 gene (mutated at L1270F) led to a decreased SHMT activity suggesting a regulation by Fd-GOGAT1 in the mitochondrion.

Further experiments demonstrated that this regulation occurred by a direct interaction with SHMT1. This interaction seemed to be required for mitochondrial SHMT activity and L1270

was important since it was absent in glu1-201 L1270F [START_REF] Jamai | Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase[END_REF].

c) Transporters of the photorespiratory cycle

Since the photorespiratory cycle spans three different subcellular compartments and the cytosol, metabolic intermediates must be transported in and out of them.

Chloroplastic decarboxylate transporters (DiT1 and DiT2.1)

The first-identified membrane-transport system involved in the photorespiratory cycle was the chloroplastic decarboxylate transporter system. It includes two transporters, DiT1 and DiT2.1 [START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF][START_REF] Renné | The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2[END_REF][START_REF] Eisenhut | Plastidial metabolite transporters integrate photorespiration with carbon, nitrogen, and sulfur metabolism[END_REF]. These two distinct transporters have overlapping substrates that are moved through the chloroplastic envelope membrane. DiT1 imports 2OG into the chloroplast in exchange for malate. Then the 2OG can be used to make glutamate by the GS2/GOGAT cycle [START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF]. The DiT2.1 transporter is specific for the same substrates as DiT1, but exports in addition glutamate from the chloroplast by the GS2/GOGAT cycle in exchange for malate [START_REF] Eisenhut | Plastidial metabolite transporters integrate photorespiration with carbon, nitrogen, and sulfur metabolism[END_REF][START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF][START_REF] Renné | The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2[END_REF]. These chloroplastic decarboxylate transporters play an important role in ammonium re-assimilation (generated by the photorespiratory cycle) via the GS/GOGAT cycle. In Arabidopsis, there is only one gene encoding DiT1 (AtpOMT1:

At5g12860) [START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF][START_REF] Renné | The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2[END_REF] and although it is believed to be important during photorespiration, the Arabidopsis DiT1 T-DNA mutant omt1 can grow in ambient air without any photorespiratory phenotype (in Class IV phenotype) [START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF]. However, the tobacco DiT1 antisense mutant exhibits slower growth and chlorosis especially around the veins in ambient air with an increase of 2OG and a decrease of amino acid contents. This phenotype is absent when the tobacco mutant is transferred to high CO 2 air [START_REF] Schneidereit | Antisense repression reveals a crucial role of the plastidic 2-oxoglutarate/malate translocator DiT1 at the interface between carbon and nitrogen metabolism[END_REF][START_REF] Eisenhut | Plastidial metabolite transporters integrate photorespiration with carbon, nitrogen, and sulfur metabolism[END_REF]. Arabidopsis has two genes encoding DiT2 (AtpDCT1 (DiT2-1): At5g64290; AtpDCT2 (DiT2-2); At5g64280) [START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF][START_REF] Renné | The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2[END_REF]. The dit2-1 knockout mutants in Arabidopsis display a typical Class II photorespiratory phenotype: they could not grow in ambient air but growth is restored in high CO 2 while dit2-2 mutants did not show any phenotype [START_REF] Renné | The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2[END_REF].

Plastidial glycolate-glycerate transporter (PLGG1)

PLGG1 was identified as a glycolate/glycerate transporter situated in the inner envelope of the chloroplast membrane [START_REF] Pick | PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters[END_REF]. It probably works as an exchange shuttle, exporting glycolate to the cytosol while importing glycerate into the chloroplast (see the review in [START_REF] Eisenhut | Plastidial metabolite transporters integrate photorespiration with carbon, nitrogen, and sulfur metabolism[END_REF]). In Arabidopsis thaliana, PLGG1 was the first core photorespiratory transporter identified and the Arabidopsis genome contains a single PLGG gene, At1g32080 [START_REF] Pick | PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters[END_REF]. A T-DNA knockout mutant plgg1-1 used to investigate its function showed a typical Class II photorespiration phenotype (retarded growth and chlorotic leaves) that was rescued in high CO 2 containing air [START_REF] Pick | PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters[END_REF]. The role of PLGG1 was tested in vitro and in vivo by transport assays using 14 C-glycerate and 18 O 2 -labelling, respectively.

For example, in WT, the 18 O-labelling of glycolate reached to a plateau after exposure to 18 O 2 for 30-s, and did not increase further in chloroplasts, and the 18 O 2 -label glycerate was increased slowly. In contrast, in plgg1-1 mutant plants, 18 O-label glycolate was continuous accumulated even after 2 mins in chloroplasts, meanwhile, the 18 O 2 -label glycerate was not observed. This demonstrated that glycolate and glycerate flux were impaired in plgg1-1 plants [START_REF] Pick | PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters[END_REF].

Bile acid sodium symporter 6 (BASS6)

A co-expression analysis using Arabidopsis gene expression data led the identification of PGGL1 (Pick et al., 2012) [START_REF] South | Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana[END_REF]. Metabolite analysis and genetic complementation demonstrated that BASS6 could transport glycolate. The bass6×plgg1 double knock-out mutant line showed a stronger growth defect, higher glycolate accumulation and a lower photosynthesis rate compared to each single mutant. It was concluded that BASS6 and PLGG1 both take part in the export of glycolate from the chloroplast, while only PLGG1 is responsible for glycerate import into chloroplast [START_REF] South | Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana[END_REF].

A BOUT DE SOUFFLE (BOU)

A BOUT DE SOUFFLE (BOU), located in the mitochondrial membrane, was another identification of a transporter based on the co-expression analysis with the photorespiratory genes in leaf tissues [START_REF] Eisenhut | Arabidopsis A BOUT de SOUFFLE is a putative mitochondrial transporter involved in photorespiratory metabolism and is required for meristem growth at ambient CO2 levels[END_REF]. BOU belongs to the mitochondrial carrier family and was previously predicted to function as a carnitine/acylcarnitine carrier [START_REF] Lawand | Arabidopsis A BOUT DE SOUFFLE, which is homologous with mammalian carnitine acyl carrier, is required for postembryonic growth in the light[END_REF]. The knockout mutant bou-2 exhibited the Class III photorespiratory phenotype, strongly reduced or fully inhibited growth and chlorotic leaves in ambient air however could be restored by the elevated CO 2 [START_REF] Eisenhut | Arabidopsis A BOUT de SOUFFLE is a putative mitochondrial transporter involved in photorespiratory metabolism and is required for meristem growth at ambient CO2 levels[END_REF]. In the bou-2 mutant, the significant accumulation of glycine and the degradation of P-protein (one subunit of glycine decarboxylase (GDC)) were reflected in strongly reduced glycine decarboxylase activity. In addition, at the shoot apical meristem organization of the seedlings, the absent of BOU protein led to the arrest of the cell division and further the plant growth arrest in ambient air condition [START_REF] Eisenhut | Arabidopsis A BOUT de SOUFFLE is a putative mitochondrial transporter involved in photorespiratory metabolism and is required for meristem growth at ambient CO2 levels[END_REF]. Up to now, although the specific substrate of the BOU transporter protein is still not clear, there enough evidence proves that the BOU protein is essential for the photorespiratory cycle, and is required for the activity of glycine decarboxylase and meristematic growth in mitochondrion [START_REF] Eisenhut | Arabidopsis A BOUT de SOUFFLE is a putative mitochondrial transporter involved in photorespiratory metabolism and is required for meristem growth at ambient CO2 levels[END_REF].

3) The variety of photorespiratory phenotypes

Over recent decades, since the first set of photorespiratory mutants was established by Chris Somerville and colleagues [START_REF] Somerville | An early Arabidopsis demonstration resolving a few issues concerning photorespiration[END_REF] who developed an experimental setup for photorespiratory mutants screen, the mutants of each photorespiratory cycle core enzyme gene and several associated processes have been identified, isolated and studied from many kinds of plants. With respect to Arabidopsis, this genetic approach allowed the identification of all core photorespiratory genes and led to a better comprehensive understanding of the photorespiratory process. A "photorespiratory phenotype" was used by Somerville as a screen to isolate the first photorespiratory mutants from EMS-treated seeds. The general phenotype used was a growth defect in ambient air (retarded growth, stunted plants, chlorosis, even lethality) that was absent in elevated CO 2 conditions [START_REF] Somerville | An early Arabidopsis demonstration resolving a few issues concerning photorespiration[END_REF]. Due to the increase in mutant availability and the diversity of photorespiratory gene mutants it became apparent that there were different degrees of photorespiratory phenotypes (see Timm and Bauwe, 2013).

Although each mutant exhibits to a certain degree the universal photorespiration phenotype, distinct phenotypic characters were observed depending on the photorespiratory gene. Based on the severity of the phenotype, four subclasses of phenotypic symptoms have been defined

(Table 2 and Fig. 4) (Timm and Bauwe, 2013).

Class I: Unconditional lethal mutants

This class contains the mutants that do not recover in elevated CO 2 . For the moment, three mutants have been reported showing an unconditional lethal phenotype. Two of them are associated with GDC, including a GDC-P double knockout mutant (gldp1xgldp2) [START_REF] Engel | Deletion of Glycine Decarboxylase in Arabidopsis Is Lethal under Nonphotorespiratory Conditions[END_REF] and the GDC-T knockout mutant (gldt1-2) [START_REF] Timm | T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis[END_REF]. The recent study on gldt1-2 showed that this mutant could not be rescued even under 1% CO 2 , while the germination could occur, the growth stopped before photosynthesis starts (Figure 4B) [START_REF] Timm | T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis[END_REF]. The third lethal mutant is the mitochondrial SHMT double mutant (shm1xshm2) (Engel et al., 2011). The double mutant shm1xshm2 was reported to present a lethal phenotype even under high-CO 2 growth condition even if it could survive on ½ MS medium containing 1% sucrose (Figure 4A). after germination and right, after 8 weeks) (Engel et al., 2011) and gldt1 mutant in high CO 2 on ½MS for 2 weeks. Class II: (C) and (D) Phenotype of mutants glyk1, after 8 weeks of growth in high CO 2 (left, 1% CO 2 ), then 2 weeks after transfer to air (right, appearance of chlorosis) (Timm et al., 2012) and the phenotype of amiRgox1/2 after 6 weeks of growth (Dellero et al., 2016a). Class III: (E) The phenotype of mutant ggt1 and hpr1 after 5 weeks of growth (ggt1, Dellero et al., 2015a and hpr1 grown in our lab). Class IV: (F) phenotype of mutant hpr2-1 after 8 weeks of growth in air [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. 4C). Glycolate oxidase double knockdown mutant (amiRgox1/2; Dellero et al., 2016a) also showed strong stunted and growth retarded in normal air, but the phenotype recovered under high-CO 2 condition (Dellero et al., 2016a) (Figure 4D).

Class III: viable in air but slowly growing

The mutants in this class include: glutamate:glyoxylate aminotransferase1 (ggat1; Dellero et al., 2015a;[START_REF] Igarashi | Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[END_REF], hydroxypyruvate reductase 1 (hpr1; [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF], catalase 2 (cat2; [START_REF] Queval | Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel[END_REF] and plastidial glycolate transporter (bass6; [START_REF] South | Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana[END_REF]. All of the mutants in this class are viable in normal air but they show only slight signs of a photorespitory phenotype (limited chlorosis and/or growth retardation in normal air but absent in high CO 2 ). In addtion, these mutants still have an altered photorespiratory metabolism and a decrease of photosynthetic CO 2 assimilation (Timm and Bauwe, 2013). For example, the ggat1 and hpr1 mutants were viable in air and only exhibited reduced growth in air that was not seen when they were grown in high-CO 2 growth condition (Dellero et al., 2015a) (Figure 4E, the hpr1 phenotype comes from our lab).

Class IV: no photorespiratory phenotype

The mutants in this class covers photorespiratory glycolate oxidase single mutants (gox1, gox2, gox3; Dellero et al., 2016a;Rojas et al., 2012 ); GDC-P single mutant (gldp1, gldp2; [START_REF] Engel | Deletion of Glycine Decarboxylase in Arabidopsis Is Lethal under Nonphotorespiratory Conditions[END_REF]; two hydroxypyruvate reductase single mutants (hpr2, hpr3; [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]; serine hydroxymethytransferase 2 (shm2; Engel et al., 2011); plastidial 2OG/malate transporter1 (omt1; [START_REF] Taniguchi | Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana[END_REF]. These mutants appear to grow normally in air.

The lack of a photorespiratory phenotype in air indicates that these enzymes either do not function in the photorespiratory cycle or have a redundant photorespiratory role due to the presence of compensatory isoforms (Timm and Bauwe, 2013). For example, the Figure 4F, hydroxypyruvate reductase 2 mutant (hpr2) can grow normally in air condition without photorespiratory phenotype [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF].

III. Interactions between photorespiration and other primary metabolic pathways

In plant leaves, photorespiration can interact with several primary metabolisms. It interacts with the Calvin-Benson cycle via RuBisCO which produces 2PG which is the initial reaction that starts the photorespiratory cycle and via the production of 3PGA that can be used to regenerate Calvin-Benson cycle intermediates. As stated earlier certain photorespiratory metabolites have the potential to inhibit certain Calvin cycle enzymes and/or directly RuBisCO (Anderson, 1971;Flügel et al., 2017;Kelly and Latzko, 1976;Chastain and Ogren, 1989;[START_REF] Cook | Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate[END_REF][START_REF] Servaites | Chemical inhibition of the glycolate pathway in soybean leaf cells[END_REF][START_REF] Gonzalez-Moro | Glycolate accumulation causes a decrease of photosynthesis by inhibiting RUBISCO activity in maize[END_REF] while reduced photorespiratory fluxes negatively impact CO 2 assimilation (e.g. Takahashi et al., 2007;Timm et al., 2012;Dellero et al., 2015a). Photorespiratory 3PGA feeds into the biosynthesis of complex sugars and other organic compounds. In mitochondria, the Krebs cycle competes with GDC for NAD + while photorespiratory NADH can inhibit NADdependent Krebs cycle enzymes. In addition, photorespiration has close relationship with C1 metabolism, N-assimilation and amino acid biosynthesis via the tetrahydrofolate (THF, essential for methylation reactions in plants), the GOGAT1/GS2 cycle, and Ser/Gly/Glu catabolism and anabolism, respectively. These different interactions are detailed in the following sections.

1) Interactions between photorespiration and photosynthesis

Among these interactions, the most obvious is photorespiration and photosynthetic CO 2 assimilation (Figure 5). A major link occurs via RuBisCO, since it is involved in both photosynthetic CO 2 assimilation and photorespiration. The balance between CO 2 assimilation and photorespiration depends on the CO 2 to O 2 ratio around the RuBisCO in the chloroplast.

Photorespiration produces one molecule of useful 3PGA from 2 molecules of toxic photorespiratory 2PG with the potential loss of a carbon (as CO 2 ) if not reassimilated by

RuBisCO. The 3PGA produced by photorespiration can return to the Calvin-Benson cycle to regenerate RuBP or be used for the biosynthesis of other organic carbon-based molecules.

It is known that the interruption of photorespiratory carbon flux dramatically reduces photosynthetic carbon flux. To date, all photorespiratory mutants with an obvious phenotype in ambient air, show a decrease, to varying degrees, of photosynthesis CO 2 assimilation rate.

For photorespiratory mutants belonging to class II, there is a significant inhibition of net CO 2 assimilation in ambient air and even in the class III, the mutants also show an obvious inhibition of net CO 2 assimilation (e.g. Somerville and Ogren, 1980, 1981[START_REF] Somerville | An Arabidopsis thaliana mutant defective in chloroplast dicarboxylate transport[END_REF]Chastain and Ogren, 1989;Timm et al., 2012;Dellero et al., 2015a;Dellero et al., 2016a). The decrease of net CO 2 fixation, to some extent, reflects a reduced carboxylase activity of RuBisCO obviously. This can occur by a number of different mechanisms, for example, a decrease of the RuBisCO activation state, a decrease of the amount of RuBisCO protein, a decrease of the CO 2 concentration around the RuBisCO or a decrease in the recycling of RuBP by the Calvin-Benson cycle. For example, in Arabidopsis thaliana, SHMT1, Fd-GOGAT1 and GDC mutants exhibit a large decrease in leaf RuBP amounts after 30 min of light when transferred to 50% O 2 in air (Chastain and Ogren, 1989). And in rice, GOX1 (GLO1) antisense lines showed a reduced net CO 2 assimilation rate in ambient air that correlated with a reduced expression of genes encoding RuBisCO activase and the subsequent decrease of the activation state of RuBisCO (Xu et al., 2009). In Arabidopsis thaliana, an artificial miRNA GOX line (amiRgox1/2), with 5% of leaf GOX activity, when transferred from high CO 2 to ambient air showed a 50% decrease of photosynthetic CO 2 assimilation compared with control plants that was associated with a decrease of the amount of RuBisCO protein (Dellero et al., 2016a).

Moreover, Dellero et al. (2015a) showed that Arabidopsis ggt1 mutants only had 50% net CO 2 assimilation rates compared to the WT, and this was also correlated with the decline of RuBisCO protein contents (Dellero et al., 2015a).

The build-up of photorespiratory intermediates can also have a negative impact on photosynthetic CO 2 assimilation. Perhaps the most "toxic" intermediate is 2PG which has been shown to inhibit a number of enzymes some of which are involved in the Calvin-Benson cycle. Recently (Flügel et al., 2017), it has been confirmed that 2PG inhibits the triosephosphate isomerase (TPI) (originally observed in vitro by Anderson, 1971) and that it also inhibits sedoheptulose 1,7-bisphosphate phosphatase (SBPase) (both Calvin-Benson cycle enzymes required for regenerating RuBP) and it is proposed that 2PG feeds back on the Calvin-Benson cycle thereby altering the allocation of photosynthates between RuBP regeneration and starch synthesis by inhibiting the two Calvin-Benson cycle enzymes (Flügel et al., 2017); furthermore, 2PG can also inhibit the glycolytic enzyme phosphofructokinase (Kelly and Latzko, 1976) that is downstream from starch degradation and this inhibition could also be important in the proposed regulation of starch synthesis by 2PG. Glyoxylate can inhibit the in vitro activity of RuBisCO purified from spinach or Arabidopsis thaliana isolated chloroplasts [START_REF] Cook | Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate[END_REF]Chastain and Ogren, 1989) while in spinach chloroplasts, glyoxylate appeared to inhibit RuBP regeneration without affecting RuBisCO activity (Mulligan et al., 1983) Another in vivo study indicated that accumulation of glyoxylate inhibited the light activation state of RuBisCO [START_REF] Campbell | Electron transport through photosystem I stimulates light activation of ribulose bisphosphate carboxylase / oxygenase (rubisco) by rubisco activase[END_REF]. Moreover, the increased glyoxylate levels of GOX-silenced transgenic rice lines were negatively correlated with the net photosynthetic CO 2 assimilation rate by deactivating RuBisCO activity (Lu et al., 2013). Glycolate leads to the inhibition of metabolic processes that impair CO 2 assimilation (González-Moro et al., 1997). More recently, experimental evidence indicates that glycolate can interact in vivo with the acceptor side of PSII to protect the plant from photoinhibition in high light. It was demonstrated that glycolate could replace bicarbonate on the non-heme iron of PSII thus leading to an increased Q A /Q A -(primary quinone acceptor) ratio. The increased redox potential of Q A /Q A -would limit O 2 -induced photodamage when chloroplastic CO 2 becomes limiting such as when plants are subjected to excess light or drought stress conditions (Messant et al., 2018). and photoinhibition that will be exuberated by a negative effect on D1 protein synthesis.

RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5bisphosphate; 3PGA, 3-phosphoglycerate; 2PG, 2-Phosphoglycolate; ROS, Reactive oxygen species; TPI, Triose-phosphate isomerase. SBPase, sedoheptulose 1,7-bisphosphate phosphatase.

Impaired photorespiration also leads to the generation of harmful ROS. This is due to a decline of NADH and ATP consumption direct via the photorespiratory pathway and an indirect negative effect on carbon recycling for RuBP regeneration. Together, these lead to a transient accumulation of chloroplastic NADPH and ATP. The reduced NAD + and NADP + levels would induce alternative photosynthetic electron flow while the increase in ATP would maintain a high pH gradient and thus inhibit photosynthetic electron transfer. Such conditions will favor the production of ROS that can damage PSII [START_REF] Muller | Non-Photochemical Quenching. A Response to Excess Light Energy[END_REF][START_REF] Horton | Molecular design of the photosystem II light-harvesting antenna: Photosynthesis and photoprotection[END_REF]. Upon a shift from high CO 2 to ambient air, mutants impaired in SHMT1, GLYK1, Fd-GOGAT1 and DiT2 proteins in Arabidopsis showed a strong increase in Non-Photochemical Quenching (NPQ) (Takahashi et al., 2007). An increased NPQ has also been reported after a similar transfer in ggt1 and amiRgox1gox2 leaves [START_REF] Dellero | Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth[END_REF]Dellero et al., 2016a).

NPQ occurs in all photosynthetic eukaryotes to help, regulate and protect the photosynthetic light reactions in an environment where light energy absorption exceeds the capacity of light utilization [START_REF] Muller | Non-Photochemical Quenching. A Response to Excess Light Energy[END_REF]. In general, NPQ can be composed into three processes: heat dissipation (qE, associated with a pH gradient and the xanthophyll cycle), state transitions (qT, involving the reversible phosphorylation of PS-II antenna proteins (LHCII) regulating their association either with PSII or PSI), and photoinhibition (qI, associated with the degradation of PSII) [START_REF] Maxwell | Chlorophyll fluorescence-a practical guide[END_REF][START_REF] Muller | Non-Photochemical Quenching. A Response to Excess Light Energy[END_REF][START_REF] Horton | Molecular design of the photosystem II light-harvesting antenna: Photosynthesis and photoprotection[END_REF]. However, even in the presence of an increased NPQ after transfer from high CO 2 to air, an accelerated PSII photoinhibition was reported for each photorespiratory mutant (shm1, glyk1, fd-gogat1 and dit2) and this was explained by a suppressed PSII D1 protein repair attributed to the inhibition of the synthesis of D1 at the translational level (Takahashi et al., 2007). Moreover, in Arabidopsis thaliana, the over expression of certain photorespiratory proteins, for example, overexpression of the glycine decarboxylase GDC-H or GDC-L proteins, resulted in increased CO 2 assimilation and improved plant growth, which is likely due to a more intense photorespiratory activity leading to lower glycolate and glycerate contents then lower inhibition of photosynthesis (Timm et al., 2012a;[START_REF] Timm | Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana[END_REF]. Furthermore, another study on the simultaneous overexpression of GDC-H, fructose 1,6-bisphophate aldolase (FBPA) and sedoheptulose 1,7-bisphosphatase (SBPase) significantly increases the CO 2 assimilation and biomass, and also results in an increased seed yield at high light for Arabidopsis [START_REF] Simkin | Simultaneous stimulation of sedoheptulose 1,7-bisphosphatase, fructose 1,6-bisphophate aldolase and the photorespiratory glycine decarboxylase-H protein increases CO2 assimilation, vegetative biomass and seed yield in Arabidopsis[END_REF]. And recently, the overexpression of GDC-H protein in tobacco plant also improves the yield not only in glasshouse condition but also under field condition (Patricia E. [START_REF] Lopez-Calcagno | Overexpressing the H-protein of the glycine cleavage system increases biomass yield in glasshouse and field-grown transgenic tobacco plants[END_REF].

2) Interactions between photorespiration and the Krebs cycle

In the light, 3PGA is produced by RuBisCO carboxylase and oxygenase activities, corresponding to photosynthesis and photorespiration, respectively. This 3PGA can enter the Calvin-Benson cycle where it is converted into glyceraldehyde-3-phosphate (G3P) which can be used to make sucrose in the cytosol, and starch in chloroplasts (see [START_REF] Ruan | Sucrose Metabolism: Gateway to Diverse Carbon Use and Sugar Signaling[END_REF]. They can also feed into the glycolytic pathway to make pyruvate which is transported from the cytosol into the mitochondrial matrix where it is metabolized to acetyl-CoA for the Krebs cycle. This (Figure 6). The Krebs cycle also produces 2OG that can be used by the Fd-GOGAT1/GS2 cycle as a substrate for the re-assimilation of ammonia released by the mitochondrial GDC complex during photorespiration.

decarboxylation
Photorespiration can also affect the Krebs cycle (Figure 7). It has been demonstrated that PDH activity was down-regulated by NH 4 + , which is a produced in high quantities within the mitochondria by the GDC complex as part of the photorespiratory cycle. It also has been

shown that PDH kinase (PDK) activity of mitochondria isolated from young pea seedlings can be enhanced in the presence of NH 4 Cl (from 100 μM) [START_REF] Schuller | Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex[END_REF]. And the data from [START_REF] Thelen | Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues[END_REF] showed that PDK of Arabidopsis thaliana could phosphorylate and inhibit maize PDH. Therefore, these data indicate that NH 4 + accumulation in mitochondria by photorespiration can down-regulate the activity of PDH via phosphorylation by PDK. Besides, the activity of several Krebs cycle enzymes can also be down-regulated in the light by the increased mitochondrial NADH/NAD + ratio due to NADH production via the GDC complex. Indeed, PDH activity from maize and pea was inhibited by high NADH concentrations [START_REF] Thelen | Partial Purification and Characterization of the Maize Mitochondrial Pyruvate[END_REF]. ICDH activity was also inhibited by high NADH/NAD + ratios under light [START_REF] Igamberdiev | Regulation of NAD-and NADPdependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves[END_REF]. These observations were consistent with potato GDC antisense lines (via 14 C-labeling experiments) that exhibited a higher Krebs cycle activity in the light compared to control plants [START_REF] Bykova | Interaction between photorespiration and respiration in transgenic potato plants with antisense reduction in glycine decarboxylase[END_REF]. So, the large amounts of NADH and NH 4 + released in mitochondria by the GDC activity of the photorespiratory cycle can inhibit PDH and ICDH enzymes (and potentially the activity of all Krebs cycle dehydrogenases).

That said, contradictory results have been obtained from experiments feeding 13 C-labeled pyruvate to Xanthium strumarium leaves. It was found that under low CO 2 condition (high photorespiration flux) that brought about a higher photorespiration rate, a higher decarboxylation rate associated with several enzymes of the Krebs cycle such as PDH, ICDH, and 2OGDH suggested an increase in their activities. The 13 C-labelling of organic acids also suggested an increase in Krebs cycle activity under high photorespiratory conditions (Tcherkez et al., 2008). This was explained by the requirement to produce more 2OG to reassimilate photorespiratory ammonia via the Fd-GOGAT1/GS2 cycle, and thus a higher activity of the Krebs cycle, which is contrary to that observed in the potato GDC antisense lines [START_REF] Bykova | Interaction between photorespiration and respiration in transgenic potato plants with antisense reduction in glycine decarboxylase[END_REF].

3) Interactions between photorespiration and nitrogen assimilation

Plants with the exception of legumes absorb nitrogen from the soil in the form of ammonia and nitrate by ammonia transporters (AMT, [START_REF] Weidinger | Functional and physiological evidence for a Rhesus-type ammonia transporter in Nitrosomonas europaea[END_REF][START_REF] Xu | Plant Nitrogen Assimilation and Use Efficiency[END_REF]) and nitrate transporters (NRT, [START_REF] Lezhneva | The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogenstarved plants[END_REF][START_REF] Xu | Plant Nitrogen Assimilation and Use Efficiency[END_REF])), respectively. Nitrate is transported from the roots to the leaves via the xylem. Nitrate reduction is mainly carried out in the leaf and only a small part in roots [START_REF] Xu | Plant Nitrogen Assimilation and Use Efficiency[END_REF]. In the leaf, nitrate can be stored in the vacuole or reduced to ammonium to be assimilated to produce organic N-containing molecules. Firstly, nitrate is reduced to nitrite in the cytosol by nitrate reductase (NR) with consumption of NADH. The nitrite is transported into the chloroplast where it is reduced to ammonium by nitrite reductase (NiR) using reduced Ferredoxin [START_REF] Xu | Plant Nitrogen Assimilation and Use Efficiency[END_REF]. Then, ammonium can enter the GS / Fd-GOGAT cycle. Firstly, GS incorporates ammonium to glutamate, with ATP consumption, to produce glutamine. Then, Fd-GOGAT generates two glutamate molecules from glutamine and 2OG [START_REF] Xu | Plant Nitrogen Assimilation and Use Efficiency[END_REF]. The GS-GOGAT cycle can be found in plastids (GS2/Fd-GOGAT) or in the cytosol (GS1/NADH-GOGAT).

There are many links between photorespiration and nitrogen assimilation. The photorespiratory GDC complex activity produces 10 times more ammonia than nitrate reduction, and it is mainly re-assimilated by the GS2 / Fd-GOGAT1 cycle in leaves (Coschigano, 1998, Somerville andOgren, 1980b) (Figure 8). In addition, photorespiratory GGT1 requires glutamate as a substrate to produce 2OG, which is simultaneous substrate for the Fd-GOGAT1. Glutamate is an important amino acid used to produce other amino acids via different transamination reactions, such as used by GS2 to assimilate ammonium therefore photorespiratory GGT could negatively impact such processes. In Arabidopsis thaliana, ggt1

mutants showed a decreased of RuBisCO content in leaf resulting in a C-limitation that negatively impacts N-assimilation in plant indicates that photorespiration has an indirect impact on nitrogen content in overall plant through RuBisCO content [START_REF] Dellero | Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth[END_REF].

Over the years, increasing evidence through measuring nitrogen assimilation confirm that elevated (elevated ~ 567 ppm and ambient ~ 365 ppm) CO 2 levels in air can inhibit leaf NO 3 assimilation thus indicating that naturally occurring low photorespiratory conditions inhibit nitrate assimilation in plants (Bloom, 2015). To date, three physiological mechanisms have been proposed to explain the observed inhibition of nitrate assimilation in elevated CO 2 compared to air (Bloom, 2015).

 Inhibition of nitrite transport into chloroplasts [START_REF] Bloom | Nitrogen assimilation and growth of wheat under elevated carbon dioxide[END_REF].

 A reduced NiR activity in the chloroplast stroma, via a decreased Fd availability due to an increased requirement to make NADPH via FNR. [START_REF] Bloom | Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis[END_REF].

 A limitation in cytosol NADH that limits NR activity due to a reduced photorespirationdependent export of malate to the cytosol from the chloroplast. This malate valve increases cytosolic NADH.

Finally, [START_REF] Bloom | a Photorespiratory Pathway That Increases the Energy Efficiency of Photosynthesis[END_REF] have proposed an alternative photorespiratory pathway that involves RuBisCO-oxygenase activity produced malate depending on an association with Mg 2+ or Mn 2+ cations. In the chloroplast, photorespiration is launched by RuBisCO that contains a metal-binding site shared by Mn 2+ and Mg 2+ . When this site is associated with Mn 2+ , RuBisCO prefers the oxygenation reaction whereas when associated with Mg 2+ , RuBisCO strongly prefers the carboxylation reacrtion. When associated with either Mn 2+ or Mg 2+ , RuBisCO can directly generate pyruvate and glycolate from RuBP using O 2 and NADP, then the pyruvate is further converted to malate via the malic enzyme (in the presence of Mn 2+ ) thus consuming CO 2 and producing NADPH. In this way photorespiration can generate additional malate and reducing power for NO 3 -assimilation [START_REF] Bloom | a Photorespiratory Pathway That Increases the Energy Efficiency of Photosynthesis[END_REF].

This hypothesis now requires solid experimantal data to be confirmed since data is sparse.

4) Interactions between photorespiration and C1 metabolism

C1 metabolism is essential for all organisms, as it involves the transfer of one carbon (C1)units via tetrahydrofolate (THF), an essential cofactor in important enzymatic reactions involved in the biosynthesis of amino acids (methionine, glycine and serine), purines, thymidylate, pantothenate and formylmethionyl-transferRNA [START_REF] Collakova | Arabidopsis 10-Formyl Tetrahydrofolate Deformylases Are Essential for Photorespiration[END_REF]. THF is composed of three parts, pterin, p-aminobenzoate (pABA) and glutamate moieties (Hanson and Gregory, 2011) (Figure 9a). The pterin ring of THF exists in two forms, dihydro or tetrahydro, but only the tetrahydro form has a cofactor activity (Hanson and Gregory, 2011).

In plant, C1 units exist with different levels of oxidation (formyl, methylene, methyl) which can be attached to the N5 and/or N10 of the pterin ring to form different forms of THF (5methyl-THF or 10-formyl-THF or 5,10-methenyl-THF or 5,10-methylene-THF or 5-formyl-THF) [START_REF] Cossins | Folates and one-carbon metabolism in plants and fungi[END_REF]. THF and THF C1-substituted derivatives together are termed folates (Figure 9b). These folates can participate in different enzymatic reactions to serve as C1 donors. THF and its C1 substituted derivatives are located in chloroplasts, the cytosol, mitochondria and the vacuole but it is synthesized only in mitochondria (Hanson and Gregory, 2011;[START_REF] Jardine | Integration of C1 and C2 Metabolism in Trees[END_REF].

Photorespiration interacts with C1 metabolism via the GDC complex/SHMT1 reactions (Figure 9c), during the photorespiratory cycle, this is driven by the accumulation of glycine in the chloroplast. Firstly, GDC T-protein uses THF as the cofactor to accept a methyl-group from glycine to produce 5,10-methylene-THF, then SHMT1 consumes the 5,10-methylene-THF by transferring the methyl-group to another molecule of glycine to produce serine and THF. Therefore, THF is recycled and it can be reused by the GDC complex (Moreno et al., 2005;Voll et al., 2006;Engel et al., 2011). As described above, mutants including GDC-P double knockout mutants (gldp1xgldp2) [START_REF] Engel | Deletion of Glycine Decarboxylase in Arabidopsis Is Lethal under Nonphotorespiratory Conditions[END_REF], GDC-T knockout mutant (gldt1-

2) [START_REF] Timm | T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis[END_REF] and a SHMT double mutant (shm1xshm2) (Engel et al., 2011) do not survive even under high CO 2 condition. This drastic phenotype has been explained by the close interaction between C1 metabolism and serine to glycine interconversion that appears to be in part independent of photorespiratory flux (Timm and Bauwe, 2013). The Arabidopsis thaliana genome contains two genes At4g17360 and At5g47435 encoding formyl THF cycloligase [START_REF] Hanson | Plant one-carbon metabolism and its engineering[END_REF][START_REF] Collakova | Arabidopsis 10-Formyl Tetrahydrofolate Deformylases Are Essential for Photorespiration[END_REF].

10-formyl THF deformylase (10-FDF) [START_REF] Collakova | Arabidopsis 10-Formyl Tetrahydrofolate Deformylases Are Essential for Photorespiration[END_REF], one of the folate interconversion enzymes, which metabolizes 10-formyl-THF to yield formate and THF for purine and glycine biosynthesis. T-DNA insertion mutants in these two genes showed a retarded growth phenotype leading to smaller plants compared to the WT when grown in ambient air conditions. Metabolite analyses revealed that the double mutant showed a 70-fold increase in glycine, similar to shmt1 mutants, and they also accumulated 5-and 10-formyl THF as well as 5,10-methylene-THF. These phenotypes were not observed under a high CO 2 concentration, as observed for photorespiratory mutants [START_REF] Somerville | An early Arabidopsis demonstration resolving a few issues concerning photorespiration[END_REF]. Taken together, these results confirm that the Arabidopsis thaliana genes At4g17360 and At5g47435 which play an important role in C1 metabolism, are necessary for plant photorespiratory carbon flux [START_REF] Collakova | Arabidopsis 10-Formyl Tetrahydrofolate Deformylases Are Essential for Photorespiration[END_REF].

5) The interaction between photorespiration and serine biosynthesis

In plants, serine is an essential amino acid and plays a fundamental role in protein biosynthesis, plant metabolism and development [START_REF] Benstein | Arabidopsis Phosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis[END_REF]. Serine can participate in the synthesis of many biomolecules, such as amino acids (including glycine and tryptophan), phospholipids and sphingolipids. Moreover, serine also plays an important role in C1 metabolism (see above) and in signaling pathways (Ros et al., 2014). Finally, some evidence demonstrate that serine is also involved in plant responses to biotic and abiotic stresses [START_REF] Ho | Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana[END_REF][START_REF] Kito | Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet[END_REF]. In plants, there are three different serine biosynthesis pathways. One is the photorespiratory pathway or glycolate pathway, which is believed to be the major serine biosynthesis pathway, particularly in photosynthetic tissues.

The remaining two pathways are the non-photorespiratory glycerate pathway and the phosphorylated pathway (Ros et al., 2014) (Figure 10) and both of the two pathways start with 3PGA.

1. Photorespiratory pathway (glycolate pathway): Serine is produced by the action of the photorespiratory GDC/SHMT1 complex and therefore it depends on light and RuBisCO oxygenase activity. It is believed to be the major pathway, although it is strongly limited to photosynthetic tissues. However, serine biosynthesis by this pathway alone is not sufficient for the requirement of all cells (Moreno et al., 2005;Voll et al., 2006;Engel et al., 2011).

2. Glycerate pathway: it is composed of three different reactions catalyzed by three enzymes and takes place in cytosol and in peroxisome. First, in cytosol, the 3PGA is dephosphorylated to glycerate by 3PGA phosphatase (PGAP) and then it is transported into the peroxisome, where the glycerate is further catalyzed to hydroxypyruvate by glycerate dehydrogenase (GDH). Finally, the hydroxypyruvate is converted to serine in peroxisome via the activity of alanine:hydroxypyruvate aminotransferase (AHAT)

and glycine:hydroxypyruvate aminotransferase (GHAT) either in cytosol or in peroxisome. The activities of the enzymes involved in this pathway have been demonstrated and the genes encoding these enzymes were also identified, however the functional significance of this pathway still need to be investigated (Ros et al., 2014).

3. Phosphorylated pathway: phosphorylated pathway of serine biosynthesis (PPSB) also synthesizes serine from 3PGA coming from the plastidial glycolysis in plastids [START_REF] Benstein | Arabidopsis Phosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis[END_REF] and comprises three reactions catalyzed by three enzymes:

3PGA dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT), and 3phosphoserine phosphatase (PSP). First, 3PGA is oxidized to 3phosphohydroxypyruvate (3-PHP) by PGDH, accompanied by the formation of NADH from NAD + . Successively, via the activity of PSAT, the 3-PHP and Lglutamate are converted into 3-phosphoserine (3-PS) and 2-oxoglutarate (2OG). In this reaction, the glutamate serves as an amino donor. The last step of the pathway is the conversion from 3-PS into serine via the activity of PSP (Ros et al., 2014). Based on the TAIR database, there are three genes encoding the PGDH (PGDH1 (At4g34200, formerly known as EDA9), PGDH2, (At1g17745, formerly known as PGDH), and PGDH3 (At3g19480, formerly known 3-PGDH)), two genes encoding PSAT (At4g35630, PSAT1, and At2g17630, PSAT2) and one gene encoding PSP (At1g18640). Except PSAT2, all the genes were cloned and all the activities of encoded proteins were confirmed in vitro [START_REF] Ho | Molecular characterization of plastidic phosphoserine aminotransferase in serine biosynthesis from Arabidopsis[END_REF][START_REF] Ho | Plastidic Pathway of Serine Biosynthesis[END_REF][START_REF] Ho | Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana[END_REF]Engel et al., 2011;[START_REF] Benstein | Arabidopsis Phosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis[END_REF]. Thanks to the genetic and physiological evidences of the first and last enzymes in this pathway, we have got biological significance of PPSB for the plant growth and development (see [START_REF] Benstein | Arabidopsis Phosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis[END_REF][START_REF] Cascales-Minana | The Phosphorylated Pathway of Serine Biosynthesis Is Essential Both for Male Gametophyte and Embryo Development and for Root Growth in Arabidopsis[END_REF][START_REF] Toujani | Functional Characterization of the Plastidial 3-Phosphoglycerate Dehydrogenase Family in Arabidopsis[END_REF].

Figure 10. Plant serine biosynthesis pathways

Phosphorylated pathway: PGDH, 3-phosphoglycerate dehydrogenase; PSAT, 3phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Glycerate pathway:

PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, alaninehydroxypyruvate aminotransferase. Glycolate pathway (Photorespiratory pathway): GDC, glycine decarboxylase complex; SHMT, serine hydroxymethyltransferase (Ros et al., 2014).

IV. Photorespiration and abiotic/biotic stress

Over the years and more recently, many studies provide evidence that photorespiration plays an important role in plant response to both abiotic and biotic stresses such as high temperature, drought/water stress, salinity, high light [START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF]. The afore-mentioned stresses induce stomatal closure for instance to prevent water loss or to reduce bacterial infection.

However, this will also give rise to a limitation of leaf internal CO 2 and thus reduce CO 2 availability for RuBisCO which in turn leads to a reduction of photosynthesis and an increase of photorespiration [START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF]. Therefore, under such stress conditions, the capacity of photorespiration to detoxify 2PG and recycle photorespiratory carbon will have an increased importance for plant survival.

A major, rapid plant response to harmful stress conditions is an increase in reactive oxygen species (ROS). As the highly reactive and readily diffusible molecular, on one hand, ROS can act as the signaling molecules to regulate many processes such as plant growth/development and acclimation responses to stress [START_REF] Apel | REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction[END_REF][START_REF] Foyer | Redox Homeostasis and Antioxidant Signaling: A Metabolic Interface between Stress Perception and Physiological Responses[END_REF][START_REF] Gara | Redox homeostasis in plants. The challenge of living with endogenous oxygen production[END_REF]. On the other hand, high levels of ROS lead to cellular damage and are involved in programmed cell death mainly under the biotic or abiotic stress conditions [START_REF] Apel | REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction[END_REF]Pavet et al., 2005;[START_REF] Van Breusegem | Reactive Oxygen Species in Plant Cell Death[END_REF]. Among the different forms of ROS, H 2 O 2 can be generated via different metabolic mechanisms in different cellular compartments:

in the chloroplasts, the H 2 O 2 can be generate through the photosynthetic electron transport (PET) chain [START_REF] Asada | Production and Scavenging of Reactive Oxygen Species in Chloroplasts and Their Functions[END_REF]Takahashi et al., 2007); in peroxisomes, the H 2 O 2 can be generated via photorespiratory glycolate oxidase reaction [START_REF] Bauwe | Photorespiration has a dual origin and manifold links to central metabolism[END_REF][START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF][START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF]; and in mitochondrion through electron transport [START_REF] Rhoads | Mitochondrial Reactive Oxygen Species. Contribution to Oxidative Stress and Interorganellar Signaling[END_REF]. In addition, on the plasma membrane, NADPH oxidases generated H 2 O 2 playing a key role in ROS generation [START_REF] Sagi | Production of Reactive Oxygen Species by Plant NADPH Oxidases[END_REF]. While under conditions of high light or when photorespiration is inhibited, the photorespiratory cycle will be the main source of H 2 O 2 via the action of GOX in peroxisome [START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF]. However, the photorespiration also plays an important role in preventing ROS accumulation. The photorespiratory cycle can help to maintain sufficient NADP + /NAD + levels and ATP consumption to limit photosynthetic ROS generation either directly or indirect (Eisenhut et al., 2017). First, photorespiration consumes both ATP and NADH to produce ADP (via the action of GLYK in the chloroplasts)

and NAD (via the HPR reaction in the peroxisomes or cytosol) [START_REF] Bauwe | Photorespiration has a dual origin and manifold links to central metabolism[END_REF]). An indirect pathway involves the re-assimilation of NH 4 + , released by the GDC complex in mitochondria, by the GOGAT1/GS2 cycle that also consumes reducing equivalents (reduced ferredoxin) and ATP [START_REF] Coschigano | Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primar y Nitrogen Assimilation[END_REF][START_REF] Wallsgrove | Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis[END_REF]. In addition, CO 2 and 3PGA produced by the photorespiratory cycle can both enter the Calvin-Benson cycle allowing the recycling of reducing equivalents and ADP. So, under stressful condition, photorespiration is a major sink for reducing equivalents as well as ATP, thus allowing photosynthesis to continue efficiently [START_REF] Muller | Non-Photochemical Quenching. A Response to Excess Light Energy[END_REF][START_REF] Horton | Molecular design of the photosystem II light-harvesting antenna: Photosynthesis and photoprotection[END_REF]Takahashi et al., 2007). Thus, it was proposed that photorespiration could maintain the optimal redox state during the different stress situations that could alter the photorespiration flux by combining the increasing and decreasing ROS production [START_REF] Scheibe | Strategies to maintain redox homeostasis during photosynthesis under changing conditions[END_REF][START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF][START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF].

1) Photorespiration and abiotic stress

Drought stress

Drought is a major environment factor that negatively impacts plant growth and crop yield.

As mentioned earlier, drought or water stress leads to stomatal closure that will decrease the CO 2 /O 2 ratio around the RuBisCO in leaves. This decrease of cellular CO 2 concentration will in turn decrease photosynthetic carbon assimilation and facilitate an increase in photorespiration [START_REF] Bauwe | Photorespiration has a dual origin and manifold links to central metabolism[END_REF]Abadie et al., 2016;[START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF]. In photorespiratory mutants, the required stress-induced increase in photorespiratory flux is not possible and therefore they will show stress-related symptoms. For example, hpr1 mutants that exhibit a type III photorespiratory phenotype indicating only a mild disturbance of photorespiratory cycle flux still exhibit an increased sensitivity to a drought stress (Li and Hu, 2015). In another study, barley photorespiratory GDC and SGAT mutants showed a larger accumulation of glycine under drought stress conditions when compared to WT. Since the accumulation of glycine is considered to reflect photorespiratory cycle intensity, it was proposed that a drought stress increased RuBisCO oxygenase activity and enhanced photorespiration in these mutants [START_REF] Wingler | The role of photorespiration during drought stress: An analysis utilizing barley mutants with reduced activities of photorespiratory enzymes[END_REF]. A study of the drought tolerant plant Pancratium maritimum, showed that photorespiration was higher when water-stressed as indicated by the increased expression level of GDC-H and SGAT as well as the increased level of glyoxylate and the Gly/Ser ratio [START_REF] Abogadallah | Differential regulation of photorespiratory gene expression by moderate and severe salt and drought stress in relation to oxidative stress[END_REF]. In maize, a C4 plant, metabolite analysis showed that photorespiration was important for crop yield under drought stress.

Notably, there was a significant correlation between the level of glycine and yield. Indeed, when maize was subjected to a drought stress, leaf glycine content increased by 3-4 fold compared to non-stressed plants [START_REF] Obata | Metabolite profiles of maize leaves in drought, heat and combined stress field trials reveal the relationship between metabolism and grain yield[END_REF]. A possible explanation is that glycine, mainly generated by photorespiration, is required for the biosynthesis of glutathione, which is an important antioxidant [START_REF] Foyer | Oxygen processing in photosynthesis: Regulation and signalling[END_REF]. During water stress, photorespiration can act as an electron sink to consume excess reducing equivalents. For example, under drought stress, consumption of reducing energy by CO 2 assimilation is decreased, however consumption by the photorespiration increased from 23% to 40% compared to control conditions. In this way, photorespiration can play a major role in preventing photodamage under drought stress, especially when CO 2 becomes extremely limiting [START_REF] Haupt-Herting | Oxygen exchange in relation to carbon assimilation in water-stressed leaves during photosynthesis[END_REF]). This appears to agree with the observation that photorespiration increased under drought stress, and helped to maintain PSII activity and thus allow plants to rapidly recover their normal photosynthetic capacity after re-watering [START_REF] Guan | Photoprotective function of photorespiration in several grapevine cultivars under drought stress[END_REF]. The impact of an impaired photorespiratory pathway on photoinhibition using 4 Arabidopsis photorespiratory enzyme mutants (Fd-GOGAT1, SHMT1, DiT2 and GLYK1) confirmed that impairment of photorespiration facilitates the photoinhibition of PSII by suppressing photodamage repair (Takahashi et al., 2007).

Salt stress

Together with drought stress, salinity stress is another major environmental factor that impacts plant growth and development. H 2 O 2 is an essential part of the signal transduction cascade induced under salt stress condition. It has been reported for C3 and C4 plants, that H 2 O 2 is produced and increased under salt stress due to a reduction in water availability and CO 2 assimilation. It is accompanied by the induction and expression of ROS scavenging enzymes such as catalases (CAT), peroxidases (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) [START_REF] Noctor | Drought and oxidative load in the leaves of C3plants: A predominant role for photorespiration?[END_REF][START_REF] Noctor | Climate Change, CO2, and Defense: The Metabolic, Redox, and Signaling Perspectives[END_REF]. As stated above, in plants, photorespiration plays an important role in maintaining the redox state in intercellular under stressful conditions including salt stress by decreasing the ROS generation through direct and indirect pathways as detailed above [START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF].

In order to demonstrate the attribution of photorespiration to oxidative load when plants were undergoing salt-stress, 7-week-old salt-tolerant Pancratium maritimum L. (a C3 plant) was subjected to either 150 mM or 300 mM NaCl. It was found that photorespiratory rate was remarkably higher under a moderate salt treatment (as indicated by higher Gly/Ser ratio and glyoxylate content, higher expression of photorespiratory enzyme genes such as GDC-H, SGAT and GS2). In addition, the efficient up-regulation of ROS scavenging enzymes such as catalases and peroxidases were also observed in plant leaves. It was concluded that photorespiration played an important role in dealing with the salt stress [START_REF] Abogadallah | Differential regulation of photorespiratory gene expression by moderate and severe salt and drought stress in relation to oxidative stress[END_REF]).

An increasing amount of evidence suggests that photorespiratory SHMT1 plays a vital role in salt stress in plants (Moreno et al., 2005;Zhou et al., 2012;[START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF][START_REF] Kito | Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet[END_REF]. Indeed, Arabidopsis shm1-3 mutant showed a severe retarded growth phenotype, a large accumulation of ROS and an increased sensitivity to a 50 mM salt treatment compared to wild-type plants (Moreno et al., 2005). Furthermore, it was found that shm1-1 displayed an increased accumulation of salt-induced H 2 O 2 (100 mM NaCl) and a higher sensitivity to salt treatment (50 mM and 100 mM NaCl) compared to WT plants (Zhou et al., 2012) and after 24 h treatment of 50 mM NaCl, cell death detected by trypan blue staining showed that cell death was dramatically induced in shm1-1 plants (Zhou et al., 2012). Thus, these results support the notion that photorespiration play an important role in decreasing the production of the ROS and mitigating oxidative damage at the salt stress. In another study in the halophytic grass Puccinellia tenuiflora [START_REF] Yu | Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuiflora[END_REF] showed that the plant developed some ROS scavenging mechanisms to cope with mild salt stress including increased photorespiratory activity (increased GOX and catalase activities) suggesting increased glycolate oxidation as well as H 2 O 2 scavenging. Meanwhile, the up-regulated expression of SHMT1 in plant of this study also indicated an enhanced photorespiratory pathway involved in the enhanced salt tolerance [START_REF] Yu | Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuiflora[END_REF].

Thus, the photorespiration plays an important role in maintaining the redox state by decreasing the production of ROS and also in mitigating oxidative damage of the salt stress.

High temperature stress

High temperature can cause a decrease in photosynthetic CO 2 assimilation and an increase of photorespiration that lead to severe cellular damage in plants [START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF]. With increasing temperature, CO 2 solubility in water is decreased more than the solubility of O 2 , leading to a lower CO 2 /O 2 ratio at the active site of RuBisCO. Under high temperatures when it is within the permission range of plant growth (until ≈40°C), RuBisCO prefers to perform RuBP oxygenation, namely the plants favor photorespiration over CO 2 fixation [START_REF] Foyer | Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox Signaling[END_REF]. Moreover, if the high temperature is out of the permission range, it also could lead to stomatal closure to not lose water by transpiration which in turn further decreases the CO 2 assimilation, and then plant yields decline. For example, [START_REF] Sicher | Temperature shift experiments suggest that metabolic impairment and enhanced rates of photorespiration decrease organic acid levels in soybean leaflets exposed to supra-optimal growth temperatures[END_REF] showed that increased temperature (36/28 °C compared to 28/20 °C) enhanced photorespiration, indicated by increased glycine decarboxylase activity, and also led to a reduction in leaf organic acid levels [START_REF] Sicher | Temperature shift experiments suggest that metabolic impairment and enhanced rates of photorespiration decrease organic acid levels in soybean leaflets exposed to supra-optimal growth temperatures[END_REF]. Several studies have reported that plants are often subjected to a combination of different abiotic stresses, and abiotic stresses are often interrelated, either individual or in combination [START_REF] Zandalinas | Plant adaptations to the combination of drought and high temperatures[END_REF]. For example, when Nicotiana tabacum was subjected to a combination of drought and heat stress, plants displayed a decrease of photosynthetic rate, an increase of respiration rate and stomatal closure [START_REF] Rizhsky | The Combined Effect of Drought Stress and Heat Shock on Gene Expression in Tobacco\r10.1104[END_REF]. Metabolite profiles analysis in maize leaves subjected to a combination of drought and temperature stress showed an accumulation of both glycine and serine levels indicated that can be interpreted as a change of photorespiratory flux [START_REF] Obata | Metabolite profiles of maize leaves in drought, heat and combined stress field trials reveal the relationship between metabolism and grain yield[END_REF].

In a recent study on rice, four independent overexpressing glycolate oxidase (GLO) lines with an increasing gradient of GLO activity (60%, 100%, 150% and 200%) were generated. Plant growth was improved when GLO activity was increased by 60 or 100%, while growth was reduced when GLO activity was further increased. These lines were used for stress experiments where they were first grown in a greenhouse under normal conditions and then transferred to a controlled chamber with high temperature and light intensity (40 ℃ (day)/30 ℃ (night) and 900 µmol m -2 s -1 of light). After a 3-day treatment, the photosynthesis was significantly increased in all the overexpressing lines, but not under normal condition. Thus, rice GLO may play an important role in tolerance to a multiple high light and high temperature stress (Cui et al., 2016).

High light stress

The photosynthetic electron transport chain (ETC) is a major source of ROS in plant cells and under high light stress, ROS is significantly produced in this way. At the same time, the excess light energy also leads to photoinhibition [START_REF] Asada | THE WATER-WATER CYCLE IN CHLOROPLASTS: Scavenging of Active Oxygens and Dissipation of Excess Photons[END_REF]. Photorespiration, which acts as a sink for stably consuming the excess light energy such as ATP and NADPH, is proposed to be important for avoiding photoinhibition of photosystem II (PSII) [START_REF] Kozaki | Photorespiration protects C3 plants from photooxidation[END_REF][START_REF] Wingler | Photorespiration: Metabolic pathways and their role in stress protection[END_REF], especially under the conditions that lead to stomatal closure to reduce consumption of light energy by CO 2 assimilation. Takahashi et coworkers (2007) found that the impairment of photorespiratory pathway in shm1, glyk1 and pglp1 accelerated photoinhibition not by accelerating photodamage to PSII but by suppressing the repair of photodamaged PSII (Takahashi et al., 2007). In rice leaves exposed to low / high light (100/700 μmol photon m -2 s -1 ) treatment, the significant increases of GDC-P, GDC-T subunits and SHMT protein were observed in response to light. While GDC-L subunit was decreased to half of its "normal" content and GDC-H was unaffected under high light. These results indicated that a high light treatment can alter the stoichiometry of GDC subunits leading to a decrease of photorespiration flux [START_REF] Huang | Differential induction of mitochondrial machinery by light intensity correlates with changes in respiratory metabolism and photorespiration in rice leaves[END_REF].

Analysis based on the measurement of photosynthetic parameters and chlorophyll fluorescence in tobacco leaves indicated that, compared to leaves exposed to a constant high light (1200 μmol photon m -2 s -1 ), those exposed to 1200 μmol photon m -2 s -1 of fluctuating light (2-min intervals while irradiance levels alternated between 100 and 1200 μmol photons m -2 s -1 ), the electron flow devoted to RuBP carboxylation was limited by a low chloroplastic CO 2 level, whereas the electron flow devoted to RuBP oxygenation remained high. So, this could indicate that photorespiration plays an important role in regulating photosynthetic ETC flow under 1200 μmol photon m -2 s -1 fluctuating light (Huang et al., 2015a). Recently, a new study on transgenic rice pointed that, under conditions where CO 2 assimilation is limited such as during stomatal closure, photorespiration which acts as a sink for excess light energy is proposed to not only be important for avoiding photoinhibition of photosystem II (PSII), but also for protecting the photosystem I (PSI) by oxidizing its reaction center chlorophyll P700. when both were compared to wild-type plants. When these plants were subjected to a light stress under low-CO 2 , in wild-type and RBCS-sense plants the oxidation of P700 was enhanced and PSI was not damaged by the excess light stress induced by repetitive saturated pulsed-light (rSP) (300 ms, 20,000 μmol photon m -2 s -1 every 10 s for 60 min in the presence of the actinic light). However, under the same stress conditions, P700 was strongly reduced in RBCS-antisense plants and PSI became damaged. These results prove new evidence that photorespiration plays a critical role in response to excess light stress [START_REF] Wada | Effects of genetic manipulation of the activity of photorespiration on the redox state of photosystem I and its robustness against excess light stress under CO2-limited conditions in rice[END_REF].

2) Photorespiration and biotic stress

Photorespiration can also play a role in plant pathogen defense (Rojas et al., 2012;Sørhagen et al., 2013). In the literature, a major photorespiratory enzyme associated with pathogen attack is peroxisomal GOX that produces H 2 O 2 when converting glycolate to glyoxylate (Dellero et al., 2016;[START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF]. Because H 2 O 2 is an essential signal molecule involved in plant response to many different stress conditions including plant pathogen defense (Noctor et al., 2015). In Nicotiana benthamiana, GOX was shown to be involved in a non-host resistance response against Pseudomonas syringae pv tabaci and involved in the induction of the hypersensitive response. Similar results were found in Arabidopsis, where single mutants for each GOX gene led to a reduced resistance to the non-host pathogen P.

syringae (Rojas et al., 2012). Furthermore, in tobacco, GOX and several other And the expression analysis of genes involved in defense against pathogens showed that NbGOX were differentially involved in defense gene expression. In addition, by analyzing the effect of silencing of single gene on expression of the other two genes, the results showed that silencing of NbHAOX8 or NbGOX1 individually did not affect the expression of the other two genes, however, silencing of NbGOX4 strongly enhanced expression of NbHAOX8 and NbGOX1 genes (12-and 9-fold, respectively). Thus, these results suggested that NbGOX4 might function through the repression of NbHAOX8 and NbGOX1 (Xu et al., 2018). However, a study in rice showed contradictory results. [START_REF] Chern | Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice[END_REF] reported that induced silencing of a rice GOX gene GLO1 enhanced rice resistance to another bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) together with an increase of defense regulator genes expression. Thus GLO1 appeared to play a negative role in this host resistance [START_REF] Chern | Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice[END_REF]. Several other photorespiratory enzymes have been associated with plant disease resistance including SHMT, HPR, SGT and GDC-P/H. In Arabidopsis, the shm1-3 mutant showed a higher sensitivity to biotrophic and necrotrophic pathogens compared to control plants (Moreno et al., 2005). In melon, SGT activity remained higher resistant lines compared to a susceptible line upon oomycete Pseudoperonospora cubensis infection [START_REF] Taler | Plant eR Genes That Encode Photorespiratory Enzymes Confer Resistance against Disease[END_REF].

Recently, a new study revealed that three photorespiratory enzymes (GOX, SGT and SHMT)

were involved in tomato pathogen resistance against Pseudomonas syringae. After inoculation of leaves with P. syringae, photorespiration rate and the expression of GOX2, SHMT1 and SGT were increased. In addition, silencing of either GOX2, SHMT1 or SGT genes in tomato decreased the photorespiration rate and increased the susceptibility to P. syringae, whereas, the transient overexpression of GOX2, SHMT1 and SGT increased the resistance to P.

syringae [START_REF] Ahammed | Tomato photorespiratory glycolate-oxidase-derived H2O2production contributes to basal defence against Pseudomonas syringae[END_REF]. Finally, it was shown that HPR which could interact with an elicitor receptor of syringolide in soybean was proposed to be a potential second message to induce the hypersensitive response in soybean [START_REF] Okinaka | The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme, NADHdependent hydroxypyruvate reductase[END_REF].

V. SHMT, serine hydroxymethyltransferase

SHMT, serine hydroxymethyltransferase, depending on tetrahydrofolate (THF), catalyzes the reversible hydroxymethyl group transfer from serine to glycine in either a tetrahydrofolatedependent or -independent manner (Chen and Schirch, 1973a;[START_REF] Ruszkowski | Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula : A Structural Characterization[END_REF]. It uses a pyridoxal-5-phosphate (PLP) as co-factor [START_REF] Alexander | Evolutionary relationships among pyridoxal-5′-phosphate-dependent enzymes: Regio-specific α, β and γ families[END_REF] and in addition, it also catalyzes the THF-independent cleavage of many 3-hydroxyamino acids [START_REF] Ogawa | Serine hydroxymethyltransferase and threonine aldolase: are they identical?[END_REF][START_REF] Webb | 4-Chlorothreonine is substrate, mechanistic probe, and mechanism-based inactivator of serine hydroxymethyltransferase[END_REF]. It is a key enzyme associated with one-carbon metabolism and has already been investigated in many organisms from bacteria to higher plants and mammals [START_REF] Renwick | The crystal structure of human cytosolic serine hydroxymethyltransferase: A target for cancer chemotherapy[END_REF][START_REF] Douce | The glycine decarboxylase system: A fascinating complex[END_REF]Simic et al., 2002;[START_REF] Daidone | In silico and in vitro validation of serine hydroxymethyltransferase as a chemotherapeutic target of the antifolate drug pemetrexed[END_REF].

Except its function as a donor for activated one-carbon units, SHMT also plays a key role in serine biosynthesis as mentioned above (Chen and Schirch, 1973a;Ros et al., 2014). In Arabidopsis, there are seven SHMT genes (AtSHMT1-7) which have different cellular localization: mitochondrial, cytosolic, chloroplastic and nuclear (Zhang et al., 2010). Among these genes, SHMT1, localized in mitochondrion and playing an important role in the photorespiratory pathway (Somerville and Ogren, 1981;Voll et al., 2006;Engel et al., 2011), is the major part of my research during my PhD and will be detailed here.

1) Reaction mechanism of SHMTs

SHMTs are a-class PLP-dependent enzymes and can tightly bind PLP and substrates to form an aldimine in the active center [START_REF] Alexander | Evolutionary relationships among pyridoxal-5′-phosphate-dependent enzymes: Regio-specific α, β and γ families[END_REF]. SHMTs catalyze the reversible interconversion of serine to glycine with THF-dependent/THF-independent and also catalyze the THF-independent b-hydroxy amino acids reactions (Chen and Schirch, 1973b;[START_REF] Ogawa | Serine hydroxymethyltransferase and threonine aldolase: are they identical?[END_REF][START_REF] Webb | 4-Chlorothreonine is substrate, mechanistic probe, and mechanism-based inactivator of serine hydroxymethyltransferase[END_REF][START_REF] Ruszkowski | Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula : A Structural Characterization[END_REF]. Considerable efforts have been devoted to clarify how serine is catalyzed to glycine. Based on structural data and the properties of several active site mutants of SHMTs in both eukaryote and prokaryote, the reaction mechanism of SHMTs is gradually being unraveled. Concerning the THF-dependent Ser-to-Gly conversion reaction, the current hypothesis is that the reaction involves a nucleophilic displacement via the N 5 of THF, in which the latter undertakes a nucleophilic attack on the C b of PLP-Ser external aldimine (PLP-Ser) with displacement of the C a of Gly without the generation of the formaldehyde intermediate [START_REF] Schirch | Serine hydroxymethyltransferase revisited[END_REF]. This nucleophilic displacement mechanism satisfies most of the available experimental evidence, however, the other retro-aldol mechanism that the reaction involved a retro-aldol cleavage of PLP-Ser to generate a formaldehyde intermediate which subsequently reacted with THF still not be excluded [START_REF] Szebenyi | Serine hydroxymethyltransferase: Role of Glu75 and evidence that serine is cleaved by a retroaldol mechanism[END_REF][START_REF] Schirch | Serine hydroxymethyltransferase revisited[END_REF][START_REF] Ruszkowski | Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula : A Structural Characterization[END_REF].

Indeed, it has been reported that the retro-aldol cleavage mechanism is plausible for the THFindependent reaction where THF is not an obligate substrate and the reaction can occur without THF (Figure 11) (Chen and Schirch, 1973b;[START_REF] Ruszkowski | Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula : A Structural Characterization[END_REF].

Moreover, in both eukaryote and prokaryote, SHMTs can accept various 3-hydroxy amino acids as substrates that undergo the cleavage of their 3-hydroxy (such as threonine) to produce glycine and aldehydes and this THF-independent reaction mainly occurs via the retro-aldol mechanism [START_REF] Chiba | Mechanism for folate-independent aldolase reaction catalyzed by serine hydroxymethyltransferase[END_REF]. More recently, an analysis of the crystal structure of chloroplast-targeted SHMT from Medicago truncatula (MtSHMT3) showed that in the absence of THF, MtSHMT3 could catalyze the conversion from serine to glycine with the release of formaldehyde [START_REF] Ruszkowski | Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula : A Structural Characterization[END_REF]. In conclusion, even after over 50 years of studying SHMT, the catalytic reaction mechanism is still not clearly elucidated [START_REF] Ruszkowski | Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula : A Structural Characterization[END_REF].

Structural studies of prokaryote SHMT proteins have shown that the enzyme is present as a dimer which contains one or two PLP molecules, although Trypanosoma cruzi SHMT is an exception as it appears to be a monomer [START_REF] Capelluto | Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi[END_REF]. In contrast, in most eukaryotes, SHMTs are homotetrameric with one PLP molecule per subunit. According to previous studies, dimeric forms of SHMT are the minimum catalytic unit in Eukaryotes, and a dynamic balance between dimeric and tetrameric oligomerization states has been reported to alter SHMT catalytic activity [START_REF] Jala | Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus[END_REF][START_REF] Zanetti | Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers[END_REF]. However, while the mechanism of the change between the different tertiary states is still not known, it has been speculated that different SHMT oligomerization states may be responsible for different cellular metabolic processes [START_REF] Giardina | How pyridoxal 5′-phosphate differentially regulates human cytosolic and mitochondrial serine hydroxymethyltransferase oligomeric state[END_REF].

2) Arabidopsis SHMT1 in the photorespiration

In Arabidopsis, SHMT is encoded by seven genes (AtSHMT1-7, see Table 4). Based on previous studies and bioinformatics predictions, the respective subcellular localization for each SMLT protein is as follows: AtSHMT1 and AtSHMT2 are located in mitochondrion (Voll et al., 2006;Engel et al., 2011); AtSHMT3 is located in plastids (Zhang et al., 2010); is required for the biosynthesis of S-adenosylmethionine and DNA methylation (Huang et al., 2016). The biological function(s) of SHMT4, SHMT5 and SHMT6 are still unknown (see Table 4). In mitochondria, SHMT1 is the main isozyme which has been confirmed to be involved in the photorespiratory cycle (Engel et al., 2011;Voll et al., 2006;Engel et al., 2011).

During photorespiration, SHMT1 together with GDC, catalyzes the biosynthesis of serine from glycine and 5,10-THF, with the formation of THF. Firstly, one molecule of glycine is decarboxylated and deaminated by the GDC complex to produce CO 2 , NH 4 + and the C1 carrier, 5,10-THF. Subsequently, 5,10-THF is used by SHMT1 to transfer the activated C1 unit to a second molecule of glycine to produce one molecule of serine and THF [START_REF] Douce | Biochemical dissection of photorespiration[END_REF].

The role of SHMT1 in the photorespiratory cycle was shown from analyses of the EMSproduced shm1-1 mutant, one of the first generation photorespiratory mutants identified in Arabidopsis (Somerville and Ogren, 1981). It displays a class II photorespiratory phenotype since the mutation is lethal in ambient air but rescued at elevated CO 2 . In the mutant, SHMT1 activity was not detected, and serine biosynthesis was significantly reduced. Later, a weak shm1 allele mutant was isolated by Moreno (2005) (shm1-3), although this weak EMS mutant was chlorotic and exhibited retarded growth it was viable in ambient air condition. Due to the disturbance of photorespiration, this weak shm1-3 mutant accumulated more ROS in cells and it was more susceptible to salt stress and pathogen infection than the wild-type (Moreno et al., 2005). Subsequently, via positional cloning and molecular characterization, the SHMT1 gene (At4g37930) was mapped in the EMS mutant shm1-1 and shown to be the defective SHMT1 gene as well as the major gene encoding an SHMT isozyme in the mesophyll. However, SHMT2 was mainly expressed in roots and shoot apical meristems. Moreover, overexpression of SHMT2 failed to complement the lethal photorespiratory shm1-1 phenotype (Voll et al., 2006). Later, SHMT2 was demonstrated to be a functional serine hydroxymethyltransferase confined to leaf vascular tissues and the shm2-1 mutant did not show any visible defects in normal air. However, as described earlier (The variety of photorespiratory phenotypes), the isolated double mutant (shm1-2 x shm2-2) in the class I had an unconditional lethal phenotype except when grown in vitro on sucrose-supplemented medium in the presence of 1% CO 2 (Engel et al., 2011) (Table 5). In addition, CO 2 transition experiment (high CO 2 plants transferred to normal air for 2 weeks) showed that after the transfer, the rosette leaves of shm1-1 were not completely bleached and young leaves of the inner rosette remained green and grew normally compared with glyk1 and pglp1 mutant which also show the class II phenotype so that the mutant could survive and produce fertile seeds (Figure 12). These observations indicated that under such condition, the remaining SHMT2 activity probably represented a compensation mechanism. However, this phenomenon was very strict to the plant age and only observed when the shm1-1 mutant grown in high CO 2 up to developmental stage 5.1 (first flower buds visible) (Boyes et al., 2001) and then transferred to ambient air.

Indeed, younger plant at developmental stage 1.0 (cotyledons fully opened) quickly bleached and could not survive when they were transferred to ambient air (Timm et al., 2012). and young leaves of the inner rosette remained green. B: after acclimation to ambient air condition, the shm1 mutant could survive and produce fertile seeds (Timm et al., 2012a).

3) The role of SHMT1 in response to biotic and abiotic stress

An increasing amount of evidence strongly suggests that plant and cyanobacterial SHMT1 plays a role in abiotic stress responses such as high salt treatment (Moreno et al., 2005;Waditee-Sirisattha et al., 2012;Zhou et al., 2012;[START_REF] Voss | Emerging concept for the role of photorespiration as an important part of abiotic stress response[END_REF][START_REF] Kito | Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet[END_REF]Waditee-Sirisattha et al., 2016;Mishra et al, 2016). It has been reported that Arabidopsis shm1-3 mutant plants accumulated more H 2 O 2 in cells and were hypersensitive to salt stress (Moreno et al., 2005). To test whether shm1-3 displayed a higher sensitivity to salt stress, wild-type and mutant plants were grown in vitro with or without NaCl (25 and 50 mM). After 2 weeks, for the WT plant, when compared to the absence of NaCl treatment, the salt treatment only slightly affected WT growth, while for the shm1-3 mutant, although the plant already showed chlorotic and stunted phenotype in air condition, after salt treatment, the mutant showed a much more severe stunted and further chlorotic phenotype; and chlorophyll content was also significantly reduced. Histochemical analyses after DAB staining revealed that the salt-induced damage was correlated with a large accumulation of ROS. Indeed, a 50 mM salt treatment, led to a significant accumulation of DAB precipitation, especially in the leaves and roots of shm1-3 whereas control plants exhibited a less abundant DAB staining that was mainly located in roots (Moreno et al., 2005).

Zhou and coworkers (2012) found that SHMT1 could be ubiquitinated and degraded via a 26S proteasome-dependent process. A ubiquitin-specific protease (UBP16) could interact with and stabilize SHMT1 by removing conjugated ubiquitin. Consistent with this, it was found that SHMT enzymatic activity was reduced in the ubp16 mutant compared to Col-0 seedlings after 12h of 100 mM NaCl treatment. Both shm1-1 and ubp16 mutants displayed a more increased accumulation of salt-induced H 2 O 2 (100 mM NaCl), and a higher sensitivity to salt treatment (50 mM and 100 mM NaCl) compared with wild-type plants. The changes observed in ubp16

were correlated with an increased degradation of SHMT1. In contrast, the overexpression of SHMT1 in ubp16 only partially rescued the salt-sensitive phenotype of the mutnat. Therefore, it appears that SHMT1 plays an important role in plant salt tolerance and UBP16 participates in this process by modulating SHMT1 stability.

A mutant in the rice SHMT1 gene (OsSHMT1), an orthologue of Arabidopsis SHMT1, had an obvious photorespiratory phenotype, chlorotic lesion, growth delay and lethal under ambient air condition that could be restored under elevated CO 2 conditions (0.5% CO 2 ). Histochemical analyses and antioxidant enzyme activities measurement suggested that accumulation of both O 2 -and H 2 O 2 were higher in osshm1 compared to WT plants. At the same time, the increased ROS content induced the activity of antioxidant enzymes such as superoxide dismutase and peroxidase however this increased antioxidant activity in the mutant did not inhibit ROS accumulation. Thus, osshm1 was subjected to a significant oxidative stress and indicated that OsSHMT1 may play an important role in participating the antioxidant stress and to limit ROS-damage [START_REF] Wang | Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice[END_REF].

In another study of rice, ten-day-old seedlings including the salt-tolerant CSR27 line and saltsensitive MI48 line were subjected to 150 mM NaCl for 24h to investigate plant metabolic pathways involved in salt stress tolerance. RT-PCR analyses confirmed an induction of SHMT1 gene transcription in the salt-tolerant CSR27 line that was particularly higher in shoots compared with the salt-sensitive MI48 line under a salt stress. Similarly, amino-acid analyses revealed that salt tolerant lines had a higher level of methionine and proline in response to salt stress. Such data suggests that SHMT1 plays a role in plant response to salt treatment (Mishra et al., 2016).

SHMT in microorganisms also plays an important role in response to salt stress. For example, the SHMT gene isolated from a halotolerant cyanobacterium Aphanothece halophytica (ApSHMT) led to salt-tolerance when overexpressed in Escherichia coli. Furthermore, biochemical and molecular analyses indicated that under high salinity (300 mM, 500 mM, and 700 mM NaCl), the growth rate of ApSHMT-expressing cells was faster compared to control cells. In addition, ApSHMT transcription was strongly induced within an hour after a high salinity treatment (from 500 mM NaCl to 2.5M NaCl). Moreover, ApSHMT activity in cells increased approximately two-fold in response to increasing salinity from 0.5M NaCl to 2.5M

NaCl (Waditee-Sirisattha et al., 2012). A role of SHMT1 in salt stress was further shown when S. elongatus cells harboring either an empty vector or an ApSHMT-expressing vector were grown under salt stress condition (300 mM NaCl). Indeed, S. elongatus cells expressing

ApSHMT could grow in the salt-treatment conditions while growth was severely inhibited when S. elongatus contained the empty vector (Waditee-Sirisattha et al., 2016).

In Arabidopsis, the shm1-3 mutant showed a higher sensitivity to biotrophic and necrotrophic pathogens compared to control plants (Moreno et al., 2005). 4-week-old shm1-3 and WT plants were inoculated either with suspensions of Pseudomonas syringae pv. tomato (Pst) DC3000 avrRPM1 or Pst DC3000. The shm1-3 mutant showed a higher bacteria growth rate compared with WT plants and they displayed differential symptoms; 80% WT leaves showed a typical HR (hypersensitive response) response and only a slight yellowing around the necrotic lesions in the remaining 20% whereas 60% of the shm1-3 leaves developed severe symptoms and the remaining 40% exhibited death by uncontrolled necrosis. Similar consequences were observed when shm1-3 and WT plants were infected with the fungal pathogen Alternaria brassicicola. Four days after inoculation, WT leaves were resistant to A.

brassicicola infection with no obvious symptom, on the other hand 40% of shm1-3 leaves displayed necrosis and chlorosis (Moreno et al., 2005). Recently, a study of the contribution of photorespiration to tomato resistance against Pseudomonas syringae revealed that three photorespiratory enzymes (GOX, SGT and SHMT) were involved in pathogen defense. After inoculation of leaves with P. syringae, photorespiration rate and the expression of GOX2, SGT and SHMT1were increased. In addition, silencing of GOX2, SGT or SHMT1 genes in tomato decreased the photorespiration rate and increased susceptibility to P. syringae, whereas, the transient overexpression of these three genes independently increased resistance to P. syringae [START_REF] Ahammed | Tomato photorespiratory glycolate-oxidase-derived H2O2production contributes to basal defence against Pseudomonas syringae[END_REF].

VI. HPR, hydroxypyruvate reductase

Hydroxypyruvate reductase catalyzes the NADH/NADPH-dependent conversion of hydroxypyruvate to glycerate. In Arabidopsis, 3 HPR-encoding genes have been identified:

AtHPR1, At1g68010; AtHPR2, At1g79870; AtHPR3, At1g12550. AtHPR1 protein (42 kDa) is peroxisomal, AtHPR2 (34 kDa) is cytosolic and AtHPR3 (35 kDa) is plastidial [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. HPR1 is the major core enzyme of the photorespiratory cycle, since the loss of HPR1 led to a slight growth retardation when hpr1 mutant plants were grown in normal air that was absent when plants were grown in elevated CO 2 (0.15-0.2% CO 2 ). In addition, based on the isotope assay, it was found that the accumulation of hydroxpyruvate was over 103 times in hpr1 whereas hpr2 accumulated 3 times more compared with WT indicating that HPR1 played a dominant role in photorespiration [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. For most photorespiratory enzymes, the deletion of them typically leads to strong oxygen sensitivity [START_REF] Igarashi | Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis[END_REF]Boldt et al., 2005;Voll et al., 2006;[START_REF] Schwarte | Identification of the Photorespiratory 2-Phosphoglycolate Phosphatase, PGLP1, in Arabidopsis[END_REF], however, the hpr1

showed only minor phenotypic and metabolic alterations when grown in ambient air in moderate photoperiod. Though the effect on metabolites was minor in hpr1, it was still more severe than in hpr2 and hpr3, and it accumulates hydroxpyruvate and glycerate for example [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF][START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. Moreover, the hpr1 phenotype was affected by different photoperiod, since this mutant showed the strongest phenotype when grown in shortday conditions (8-10 h light) rather than a 12 h:12 h day-/night-cycle [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. It was consistent to the observations that after the transition from high CO 2 to ambient air condition, the pglp1, shm1 and pglk all showed stronger photorespiratory phenotype in long day condition than short day condition, except hpr1 (Timm and Bauwe, 2013). And according to Timm and Bauwe (2013), the hpr1 phenotype was also affected by light intensity and sucrose-supplemented in in vitro culture condition.

The weak type III photorespiratory phenotype of hpr1 in normal air was shown to reflect the compensatory, redundant photorespiratory function of HPR2 and HPR3 and thus the flexibility of hydroxypyruvate to glycerate interconversion (Timm et al., 2012;Timm and Bauwe, 2013). The compensatory extent of the two different HPR-bypass reactions was: chloroplastic HPR3 < cytosolic HPR2. It should be noted that HPR2 and HPR3 single knockout mutants do not show noticeable growth impairment to corresponding wild type in ambient air condition, and only led to slight increase of photorespiratory intermediates. The absence of HPR2, the major enzyme of the extraperoxisomal hydroxypyruvate-reducing pathway in Arabidopsis, led to an elevated level of hydroxypyruvate and glycerate [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF]. HPR3 only contributed for a small part to glycerate formation since in the presence of HPR1 and HPR2, it only plays a minor role in the photorespiratory process and its absent led to a slight increase of hydroxypyruvate, glycerate and glycine [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. In addition, while the hpr2xhpr3 double mutant did not show growth retardation compared to WT, the double mutant of hpr1xhpr2 resulted in a pronounced air-sensitivity phenotype and exhibits a dramatic reduction in growth that was not a fully lethal photorespiratory phenotype.

Moreover, hpr1xhpr3 showed the same retarded growth phenotype as hpr1 alone which was consistent with its minor role in the photorespiratory cycle. In addition, the combined mutation in HPR1, HPR2 and HPR3 resulted in an increased growth retardation, a decreased photochemical efficiency and a decreased in CO 2 assimilation compared with hpr1xhpr2 [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. The brief phenotypic features of HPR mutants in Arabidopsis are given in Table 6.

Studies have demonstrated that NADH/NADPH-dependent hydroxypyruvate-reducing enzymes can also use glyoxylate as a substrate [START_REF] Kleczkowski | Purification and characterization of a novel NADPH ( NADH ) -dependent glyoxylate reductase from spinach leaves[END_REF]. The analysis of the enzymatic properties of different Arabidopsis HPR enzymes in the presence of different substrate-cofactor combinations in vitro revealed that HPR1 and HPR2 are the major hydroxypyruvate-reducing enzymes in leaves. HPR1 was found to be an NADH-dependent hydroxypyruvate reductase that showed a relaxed substrate and cofactor specificity while it was slightly inhibited by oxalate. HPR2 appeared to be an NADPH-dependent hydroxypyruvate reductase that also showed relaxed substrate and cofactor specificities, but strongly inhibited by oxalate [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF]. In contrast, HPR3 showed a more specific activity with glyoxylate and NADPH as substrates and it was inhibited by oxalate [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF] Furthermore, HPR1 expression can be induced by drought stress [START_REF] Seki | Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray[END_REF][START_REF] Rizhsky | When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress[END_REF][START_REF] Wang | Shoot-specific down-regulation of protein farnesyltransferase (α-subunit) for yield protection against drought in Canola[END_REF]Li and Hu, 2015). Co-expression analyses and stress-based screens have identified a significant up-regulation of HPR1 transcript levels by a drought stress. This was confirmed when hpr1 was used in drought stress assays where 3.5 week-old plants grown in pots were subjected to drought stress for 18 days. After the drought stress, hpr1 showed an enhanced susceptibility to drought compared with WT plants. Indeed, the mutant exhibited a reduced anthocyanin content, an accelerated chlorophyll degradation and a lower relative water content compared to WT plants (Li and Hu, 2015). In another study, 3week-old WT Arabidopsis were subjected to a 6-day drought stress, and as drought stress progressed, HPR1 expression was induced from the second day, and reached a maximum at the 4 th day with a 2-fold expression when compared with control conditions. Subsequently, HPR1 expression began to decline gradually to reach the basal level over the next 2 days. In addition, a core motif of a dehydration-responsive cis-acting element has been identified in the promoter region of HPR1. The HPR1 drought-inducible promoter was used to drive an RNAi construct of FTA (a-subunit of farnesyltransferase, key negative regulator controlling ABA sensitivity in guard cells) in Canola (Bassica napus), to successfully down-regulate the expression of FTA. The drought-induced down-regulation of FTA in Canola successfully increased the drought tolerance of this crop [START_REF] Wang | Shoot-specific down-regulation of protein farnesyltransferase (α-subunit) for yield protection against drought in Canola[END_REF]. It has also been reported that AtHPR1 is able to interact with an elicitor receptor to participate to the plant-pathogen response [START_REF] Okinaka | The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme, NADHdependent hydroxypyruvate reductase[END_REF]. Therefore, it appears that HPR1 can play a role in plant responses to biotic or abiotic stress.

VII. Phosphorylation of photorespiratory enzymes

The photorespiratory cycle interacts with several major primary metabolic pathways (Hodges et al., 2013). In order to coordinate these multiple interactions, it is probable that the plant has evolved a complex regulatory network. However, knowledge about how the photorespiratory cycle is regulated and how this is coordinated with other pathways is still poorly understood.

Facing complex and changing environmental conditions, plants must be able to rapidly respond and acclimate to them. The initial plant response to an environmental stimulus can involve the rapid post-translational modification of proteins that include acetylation, methylation, glutathionylation, nitrosylation, ubiquitylation and phosphorylation.

1) Phosphorylation sites of the photorespiratory enzymes

Protein phosphorylation is a wide-spread post-translational modification. It is estimated that in Eukaryotes, between 1/3 to 2/3 of proteins can be phosphorylated [START_REF] Philip | The origins of protein phosphorylation[END_REF][START_REF] Vlastaridis | Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes[END_REF]. Phosphorylation occurs in both Prokaryotic and Eukaryotic organisms and plays a critical role in the regulation of cell function and many biological processes including metabolism, cell cycle, growth, abiotic and biotic responses. Protein phosphorylation is a reversible process involving a protein kinase and a protein phosphatase. It can cause a conformational change in the structure of protein that affects the protein functions, such as activity, subcellular localization, capacity to interact with other proteins and stability.

Phosphorylation can occur on several specific amino acids; serine, threonine, tyrosine and histidine. It leads to the covalent attachment (phosphoester bond formation) of a phosphoryl group to the amino acid. Serine phosphorylation is the most common, followed by threonine while tyrosine and histidine phosphorylations are relatively rare. Phosphoproteomic analyses have allowed the discovery of thousands of phosphorylated proteins providing a global view of the phosphorylation status of a whole seedling, a plant organ or a purified plant organelle [START_REF] Nakagami | Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites in Plants[END_REF][START_REF] Kline-Jonakin | Quantitative plant phosphoproteomics[END_REF] and allowed (semi-) quantitative and comparative analyses, for example, of plants grown under different environmental conditions or between wild-type and mutant plants [START_REF] Nakagami | Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites in Plants[END_REF][START_REF] Kline-Jonakin | Quantitative plant phosphoproteomics[END_REF].

Phospho-peptide deposits in the PhosPhAt database (http://phosphat.mpimp-golm.mpg.de/phosphat.html) indicate that all but one (the glycerate kinase) of the 8 core photorespiratory enzymes appear to be phosphorylated, thus suggesting that protein phosphorylation could be a key regulatory component of the photorespiratory cycle (Hodges et al., 2013;Abadie et al., 2016). A list of phosphopeptides associated with photorespiratory or photorespiratory-associated enzymes/proteins detected in published phosphoproteomic studies (see the review, Hodges et al., 2013) and/or taken from the Arabidopsis phosphorylation site database PhosPhAt are given in Table 7.

However, to date, a role for phosphorylation in regulating the photorespiratory cycle is only based on the phosphopeptides detected by the bioinformatics analyses of phosphoproteomics data. Biological and genetic evidence are now required to explain how the protein phosphorylation affects photorespiratory enzyme activity, protein localization or proteinprotein interactions and how protein phosphorylation impacts plant physiology and metabolism. For each photorespiratory enzyme, site-directed mutagenesis at each reported phosphorylation site could be carried out to mimic a constitutively phosphorylated protein (by converting the phospho-amino acids into aspartic acid) or a constitutively nonphosphorylatable protein (by converting the phospho-amino acids into alanine/valine).

Subsequently, it is possible to produce both wild-type and phospho-site-mutated recombinant proteins to test in vitro enzymatic activities and properties. Further, in order to investigate the impact of phosphorylation/non-phosphorylation on plant physiology and metabolism in planta, it is possible to perform plant complementation experiments using knockout mutants and genes to express mutated proteins. The analysis of such transgenic plants (such as growth, development, metabolite level, metabolic fluxes, etc…) could help to explain the role of each phosphorylation event. It is also important to note that the protein phosphorylation is a rapid, temporary and reversible response to environmental stimuli. The phosphorylation state of a given protein will depend on the state and origin of the plant material (whole seedlings, plant organs …) used for phosphoproteomic analyses, and the phosphorylation state will probably depend on growth condition, plant age, organ type. It is therefore important to discover when a specific phosphorylation event is occurring. Furthermore, the list of phosphopeptides associated with photorespiratory enzymes or photorespiratory related enzymes/proteins described in Table 7 may not be exhaustive (Hodges et al., 2013)

2) Objectives of my thesis

Photorespiration has become a target to improve plant photosynthesis and, as a consequence, plant yield [START_REF] Kebeish | Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana[END_REF]2014). To date, although each enzyme of the photorespiratory cycle is known, information about the regulation of the cycle is lacking.

Recent reports and data in the PhosPhAt database show that all core photorespiratory enzymes except the glycerate kinase are associated with at least one phopho-peptide (Table 7)

suggesting that protein phosphorylation could be a potential key regulatory component of the photorespiratory cycle (Hodges et al., 2013). My thesis work was to unravel the consequences of SHMT1 and HPR1 phosphorylations. At the beginning of my project, a single SHMT1 phospho-peptide (pS)LPSEAVDEKER had been identified in rosette leaves [START_REF] Reiland | Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase Substrates and Phosphorylation Networks[END_REF] The phosphoserine was described as located at position 31 of the SHMT1 protein sequence before the removal of the mitochondrial transit peptide and a further N-terminal processing (Huang et al., 2015b). The major photorespiratory HPR1 enzyme was associated with two phospho-peptides, EGMA(pT)LAALNVLGR containing a phosphorylated threonine at position 335 [START_REF] Aryal | Identification of phosphoproteins in arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metalion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry[END_REF], and AS(pS)MEEVLR containing a phosphorylated serine at position 229 [START_REF] Roitinger | Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana[END_REF]. They were detected in cauline leaves and rosettes, respectively.

The major objective of my project was to understand the function and the role of the selected SHMT1 and HPR1 phosphorylation events on enzyme properties (for HPR1) and plant growth and development (for both enzymes). The general strategy to achieve this objective was to study phosphorylation-site mutated recombinant proteins produced either in E. coli or in planta using complemented mutants (hpr1 and shm1-1). In both cases, we compared phosphorylation-mimetic enzymes (where the serine/threonine was changed to an aspartate) with non-phosphorylatable enzymes (where the serine/threonine was changed to an alanine) and wild-type enzymes.

For each strategy the following was carried out:

The characterization of recombinant phospho-site mutated HPR1 produced in E. coli

In order to investigate the effects of HPR1 S229 and T335 phosphorylation on in vitro enzyme activities, the following was performed:

 Recombinant proteins including wild-type HPR1, HPR1-T335A/D and HPR1-S229A/D proteins were expressed in E. coli and purified using the pBAD/His expression system.

 The enzymatic activities of the different recombinant proteins were measured in vitro in the presence of different substrate (hydroxpyruvate or glyoxylate)/cofactor (NADH or NADPH) combinations.

The functional complementation of KO mutants with synthetic genes expressing mutated proteins.

In order to investigate the impact of the SHMT1 and HPR1 phosphorylation/nonphosphorylation on plant physiology and metabolism in planta, Arabidopsis shm1-1 and hpr1-1 mutants were complemented to express different mutated forms of Arabidopsis HPR1 and SHMT1 (for HPR1: HPR1-T335A/D, HPR1-S229A/D and wild-type HPR1; for SHMT1: SHMT1-S31A/D and wild-type SHMT1), under the control of an SHMT1 promoter region.

This led to the production of the following transgenic lines named Compl-HPR1, Compl-HPR1-S229A/D, Compl-HPR1-T335A/D and Compl-SWT, Compl-S31D, Compl-S31A.

The impact of phosphorylation/non-phosphorylation on plant growth and metabolism was assessed.

For HPR1, including Compl-HPR1-S229A/D, Compl-HPR1-T335A/D and Compl-HPR1:

 Phenotypic characterization of hpr1-complemented lines grown in ambient air shortday conditions was carried out by measuring the fresh and dry weight of reosettes.

For SHMT1, including Compl-SWT, Compl-S31D and Compl-S31A:

 Growth phenotype analyses of the shm1-1 complemented transgenic lines was carried out on soil-grown plants in short-day ambient air conditions and in response to salt stress (in vitro and soil-grown plants in ambient air and high CO 2 conditions) and drought stress recovery (soil grown in ambient air conditions).

 Physiological and metabolic phenotyping of the shm1-1 complemented transgenic lines in control and/or in salt stress conditions were determined by gas exchange, stomatal aperture measurements, gene expression analysis, H 2 O 2 amounts and GC-Ms metabolite profiling.

 Characterization of SHMT1 protein and activity levels of the different mutated SHMT1 proteins (SHMT1-31D, SHMT1-31A and SHMT1) by western-blotting and SHMT activities of soluble protein extracts from rosette leaves.

 The role of UBP16 was assessed by yeast-two-hybrid assays and cell-free degradation assays.

 Modifications in the response of the shm1-1 complemented transgenic lines to ABA response were studied with respect to stomatal closure and germination.

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The photorespiratory activity of serine hydroxymethyltransferase 1 (SHMT1) impacts stomatal closure and contributes to abiotic stress tolerance in Arabidopsis thaliana

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The photorespiratory activity of serine hydroxymethyltransferase 1 (SHMT1) impacts stomatal closure and contributes to abiotic stress tolerance in Arabidopsis thaliana

Yanpei Liu 1 , Caroline Mauve 1 , Nathalie Glab 1 , Michael Hodges

INTRODUCTION

Photorespiration is a key primary metabolic pathway that starts with the fixation of O 2 through the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) that leads to the synthesis of one 2-phosphoglycolate (2PG) and one 3phosphoglycerate (3PGA) (Bauwe et al., 2010). However, 2PG, and other intermediates of photorespiration such as glycolate have been shown to reduce photosynthesis by inhibiting triose-phosphate isomerase and RuBisCO, respectively (Anderson, 1971;Kelly and Latzko, 1976;Flügel et al., 2017;Messant et al., 2018). Therefore, the photorespiratory cycle is crucial to remove these toxic metabolites and to recover the carbon as 3PGA. It takes place in four cell compartments (cytosol, chloroplasts, peroxisomes and mitochondria) and involves eight core enzymes and several transporters. Together they convert two molecules of 2PG into one molecule of 3PGA, and in doing so consume one molecule of NADH and ATP and produce one molecule of CO 2 , NH 3 , NADH and ADP (Bauwe et al., 2010). The photorespiratory cycle interacts with a number of primary metabolic processes such as photosynthesis, nitrogen assimilation and amino acid metabolism, the TCA cycle, and C1metabolism (Takahashi et al., 2007;Tcherkez et al., 2008;Hanson and Gregory, 2011;Ros et al., 2014;Bloom, 2015). It also impacts pathogen defense (see Sørhagen et al., 2013) and more recently it has been proposed to affect stomatal aperture (Eisenhut et al., 2017). The importance of the photorespiratory cycle is highlighted by the strong phenotypes exhibited by air-grown knock-out mutants of photorespiratory enzymes and transporters that include reduced growth, chlorosis, and even lethality. Although the phenotypic severity depends on the affected protein, all photorespiratory mutants can be rescued under high CO 2 growth conditions (see Timm and Bauwe, 2013).

Amongst the first photorespiratory cycle mutants identified from EMS-mutated Arabidopsis thaliana were three allelic stm (later renamed shm) mutants affected in mitochondrial serine hydroxymethyl transferase (EC 2.1.2.1; SHMT) activity (Somerville and Ogren, 1981). In A.

thaliana, two of the seven putative SHMT genes appear to encode mitochondrial SHMT isoforms (AtSHMT1, At4g37930; AtSHMT2, At5g26780) (McClung et al., 2000;Engel et al., 2011). The shm1-1 (CS8010) mutant of Somerville and co-workers was mapped to the SHMT1 gene (At4g37930) (Voll et al., 2006). It displays a strong-to-intermediate photorespiratory phenotype that is not observed under high CO 2 (Timm and Bauwe, 2013).

This phenotype shows that SHMT1 has a non-redundant photorespiratory role in which it catalyzes the transfer of a hydroxymethyl group from 5,10-methylene-THF produced by the

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81 glycine decarboxylase complex (GDC), to a glycine to form serine. Interestingly, a shm1-1 x shm2-1 double mutant lacking both mitochondrial SHMT isoforms was unable to survive even in CO 2 -enriched air probably because of the impairment of the additional role of SHMT in C1-metabolism (Douce et al., 200;Engel et al., 2011).

Photorespiratory enzymes also appear to play important roles in plant responses to biotic and abiotic stresses including high temperature, drought, salinity and pathogen attack (Moreno et al., 2005;Rojas et al., 2012;Li and Hu, 2015;Noctor et al., 2015;Cui et al., 2016;Xu et al., 2018). In response to salt stress and pathogen attack, sufficient SHMT1 activity is a key element. Indeed, shm1-2 mutant plants are less resistant to pathogens and hypersensitive to salt and both have been associated with an accumulation of reactive oxygen species (ROS) (Moreno et al., 2005). More recently, a salt tolerant rice variety was found to have a high SHMT expression associated to a strong accumulation of serine in response to salt stress when compared to a salt sensitive variety (Mishra et al., 2016). The overexpression of SHMT from the halotolerant cyanobacteria Aphanothece halophytica either in E. coli or in the fresh water cyanobacteria Synechococcus elongates conferred a better tolerance to salinity (Waditee-Sirisattha et al., 2012;Waditee-Sirisattha et al., 2016).

Although SHMT1 has a key role in the photorespiratory cycle and it has been shown to be involved in abiotic and biotic stress responses, the regulation of SHMT1 activity remains to be elucidated. To rapidly respond to changing conditions, enzymes are often posttranslationally modified and several reports suggest that this could be the case for SHMT1.

Indeed, SHMT1 is a potential target of thioredoxin-dependent redox regulation (Marchand et al., 2004). SHMT1 was found to be degraded in a 26S proteasome-dependent process, and ubiquitin-specific protease 16 (UBP16) stabilized SHMT1 during a salt stress by removing conjugated ubiquitin (Zhou et al., 2012). Pea mitochondrial SHMT was found to be nitrosylated under control conditions but not during a salt stress (Camejo et al., 2013).

According to the Arabidopsis phosphopeptide database, PhosPhAt (http://phosphat.unihohenheim.de/), 8 residues of SHMT1 can be phosphorylated. Previously, we reported that the phosphorylation of serine 31 (S31) of the phosphopeptide (pS)LPSEAVDEKER remained unchanged in Arabidopsis rosettes when treated with varying O 2 /CO 2 conditions suggesting that this phosphorylation event was not correlated to leaf photorespiratory activity (Abadie et al., 2016). It should be noted however, that after removal of the mitochondrial signal peptide 82 and a further processing by intermediate cleavage peptidase 55 (ICP55; Huang et al., 2015), S31 become S1 of the mature SHMT1 protein.

In this work, the function of Arabidopsis thaliana SHMT1 S31 phosphorylation was investigated by studying shm1-1 plants complemented with either wild-type, phosphorylation mimetic (S31D) or non-phophorylatable (S31A) SHMT1. In normal air-grown conditions, no significant differences in growth were observed between the different lines, although they were slightly smaller than WT Col-0 plants. However, when subjected to either a salt or a drought stress, Compl-S31D lines exhibited a further reduction in growth. In the case of a salt treatment, the increased sensitivity was associated with altered phosphorylation-mimic S31D SHMT1 protein and activity levels. Our data will be discussed in terms of the importance of photorespiratory SHMT1 activity in plant abiotic stress tolerance and ABA-induced stomatal movements, and the possible role of S31 phosphorylation in modulating SHMT1 protein stability.

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83

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Complementation of the shm1-1 photorespiratory growth phenotype by S31-mutated

SHMT1

To evaluate the impact of SHMT1 S31 phosphorylation on plant physiology and metabolism, site-directed mutagenesis was undertaken using the Arabidopsis cDNA of wild-type SHMT1, so that S31 was replaced either by a phosphomimetic aspartic acid (SHMT1-S31D) or by a non-phosphorylatable alanine (SHMT1-S31A) (Figure 1A). Subsequently, the resulting constructs were placed under the control of an AtSHMT1 promoter region and used to transform high CO 2 -grown shm1-1 plants to generate complemented plants named Compl-SWT, Compl-S31A, and Compl-S31D (Figure 1B). Homozygous lines containing a single insertion for the transgene were selected for further analysis. While shm1-1 was unable to grow in air, this was not the case for the complemented lines although they were slightly smaller compared to wild-type (WT, Col-0) control plants (Figure 1B). After 4 weeks on soil, the development of each transgenic line was similar when compared to each other (Figure 1 B).

N-terminal peptide analyses were performed by LC-MS/MS using extracts from rosettes of complemented lines (Compl-SWT-5, Compl-S31A-7 and Compl-S31D-5) to determine whether ICP55 processing was modified. In Compl-SWT-5 and Compl-S31A-7, the identified N-terminal peptides showed that ICP55 processing still took place however this was not the case for SHMT1 of Compl-S31D-5 since only a SDLPSEAVDEK peptide was observed (Supplemental Figure S1).

Western blot analyses were carried out to compare rosette leaf SHMT protein amounts between the different lines (Figure 1C). To allow the shm1-1 mutant to grow, plants were grown in high CO 2 (3000 µL.L -1 ). As expected, almost no SHMT protein was detected in shm1-1 rosettes while complemented lines exhibited different amounts of SHMT protein (between 40% and 70% of Col-0 SHMT protein levels) (Figure 1C and Supplemental Figure S2). Moreover, in accordance with SHMT protein levels in the complemented lines, SHMT activity analysis of high-CO 2 -grown rosette leaves showed that they all had a 40-60 % reduced activity compared to Col-0 leaves (Figure 1D).

Since a reduced photorespiratory cycle activity has a negative impact on photosynthetic CO 2 assimilation (Somerville and Ogren, 1980, 1981, 1982, Chastain and Ogren, 1989;Timm et al., 2012) and electron transfer properties (Dellero et al., 2015(Dellero et al., , 2016)), gas exchange and

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84 chlorophyll fluorescence parameters of fully expanded leaves were compared between 6week-old WT plants and complemented lines with lower SHMT1 activities. In the three transgenic lines Compl-SWT-5, Compl-S31D-5 and Compl-S31A-7, net CO 2 assimilation rate (A n ) was reduced by 20% compared to control Col-0 plants while stomatal conductance (g s ), transpiration (Tr), photosynthetic electron transfer rate (ETR), non-photochemical chlorophyll fluorescence quenching (NPQ) and Fv/Fm ratios were not significantly different plants (Supplemental Table S1). These results suggest that the smaller size of the complemented lines could be due to their lower capacity to assimilate CO 2 arising from their lower SHMT1 activity leading to an impaired photorespiratory cycle.

Col-0 Compl-SWT-5 Compl-S31A-7 Compl-S31D-5

A n (µmol CO 2 m -2 s -1 ) 7.50 ± 0.45 5.99 ± 0.57** 6.09 ± 0.90** 6.52 ± 0.25** g s (mmol H 2 O m -2 s -1 ) 0.156 ± 0.028 0.123 ± 0.021 0.139 ± 0.029 0.128 ± 0.040

Tr (mmol H 2 O m -2 s -

Compl-S31D lines are hypersensitive to salt stress in air-grown conditions but not in high CO2-growth conditions

Since the different complemented lines did not show any distinct growth and photosynthetic differences between them, it was decided to test their response to abiotic stress. Indeed, it had been previously reported that the shm1-3 mutant was more sensitive to salt stress (Moreno et al., 2005) therefore in vitro grown one-week-old WT, Compl-SWT-5, Compl-S31D-5 and

Compl-S31A-7 seedlings were transferred to 50 mM NaCl or 100 mM mannitol for 3 weeks.

In the absence of NaCl, no difference was observed between the complemented lines although they presented a growth deficiency compared to Col-0 seedlings (Figure 2A). This resulted in a 50% decrease of seedling fresh weight (Figure 2B) and a 20% decrease of primary root length (Supplemental Figure S3A). When transferred to mannitol, no significant differences were observed between the complemented lines but they all exhibited a 40% reduction of fresh weight compared to Col-0 (Figure 2A and2B). However, in the presence of 50 mM NaCl, primary root length and seedling fresh weigh of the Compl-S31D-5 line showed significant reductions compared to Compl-S31A-7, Compl-SWT-5 and wild-type lines (Figure 2A and Supplemental Figure S3A). While Col-0 seedlings were slightly affected by the salt treatment, the Compl-S31D-5 fresh weight was decreased by 50% (Figure 2B) and primary root length was decreased by almost 30% (Supplemental Figure 3A) when compared to

Compl-S31A-7 and Compl-SWT-5 lines. Similar results were obtained for two other independent Compl-S31D lines (S31D-4 and S31D-7) when treated with salt, both presenting a larger decrease in seedling fresh weight and primary root length on 50mM NaCl (Supplemental Figure S4A and S4B). To determine whether the observed salt-induced growth phenotypes of Compl-S31D lines were associated with photorespiration, experiments were repeated in high CO 2 -growth conditions (3000 µL CO 2 .L -1 ) and included the shm1-1 mutant as a control. After 4 weeks, Compl-S31D-5 seedlings did not display any differences in growth (seedling fresh weight and primary root length) compared to shm1-1, Compl-SWT-5

and Compl-S31A-7 lines whether grown with or without 50 mM NaCl (Figure 2C and 2D, Supplemental Figure S3). Again, these observations were confirmed with the other Compl-S31D lines (Supplemental Figure S4C andS4D). Taken together, our data show that Compl-S31D lines are more sensitive to salt stress and that this is dependent on photorespiration.

Furthermore, the growth differences between air-grown Col-0 WT plants and the different transgenic lines also appear to be photorespiration-dependent whether under control or saltstress conditions (Figure 2).

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SHMT1 protein is less stable in the Compl-S31D-5 line in response to salt stress leading to a lower SHMT activity

To determine whether the salt-induced Compl-S31D-5 growth was linked to SHMT activity, this was measured using in vitro grown Col-0, Compl-SWT-5, Compl-S31D-5 and Compl-S31A-7 lines under either control or a 3-week salt treatment (Figure 3A). Leaf total soluble proteins were extracted and SHMT activity was measured as the conversion of serine to glycine using an HPLC based method (Simic et al., 2002). The salt treatment led to a significant 40% decrease in SHMT activity of Compl-S31D-5 while a much smaller decrease was measured for Compl-SWT-5 and Compl-S31A-7 (reduction of 13% and 15%, respectively) (Figure 3A). These activity changes appeared to mirror leaf SHMT protein levels as determined by Western blot analyses (Figure 3B and3C). Indeed, the SHMT protein level of Compl-S31D-5 was significantly reduced by 35% after a salt stress compared to Compl-SWT-5 and Compl-S31A-7 (non-significant 11% and 15% reduction, respectively) (Figure 3B and 3C). Similar results were obtained for the other Compl-S31D lines (4 and 7) compared to the Compl-SWT (1 and 7) and Compl-S31A (4 and 6) lines (Supplemental Table 2). Taken together, it appears that leaf SHMT1 protein of Compl-S31D-5 is less stable in response to the NaCl treatment leading to a lower SHMT activity and thus giving rise to a more severe saltinduced growth phenotype (Figure 2).

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Metabolic responses of Col-0, Compl-SWT-5, Compl-S31A-7 and Compl-S31D-5 to salt stress

In order to explore the metabolic consequences of the reduced SHMT activity of Compl-S31D-5 in response to salt stress, relative metabolite levels of whole rosettes of seedlings grown in vitro with or without 50 mM NaCl were analyzed by GC-MS (Supplemental Table S3). In control conditions, a limited number of similar differences were observed in all of the transgenic lines compared to Col-0. These included certain amino acids, organic acids and sugar levels. Indeed, asparagine, glutamate, glutamine, homoserine and tryptophan as well as the photorespiratory metabolites glycine, serine and glycerate accumulated in the complemented lines (Figure 4). Krebs cycle-associated organic acids including citrate, malate, and α-ketoglutarate as well as glucose and fructose levels also increased while dehydroascorbate and glycerol-3-phosphate decreased in the complemented lines Col-0 (Figure 4). However, glucose-6-phosphate and fructose-6-phoshate were only higher in the Compl-S31D-5 line (Figure 4). In response to 50 mM NaCl, while the relative levels of asparagine, glycine, glutamate and glutamine decreased in Col-0, Compl-SWT-5 and Compl-S31A-7, these amino acids remained at a higher level in Compl-S31D-5. On the other hand, levels of metabolites associated with stress protection such as proline, galactaric acid, and myo-inositol increased in all lines except Compl-S31D-5 in response to salt stress (Figure 4).

Citrate and glycerol-3-phosphate levels did not change in response to salt stress in Compl-SWT-5 and Compl-S31A-7 but decreased in Compl-S31D-5. Interestingly, major metabolic differences concerned the levels of polyamines (spermidine and putrescine) and their precursors, ornithine and citrulline, that increased by 3-fold (2-fold for putrescine) in response to salt stress only in Compl-S31D-5 (Figure 4). Hydrogen peroxide is a key element of stressinduced signaling pathways and Arabidopsis lacking SHMT1 exhibit a large accumulation of H 2 O 2 in their leaves in response to salt stress (Moreno et al., 2005). In order to determine if the complemented lines exhibited altered leaf H 2 O 2 contents, control and salt stressed Col-0, Compl-SWT-5, Compl-S31D-5 and Compl-S31A-7 seedlings where stained with DAB. The H 2 O 2 -induced brown deposit in Compl-S31D-5 was more important than Col-0, Compl-SWT-5 and Compl-S31A-7 plants after the stress treatment indicating higher H 2 O 2 levels while no differences were observed under control conditions (Figure 5A). A quantitative analysis of H 2 O 2 content carried out on the same samples showed that all lines exhibited an increase in H 2 O 2 increased after the salt treatment, but the increase was 2-fold more important in Compl-S31D-5 rosettes (Figure 5B and Supplemental Figure S5).

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Compl-S31D plants are unable to recover from a drought stress in ambient air

Since our data indicated that Compl-S31D-5 plants were more sensitive to a salt stress, it was decided to test their sensitivity to a drought stress. To achieve this, Col-0, Compl-SWT, Compl-S31A and Compl-S31D plants were grown on soil for 3 weeks in well-watered conditions, and then they were subjected to a drought stress by withholding water for 3 weeks before being re-watered. Under such conditions, the Compl-S31D lines were unable to recover from the drought stress (Figure 6A). Moreover, detached leaves of Compl-S31D lines showed a faster initial water loss ratio compared to the other lines (Figure 6B). It should be noted that all plant lines had similar initial water contents (Supplemental Figure S6).

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Compl-S31D plants have altered ABA-induced gene expression under salt treatment

Since Compl-S31D lines were more sensitive to drought and salt stresses and they exhibited a reduced response to NaCl-induced and ABA-induced stomatal closure, it was decided to further explore the ABA signaling responses of the plant lines. Therefore, the expression of several ABA-responsive genes was examined by qPCR, before and after a 6 h salt treatment of 2-week-old in vitro grown Col-0, Compl-SWT-5, Compl-S31A-7 and Compl-S31D-5

seedlings. The expression of the ABA-responsive genes RD29A (Lee et al., 2016), RD29B (Nordin et al., 1993) and P5CS1 (Ábrahám et al., 2003) increased after the salt treatment in all lines (Figure 8). Whereas RD29B expression did not differ between lines, salt treatment led to

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103

DISCUSSION

The reduced SHMT1 activity of shm1-1 complemented lines negatively impacts

photosynthetic CO 2 assimilation and plant growth

The molecular mechanisms by which photorespiratory enzymes are regulated are still not known however, phosphorylation is a good candidate since seven of the eight core photorespiratory cycle enzymes are associated with phosphopeptides (Hodges et al., 2013).

Among these enzymes, SHMT1 undergoes a photorespiration-independent phosphorylation at S31 (Abadie et al., 2016) that therefore could be involved in a different physiological response. In order to evaluate the impact of SHMT1 S31 phosphorylation on plant metabolism and growth, the shm1-1 mutant was complemented with SHMT1-S31D

(phospho-mimetic), SHMT1-S31A (non-phosphorylatable) or SHMT1-SWT (non-mutated) forms (Figure 1A). In air-grown conditions, each recombinant SHMT1 form reversed the shm1-1 photorespiratory growth phenotype however they remained smaller than wild-type Col-0 plants (Figure 1B,2A and 2B). This observation was associated with a 50% lower SHMT activity in all complemented rosettes compared to Col-0 controls and correlated with reduced SHMT1 protein levels (Figure 1C, 1D and Supplemental Figure S2). Therefore, if the phospho-mimetic SHMT1 S31 is indeed equivalent to phosphorylation, it does not appear to regulate maximal SHMT1 activity. As might be expected, the reduced SHMT activity plants also exhibited lower photosynthetic CO 2 assimilation rates when compared with Col-0 (Supplemental Table S1). This suggested that under standard air-grown conditions, SHMT1 activity had become limiting for the photorespiratory cycle requirements of the transgenic plants, probably leading to toxic 2PG accumulation and/or limited C-recycling that would negatively impact Calvin Cycle activity and CO 2 assimilation. Indeed, an inhibition of photosynthesis is a common feature of a blocked photorespiratory cycle as seem by transferring photorespiratory mutants, including shm1-1, from high CO 2 conditions to air (Somerville and Ogren, 1981;Chastain and Ogren, 1989;Timm et al., 2012). The photorespiratory nature of the observed growth phenotype of our complemented lines was confirmed by the absence of any growth phenotype under high CO 2 conditions (Figure 2C and 2D). The non-optimal SHMT1 activity also had an impact on rosette metabolite levels in a similar manner (albeit less important) to those reported for shm1-1 when transferred from high to normal CO 2 air (Kuhn et al., 2013). Major changes were observed in several photorespiratory metabolite levels, as well as certain sugars, amino and organic acids. Indeed, the 5-to-6 fold increase in glycine is a good indicator of the limiting SHMT1 photorespiratory

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104 activity in the complemented lines. The higher serine levels (2-3 fold) might be due to alternative serine biosynthesis pathways such as the phosphorylated pathway and the glycerate pathway (Ros et al., 2014). Glucose and fructose increased by 2-to-3 fold (Figure 4) and this could reflect an altered starch metabolism as recently reported in PGLP1 knock-down lines (Flügel et al., 2017) to allow the production of soluble sugars for cell metabolism when photosynthesis is impacted by an ineffcient photorespiratory cycle. Perhaps surprisingly, the limiting SHMT1 activity had only small effects on amino and organic acid levels although proline content was 2-fold reduced while -ketoglutarate amounts were increased by 4-to-6 fold (as already reported by Kuhn et al., 2013). Both of these metabolites are associated with glutamate metabolism, however glutamate levels only increased by 40% while glutamine increased by 70-90% thus suggesting a limited impact of reduced SHMT1 activity on GS/GOGAT cycle functioning and nitrogen assimilation.

The sensitivity of Compl-S31D lines to salt stress in air-grown conditions is due to a further reduction of SHMT1 protein and activity

Previous studies have shown shmt1 mutants to be more sensitive to NaCl stress (Moreno et al., 2005;Zhou et al., 2012). Interestingly, Compl-S31D lines were more sensitive to a salt stress,

showing reduced growth and yellow rosettes as well as a lower fresh weight and shorter primary root growth compared to the other lines when grown in air (Figure 2 and S3A). Plant response to salinity occurs in two steps: a rapid response to an increase of external osmotic pressure, and a slower response due to the accumulation of toxic ions (Munns and Tester, 2008). No differences were observed between the complemented lines in response to a mannitol-generated osmotic stress. Indeed, the transgenic lines remained smaller than Col-0 even though plant growth was severely reduced compared to control conditions (Figure 2A, 2B). Thus, the phenotype observed in Compl-S31D lines can be attributed to the ionic phase of the salinity response. Interestingly, this phenotype disappeared when plants were grown under high-CO 2 conditions (Figure 2C, 2D and S3B) thus showing that the salt stress phenotype of Compl-S31D lines was linked to photorespiration. Since the transgenic lines had only 50% of normal Col-0 SHMT activity and already exhibited a growth phenotype in air, it was possible that the additional salt-effect on Compl-S31D lines might reflect a further inhibition of SHMT1 activity. This hypothesis appeared to be correct since there was a further decrease in SHMT activity (of about 43%) in Compl-S31D-5 seedlings when salt stressed compared to control conditions while no significant differences were observed for Compl-

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105 SWT-5 and Compl-S31A-7 lines (Figure 3A and Table S2). This decrease in activity correlated with a further reduction in SHMT1 protein (of about 35%) (Figure 3B,C). So after a salt stress, Compl-S31D rosettes contained only 33% of WT Col-0 SHMT activity (Figure 3A)

and exhibited a severe growth inhibition phenotype compared to the other lines (Figure 2, S2 andS3). This suggested that a critical SHMT1 activity had been reached that no longer allowed the plants to cope with a salt stress-induced increase of photorespiration. Of course, it should be noted that transgenic plants with only 50% SHMT activity already showed a small growth phenotype in non-stressed air-grown conditions. The need to reduce a photorespiratory enzyme activity below a certain level before observing a phenotype is common. Rice glycolate oxidase (GOX) activity had to be reduced below 60% to observe a reduced photosynthetic activity (Xu et al., 2009). Arabidopsis gox1-1 showed no growth phenotype with only 27% of WT leaf GOX activity, while a photorespiratory phenotype required the activity to be reduced to a residual 5-10% (Dellero et al., 2016a). Tobacco leaf GOX activity had to be decreased to 36% in order to induce photorespiratory growth symptoms (Yamaguchi and Nishimura, 2000) while less than 10% GOX activity was required to affect maize (Zelitch et al., 2009). So, we conclude that the salt-induced sensitivity phenotype of Compl-S31D lines was due to their low photorespiratory SHMT activity brought about by a salt-induced decrease in SHMT1 protein abundance. Recently, it was reported that ubiquitin-specific protease 16 (UBP16) modulates salt tolerance by regulating SHMT1 stability (Zhou et al., 2012). It was proposed that during salt stress, UBP16-dependent SHMT1 stability reduced the accumulation of reactive oxygen species thus increasing the activity of a Na + /H + antiporter (Zhou et al., 2012). Therefore, it is possible that a low SHMT1 activity could reduce cell Na + export thus leading to salt stress sensitivity. Perhaps, the action of UBP16 is altered by phospho-mimetic S31 SHMT1 in Compl-S31D lines thus leading to decreased protein stability under salt conditions. Modification of the N-terminal of SHMT1 has also been shown to alter SHMT1 stability. In Arabidopsis, intermediate cleavage peptidase55 (ICP55) removes the N-terminal S30 of "mature" SHMT1 thus making it less stable due to an increased turnover rate (Huang et al., 2015). It was found that S30 was correctly removed from SHMT1 in Compl-SWT-5 and Compl-S31A-7 rosettes but not in the Compl-S31D-5 line (Supplemental Figure S1). Surprisingly, SHMT1 S31D protein abundance appeared to be similar to SHMT1 S31A and SHMT SWT proteins in normal growth conditions (soil and in vitro) while it decreased in response to salt stress (Figure 1C, 3B, 3C

and Supplemental Figure S2). Thus, the presence of S30-containing SHMT1 S31D protein did

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106 not appear to impact SHMT1 stability under non-stress conditions. In WT plants, the S31containing phosphopeptides never contained S30 and therefore it has been processed by ICP55, however S31 phosphorylation was not required for ICP55 processing since this still occurred when phosphorylation was not possible, as observed for SHMT1 S31A

(Supplemental Figure 1). Further evidence suggests that the N-terminal sequence of SHMT1 is important for stability since a deletion of the first six or more amino acids of sheep SHMT decreased the heat-stability of its oligomeric structure (Jagath et al., 1997). SHMT1 is a tetramer (Wei et al., 2013) although a dimer organization is the minimum catalytic unit (Zhang et al., 2018a) while monomeric SHMT1 is inactive. Perhaps the additional negative charges of a phosphate (or an aspartate) at the N-terminus of the protein could result in a less stable SHMT1 under salt stress conditions.

Decreased SHMT1 activity in phospho-mimetic Compl-S31D alters metabolic responses to salt stress

In response to salt stress, a number of metabolite levels were seen to change in all plant lines studied including decreases in glutamine, asparagine, tryptophan, -ketoglutarate, glucose, fructose (Figure 4) and glucose-6-phosphate (Supplemental Table S3) while serine, myoinositol (Figure 4), urea and nicotinic acid (Supplemental Table S3) increased. The limited number of metabolic changes suggested that our salt treatment was rather mild. Globally, the SHMT1 transgenic lines that maintained 50% of WT Col-0 SHMT activity underwent similar salt-stress metabolic rearrangements as the WT Col-0 plants thus indicating that at this low SHMT1 activity additional adverse affects on plant metabolism where not induced by a 50mM NaCl treatment. This was perhaps unexpected since salt stress led to stomatal closure (Figure 7A, 7B) that should increase photorespiration and a requirement for a higher SHMT1 activity. Therefore, it was surprising to observe a salt-stress induced decrease of glycine (Figure 4) that was negatively correlated with SHMT activity (Figure 3A) suggesting that glycine was being mobilized for other processes such as the biosynthesis of glutathione, an important stress-induced molecule involved in ROS-detoxification (Noctor et al., 2012).

Indeed, a trend was observed of increased metabolite levels associated with stress responses including proline and myo-inositol while galactaric acid increased in all lines except Compl-S31D-5 (Figure 4). The latter has been reported to have a role in plants under stress conditions (Perlikowski et al., 2016;Sprenger et al., 2018), proline is an essential osmoprotectant in response to salt stress (Szabados and Savouré, 2009) and myo-inositol is important for plant

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107 tolerance to drought and salt stresses (Kaur et al., 2013). The reduced levels of these three metabolites in salt-stressed Compl-S31D-5 plants might partially explain their increased sensitivity to salt and drought stresses. Compl-S31D-5 rosettes were also different compared to the other lines with respect to the accumulation of polyamines such as spermidine (3-fold more) and putrescine (2-fold more) (Figure 4) while ornithine also accumulated in Compl-S31D-5 in response to a NaCl treatment and citrulline remained at a higher level compared to the other lines (Figure 4). Therefore, salt-stressed Compl-S31D-5 plants appeared to modify their metabolism to favor polyamines. The reason for this is unknown but it might reflect the biosynthesis of nitrogen-rich compounds at a lower carbon cost due to limited carbon resources arising from an inability to maintain sufficient photorespiratory carbon recycling and a further reduced photosynthetic capacity.

Compl-S31D lines exhibit an altered stomatal response to ABA and salt stress

Compl-S31D plants were not only more sensitive to salt stress but also to drought stress recovery while Compl-S31D detached rosettes exhibited a higher and faster water loss compared to the other lines (Figure 6A and 6B). The water loss phenotype was not due to a defect in initial water content (Supplemental Figure S5) and it probably reflected the altered stress-induced stomatal movements of Compl-S31D lines (see Figure 7). Indeed, both salt and drought stresses lead to stomatal closure to reduce transpiratory water losses however the reduction of leaf internal CO 2 levels should reduce photosynthesis while increasing photorespiration (Chastain et al., 2014;Fricke et al., 2004). By studying photorespiratory mutants, including shm1, it was recently shown that photorespiration is essential for stomatal movements (Eisenhut et al., 2017). Since no differences in stomatal conductance or aperture were detected between our SHMT1 lines in air-grown conditions (Figure 7 and Supplemental Table S1), it appeared that 50% of WT Col-0 SHMT1 activity was enough to maintain stomatal aperture. However, after a salt stress or an ABA treatment that led to stomatal closure, the stomata of Compl-S31D-5 remained more open (Figure 7) while SHMT activity was further reduced to only 35% of the Col-0 value. This decreased stomatal closure in response to salt and to ABA (Figure 7C and 7D) could impact the sensitivity of Compl-S31D-5 to salt stress and drought recovery. Since mesophyll organic acid metabolism is involved in regulating stomatal aperture (Araújo et al., 2011) , metabolic changes in Compl-S31D rosettes might help explain the observed differences. However, malate and fumarate levels were not significantly different between Compl-S31D and WT Col-0 lines. The plant hormone, ABA, is

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108 involved in stomatal closure during both salt and drought stresses and Compl-S31D plants were less sensitive to ABA (Figure 7C, 7D). This is not the first time that photorespiration has been implicated in ABA signaling. A screen of EMS-mutagenized plants for an altered expression of the ABA and NaCl induced gene RD29A (Responsive to Dessication 29A) led to the identification of two glutamate:glyoxylate transferase 1 (GGT1) alleles (Verslues et al., 2007). However, while ggt1 mutants were ABA desensitized due to a basal H 2 O 2 accumulation, this was not the case for Compl-S31D lines as their rosettes contained similar amounts of H 2 O 2 when compared to the other lines under control conditions and only a small 20% difference in rosette H 2 O 2 levels was measured in response to salt stress (Figure 5 and Supplemental Figure S4). To examine whether ABA signaling was involved in the differential stomatal responses, the expression of several stress marker genes, RD29A, RD29B and P5CS1, was tested in response to NaCl treatment. RD29B expression is regulated by ABA while RD29A expression is regulated by ABA and by an unknown pathway activated by NaCl stress (Sakuma et al., 2002;Yamaguchi-Shinozaki and Shinozaki, 1993) and salt-induced P5CS1 expression can involve either ABA or an ABA-independent phospholipase C (PLC) pathway (Strizhov et al., 1997;Parre et al., 2007). Since RD29A and RD29B were induced in response to our NaCl treatment in all lines (Figure 8), the ABA signaling pathway appeared unaffected at the whole rosette level. However, a possible role of the PLC pathway was suggested by the lower salt stress induction of P5CS1 in Compl-S31D-5 (Figure 8). Myo-inositol together with glycerol-3-phosphate are essential metabolites for phosphoglycerolipid biosynthesis (Janda et al., 2013) and both metabolites were not induced by a salt stress in Compl-S31D-5 when compared to the other lines (Figure 4). Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) can be hydrolyzed by phospholipase C (PLC) to generate the secondary messenger inositol 1,4,5triphosphate (IP 3 ) (Janda et al., 2013) that participates in stomatal closure (Glilroy et al., 1990). Recently, an Arabidopsis PLC mutant (plc3) was found to exhibit a stomatal response phenotype similar to Compl-S31D-5 as no significant differences were observed in stomatal aperture between wild-type and plc3 but the latter exhibited a reduced sensitivity to ABAinduced stomatal closure (Zhang et al., 2018b). In this scenario, if PIP 2 synthesis is deficient in response to a salt stress in Compl-S31D-5, stomatal closure will be slower or reduced.

Finally, spermidine accumulation which can occur rapidly (Podlešáková et al., 2018), can lead to the accumulation of 1,3-diaminopropane (DAP) (Cona et al., 2006). Acetyl-DAP has been shown to antagonize ABA and to slow down stomatal closure to maintain CO 2 uptake during a mild drought stress (Jammes et al., 2014). Salt stress led to an accumulation of spermidine 109 in Compl-S31D-5 (Figure 4) that could eventually bring about a higher production of acetyl-DAP and thus reduce Compl-S31D stomatal closure.

In conclusion, we have shown that photorespiration is an important metabolic process when plants undergo salt stress. Indeed, the absence/reduction of photorespiration brought about by high CO 2 conditions almost abolished the salt-induced growth defects. In air, plants must maintain an efficient photorespiratory cycle flux since we observed that a low SHMT1 activity increased salt stress sensitivity. With respect to S31 phosphorylation of SHMT1, based on the position of S31 in the observed phosphopeptides, it is not involved in the removal of the mitochondrial signal peptide nor N-terminal processing by ICP55. Since the phospho-mimetic SHMT1 did not exhibit an altered maximal SHMT activity, S31 phosphorylation does not appear to be directly involved in modulating enzymatic activity.

However, the stability of S31 phospho-mimetic SHMT1 appeared to be reduced under mild salt stress conditions, although we would not expect SHMT1 S31 phosphorylation to be induced under such conditions due to its detrimental effect. Finally, photorespiratory cycle flux efficiency appears to influence stomatal movements in a yet to be discovered mechanism that could involve a common metabolic component involved in stomatal closure.

MATERIALS AND METHOD

Plants material and growth conditions

Arabidopsis thaliana Columbia (Col-0) accession was used as a wild-type control in all experiments. The shm1-1 mutant (Somerville and Ogren, 1981;Voll et al, 2006) was provided by Andreas Weber (University of Dusseldorf, Germany).

For in vitro experiments, sterilized seeds were sown on ½MS medium (Murashige and Skoog, 1962), supplemented with 0.8% agar (m/v), and placed at 4°C in the dark for 48 h to promote germination. Plants were grown in short day conditions (8h light/16h dark, 160 μmol of photons.m -2 .s -1 , 65% humidity, with a temperature of 20°C day/18°C night). For the salt treatment, one-week-old seedlings were grown vertically on ½MS medium containing 0.8% agar (m/v) before their transfer to new plates with or without 50 mM NaCl or 100 mM mannitol. For soil-grown experiments, Arabidopsis seeds were germinated on potting soil for 10 days in short days (8h light/16h dark, 200 μmol of photons.m -2 .s -1 , 65% humidity, 20°C day/18°C night). Subsequently, seedlings were transplanted to individual pots and grown further in short day conditions either in air (CO 2 concentration around 400 μL.L -1 ) or high CO 2 (3000 μL.L -1 ) enriched air (HC). For the drought treatment, Arabidopsis seeds were germinated on potting soil for 10 days in short day conditions, transplanted and grown for 2 weeks before being subjected to water deprivation for 3 weeks then re-watering for 3 days.

Plasmid construction, site-directed mutagenesis and generation of complemented lines

Genomic DNA was extracted from A. thaliana Col-0 leaves according to Edwards et al., 1991, and the SHMT1 promoting region (-1000 to +55) was PCR-amplified using primers (promSHMT1-F/promSHMT1-R) described in Supplemental Table S4. The resulting DNA (SHMT1 promoter) was cloned into pGEM-T Easy® and sequenced (by MWG Operon, Eurofins). The SHMT1 cDNA in pUNI51 (clone U21423) was obtained from the Arabidopsis Biological Resource Center (ABRC) (https://abrc.osu.edu/). SHMT1 cDNA was PCRamplified using primers cDNA-SHMT1-F and GW-SHMT1-R (Supplemental Table S4) and the SHMT1 promoter region was re-amplified with the primers described above. The two resulting DNA fragments (SHMT1 cDNA and SHMT1 promoter), with an overlapping region (0 to +55), were used as templates for two consecutive PCR amplifications (using primers GW-SHMT1-F/GW-SHMT1-R followed by GateWay-F/GateWay-R, see Supplemental Table S4) in order to join the two DNAs and tto add attB Gateway® recombination sites to the 5' and 3' ends. The resulting DNA (promSHMT1::SHMT1) was further sequenced before being cloned into pDONR207 using the Gateway® cloning system to generate pDONR207-promSHMT1::SHMT1.

To introduce the required point mutations into pDONR207-promSHMT1::SHMT1, primer pairs (see Supplemental Table S4) were selected and used with the QuikChange® II XL Site-Directed Mutagenesis Kit (Agilent®), according to the manufacturer's instructions to obtain pDONR207-promSHMT1::SHMT1-S31A and pDONR207-promSHMT1::SHMT1-S31D. Then, promSHMT1::SHMT1, promSHMT1::SHMT1-S31A and promSHMT1::SHMT1-S31D were subcloned into pH7WG using the Gateway® cloning system to generate the plant transformation constructs pH7WG-promSHMT1::SHMT1, pH7WG-promSHMT1::SHMT1-S31A and pH7WG-promSHMT1::SHMT1-S31D. They were used to transform HC-grown shm1-1 plants by Agrobacterium-mediated floral dip (Clough and Bent, 1999). Hygromycinresistant seeds were selected and homozygous transgenic lines containing a single insertion were selected by successive rounds of self-crossing and antibiotic selection on ½MS medium agar plates.

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111

Analysis of N-terminal sequence of SHMT1 protein

Arabidopsis rosette leaves (100 mg fresh weight) were rapidly frozen in liquid nitrogen and stored at -80°C until extraction. Protein extraction, LC-MS/MS analysis and identification of SHMT1 peptides were carried out as previously described (Boex-Fontvieille et al., 2013).

Leaf gas exchange and chlorophyll fluorescence measurements

Leaf gas exchange and chlorophyll fluorescence measurements were carried out on 2 cm 2 of fully expanded leaves in a gas exchange chamber (LCF 6400-40, LiCOR) connected to a portable measuring system (LI 6400XT, LiCOR) [START_REF] Dellero | Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth[END_REF]. Standard measuring conditions were: 200 μmol photons m -2 s -1 of light intensity, a leaf temperature of 21 °C, 60-70% relative humidity [vapor pressure deficit (VPD) of the leaf approximately 1], 400 μmol mol -1 CO 2 and 21% O 2 . For chlorophyll fluorescence parameters, plants were adapted for 30 min to darkness before measuring the Fo and Fm levels. Parameters were calculated as follows: Fv=Fm-Fo, NPQ=(Fm-Fm´)/Fm´, ETR=[(Fm´-Fv)/Fm´]0.5×I×αleaf, with I corresponding to the irradiance in μmol photons m -2 s -1 and αleaf to the light absorption coefficient of a leaf=0.85.

SHMT activity assays

SHMT activity was determined by following the production of glycine from serine as measured by HPLC. 400 mg of rosette or seedlings leaves were harvested and ground into a fine powder in liquid nitrogen. Total soluble protein was extracted in 500 µl buffer containing 50 mM NaH 2 PO 4 , anti-protease cocktail (Complete-Mini Free-EDTA, Roche®), 1 mM βmercaptoethanol, pH 8.0. Extracts were clarified by centrifugation at 16000xg for 30 min at 4°C, and the resulting supernatant was desalted by NAP-5 size-exclusion filtration after equilibration with 50 mM NaH 2 PO 4, pH 8.0. Protein concentration was determined as described previously (Bradford, 1976) using Bradford reagent (B6916, Sigma-Aldrich). Each reaction was composed of 50 mM NaH 2 PO 4 , pH 8.0, 0.25 mM pyridoxal-5-phosphate, 2 mM 5,10-methylene tetrahydrofolate, 20 mM serine, 0.15 mM α-amino-n-butyric acid, 400 µg of total soluble protein in a final volume of 500 µl and incubated at 25°C for 5 min (a time that was shown to be in the linear phase of the reaction). The reaction was stopped by adding 175 µL of trichloroacetic acid 25% (v/v) followed by incubated on ice for 15 min. After a centrifugation at 15000xg for 10 min at 4°C, 480 µL of the supernatant were transferred to a new tube, the pH was neutralized with 50 µL of K 2 CO 3 buffer (2.3 M K 2 CO 3 , 50 mM NaH 2 PO 4 , pH 8.0) and glycine was quantified by HPLC.

RNA isolation and real-time RT-qPCR

For RT-qPCR analysis of gene expression, two-week-old Arabidopsis seedlings were transferred to liquid ½MS medium containing 50 mM NaCl for 6h under light in short day.

Gene expression was examined using quantitative RT-PCR. The total RNA was extracted by using the nuleoSpin RNA Plus Kit, according to the manufacturer's instructions. Then, 2µg of RNA was reversed to cDNA using the reverse transcriptase (IM-promII TM ), also according to the manufacturer's instructions (Promega). Quantitative real-time PCR was conducted by using the LightCycle-480-Real-Time PCR System and SYBR green (Roche Diagnostics).

Relative-expression level was normalized by Actin-2 (At3g18780) and three independent experiments were performed. The primers used in this experiment are given in Supplemental Table S4.

Amino acid analyses by HPLC and Metabolite analyses by GC-MS

Arabidopsis rosette leaves (100 mg fresh weight) were rapidly frozen in liquid nitrogen and stored at -80°C until extraction. Quantitative amino acid amounts were measured by HPLC after cold-methanol extraction as described in Noctor et al. (2007). Relative metabolite levels of whole rosette leaves were analyzed by GC-MS using cold methanol extracts also as described in Noctor et al. (2007).

SDS-PAGE and western-blot

For SDS-PAGE, 10 µg soluble proteins were separated on an SDS-PAGE gel (10 % acrylamide) and proteins were detected by Coomassie blue staining (Laemmli UK, 1970) .

For western-blot analyses, SDS-PAGE separated proteins were transferred to nitrocellulose membranes and were blocked for 1 h at room temperature in blocking buffer (5% non-fat dry milk in TBST buffer including 50 mM Tris-base, 150 mM NaCl, 0.1 % tween 20, pH 7.4) and incubated overnight at 4°C with either a 1/3500 dilution of antibodies raised against potato SHMT1 (Engel et al., 2011) or actin antibodies (A0480, Sigma-Aldrich) diluted 1/5000. All dilutions were made using blocking buffer. The membranes were washed three times with TBST buffer and then incubated with either 1/5000 diluted anti-rabbit (A6154, Sigma-Aldrich)

or anti-mouse antibodies (A4416, Sigma-Aldrich) for 1 h at room temperature in blocking buffer. The proteins were revealed using chemoluminescent peroxidase substrate-3 (Sigma-Aldrich).

Analysis of H 2 O 2 levels

Hydrogen peroxide was measured using two methods. Seedlings were grown on ½MS plates for 14 days in short day conditions before a 14 h treatment with 150 mM NaCl. For 3,3'diaminobenzidine (DAB) staining, leaves were incubated in DAB staining buffer (50 mg DAB dissolved in 45 mL sterile H 2 O, 1 M HCl was added to help dissolve the DAB. Then 25 µl Tween 20 (v/v) and 2.5 mL of 200 mM Na 2 HPO 4 (pH 6.8) were added to adjust the pH to 5.0 before leaf staining for 8 h, then DAB staining buffer was removed and replaced by a bleaching solution of ethanol:acetic acid:glycerol (v:v:v = 3:1:1). Bleaching was carried out in a boiling water bath for 15 min for removing the chlorophyll in the leaves and then replace the bleaching solution with fresh bleaching solution with shaking for 30 min at 100 rpm at roomtemperature. For H 2 O 2 quantification, seedlings were treated as above but H 2 O 2 content was measured by colorimetric assay using the Hydrogen Peroxide Assay Kit according to the manufacturer's instructions (Abcam®). 100 µl of reaction mix in 96 well plate was incubated at room temperature for 10 min protected from light and then absorbance was measured at OD=570 nm by Thermo Scientific Multiskan® Spectrum.

Water content and water loss assays

Water content was measured using leaves of 3-week-old seedlings by comparing the fresh and dry weights based on the equation: water content (%) = 100 × (fresh weight-dry weight) / fresh weight. Dry weight was measured after lyophilization in a Christ Alpha 2-4 (PCR-EN7049) for 72 h. Water loss assays were carried out in a growth chamber where rosettes of six-week-old plants were cut and weighed at different times. Water loss is presented as the percentage of the initial fresh weight at the indicated time.

Stomatal aperture assays

Stomatal aperture was measured according to the method previously described (Merlot et al., 2007). Briefly, for the ABA-induced stomatal closure assay, leaves of 5 to 6 week-old plants were harvested in darkness at the end of the night period. The abaxial side of leaves were glued to a coverslip using non-toxic medical adhesive (Hollister Medical Adhesif (7730 Newpharma)). To obtain isolated epidermal cells, leaves were peeled gently and coverslips

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114 with glued epidermis were placed in small 4.5 cm-diameter plastic petri-dishes containing 50 mM KCl, 0.1 mM CaCl 2 , 10 mM MES-KOH, pH 6 for 1 h in the dark to ensure closed stomata. Then the epidermis was incubated under light (200 μmol.m -2 .s -1 ) for 4 h to ensure stomatal opening. Subsequently, leaf epidermis was incubated under light a further 4 h in the presence of 50µM ABA to induce stomatal closure. Photographs of stomata were made using a microscope DMI 6000 (Leica). Stomatal pore area (in µm 2 ) was measured using ImageJ software. For the NaCl-induced stomatal closure assay, plants were exposed to light (200 μmol.m -2 .s -1 ) for 2.5 h before being watered with or without 300 mM NaCl for 4 h under light.

Leaves were harvested and stomatal aperture determined using isolated epidermal cells as described above. Photographs were taken under a microscope AxioImager Z2 (Zeiss).

Stomatal aperture (width/length) was measured using ImageJ software.

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115 Table S3. Relative metabolite levels of 3-week old Col-0, Compl-SWT-5, Compl-S31D-5

and Compl-S31A-7 rosette leaves grown on 1/2Ms with or without 50mM NaCl, related to Figure 4.

Name 1,6-anhydro-glucose 1,00 ± 0,08 0,93 ± 0,04 0,85 ± 0,04 0,81 ± 0,14 1,01 ± 0,09 0,85 ± 0,05 0,99 ± 0,13 0,99 ± 0,15 2-hydroxypyridine 1,00 ± 0,08 0,98 ± 0,03 0,97 ± 0,02 0,98 ± 0,16 0,96 ± 0,01 0,90 ± 0,04 0,71 ± 0,05 0,99 ± 0,06 3,5-dimethoxy-4-hydroxycinnamic 1,00 ± 0,03 0,69 ± 0,08 0,47 ± 0,07 0,66 ± 0,17 0,81 ± 0,09 0,56 ± 0,02 0,34 ± 0,04 0,59 ± 0,14 3-aminoisobutyric acid 1,00 ± 0,05 0,87 ± 0,11 1,14 ± 0,14 0,98 ± 0,28 0,81 ± 0,14 1,03 ± 0,14 0,97 ± 0,08 0,77 ± 0,24 3-indoleacetonitrile 1,00 ± 0,04 0,89 ± 0,10 0,86 ± 0,16 0,88 ± 0,14 1,37 ± 0,24 1,06 ± 0,08 1,05 ± 0,05 1,59 ± 0,11 3-phosphoglyceric acid 1,00 ± 0,09 1,24 ± 0,24 1,56 ± 0,51 1,09 ± 0,32 0,76 ± 0,69 0,52 ± 0,09 0,34 ± 0,06 0,74 ± 0,31 adenosine 5'-diphosphate 1,00 ± 0,09 1,12 ± 0,20 1,77 ± 0,56 1,19 ± 0,42 1,21 ± 0,52 0,89 ± 0,08 0,68 ± 0,12 1,52 ± 0,52 alanine 1,00 ± 0,07 1,18 ± 0,15 1,26 ± 0,20 1,16 ± 0,31 1,35 ± 0,12 1,52 ± 0,12 1,71 ± 0,22 1,55 ± 0,31 allantoin 1,00 ± 0,11 0,64 ± 0,21 0,56 ± 0,09 0,85 ± 0,30 0,62 ± 0,10 0,49 ± 0,05 0,58 ± 0,04 0,43 ± 0,16 alpha ketoglutaric acid 1,00 ± 0,27 4,41 ± 2,19 6,37 ± 1,59 5,94 ± 2,41 0,71 ± 1,00 2,34 ± 0,52 * 1,79 ± 0,48 * 1,83 ± 1,87 * altrose 1,00 ± 0,05 1,07 ± 0,09 1,06 ± 0,08 1,05 ± 0,11 0,77 ± 0,14 0,76 ± 0,09 0,87 ± 0,12 0,94 ± 0,31 arachidic acid 1,00 ± 0,18 0,97 ± 0,14 1,21 ± 0,56 1,09 ± 0,69 1,48 ± 0,40 0,73 ± 0,10 0,79 ± 0,13 1,60 ± 0,88 ascorbic acid 1,00 ± 0,10 1,36 ± 0,31 1,94 ± 0,68 1,70 ± 0,55 0,66 ± 0,29 1,39 ± 0,62 1,36 ± 0,32 1,17 ± 0,39 asparagine 1,00 ± 0,10 1,29 ± 0,48 1,54 ± 0,15 1,74 ± 0,31 0,57 ± 0,32 * 0,53 ± 0,10 * 1,22 ± 0,24 0,49 ± 0,13 * aspartic acid 1,00 ± 0,06 1,10 ± 0,09 1,00 ± 0,19 1,03 ± 0,17 0,95 ± 0,09 0,72 ± 0,06 * 0,82 ± 0,09 0,94 ± 0,34 beta-alanine 1,00 ± 0,05 0,87 ± 0,07 0,98 ± 0,06 0,91 ± 0,07 0,90 ± 0,03 0,96 ± 0,06 0,96 ± 0,06 0,96 ± 0,04 beta-glycerolphosphate 1,00 ± 0,05 0,88 ± 0,04 0,85 ± 0,14 0,84 ± 0,20 1,15 ± 0,11 1,01 ± 0,05 0,76 ± 0,05 0,83 ± 0,30 beta-sitosterol 1,00 ± 0,05 1,04 ± 0,09 1,00 ± 0,06 1,04 ± 0,13 1,21 ± 0,04 1,19 ± 0,07 1,01 ± 0,08 1,15 ± 0,04 cholesterol 1,00 ± 0,08 1,01 ± 0,07 0,80 ± 0,09 0,93 ± 0,03 1,16 ± 0,11 0,94 ± 0,04 0,57 ± 0,04 1,08 ± 0,03 citric acid 1,00 ± 0,05 1,39 ± 0,23 1,64 ± 0,18 1,37 ± 0,29 1,19 ± 0,18 1,40 ± 0,18 0,83 ± 0,18 * 1,42 ± 0,37 citrulline 1,00 ± 0,07 0,69 ± 0,20 0,98 ± 0,26 0,91 ± 0,18 0,31 ± 0,30 0,23 ± 0,05 0,91 ± 0,24 0,22 ± 0,09 cysteinylglycine 1,00 ± 0,02 1,17 ± 0,12 1,33 ± 0,17 1,18 ± 0,08 0,66 ± 0,24 * 0,81 ± 0,09 * 0,58 ± 0,10 * 0,84 ± 0,26 * dehydroascorbic acid 1,00 ± 0,07 0,60 ± 0,20 0,61 ± 0,15 0,72 ± 0,10 0,87 ± 0,08 0,75 ± 0,10 0,54 ± 0,10 0,80 ± 0,07 fructose 1,00 ± 0,09 2,12 ± 0,12 2,73 ± 0,34 2,31 ± 0,28 0,33 ± 0,78 * 0,65 ± 0,13 * 0,44 ± 0,08 * 0,69 ± 0,28 * fructose 6-phosphate 1,00 ± 0,05 1,06 ± 0,12 1,39 ± 0,15 1,05 ± 0,19 0,51 ± 0,34 * 0,47 ± 0,05 * 0,39 ± 0,05 * 0,55 ± 0,15 * fumaric acid 1,00 ± 0,05 1,20 ± 0,12 1,02 ± 0,14 1,14 ± 0,14 0,58 ± 0,16 0,84 ± 0,05 0,47 ± 0,06 0,79 ± 0,11 galactaric acid 1,00 ± 0,07 1,27 ± 0,10 2,07 ± 0,39 1,41 ± 0,38 3,12 ± 0,17 * 2,38 ± 0,26 * 1,35 ± 0,26 * 2,67 ± 0,74 * galactinol 1,00 ± 0,11 0,81 ± 0,10 0,81 ± 0,14 0,81 ± 0,16 0,86 ± 0,09 1,02 ± 0,05 0,61 ± 0,10 * 0,92 ± 0,21 galactonic acid 1,00 ± 0,02 1,07 ± 0,07 1,14 ± 0,11 1,12 ± 0,08 0,75 ± 0,13 0,77 ± 0,02 0,63 ± 0,03 0,78 ± 0,06 gamma-aminobutyric acid 1,00 ± 0,08 1,31 ± 0,26 1,43 ± 0,17 1,29 ± 0,20 1,06 ± 0,12 0,94 ± 0,07 0,92 ± 0,10 1,78 ± 1,11 glucose 1,00 ± 0,19 1,74 ± 0,08 2,75 ± 0,50 1,80 ± 0,39 0,20 ± 0,03 * 0,46 ± 0,22 * 0,28 ± 0,04 * 0,40 ± 0,15 * glucose-6-phosphate 1,00 ± 0,04 1,03 ± 0,12 1,45 ± 0,21 1,04 ± 0,19 0,53 ± 0,38 * 0,49 ± 0,05 * 0,44 ± 0,06 * 0,56 ± 0,15 * glutamic acid 1,00 ± 0,02 1,41 ± 0,18 1,39 ± 0,05 1,35 ± 0,14 0,78 ± 0,02 * 0,78 ± 0,01 * 1,02 ± 0,12 * 0,83 ± 0,04 * glutamine 1,00 ± 0,09 1,80 ± 0,65 1,93 ± 0,24 1,72 ± 0,34 0,27 ± 0,03 * 0,28 ± 0,05 * 0,96 ± 0,38 * 0,26 ± 0,09 * glyceric acid 1,00 ± 0,03 1,50 ± 0,13 1,84 ± 0,38 1,50 ± 0,24 1,07 ± 0,23 1,14 ± 0,03 * 0,90 ± 0,06 * 1,17 ± 0,16 * glycerol 1,00 ± 0,22 0,73 ± 0,03 0,71 ± 0,04 0,65 ± 0,07 0,57 ± 0,07 * 0,59 ± 0,02 * 0,58 ± 0,05 * 0,70 ± 0,09 * glycerol 3-phosphate 1,00 ± 0,04 0,85 ± 0,05 0,76 ± 0,10 0,77 ± 0,12 1,25 ± 0,12 0,94 ± 0,05 0,56 ± 0,09 0,93 ± 0,20 glycine 1,00 ± 0,14 4,92 ± 2,00 6,02 ± 1,52 5,00 ± 1,53 0,05 ± 0,01 * 1,35 ± 0,51 * 3,26 ± 0,55 * 1,34 ± 1,20 * glycolic acid 1,00 ± 0,06 1,02 ± 0,07 1,02 ± 0,07 1,01 ± 0,10 1,04 ± 0,06 1,11 ± 0,07 1,08 ± 0,07 1,04 ± 0,06 glycylproline 1,00 ± 0,14 1,38 ± 0,20 1,49 ± 0,13 1,40 ± 0,38 0,87 ± 0,12 1,12 ± 0,16 0,88 ± 0,09 1,12 ± 0,42 glyoxylic acid 1,00 ± 0,15 0,94 ± 0,08 0,89 ± 0,17 0,74 ± 0,20 0,81 ± 0,14 1,07 ± 0,09 1,02 ± 0,09 0,71 ± 0,21 heptadecanoic acid 1,00 ± 0,17 1,01 ± 0,17 1,27 ± 0,62 1,15 ± 0,67 1,47 ± 0,44 0,74 ± 0,12 0,81 ± 0,13 1,70 ± 0,95 hexanoic acid 1,00 ± 0,07 1,06 ± 0,10 1,05 ± 0,12 1,02 ± 0,18 0,96 ± 0,09 1,06 ± 0,08 0,80 ± 0,07 0,92 ± 0,07 histidine 1,00 ± 0,12 1,36 ± 0,78 1,24 ± 0,24 1,75 ± 0,27 0,13 ± 0,03 0,12 ± 0,02 1,29 ± 0,37 0,11 ± 0,06 homoserine 1,00 ± 0,13 2,05 ± 0,47 3,74 ± 0,37 2,91 ± 0,37 1,38 ± 0,25 3,65 ± 0,18 * 3,53 ± 0,45 3,50 ± 0,95 iminodiacetic acid 1,00 ± 0,03 1,91 ± 0,51 1,72 ± 0,20 1,80 ± 0,68 0,57 ± 0,39 * 0,90 ± 0,15 * 1,16 ± 0,07 * 0,60 ± 0,18 * isoleucine 1,00 ± 0,14 1,82 ± 0,91 1,69 ± 0,60 2,30 ± 0,77 0,94 ± 0,10 1,22 ± 0,15 1,25 ± 0,19 1,01 ± 0,63 lactobionic acid 1,00 ± 0,05 0,92 ± 0,07 1,04 ± 0,22 1,02 ± 0,30 0,79 ± 0,22 1,11 ± 0,14 0,79 ± 0,13 0,91 ± 0,36 lactose 1,00 ± 0,09 0,97 ± 0,12 1,16 ± 0,36 1,09 ± 0,14 0,88 ± 0,26 0,98 ± 0,02 0,89 ± 0,08 0,94 ± 0,30 lauric acid 1,00 ± 0,12 1,12 ± 0,10 1,17 ± 0,26 1,02 ± 0,25 0,97 ± 0,10 0,99 ± 0,07 0,75 ± 0,09 1,19 ± 0,28 leucrose 1,00 ± 0,02 0,79 ± 0,04 0,88 ± 0,09 0,80 ± 0,05 1,10 ± 0,08 1,03 ± 0,04 0,72 ± 0,06 1,00 ± 0,15 lysine 1,00 ± 0,07 0,84 ± 0,37 1,14 ± 0,21 1,10 ± 0,28 0,48 ± 0,06 0,58 ± 0,11 1,07 ± 0,12 0,51 ± 0,29 maleic acid 1,00 ± 0,02 0,96 ± 0,06 0,76 ± 0,07 0,92 ± 0,19 0,72 ± 0,07 * 0,75 ± 0,04 * 0,55 ± 0,04 * 0,64 ± 0,22 * malic acid 1,00 ± 0,07 1,36 ± 0,08 1,45 ± 0,13 1,39 ± 0,14 0,87 ± 0,18 1,19 ± 0,04 0,72 ± 0,11 * 1,02 ± 0,13 * malonic acid 1,00 ± 0,04 1,39 ± 0,10 1,92 ± 0,42 1,47 ± 0,39 0,89 ± 0,13 1,31 ± 0,04 1,05 ± 0,14 * 1,59 ± 0,50 maltose 1,00 ± 0,22 1,40 ± 0,48 1,03 ± 0,05 0,86 ± 0,35 1,56 ± 0,25 1,89 ± 0,36 1,87 ± 0,35 2,87 ± 1,36 mannose 1,00 ± 0,06 1,11 ± 0,09 1,26 ± 0,15 1,14 ± 0,07 0,54 ± 0,24 0,58 ± 0,03 0,60 ± 0,04 0,66 ± 0,10 melibiose 1,00 ± 0,13 0,80 ± 0,08 0,96 ± 0,08 0,81 ± 0,10 1,59 ± 0,24 1,19 ± 0,24 0,82 ± 0,08 1,57 ± 0,90 methionine sulfoxide 1,00 ± 0,05 1,29 ± 0,25 1,00 ± 0,14 1,08 ± 0,22 0,91 ± 0,08 0,96 ± 0,10 0,85 ± 0,10 1,13 ± 0,27 myo-inositol 1,00 ± 0,09 0,63 ± 0,16 0,53 ± 0,10 0,55 ± 0,19 2,10 ± 0,14 * 1,44 ± 0,18 * 0,80 ± 0,08 * 1,62 ± 0,38 * myristic acid 1,00 ± 0,30 0,94 ± 0,06 0,75 ± 0,12 1,22 ± 1,03 0,97 ± 0,08 1,40 ± 0,40 1,39 ± 0,54 1,53 ± 0,54 nicotinic acid 1,00 ± 0,04 1,00 ± 0,08 0,95 ± 0,08 1,12 ± 0,26 2,20 ± 0,30 2,32 ± 0,14 2,09 ± 0,10 2,07 ± 0,44 norleucine 1,00 ± 0,14 1,40 ± 0,60 1,22 ± 0,34 1,78 ± 0,55 0,90 ± 0,07 1,13 ± 0,12 0,96 ± 0,17 0,97 ± 0,40 oleic acid 1,00 ± 0,21 1,05 ± 0,20 1,31 ± 0,66 1,17 ± 0,80 1,73 ± 0,45 0,68 ± 0,08 0,79 ± 0,13 1,76 ± 1,06 O-phosphocolamine 1,00 ± 0,05 0,96 ± 0,06 0,86 ± 0,07 1,00 ± 0,06 0,92 ± 0,03 0,93 ± 0,05 0,71 ± 0,06 0,92 ± 0,14 O-phospho-L-serine 1,00 ± 0,11 1,31 ± 0,23 1,22 ± 0,11 1,48 ± 0,10 1,21 ± 0,11 0,90 ± 0,09 0,81 ± 0,08 1,10 ± 0,36 ornithine 1,00 ± 0,07 0,68 ± 0,27 1,06 ± 0,25 1,15 ± 0,32 0,42 ± 0,05 * 0,31 ± 0,07 * 1,87 ± 0,56 * 0,44 ± 0,25 * palatinitol 1,00 ± 0,11 1,00 ± 0,10 1,03 ± 0,33 1,27 ± 0,68 0,86 ± 0,20 1,57 ± 0,27 0,58 ± 0,24 0,66 ± 0,30 palmitic acid 1,00 ± 0,14 0,99 ± 0,11 1,23 ± 0,52 1,11 ± 0,56 1,46 ± 0,29 0,80 ± 0,07 0,85 ± 0,11 1,54 ± 0,72 pantothenic acid 1,00 ± 0,06 1,11 ± 0,13 1,07 ± 0,15 1,09 ± 0,12 1,06 ± 0,05 1,04 ± 0,03 0,85 ± 0,03 1,05 ± 0,13 phosphoenolpyruvic acid 1,00 ± 0,11 1,04 ± 0,15 1,42 ± 0,13 0,98 ± 0,20 0,65 ± 0,08 * 0,41 ± 0,08 * 0,25 ± 0,07 * 0,61 ± 0,16 * phosphoric acid 1,00 ± 0,04 1,00 ± 0,08 1,18 ± 0,10 1,00 ± 0,05 0,56 ± 0,05 0,53 ± 0,04 0,42 ± 0,08 0,67 ± 0,13 phytol 1,00 ± 0,07 1,10 ± 0,13 1,50 ± 0,49 1,51 ± 0,36 1,02 ± 0,04 1,13 ± 0,07 0,54 ± 0,07 * 1,02 ± 0,17 proline 1,00 ± 0,15 0,26 ± 0,12 0,05 ± 0,02 0,60 ± 0,24 1,76 ± 0,19 * 2,29 ± 0,37 * 0,78 ± 0,08 * 2,60 ± 0,56 * putrescine 1,00 ± 0,04 1,07 ± 0,28 1,03 ± 0,11 1,33 ± 0,27 0,70 ± 0,06 0,82 ± 0,06 1,30 ± 0,14 * 0,88 ± 0,15 rhamnose 1,00 ± 0,02 0,81 ± 0,08 0,90 ± 0,08 0,94 ± 0,11 1,20 ± 0,01 1,06 ± 0,03 0,93 ± 0,07 0,91 ± 0,07 ribose 1,00 ± 0,05 0,93 ± 0,03 0,89 ± 0,08 1,00 ± 0,11 1,02 ± 0,02 0,98 ± 0,02 0,74 ± 0,05 1,00 ± 0,12 serine 1,00 ± 0,24 2,37 ± 0,14 3,08 ± 0,24 2,76 ± 0,58 2,61 ± 0,31 * 6,75 ± 0,68 * 5,77 ± 1,08 * 5,46 ± 2,36 * shikimic acid 1,00 ± 0,05 0,84 ± 0,14 0,77 ± 0,14 0,76 ± 0,12 1,20 ± 0,08 * 0,88 ± 0,07 0,58 ± 0,09 * 1,06 ± 0,17 * spermidine 1,00 ± 0,04 1,14 ± 0,16 1,31 ± 0,18 1,54 ± 0,17 0,78 ± 0,04 0,97 ± 0,20 3,16 ± 0,69 * 0,90 ± 0,41 * stearic acid 1,00 ± 0,20 0,99 ± 0,16 1,22 ± 0,60 1,13 ± 0,74 1,52 ± 0,36 0,68 ± 0,11 0,81 ± 0,14 1,65 ± 0,89 stigmasterol 1,00 ± 0,06 0,99 ± 0,22 0,97 ± 0,09 1,03 ± 0,30 0,87 ± 0,15 0,86 ± 0,06 0,98 ± 0,07 0,93 ± 0,05 succinic acid 1,00 ± 0,64 5,63 ± 3,02 1,31 ± 0,29 4,33 ± 4,20 0,24 ± 0,09 * 0,86 ± 0,09 * 3,54 ± 0,97 * 1,99 ± 1,73 sucrose 1,00 ± 0,04 0,93 ± 0,05 0,90 ± 0,09 0,90 ± 0,14 1,05 ± 0,09 1,03 ± 0,07 0,92 ± 0,12 0,92 ± 0,19 tagatose 1,00 ± 0,04 0,83 ± 0,05 0,79 ± 0,08 0,89 ± 0,08 0,85 ± 0,02 0,78 ± 0,01 0,58 ± 0,03 0,79 ± 0,04 threitol 1,00 ± 0,09 0,63 ± 0,04 0,64 ± 0,05 0,73 ± 0,06 1,34 ± 0,18 * 1,11 ± 0,05 * 0,83 ± 0,06 * 1,15 ± 0,09 * threonic acid 1,00 ± 0,08 1,17 ± 0,12 0,94 ± 0,13 0,95 ± 0,17 0,99 ± 0,05 0,87 ± 0,05 0,76 ± 0,06 1,04 ± 0,28 threonine 1,00 ± 0,18 1,28 ± 0,30 1,56 ± 0,15 1,76 ± 0,17 2,04 ± 0,30 * 2,48 ± 0,13 * 1,55 ± 0,22 2,39 ± 0,73 trans-13-octadecenoic acid 1,00 ± 0,19 0,96 ± 0,16 1,33 ± 0,77 1,04 ± 0,75 1,57 ± 0,45 0,62 ± 0,09 0,67 ± 0,13 1,60 ± 0,90 tryptophan 1,00 ± 0,18 2,17 ± 1,12 1,71 ± 0,36 3,09 ± 0,83 0,41 ± 0,03 * 0,72 ± 0,17 * 0,92 ± 0,08 * 0,57 ± 0,34 * tyramine 1,00 ± 0,04 1,16 ± 0,08 1,27 ± 0,07 1,24 ± 0,07 1,51 ± 0,09 * 1,54 ± 0,09 * 1,58 ± 0,13 * 1,65 ± 0,12 * tyrosine 1,00 ± 0,10 1,52 ± 0,69 1,46 ± 0,32 2,10 ± 0,55 0,74 ± 0,08 0,84 ± 0,16 0,83 ± 0,09 0,83 ± 0,43 urea 1,00 ± 0,01 1,09 ± 0,06 0,99 ± 0,04 0,94 ± 0,09 6,39 ± 0,25 * 6,41 ± 0,30 * 6,31 ± 0,73 * 5,51 ± 2,66 * uric acid 1,00 ± 0,06 1,02 ± 0,28 0,98 ± 0,10 1,28 ± 0,43 0,44 ± 0,03 0,66 ± 0,09 0,49 ± 0,04 0,72 ± 0,11 valine 1,00 ± 0,06 1,08 ± 0,27 1,04 ± 0,18 1,29 ± 0,25 1,12 ± 0,06 1,12 ± 0,04 1,17 ± 0,08 1,05 ± 0,18 xanthosine 1,00 ± 0,07 0,96 ± 0,22 1,28 ± 0,24 1,33 ± 0,21 0,55 ± 0,11 0,73 ± 0,12 0,71 ± 0,13 0,65 ± 0,34 xylose 1,00 ± 0,01 0,85 ± 0,09 0,84 ± 0,08 0,88 ± 0,04 0,96 ± 0,05 0,97 ± 0,02 0,70 ± 0,03 * 1,07 ± 0,10 xylulose 1,00 ± 0,07 0,92 ± 0,07 0,83 ± 0,08 0,96 ± 0,18 1,29 ± 0,03 1,23 ± 0,04 0,96 ± 0,03 1,17 ± 0,08

Compl

RESULTS

CHAPTER I

117 Table S4. Primers for PCR and cloning.

Primers name

Sequence (5' to 3')

Cloning promSHMT1-F CAGCCTGAACATTCCGATTA promSHMT1-R GAATGGGTTTGTCAATTGAA cDNA-SHMT1-F ATGGCGATGGCCATGGCTCT GW-SHMT1-F TACAAAAAAGCAGGCTTCCAGCCTGAACATTCCGATTA GW-SHMT1-R CAAGAAAGCTGGGTTTTAGTTCTTGTACTTCATGG GateWay-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC GateWay-R GGGGACCACTTTGTACAAGAAAGCTGGGTC Site-directed mutagenesis SHMT1-S31A-F ACTTCATGTTACATGTCTGCTTTGCCCAGTGAAGCTG SHMT1-S31A-R CAGCTTCACTGGGCAAAGCAGACATGTAACATGAAGT SHMT1-S31D-F CCACTTCATGTTACATGTCTGATTTGCCCAGTGAAGCTGTTG SHMT1-S31D-R CAACAGCTTCACTGGGCAAATCAGACATGTAACATGAAGTGG qPCR Actin2-F GGTAACATTGTGAGTGGTGG Actin2-R AACGACCTTAATCTTCATGCT RD29a-F ATCAAACTCAAGTGGCGGGA RD29a-R TATCTTCCCCTCGTTGCTCC RD29b-F ATGAGACAAAACCAAGCACCTACA RD29b-R CTGCCCGTAAGCAGTAACAGAT P5CS1-F GGTGGACCAAGGGCAAGTAAG P5CS1-R CTCCCATGTCGGTGAATGTGAT 126 PERSPECTIVE Preamble
Here additional discussions and perspectives are developed that were not included in the research article presented in chapter 1 of the results section. Furthermore, a detailed description of the methods used for metabolite profiling by GC-MS and glycine quantification by HPLC to measure SHMT activity are also developed.

Discussions and perspectives related to SHMT1 activity and stomatal closure in response to a salt stress

Eisenhut and coworkers have studied the impact of a transfer from high CO 2 growth conditions (low photorespiration) to air-grown conditions (normal photorespiration) on rosette leaf transpiration of Col-0 and photorespiratory mutants (Eisenhut et al., 2017). The wild-type plants showed 2-fold higher transpiration rates when transferred to air suggesting stomatal opening that gradually returned to pre-transfer levels during the next 5 days. However, shm1-1 exhibited a reduced (two-fold) transpiration rate after transfer suggesting stomatal closure.

These observations showed that in photorespiratory conditions, shm1-1 was unable to carry out normal stomatal movements to response to altered external CO 2 concentration that bring about increases in photorespiratory fluxes. Interestingly after the transfer to air, the expression of ABI1 and ABI2, encoding two phosphatases which are negative regulators of ABA-induced stomatal closure (Merlot et al., 2001), were upregulated in shm1-1 (Eisenhut et al., 2017).

Thus we speculate that ABI1 and ABI2 expression could be upregulated in Compl-S31D lines in salt stress conditions thus inhibiting ABA signaling and reducing stomata closure.

Therefore, we can:

 Analyze ABI1 and ABI2 expression by qPCR in the complemented lines in response to salt stress.

 Measure ABA levels in the complemented lines to confirm that the defect in ABAinduced stomatal closure is not related to ABA content.

Myo-inositol is the precursor of inositol which itself is a precursor for many inositolcontaining compounds involved in various plant responses to biotic and abiotic stresses (den Ende, 2013) . Myo-inositol is synthetized from glucose-6-phosphate which is converted to myo-inositol-1-phosphate by myo-inositol-1-phosphate synthase (MIPS), then myo-inositol-1phosphate is dephosphorylated to myo-inositol by myo-inositol monophosphatase [START_REF] Loewus | myo-Inositol metabolism in plants[END_REF]. In Arabidopsis, overexpression of MIPS1 has been reported to improve biotic and abiotic stress tolerance in plants and MIPS1 was reported to be a key rate limiting enzyme and to have an important contribution to myo-inositol biosynthesis [START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF][START_REF] Donahue | The Arabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required for Myo-inositol Synthesis and Suppression of Cell Death[END_REF]Zhai et al., 2016). In response to salt stress, glucose-6-phosphate content was similar in all tested lines, whereas myo-inositol content increased in all lines except Compl-S31D-5. The lower myo-inositol biosynthesis could reflect a low MIPS1 gene expression or enzyme activity. Indeed, myo-inositol levels are of critical importance for drought and salt tolerance since increased levels in MIPS-overexpression lines improves stress tolerance (Das-Chatterjee et al., 2006;Joshi et al., 2013;Kaur et al., 2013;Zhai et al., 2016).

It is therefore possible that a decrease in this metabolite could have an impact on salt and drought tolerance as seen in the Compl-S31D-5 line. Myo-inositol, together with glycerol-3-P, is essential for the biosynthesis of phosphoglycerolipids (Janda et al., 2013). Among them, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is known to be involved in signaling pathways and can be hydrolyzed by phospholipase C (PLC) to generate the secondary messengers, inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol (DAG) (Janda et al., 2013). IP 3 participates in stomatal movements as microinjection of IP 3 into stomata initiated their closure (Glilroy et al., 1990). In stomata, in response to ABA, IP 3 can be converted to IP 6 causing the release of Ca 2+ from an intracellular store [START_REF] Lemtiri-Chlieh | Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells[END_REF][START_REF] Liepman | Alanine Aminotransferase Homologs Catalyze the Glutamate:Glyoxylate Aminotransferase Reaction in Peroxisomes of Arabidopsis[END_REF][START_REF] Munnik | PI-PLC: Phosphoinositide-Phospholipase C in Plant Signaling[END_REF] and thus co-activated the slow anion channel 1 (SLAC1) [START_REF] Siegel | Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+channels in Arabidopsis guard cells[END_REF] and inhibited K + influx [START_REF] Lemtiri-Chlieh | Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells[END_REF]. As a result, stomata closed because of the net efflux of K + and Cl -along with water. Interestingly, an Arabidopsis PLC mutant (plc3) showed a similar stomata phenotype to Compl-S31D-5. No significant differences were observed in stomatal aperture between the wild-type and plc3 which exhibited a reduced sensitivity to ABAinduced stomatal closure (Zhang et al., 2018). Since Compl-S31D show lower levels of myoinositol and glycerol-3-phosphate compared to the other lines, we propose that Compl-S31D lines could be deficient in PIP 2 synthesis (and probably in others phosphoglycerolipids) causing a lower IP 3 release in response to ABA and thereby impacting stomatal closure.

To help validate our hypothesis, it is possible to:

 Test MIPS1 expression and/or MIPS activity to determine if this can explain the decreased myo-inositol content in Compl-S31D.

 Measure stomatal aperture of Compl-S31D in response to ABA in the presence of IP 3 to see whether IP 3 can revert the stomata phenotype. These results will indicate whether the stomata phenotype of Compl-S31D is due to a disturbance of the PLC pathway.

RESULTS

CHAPTER I 128

Again with respect to stomata, there is a possible link between the observed accumulation of polyamines in Compl-S31D-5 during a salt stress and the stomatal phenotype. Indeed, spermidine is the direct precursor of 1,3-diaminopropane (DAP) (Cona et al., 2006).

Acetylation of DAP by NATA1 (N-acetyltransferase activity 1) produces acetyl-DAP which can antagonize ABA to slow down stomatal closure to maintain CO 2 uptake during a mild drought stress (Jammes et al., 2014). Thus, an over-production of spermidine could eventually lead to a higher biosynthesis of acetyl-DAP during salt or drought stresses. Therefore, Compl-S31D plants would be resistant to ABA-induced stomatal closure as observed during a salt stress. Unfortunately, as it is a low abundant metabolite, acetyl-DAP is not detected by standard GC-MS protocols. To explore our hypothesis, we can:

 Optimize GC-MS analysis methods to detect acetyl-DAP and then measure changes in its level in our SHMT1 transgenic lines.

 Express SHMT1-S31D in the acetyl-DAP defective nata1 mutant (Jammes et al., 2014) and measure stomatal aperture in response to ABA and a salt stress to see if the absence of acetyl-DAP can revert the stomata phenotype of Compl-S31D lines.

Guard cell photosynthesis is essential for stomatal turgor even though transgenic plant lacking chlorophyll still showed a stomatal closing response to ABA [START_REF] Azoulay-Shemer | Guard cell photosynthesis is critical for stomatal turgor production, yet does not directly mediate CO2-and ABA-induced stomatal closing[END_REF]. It is not known whether our salt-treated SHMT1 transgenic lines exhibited altered difference in photosynthetic activity. However, glucose-6-phosphate and fructose-6-phosphate were significantly more abundant in Compl-S31D-5 compared to the other lines in control conditions. Both hexose-sugars are produced by the activity of hexokinase which is now known to be involved in stomatal movements [START_REF] Kelly | Hexokinase mediates stomatal closure[END_REF][START_REF] Lugassi | Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration[END_REF]. Thus, in Compl-S31D-5, hexokinase activity appears to be enhanced which could impact the stomatal aperture in response to ABA. The slight over accumulation of glucose and fructose observed in Compl-S31D-5 and the over accumulation of hexose phosphates could reflect an increased starch degradation (or a reduced starch synthesis). Starch in guard cells is essential for stomatal movements and an impaired breakdown was accompanied by decreased stomatal opening [START_REF] Prasch | βamylase1 mutant Arabidopsis plants show improved drought tolerance due to reduced starch breakdown in guard cells[END_REF][START_REF] Horrer | Blue light induces a distinct starch degradation pathway in guard cells for stomatal opening[END_REF]. So, an altered starch metabolism could help explain the reduced stomatal closure observed in Compl-S31D-5.

It would be important to:

 Measure the starch content of complemented lines in normal and salt stress conditions.  Extraction of polar primary metabolites Rosette leaf extracts were either lyophilized for about 100 h before being ground either using 2 iron balls for 90 s at 29 Hz with an electric grinder, or freshly ground in liquid nitrogen with a mortar. Polar metabolites were extracted with a solution of methanol 80% (v / v) containing 100 μM α-aminobutyric acid (α-ABA) (internal standard for the absolute quantification of amino acids by HPLC). After a centrifugation step of 10000 g, 15 min at 4°C, 1.5 mL of supernatant was removed and 150 μL of a 200 μM solution of adonitol (internal standard for relative quantification by GC-MS) prepared in 80% methanol (v / v) were added. The mixture was vortexed, then aliquoted into 0.2 mL fractions which were vacuum-dried for 20 h using a Speedvac. A blank control, containing no extract was done in the same way for each new series of analysis.

 Relative metabolite analysis by GC-MS

For a global comparative analysis of primary polar metabolites by GC-MS, the molecules must be chemically transformed to become volatile. Aliquots of metabolites were thus incubated, with orbital shaking at 200 rpm, 90 min at 30 °C with methoxyamine and then for 30 min at 37°C with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). After standing for 2 h at room temperature, 1 μL of this solution was injected into the GC-MS. The liquid was vaporized in a chamber heated to 330 °C, and the volatilized molecules were mixed with the gaseous mobile phase (helium). Molecules were eluted through a RTX-5w / integra-Guard capillary column (30 m × 0.25 mm + 10 m integrated guard column (Restek)) by adsorption-desorption with a helium flow rate of 1mL.min -1 along a temperature gradient of 80 to 330 ° C for about 30 min (see Noctor et al., 2007). The temperature gradient breaks the interactions between the molecules and the column progressively and thus to separate the

RESULTS

CHAPTER I

130 metabolites in the mixture. The separated metabolites were then ionized by electron impact with a source at 70 eV, and the different ionic fragments resulting for each of the metabolites were propelled into a flight tube. Since this tube is under vacuum, the separation of the ions depends solely on the mass / charge ratio (m/z). The identification of metabolites was established by comparing the m/z spectra relative to reference spectra obtained from several free metabolite libraries, as well as the reference spectra of standards already injected into the device, according to their retention index. Identification of metabolites and integration of peaks were performed using the LECO Pegasus software, using the metabolites library of the Metabolism-Metabolome platform and after a manual check. The samples were compared with each other after the normalization of each calculated areas with the area of adonitol (internal standard).

 Quantification of glycine by HPLC analysis

SHMT activity measurements were determined by quantification of the glycine produced, this was achieved by HPLC. Samples of the SHMT reaction mixture were filtered through a 1 mL syringe containing a 0.2 μm filter (Minisart RC4) before being injected into the HPLC (High Performance Liquid Chromatography; Alliance Waters). 10 µL of the filtrate was incubated for 2 min in the HPLC injection loop with 10 µL of a solution containing o-phthaldialdehyde (OPA) at 7.5 gL -1 , 2% β 1, 2 -mercaptoethanol (v/v), methanol 10% (v/v) in a 500 mM borate buffer at pH 9.5. This reaction marked the amino acids fluorescent for their detection and quantification. OPA and β-mercaptoethanol reacted with the amino function of the amino acids to produce a stable fluorescent derivative. This mixture was then injected into the column and the derived glycine was separated for 42 min at 40 °C with a flow rate of 0.8 ml.min -1 according to a gradient of two buffers (buffer A: 20% methanol, 50 mM sodium acetate pH 5.9 (adjusted with glacial acetic acid), 0.1% tetrahydrofuran; buffer B: 80% methanol, 50 mM sodium acetate pH 5.9 (adjusted to glacial acetic acid), see Noctor et al., 2007. The identification of glycine and its quantification were carried out by comparing the retention times with a reference chromatogram, and the areas of the chromatograms of the We previously observed that Compl-S31D lines were more sensitive to salt stress and drought recovery and we suggested that this phenotype could be in part due to a decreased sensitivity to stomatal closure induced by salt and perhaps involving ABA. However, expression analyses of ABA-induced genes (P5CS1, RD29A and RD29B) in response to salt-treatment revealed that each transgenic line appeared to respond in a similar manner to the salt stress except for a smaller induction of P5CS1 in Compl-S31D lines. So the ABA signaling pathway appeared unaffected at the whole rosette level in all lines. Since ABA is a central hormone in regulating seed germination by maintaining seed dormancy (for a review see [START_REF] Yan | The pivotal role of abscisic acid signaling during transition from seed maturation to germination[END_REF], we decided to investigate the germination of Compl-SWT, Compl-S31 and Compl-S31D seeds in response to ABA.

GERMINATION METHOD

Seeds of Compl-SWT-5/1, Compl-S31D-5/4 and Compl-S31A-7/6 lines were sterilized for 6 min by adding 1 mL of 95% (v/v) ethanol / 7.5% (w/v) sodium hypochlorite, then they were washed twice with ethanol 95% (v/v) and dried under a hood for 6 h. Then, seeds were sown on ½MS medium plates containing or not 5 µM ABA. The plates were kept at 4°C for 2 days and transferred to short-day condition (8 h light / 16 h dark, 160 μmol of photons.m -2 .s -1 , 65% humidity, with a temperature of 20°C day/18°C night). Seed germination (emergence of radicals) was counted daily for seven days.

RESULTS

Expression of SHMT1 during seed germination

SHMT1 has been shown to be highly expressed during seed germination. Indeed, based on the data available in the Arabidopsis eFP browsers (http://bar.utoronto.ca), two seed germination studies based on microarray or RNAseq analyses revealed that SHMT1 was strongly expressed during this process [START_REF] Narsai | In-Depth Temporal Transcriptome Profiling Reveals a Crucial Developmental Switch with Roles for RNA Processing and Organelle Metabolism That Are Essential for Germination in Arabidopsis[END_REF][START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF]. In Col-0 seeds, stratified at 4°C for 48 h then transferred to continuous light for a further 48 h, SHMT1 expression increased by a factor greater than 10 (Figure 1a and 1b) [START_REF] Narsai | In-Depth Temporal Transcriptome Profiling Reveals a Crucial Developmental Switch with Roles for RNA Processing and Organelle Metabolism That Are Essential for Germination in Arabidopsis[END_REF]. Again, in Col-0 seeds soaked for 3 days, SHMT1 expression accumulated 15-fold more than in dry seeds. SHMT1 expression was found to be mainly in the cotyledons and hypocotyl of 1-dayold seedlings (Figure 1c and1d) [START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF]. This expression was even 2-fold higher than mature leaves, the major site for photorespiration (Figure 1c and1d) [START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF]. Absolute AtSHMT1 expression levels in the seed from harvest to stratification to germination.

The data are given as a color scheme (a) and a histogram (b). Col-0 seeds were harvested (H) and desiccated for 15 days in darkness (0 h). Then seeds were stratified at 4°C in the dark for 48 h prior being transferred to continuous light for 48 h [START_REF] Narsai | In-Depth Temporal Transcriptome Profiling Reveals a Crucial Developmental Switch with Roles for RNA Processing and Organelle Metabolism That Are Essential for Germination in Arabidopsis[END_REF]. Absolute AtSHMT1expression levels of germinating Col-0 seeds (soaked for 1, 2 and 3 days) and 1day-old seedlings. Expression levels are represented as a color scheme (c) and a histogram (d), [START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF]. SHMT1 expressions in the mature leaf and the root are given as positive and negative control levels, respectively. Representations of the data were taken from the Arabidopsis eFP Browses (http://bar.utoronto.ca).

CONCLUSION & PERSPECTIVES

Since ABA is known for its role in seed dormancy by inhibiting germination [START_REF] Nambara | Abscisic acid and the control of seed dormancy and germination[END_REF][START_REF] Nakashima | ABA signaling in stress-response and seed development[END_REF], we investigated whether seed germination could be impacted in our phospho-mimetic Compl-S31D lines and whether their seeds responded differently to ABA. Our results were a little surprising, since all of our SHMT1 transgenic lines germinated in a similar manner in the absence of ABA as perhaps expected since in rosette leaves they all exhibited similar SHMT activities. However, their response to ABA was different with again SHMT1-S31D containing seeds exhibiting a lower sensitivity to ABA compared to the other lines. Could this indicate that ABA preferentially alters phosphor-mimetic S31 SHMT1 activity by somehow decreasing SHMT1 protein stability? Expression analyses revealed that SHMT1 was expressed early during germination in cotyledons and hypocotyls, this indicates that photorespiration is probably important at this early development stage of the plant. However, it is possible that SHMT1 activity in germinating seeds could involve C1-metabolism. The decreased sensitivity to ABA observed for Compl-S31D lines might reflect the regulation of SHMT1 by phosphorylation with respect to a specific metabolic function during seed germination that is modulated by an ABA signaling pathway.

Based on these results it will be necessary to: Previous reports showed that SHMT1 was ubiquitinated and underwent 26S proteasomedependent degradation that was linked to the activity of UBP16 (UBiquitin-specific Protease 16). When salt-stressed, UBP16 was proposed to stabilize SHMT1 by removing ubiquitin from the protein (Zhou et al., 2012). My working hypothesis was that the phospho-mimetic SHMT1 could modify the interaction with UBP16 thus making SHMT1-S31D more sensitive to 26S proteasome degradation. This was tested by in vitro cell-free degradation assays in the presence/absence of the 26S proteasome inhibitor MG132 and by yeast two-hybrid assays between SHMT1 and UBP16 and between the mutated SHMT1 forms.

METHODS

Cell-free degradation assays using the 26S proteasome inhibitor MG132

The assay followed the method described in Zhou et al. (2012). Two-week-old Arabidopsis seedlings were ground to a fine powder in liquid nitrogen before adding degradation buffer (25 mM NaH 2 PO 4 , pH 7.5, 10 mM NaCl, 10 mM MgCl 2 , 4 mM PMSF, 5 mM DTT, 10 mM ATP) and debris was removed by 2 centrifugations at maximum speed in a bench centrifuge for 10 min at 4°C, and the supernatants were collected in new tubes. Protein concentration was determined as described in Bradford, 1976(Bradford, 1976). Total soluble protein extracts were adjusted to an equal concentration with the degradation buffer. Either 50µM MG132 (Sigma-Aldrich, a specific 26S proteasome inhibitor) or DMSO (control solvent) were added to the extracts to assay 26S proteasome-dependent SHMT1 degradation. The mixtures were incubated at 22°C for 45 min, reactions were stopped by adding 2× Laemmli buffer (containing 4% SDS (W/V), 20% glycerol (V/V), 120 mM Tris-HCL, 0.02% bromophenol blue (W/V)) and boiling for 5 min at 100°C. SHMT1 protein was determined by western-blot assays using the previously described potato SHMT1 antibodies. Protein levels were quantified using Image J software. Actin was used as a control.

Yeast two-hybrid assays to test UBP16-SHMT1 and SHMT1-SHMT1 interactions

The plasmids of pGADT7, pGBKT7, pGBKT7-UBP16, pGADT7-UBP16C and pGADT7-SHMT1 were kindly provided by Yan GUO (College of Biological Sciences, China Agricultural University, Beijing 100193, China). pGADT7-SHMT1-S31A and pGADT7-SHMT1-S31D were made using the QuikChange® II XL Site-Directed Mutagenesis Kit (Agilent®), according to the manufacturer's instructions, and pGADT7-SHMT1 as the template. Primers SHMT1-S31A-F and SHMT1-S31A-R were used to generate AD-SHMT1-S31A, primers SHMT1-S31D-F and SHMT1-S31D-R were used to generate AD-SHMT1-S31D (see below for the primers sequences). To generate BD-SHMT1-S31A, BD-SHMT1-S31D and BD-SHMT1 plasmids, restriction enzyme sites were added to the cDNA of SHMT1-S31A, SHMT1-S31D by PCR with SHMT1-BamHI-F and SHMT1-PstI-R as primers (see below for the primers sequences) and pGADT7-SHMT1-S31A, pGADT7-SHMT1-S31D and pGADT7-SHMT1 as templates. The different SHMT1 cDNAs were then transferred into pGBKT7 by restriction enzyme cloning. The constructs were co-transformed into the yeast strain AH109 according to the protocol of MATCHMAKER Two-Hybrid-System (Clontech). The interaction of two proteins were determined by growing serial dilutions of transformants on SD synthetic complete medium without Trp, Leu and His but with or without an indicated concentration of 3-amino-1,2,4-triazole (SC-T/L/H, SC-T/L/H + 3-AT).

Primers name

Sequence (5' to 3')

BD plasmids SHMT1-BamHI-F GCGGATCCTTATGGCGATGGCCATGG SHMT1-PstI-R GATCCTGCAGTTAGTTCTTGTACTTCATGGTTTC AD plasmids (Site-directed mutagenesis) SHMT1-S31A-F ACTTCATGTTACATGTCTGCTTTGCCCAGTGAAGCTG SHMT1-S31A-R CAGCTTCACTGGGCAAAGCAGACATGTAACATGAAGT SHMT1-S31D-F CCACTTCATGTTACATGTCTGATTTGCCCAGTGAAGCTGTTG SHMT1-S31D-R CAACAGCTTCACTGGGCAAATCAGACATGTAACATGAAGTGG

RESULTS

SHMT1-S31D protein is sensitive to degradation by the 26S proteasome

In order to examine the potential mechanisms involved in reducing SHMT1-S31D protein levels in response to a salt stress, it was decided to investigate the reported degradation of SHMT1 by the 26S proteasome. To achieve this, a cell-free-degradation assay was carried out using total soluble extracts from the rosettes of 2-week-old Compl-SWT, Compl-S31D and

Compl-S31A transgenic seedlings grown in normal short-day conditions. Protein extracts were treated with/without either 50µM MG132 (a specific 26S proteasome inhibitor, dissolved in DMSO) or DMSO (mock-treated) and incubated at 22°C for 45 min. SHMT1 protein abundance was then determined by western-blot assays using SHMT1 antibodies. After 45 min, the abundance of SHMT1 protein extracted from Compl-S31D lines and treated without MG132 decreased (from 20% to 40%) compared to the same extract at the beginning of the treatment or in the presence of MG132 (Figure 3). SHMT1 protein extracted from the two other lines Compl-SWT and Compl-S31A did not exhibit any significantly changes in their amounts (Figure 3). Therefore, we did not find the same changes described in the work of Zhou et al (2012), since we did not observe a 26S proteasome-dependent degradation of SHMT1-WT (or SHMT1-S31D) in our assay condition. However, it appeared that the phospho-mimetic SHMT1-S31D exhibited a higher sensitivity to this degradation pathway even when the assays were carried out using extracts from plants grown in normal short-day growth conditions.

SHMT1 does not interact with UBP16 and Ser31 phosphosite mutations do not modify

SHMT1 subunit interactions

The 26S proteasome-dependent degradation of SHMT1 was shown to be UBP16-dependent and UBP16 activity was important for maintaining SHMT1 activity under salt stress conditions (Zhou et al (2012)). Indeed, Arabidopsis plants lacking UBP16 led to less SHMT1 protein, lower SHMT activity and salt-induced H 2 O 2 accumulation (Zhou et al., 2012). We hypothesized that SHMT1-S31D protein interaction with UBP16 could be modified in our

Compl-S31D lines thus leading to the observed decrease in both SHMT1 protein and activity.

We also decided to test whether S31A/S31D mutations modified SHMT1 subunit interactions.

To verify our hypotheses, yeast-two-hybrid assays were performed using the same experimental conditions and with the vectors published in Zhou et al (2012). Different combinations of UBP16/UBP16C and SHMT1-SWT or SHMT1-S31A or SHMT1-S31D were co-transformed into the yeast strain AH109. Protein interaction were determined by growing transformed yeast on SD synthetic complete medium without Trp, Leu and His and with or without 3-amino-1,2,4-triazole (3-AT) (SC-T/L/H, SC-T/L/H + 5 or 10mM 3-AT ) (Figure 4 and 5). On SC-T/L/H, three constructs (AD-SHMT1-S31D, AD-UBP16C and BD-UBP16)

when co-transformed with empty plasmids pGBKT7 (BD-V) or pGADT7 (AD-V) showed a weak self-activation that allowed the yeast to grow (Figure 4), this residual growth disappeared in the presence of 5 mM and 10 mM 3-AT (Figure 4). Unfortunately, in this experiment that was repeated several times, and even though we used their original plasmids, we could not repeat the results of Zhou et al. (2012). Indeed, none of our SHMT1 proteins (SHMT1-SWT / SHMT1-S31A / SHMT1-S31D) interacted with either the full length UBP16

or C-terminus part of UBP16 (Figure 4). On the other hand, the SHMT1 interaction analyses showed that the different SHMT1 protein combinations exhibited similar interactions, whatever the combination tested (Figure 5). However, it should be noted that the pGADT7-SHMT1-S31D (with the activation domain) always presented a weaker interaction on SC-T/L/H + 10 mM 3AT whatever version of SHMT1 was associated with the binding domain (Figure 5). Since, pGBKT7-SHMT1-S31D co-transformed with either pGADT7-SHMT1 or pGADT7-SHMT1-S31A allowed a similar yeast growth on 10mM 3AT, we concluded that the mutation of S31 did not impact SHMT1 interactions.  Carry out the yeast double-hybrid experiments using constructs that produce mature SHMT1 fusion proteins since the presence of the mitochondrial transit peptide could alter SHMT1 protein conformation. Of course, this does not explain our inability to find the same results as Zhou etal (2012).

 Analyze the role of SHMT1 polyubiquitination. According to the UbPred predictor of potential ubiquitination sites (http://www.ubpred.org/), SHMT1 contains 3 potential sites: K40, K49 and K473. Two of these predicted sites are close to the N-terminal region of SHMT1 and therefore to S31. By mutation of these lysine residues associated or not with the S31 mutations and their expression in shm1-1, it will be possible to see if ubiquitination and phosphorylation are linked and whether ubiquitination of SHMT1 is involved in SHMT1 stability in salt stress conditions. It would be possible to immunoprecipitate SHMT1-31D in Compl-S31D lines using SHMT1 antibodies followed by western-blot detection of ubiquitin with ubiquitin antibodies to determine if S31D impacts the ubiquitination/deubiquitination process in Compl-SHMT1-S31D lines.

INTRODUCTION

The photorespiratory C-2 cycle is an essential pathway for normal plant growth and development in air and it represents the most important carbon metabolism process after photosynthesis (Bauwe et al., 2010;[START_REF] Peterhansel | Photorespiration Redesigned[END_REF]. The photorespiratory cycle involves eight core enzymes that are distributed in four subcellular compartments; chloroplasts, peroxisomes, mitochondria and cytosol. It is initiated in chloroplasts by the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) to produce one molecule of toxic 2-phosphoglycolate (2PG) and one molecule of 3phosphoglycerate (3PGA) [START_REF] Peterhansel | Photorespiration[END_REF]. The major roles of the photorespiratory cycle are to salvage carbon by recycling 2PG to 3PGA that can re-enter the Calvin-Cycle and at the same time to remove potentially toxic metabolites. However, the photorespiratory cycle also leads to the release of assimilated carbon and nitrogen as CO 2 and ammonia that must be either re-assimilated or lost to the atmosphere. Therefore, photorespiration reduces CO 2 assimilation and it has an important energetic cost that negatively impacts crop yields and this is expected to become more important under future climate change conditions such as high temperature and drought [START_REF] Peterhansel | Photorespiration Redesigned[END_REF][START_REF] Walker | The Costs of Photorespiration to Food Production Now and in the Future[END_REF]. The photorespiratory cycle also interacts with other metabolic pathways and processes, such as photosynthesis in chloroplasts, respiration and C1 metabolism in mitochondrion, cell redox homeostasis (H 2 O 2 ) in peroxisomes, as well as nitrogen metabolism [START_REF] Hodges | Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network[END_REF]. We now have a good understanding of the eight core photorespiratory enzymes mainly due to the sequencing of the Arabidopsis genome and the availability of mutants affected in each enzyme (Bauwe et al., 2010). However, knowledge of the coordination of the photorespiratory cycle with other pathways and how individual enzymes are regulated is limited. Recent data indicate that protein phosphorylation could be a key regulatory component of the photorespiratory cycle (Hodges et al., 2013). Protein phosphorylation leads to the covalent addition of a phosphate group to specific amino acid residues (serine, threonine, tyrosine and histidine). It often plays a key role in regulating cell function, growth and development, metabolism, and also biotic and abiotic stress responses by modulating protein activity, sub-cellular localization, stability and capacity to interact with other proteins.

Hydroxypyruvate reductase (HPR) catalyzes the NADH/NADPH-dependent conversion of hydroxypyruvate to glycerate. In Arabidopsis, three putative HPR-encoding genes have been identified: AtHPR1, At1g68010; AtHPR2, At1g79870; AtHPR3, At1g12550. AtHPR1 is a 42 kDa peroxisomal protein, AtHPR2 (34 kDa) is cytosolic and AtHPR3 (35 kDa) is plastidial [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. HPR1 is a core enzyme of the photorespiratory cycle, since hpr1 mutants exhibit a viable retarded growth phenotype when grown in air which is absent when grown in elevated CO 2 (0.15-0.2% CO 2 ) [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF]. A dominant role in photorespiration of HPR1 was further shown by isotopic assays, where a 103-fold accumulation of hydroxypyruvate was measured in hpr1 whereas it accumulated only 3-fold in hpr2 when compared with wild-type plants [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF]. The absence of HRP1 also increased the steady-state levels of other metabolites including glycerate, glycine, serine, hydroxypyruvate and glutamine [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF]. Based on the phenotypes of hpr2 and hpr3 mutants as well as double and triple hpr mutant lines, the cytosolic AtHPR2 and chloroplastic AtHPR3 isoforms appear to have a limited compensatory photorespiratory cycle by-pass function which is mainly carried out by cytosolic AtHPR2 [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. Studies have demonstrated that the weak type III photorespiratory phenotype of hpr1 in normal air was affected by photoperiod since it showed the strongest phenotype when grown in short-day conditions (8-10 h light) rather than 12 h/12 h day-/night-cycles [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]). It has also been reported that HPR1 is involved in plant response not only to biotic stress by interacting with an elicitor receptor to induce plant defense against avirulent bacteria in soybean [START_REF] Okinaka | The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme, NADHdependent hydroxypyruvate reductase[END_REF] but also to abiotic stress based on the induction of HPR1 gene expression by drought [START_REF] Seki | Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray[END_REF][START_REF] Rizhsky | When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress[END_REF][START_REF] Wang | Shoot-specific down-regulation of protein farnesyltransferase (α-subunit) for yield protection against drought in Canola[END_REF]Li and Hu, 2015). While HPR1 is a NADH-dependent hydroxypyruvate reductase, HPR2 and HPR3 are NADPH-dependent hydroxypyruvate reductase also able to use glyoxylate as substrate and NADH as cofator [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF][START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF].

Although HPR1 has been identified as the major HPR-photorespiratory enzyme, little is known about how it is regulated. In order to rapidly respond to changing conditions, enzymes are often post-translationally modified and it has been reported that pea HPR1 activity can be regulated by tyrosine nitration [START_REF] Corpas | Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration[END_REF]. In vitro, this modification led to a 65% inhibition of HPR activity, and site-directed mutagenesis showed that nitration at tyrosine 198 was responsible for the observed inhibition [START_REF] Corpas | Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration[END_REF]. Furthermore, phosphoproteomic studies have indicated that Arabidopsis HPR1 can be phosphorylated at two sites; serine 229 [START_REF] Roitinger | Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana[END_REF][START_REF] Umezawa | Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in arabidopsis thaliana[END_REF] In this study, the effect of HPR1 Ser229 and Thr335 phosphorylation on enzymatic activities and kinetic properties were compared using recombinant non-phosphorylatable (S229A and S335A) and phosphorylation-mimetic (S229D and S335D) proteins. To evaluate their role in planta, the hpr1 mutant was complemented with either phosphorylation-mimetic HPR1 (S229D and S335D) or non-phophorylatable HPR1 (S229A and S335A). We found that the phosphorylation-mimic HPR1-T335D recombinant protein exhibited a 51% inhibition of maximal NADH-hydroxypyruvate-dependent activity compared to wild-type HPR1 recombinant protein. In accordance with this lower activity, hpr1 expressing this HPR1 form (Compl-HPR1-T335D) had smaller rosettes when grown in air. Based on our initial studies, we wanted to investigate whether the phosphorylation-mimetic HPR1 proteins showed altered activities with other known HPR substrates and cofactors.

Indeed, HPR1 has been shown to use glyoxylate as a substrate and NADPH as cofactor [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF][START_REF] Kleczkowski | Purification and characterization of a novel NADPH ( NADH ) -dependent glyoxylate reductase from spinach leaves[END_REF]. Therefore, recombinant WT HPR1, HPR1-T335A and HPR1-T335D protein activities were compared using four different substratecofactor combinations (Figure 2). Consistent with a previous report [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF], all recombinant HPR1 proteins were more active with the HP-NADH combination (Figure 2a)

when compared to the measured HP-NADPH activity (Figure 2b). When glyoxylate was tested as substrate, the NADH-dependent activity was 5-fold reduced (Figure 2c) and it was even lower when NADPH was used as cofactor (Figure 2d). Such general trends were observed for all recombinant HPR1 proteins, however certain kinetic parameters of HPR1-T335D were modified compared to wild-type HPR1 and HPR1-T335A values depending on the substrate-cofactor combinations (Figure 2e and2f). When compared to wild-type HPR1, as already described above, the V max of HPR1-T335D was reduced by about 50% with the HP-NADH combination (Figure 2f), while the V max was significantly increased by either the HP-NADPH and glyoxylate-NADPH substrate-cofactor combinations when compared to HPR1-WT (Figure 2f). Moreover, the K m for glyoxylate of HPR1-T335D decreased by 44% compared to wild-type HPR1 with NADPH as the co-factor whereas the Km substrate values remained unchanged with the other substrate-cofactor combinations (Figure 2e). Taken together, these results indicate that in planta phosphorylation at Thr335 could modify HPR1 activities and substrate specificities since the T335D mutation alters the NADH-NADPH activity balance in favor of NADPH while improving the use of glyoxylate. NADPH-dependent reaction with either hydroxypyruvate or glyoxylate compared to wildtype HPR1 (Figure 2b, 2d and 2f). Furthermore, although the activity was not different compared to wild-type HPR1 in the presence of glyoxylate-NADH (Figure 2c and 2f), the HPR1-T335D K m for glyoxylate was reduced by 44% compared with HPR1-T335A and wildtype HPR1 (Figure 2e). Interestingly, cytosolic HPR2 [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF] and HPR3 [START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF], a potential chloroplast HPR, both display similar catalytic properties, preferring NADPH as co-factor [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF][START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. These two isoforms also use glyoxylate more efficiently than HPR1. A protein sequence alignment of HPR1, HPR2 and HPR3 showed that Thr335 of HPR1 was replaced by an aspartic acid (D293) in HPR2 (Figure 6). HPR1-T335D exhibited a shift in co-factor specificity, with a decrease in NADH-dependent activity and an increase in NADPH-dependent activity, as well as an improved glyoxylate-NADH-dependent activity.

Such properties are similar albeit much more important in HPR2, and indicate that HPR1

Thr335 phosphorylation might alter the kinetic properties of peroxisomal HPR1 towards those of cytosolic HPR2. As peroxisomal NADH generation is considered a limiting step in serine to glycerate conversion [START_REF] Yu | Conversion of serine to glycerate in intact spinach leaf peroxisomes: Role of malate dehydrogenase[END_REF], phosphorylation of HPR1 at Thr335 could reduce NADH use in favor of NADPH. This mechanism could intervene when metabolic NADH/NADPH balance is altered by changing environmental conditions thus conferring cofactor flexibility to maintain the required HPR1 activity for cellular photorespiratory flux.

In planta, only Compl-HPR1-T335D lines displayed retarded growth compared to Col-0, Compl-HPR1-T335A and Compl-HPR1-S229 lines (Figure 3d and4). This suggested that under our standard growth conditions, Compl-HPR1-T335D rosettes had a lower HPR1 activity and that the observed in vitro changes were not beneficial. The most likely explanation for the reduced plant growth is that the low HPR1 activity of Compl-HPR1-T335D lines was not sufficient to maintain the required photorespiration flux and this negatively impacted photosynthetic activity and growth (Somerville and Ogren, 1980;1981;1982;Chastain and Ogren, 1989;Timm et al., 2012). However, perhaps this was not the reason for the retarded growth of the Compl-HPR1-T335D lines since the Compl-HPR1-S229A lines grow normally (Figure 3c) however recombinant HPR1-S229A had a lower NADH-hydroxypyruvate-dependent activity (Table 1) and the plants appeared to contain less HPR1 protein compared to Compl-HPR1-T335D lines (Figure 3b).  Grow the complemented lines in high CO 2 conditions to determine whether the phenotype of Compl-HPR1-S335D lines in ambient air is related to photorespiration.

 Analyze plant metabolite levels by GC-MS to determine whether metabolic pathways are differentially affected between the lines  Analyze CO 2 assimilation rates and photosynthetic electron transfer parameters by leaf gas-exchange to determine whether the low HPR1 activity impacts photosynthesis.

HPR1 expression is induced by a drought stress [START_REF] Seki | Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray[END_REF][START_REF] Rizhsky | When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress[END_REF][START_REF] Wang | Shoot-specific down-regulation of protein farnesyltransferase (α-subunit) for yield protection against drought in Canola[END_REF]Li and Hu, 2015) while it has been reported that the hpr1 growth phenotype depends upon photoperiod, therefore the Compl-HPR1-S335A/D and Compl-HPR1 lines will be used to:

 Test whether HPR1 phosphorylation affects plant response to abiotic stresses, such as drought and NaCl stress.

 Test plant growth under different photoperiods to determine whether the phenotype of Compl-HPR1-T335D lines is still impacted.

Since HPR1 Ser229 is replaced by an aspartate in HPR3, it would be interesting to test the different substrate-cofactor combinations on the different HPR1-Ser229 recombinant proteins.

Furthermore, it would also be interesting, since NADPH/NADH-dependent activities are modified, to study the Km values for these co-factors using the different recombinant HPR1

proteins.

Finally, it would be interesting to make and study the double phospho-mimetic HPR1 recombinant protein and complement hpr1 with double mutated proteins (phospho-mimetic / non-phosphorylatable).

pH7WG-promSHMT1::HPR1-S229A, pH7WG-promSHMT1::HPR2-S229D, pH7WG-promSHMT1::HPR1-T335A and pH7WG-promSHMT1::HPR2-T335D.

All plasmids were transformed into Agrobacterium strain GV3101 and Arabidopsis hpr1complemented transgenic lines were generated by the Agrobacterium-mediated floral dip method (Clough and Bent, 1998). Transformed lines were selected by their resistance to hygromycin on ½ MS medium containing 0.8% agar (w / v), 1% sucrose (w / v), 15 µg ml -1 of hygromycin as described by [START_REF] Harrison | A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation[END_REF]. Homozygous lines containing a single insertion were selected by successive rounds of self-crossing and antibiotic selection on ½MS medium agar plates.

Expression and purification of recombinant HPR1 proteins

The pBAD/His expression system was used to produce recombinant N-terminal 6×His tagged HPR1 proteins [START_REF] Lee | Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter[END_REF]. All HPR1 proteins were produced using E. coli strain LMG194 that can grow on RM medium (minimal medium) which ensures a low basal expression from the pBAD promoter. The plasmids pBAD-AtHPR1, pBAD-AtHPR1-S229A / D and pBAD-AtHPR1-T335A / D were transformed into E. coli LMG194 by electroporation.

Individual transformed colonies were used to inoculate a 10 mL RM medium preculture containing 0.2 % glucose and 50 µg/ml ampicillin that was left overnight at 37°C. The next day, this was used to inoculate 200 mL RM medium containing 0.2 % glucose and 50 µg/ml ampicillin at 37°C. All bacterial cultures were shaken at 200 rpm (1.5xg). When the OD 600 was ~0.7, protein production was induced by adding 0.2% (w/v) L-arabinose (Sigma, A3256).

After 20 h at 37°C, cells were harvested by centrifugation at 4000xg for 30 min at 4°C.

For protein purification, His-Select nickel affinity resin (His-Select ® Nickel Affinity, P6611, Sigma-Aldrich) was used and His-tag protein purification was carried out as in (Dellero et al., 2015a). The bacterial pellets were re-suspended in 3 mL lysis buffer (50mM NaH 2 PO 4 , pH 8.0, 300mM NaCl, 5mM imidazole and a protease inhibitor mixture (Complete Mini, EDTAfree, Roche Applied Science)). Cells were disrupted three times through a French-Press at 600

Pa. Soluble proteins were collected by centrifugation at 15000xg for 30 min at 4°C. At the same time, 2 mL of affinity resin was washed twice with 50 mL of washing buffer (lysis buffer supplemented with 20 mM imidazole), each time centrifuged at 4000xg for 10 min at 4 °C and then re-suspended in 10 mL of washing buffer. ~ 2.5 mL of soluble protein supernatant and 10 mL of resuspended affinity resin were mixed and incubated at 4°C for 2 h on a rotating wheel. The resin-protein mixture was poured into a syringe to make an affinity column and washed with 50 mL washing buffer to remove non-interacting proteins.

Recombinant his-tagged protein was then eluted with 3 mL of elution buffer (lysis buffer supplemented with 250 mM imidazole), the eluted proteins were directly desalted using PD10 column previously equilibrated by 50 mM NaH 2 PO 4 , pH 8.0 following the manufacturer's instructions (GE Healthcare). Protein concentrations were determined using Bradford reagent (B6916, Sigma-Aldrich) (Bradford, 1976), using BSA as a standard and stored at -80°C for further experiments.

 RM medium (with ampicillin): for 1 L of RM medium, mix 20 g Casamino Acids and 890 mL deionized water for autoclave. After the autoclaved solutions were cooled, add 10×M9 Salts 100 mL, 1 M MgCl 2 1mL, 20% glucose 10 mL, 100 mg/ml ampicillin 0.5 mL. Mix well and store at 4°C for one month.

 10×M9 Salts: for 1 L, Na 2 HPO 4 60 g, NaH 2 PO 4 30 g, NaCl 5 g, NH 4 Cl 10 g, water 900 ml. Dissolve reagents in water and adjust the pH to 7.4 with 10 M NaOH. Add water to 1 L and autoclave for 20 min. Cool and add 1 mL of 1 M thiamine (filtersterilized, Sigma-Aldrich) and store at room temperature.

Measurement of HPR1 activity

HPR1 enzyme activity measurements were performed according to previous reports [START_REF] Tolbert | Localization Reductases and Properties in Spinach Leaf of Hydroxypyruvate Particles *[END_REF][START_REF] Kleczkowski | Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases[END_REF]. Briefly, 1 mL of reaction mixture contained 50 mM phosphate buffer pH 6.2, 0.2 mM NADH (N8129, Sigma-Aldrich) or NADPH (N1630, Sigma-Aldrich), 5 µg of purified recombinant HPR1 protein, and increasing amounts of hydroxypyruvate hydrate (06367, Sigma-Aldrich) or glyoxylate (G4502, Sigma-Aldrich). The reactions were initiated by adding the recombinant HPR1 at 25°C, the NADH/NADPH oxidation was monitored using a Varian Cary 50 spectrophotometer at 340 nm. The enzyme activity was defined as µmol of oxidized NADH or NADPH catalyzed by 1 mg protein per minute.

K m and V max values were calculated using SigmaPlot 13.0 software, based on the curve fitting Michaelis-Menten equation: v 0 =V mas [S]/ (K m +[S]).

For the hydroxpyruvate and NADH (substrate-cofactor) combination, the increasing hydroxypyruvate concentrations were: 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM and 2000 µM.

For the hydroxpyruvate and NADPH (substrate-cofactor combination), the increasing hydroxpyruvate concentrations were: 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM, 3000µM, 4000µM and 5000 µM.

For the glyoxylate and NADH (substrate-cofactor) combination, the increasing glyoxylate concentrations were: 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM, 20 mM, 30 mM and 40 mM.

For the glyoxylate and NADH (substrate-cofactor combination), the increasing glyoxylate concentrations were: 5 mM, 10 mM, 20 mM, 40 mM, 80 mM, 100 mM and 150 mM.

Protein computing

The 3-D structure of HPR1 was generated using the open-server I-TASSER (http://zhanglab.ccmb.med.umich.edu/) [START_REF] Yang | I-TASSER server: New development for protein structure and function predictions[END_REF] based on crystallized Pyrococcus HPR1 protein [START_REF] Yoshikawa | Structure of archaeal glyoxylate reductase from Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate[END_REF]. Since the late 70s and the publication of the first photorespiratory mutant [START_REF] Somerville | A phosphoglycolate phosphatasedeficient mutant of Arabidopsis[END_REF], extensive efforts have been made to discover the different players and partners taking part in photorespiration (Bauwe et al., 2010). Now, except for certain transporters, most of the key players have been identified and all of the core photorespiratory enzymes have been characterized, although their post-translational regulation still remains unclear.

Even if it has been shown that the activity of some photorespiratory enzymes can be modified by nitrosylation [START_REF] Palmieri | Regulation of Plant Glycine Decarboxylase by S-Nitrosylation and Glutathionylation[END_REF][START_REF] Ortega-Galisteo | S-Nitrosylated proteins in pea (Pisum sativum L.) leaf peroxisomes: Changes under abiotic stress[END_REF]Camejo et al., 2013;[START_REF] Corpas | Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration[END_REF], that they could be targets of thioredoxin-dependent redox regulation (Marchand et al., 2004), that SHMT1 is ubiquitinated (Zhou et al., 2012) and all but one of the core enzymes are probably phosphorylated (Hodges et al., 2013), many questions remain unanswered. To date, we still do not know the consequences of the protein modifications cited Before discussing the major results of my PhD, I would like to discuss some general problems and perspectives of working on protein phosphorylation. My project was based on the identification of phosphopeptides from phosphoproteomic studies. It would be interesting to show that SHMT1 and HPR1 are actually phosphorylated, to discover when they are phosphorylated and eventually try to identify the kinases involved. Perhaps, the first step should be to identify under which conditions the phosphorylation events actually occur. Since protein phosphorylation is a rapid, temporary and reversible response to environmental stimuli, experiments should be now conducted to detect when photorespiratory enzymes are phosphorylated in response to different conditions that are expected to modulate photorespiratory flux (for example: day/night cycle, high light, high CO 2 , heat stress, salt stress, drought stress, pathogen attack, etc). During my thesis, antibodies were raised against HPR1 and other photorespiratory enzymes while SHMT1 antibodies were available from Professor Bauwe's team in Germany. Therefore, it is now possible to immunoprecipite specific photorespiratory enzymes from leaf total soluble protein extract at different times after a given stress treatment or during the day/night cycle followed by detection of phosphopeptides by mass spectrometry. This approach should allow us to identify specific conditions where photorespiratory enzyme phosphorylation is induced. Such information would be not only very precious to help understand the roles of the different phosphorylation events but to determine conditions to verify phosphorylation with radiolabeled-ATP using leaf extracts and recombinant proteins (mutated or not at specific phosphorylation sites), and eventually to identify kinase candidates from proteomic analyses of organelle-enriched fractions.

Now, I would like to develop some ideas and perspectives relative to the two photorespiratory enzymes studied during my PhD.

HPR1: The enzymatic kinetic parameters of recombinant HPR1 and its various mutated forms revealed that the main modification was obtained with HPR1 T335D while the phosphomimetic mutation (S229D) had no significant effects. Indeed, the Vmax of HPR1 T335D decreased in the presence of hydroxypyruvate and NADH and increased in the presence of hydroxypyruvate and NADPH, that is to say the NADH/NADPH activity ratio of HPR1 T335D was shifted in favor of NADPH compared to WT HPR1. Furthermore, HPR1 T335D also exhibited an improved Km and Vmax with respect to glyoxylate/NADH and glyoxylate/NADPH activities, respectively. Such changes suggested that the T335D mutation modified the enzyme's kinetic properties towards those of HPR2 which prefers glyoxylate and NADPH compared to hydroxypyruvate and NADH [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF][START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF]. However, when HPR1 mutated forms were expressed in hpr1, only HPR1 T335D was unable to fully complement the photorespiratory growth phenotype of hpr1 in short-day airgrown conditions. Thus, if T335D really mimics a phosphorylation, we would expect this phosphorylation to occur under conditions where it would be more advantageous for HPR1 to use NADPH. Peroxisomes are able to produce NAPDH from NADP that is made by the action of and NAD kinase after NAD import into the peroxisome via the PXN (peroxisomal nicotinamide adenine dinucleotide carrier). The conversion of NADP to NADPH can probably be achieved by a number of peroxisomal dehydrogenases including glucose-6phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, etc (reviewed in [START_REF] Kaur | Defining the Plant Peroxisomal Proteome: From Arabidopsis to Rice[END_REF].

Using glyoxylate as a substrate instead of hydroxypyruvate by HPR1 would result in the production of glycolate that could return back into the photorespiratory cycle. This would create a glycolate/glyoxylate shunt that could enhance the production of H 2 O 2 via GOX perhaps in response to plant pathogens. Indeed, GOX activity has been shown to be important in plant pathogen resistance via its capacity to produce H 2 O 2 (Rojas et al., 2012;[START_REF] Gilbert | Characterization of the LOV1-mediated, victorininduced, cell-death response with virus-induced gene silencing[END_REF]Xu et al., 2018). Thus, we suggest that under conditions requiring a rapid and high production of H 2 O 2 , such as in a response to plant pathogen attack, HPR1 T335 becomes phosphorylated to favor glyoxylate as a substrate to produce glycolate that in turn will produce H 2 O 2 by the action of GOX. This hypothesis should be tested by comparing the efficiency of pathogen resistance between the different complemented lines expressing mutated HPR1 at T335 along with the production of H 2 O 2 .

Very recently, [START_REF] Pan | Proteomics of Peroxisomes[END_REF] compiled a list of about 200 confirmed Arabidopsis peroxisomal proteins based on data from peroxisome proteome and localization studies.

Among these proteins, 4 kinases and 8 phosphatases or phosphatase subunits were shown to be present in peroxisomes (Table 1). All of them, except GPK1 (previously shown to be in glyoxysomes of etiolated Arabidopsis cotyledons by [START_REF] Fukao | Novel Glyoxysomal Protein Kinase, GPK1, Identified by Proteomic Analysis of Glyoxysomes in Etiolated Cotyledons of Arabidopsis thaliana[END_REF], were identified by in silico analyses and confirmed to be peroxisomal by fluorescence microscopy [START_REF] Matre | Diversity in subcellular targeting of the PP2A B′η subfamily members[END_REF]Kataya et al., 2015a;Kataya et al., 2015b;[START_REF] Kataya | Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana[END_REF]. Apart from their localization, little is known about the role of these kinases and phosphatases. Using transiently transformed protoplasts, MKP1 was found to be cytosolic in non-stressed conditions while it was localized to the peroxisome under various stress conditions including salt, flagellin and oxidative stresses (Kataya et al. 2015b). Moreover, MPK1 was shown to be involved in plant stress responses as mpk1 mutants were more resistant to pathogens and produced more ROS than WT plants [START_REF] Anderson | Arabidopsis MAP Kinase Phosphatase 1 (AtMKP1) negatively regulates MPK6mediated PAMP responses and resistance against bacteria[END_REF]. CPK1 has also been shown to be involved in plant pathogen responses and salt stress and cpk1 mutants were more sensitive to pathogens while they were more resistant to salt stress [START_REF] Coca | AtCPK1 calcium-dependent protein kinase mediates pathogen resistance in Arabidopsis[END_REF][START_REF] Stagner | Working Through Postpartum Depression: A Guide for Clinicians and Couples[END_REF]. Perhaps

CPK1 phosphorylates HPR1 at T335 to stimulate our proposed glyoxylate/glycolate cycle to generate more H 2 O 2 . Based on the SUBA database (http://suba.plantenergy.uwa.edu.au/), 33 protein kinases are predicted to be addressed to the peroxisome. Thus, a strategy to identify kinases implicated in HPR1 phosphorylation would be to retrieve knock-out mutant lines for each gene encoding a peroxisomal protein kinase and compare HPR1 phosphorylation status by mass spectroscopy after HPR1 immunoprecipitation from soluble extracts from control plants and from plants treated to previously identified conditions that induce HPR1 phosphorylation.

Table 1. Confirmed Arabidopsis peroxisomal kinases and phosphatases. Adapted from [START_REF] Pan | Proteomics of Peroxisomes[END_REF]. PTS: Peroxisomal targeted signal. protein stability. Indeed, the phenotypic analyses of shm1-1 expressing SHMT1, SHMT1

S31A and SHMT1 S31D proteins in control and salt stress conditions indicated that Compl-S31D lines exhibited a higher sensitivity to salt stress due to a reduced level of SHMT1-S31D

protein that led to a further decrease in SHMT activity. Since this was not seen in nonphotorespiratory high CO 2 conditions, it appeared that this phenotype was linked to its photorespiratory function. Although several possible mechanisms were tested, based on the literature, including UBP16 deubiquitination and ICP55 N-terminal preprocessing, it was not possible to show that these processes were modified in the lines complemented with SHMT1-S31D. However, under control conditions, SHMT1-S31D containing plants exhibited a 26S proteasome-dependent degradation that was not seen with rosette extracts of the other transgenic lines. It is possible that the addition of an aspartate close to the N-terminal of SHMT1 leads to a less stable protein even under control conditions but we were unable to observe this with our experiments. Surprisingly, salt-and ABA-induced stomatal closure was also affected in the Compl-SHMT1-S31D lines when compared to the other transgenic plants.

To date, we do not know whether this has a role in bringing about the increased sensitivity of the SHMT1-S31D containing plants to be more sensitive to salt stress and less capable of recovering from a drought stress. Furthermore, it is difficult to reconcile an altered ABAsensitivity (stomatal closure and germination) and an inefficient photorespiratory cycle due to a low SHMT1 activity. To understand the link between these processes further work must be done.

SHMT1 Ser31 seems to be phosphorylated at a steady basal level in normal conditions, and it was not altered by varying the gas content of air to modulate photorespiratory activity (Abadie et al., 2016). If the aspartate mutation of S31 indeed mimics a phosphorylation, it is logical to assume that plant response to an abiotic stress that increases photorespiration (like a salt stress or drought stress) would not induce Ser31 phosphorylation of SHMT1 as it would have negative consequences. Under such unfavorable conditions, we propose that SHMT1

Ser31 would be dephosphorylated in order to maintain SHMT1 protein levels and activity to cope with reduced CO 2 availability and an increased photorespiratory flux.

As for HPR1, to better understand the consequence of SHMT1 phosphorylation, it is necessary to identify the conditions where SHMT1 Ser31 phosphorylation occurs and the kinase(s) that are involved. However, identification of SHMT1 kinase(s) will be difficult because according to the SUBA database, 20 protein kinases have been shown to be addressed to the mitochondrion while 1018 are predicted to be localized in this compartment. This means that the strategy of identifying the SHMT1 kinase by characterizing putative kinase mutants is not possible without a way of limiting the number of candidates perhaps by coexpression analyses or targeted yeast double-hybrid analyses. An alternative strategy would be to purify mitochondria from leaves (or at least carry out enrichment protocols) from plants grown under conditions known to induce SHMT1 Ser31 phosphorylation. If such extracts were able to phosphorylate Ser31 of SHMT1 then, they could be used to identify SHMT1kinase-containing fractions from selected chromatography procedures (for example, ionexchange or gel filtration) that could be used to identify the kinase by a proteomic analysis using mass spectrometry. The identified kinase(s) would then be identified as a veritable SHMT1-kinase by studying the relevant knock-out mutant.

2) Identification of new components involved in regulating the photorespiratory cycle or regulated by photorespiratory activity

As stated above, phosphorylation of photorespiratory enzymes is not the only posttranslational modification affecting these proteins since ubiquitination, S-nitrosylation and thioredoxin-dependent redox regulation have been reported. Therefore, other proteins besides kinases and phosphatases will be involved in the regulation of photorespiratory cycle.

Identification of such regulators remains difficult as they will intervene under special environmental or stress conditions and knock-out mutant of proteins involved in these processes may not have any photorespiratory phenotype in normal growth conditions.

However, to try to identify other components involved in linking SHMT1 photorespiratory activity to stomatal closure or to find proteins involved in SHMT1 stability, it could be possible to perform EMS mutagenesis on seeds from Compl-SHMT1-S31D lines. The mutagenized plants will be screened for alterations in NaCl and/or ABA-induced stomatal closure by thermal imaging and a reversal of the severe growth phenotype when salt stressed to identify possible components involved in SHMT1 stability and linking photorespiratory activity to stomatal movements. After backcrossing the selected plants with Compl-SHMT1-S31D to remove background mutations, high-throughput sequencing of nuclear DNA will be performed to identify the mutated locus. Depending on the identified genes or types of mutation (dominant/recessive), either knock-out mutants or over-expressions will be analyzed, and if necessary recombinant proteins carrying the identified mutations will be characterized to discover the mechanism by which the regulatory processes have been modified.

Although photorespiration is important with respect to plant tolerance to biotic and abiotic stresses and interacts with several primary metabolic pathways, it has become a target for crop improvement due to its compete with photosynthetic CO 2 assimilation and its high energetic cost [START_REF] Betti | Manipulating photorespiration to increase plant productivity: Recent advances and perspectives for crop improvement[END_REF]. Several strategies have been shown to improve plant growth including the expression of artificial chloroplastic glycolate by-pass pathways that compete with the photorespiratory cycle to generate glycerate from glycolate at a lower energetic cost and without the loss of nitrogen while producing CO 2 in the chloroplast where RuBisCO is located [START_REF] Kebeish | Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana[END_REF], and the overexpression of certain photorespiratory enzymes (Timm et al., 2012b;[START_REF] Wu | The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping[END_REF]. Both methods depend on creating GMO plants that are not welcome in Europe.

Perhaps the identification and modification of regulators of the photorespiratory cycle could be useful to generate plants with:

 A better resistance to different stresses (for example: salt, drought, pathogens, temperature).

 An improved biomass production (and for less inputs).

 An increase in nutritional qualities.

3) Overexpression of SHMT1 in crops

In this context of crop improvement, SHMT1 is an excellent target. We have already seen that overexpression of SHMT isolated from a halotolerant cyanobacterium Aphanothece halophytica led to salt-tolerant Escherichia coli and S. elongatus (Waditee-Sirisattha et al., 2012;Waditee-Sirisattha et al., 2016). In rice, salt-tolerant CSR27 had a high shoot induction of the SHMT1 gene leading to a better salt tolerance compared to a salt-sensitive line (Mishra et al., 2016). Moreover, rice lines overexpressing SHMT1 had a better photosynthetic efficiency and a higher seed yield compared to control plants [START_REF] Wu | The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping[END_REF].

First, overexpression of SHMT1 and our mutated forms SHMT1 S31A/S31D could be carried out in A. thaliana to see whether similar results obtained in rice are obtained and if so whether the mutations S31A and S31D amplify or decrease the beneficial effects. Second, the overexpression of the best version of SHMT1 could be undertaken in crop plants like tomato or rapeseed. However, since this strategy will involve the production of GMO plants it will be necessary to achieve the overexpression of the selected SHMT1 form by a non-transgenic method. To do this, wild-type Arabidopsis will be transformed with a construct including a reporter gene (like luciferase) under the control of the SHMT1 promoter. Subsequently, mutagenesis by EMS will be performed on seeds of the reporter line, and the offspring will be screened to identify plants with the highest reporter gene expression via luminescence (in the case of luciferase). The mutation(s) correlated with luciferase overexpression will be identified in the offspring following crosses with wild-type plants by high throughput genomic DNA sequencing. Similar mutations in the homologous genes of crops of interest will be identified in Targeting Induced Local Lesion IN Genomes (TILLING) libraries when available. If necessary, a specific bank for the plant of agronomic interest will be made if it does not exist. If the SHMT1 phosphorylation sites are found to confer interesting agronomical traits, then the TILLING libraries can be screened to identify mutations that either lead to the absence of the phosphorylation site of interest in the expressed protein or create a phospho-mimetic protein.

Figure 1 .

 1 Figure 1. The light-dependent reactions of photosynthesis. (Figure modified from Meyer et al. (2009)). Chl, Chlorophyll; LHC I and II, Light Harvesting Complex I and II; PSI and PSII, Photosystems I and II; OEC, Oxygen-evolving complex; 4Mn-1Ca, metallic catalytic center of the OEC; Tyr-z, Tyrosine of the protein D1; P680 and P700, the reaction centers of PSI and PSII, respectively; Pheo, Pheophytin; QA, Quinone A; QB, Quinone B; D1, reaction center protein of PSII (binds Chlorophyll P680, Pheophytin, Quinone and 4Mn-1Ca center); D2 reaction center protein of PSII; PQ, Plastoquinone; Cyt-b6f, Cytochrome-b6f complex; PC, Plastocyanin; PsaA and PsaB, the heterodimer of the PI; Fe-S (F X and F A F B ), iron-sulfur centers; Fd: Ferredoxin; FNR, Ferredoxin-NADP reductase; FTR: Ferredoxin thioredoxin reductase; TRX, Thioredoxin; PsaC, PSI terminal acceptor; Red dotted line, electron flow; Blue dotted line, proton flow.

Figure 2 .

 2 Figure 2. A simplified scheme of the photorespiratory cycle. The enzymes and known transporters of the cycle are represented. The photorespiratory cycle takes place in four compartments: chloroplasts, peroxisomes, mitochondrion and cytosol. Abbreviations: RuBisCO, RuBP carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; PGLP1, 2-

  This transfer also led to a rapid decrease in photosynthetic CO 2 assimilation. It was proposed that the low photosynthetic CO 2 assimilation due to an impaired photorespiratory cycle limits N-assimilation and decreases leaf RuBisCO content until plants attain a new homeostatic C/N balance thus leading to the phenotype of smaller, slower growing plants in air[START_REF] Dellero | Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth[END_REF].

Figure 3 .

 3 Figure 3. Schema of performance of GDC complex (Douce et al., 2001)

Figure 4 .

 4 Figure 4. Examples of the four classes of photorespiratory phenotypes (according to Timm and Bauwe, 2013). Class I: (A) and (B) The double mutant shm1-2 x shm2-2 in high CO 2 on potting soil after 10 weeks of growth (top) or on ½ MS medium + 1% sucrose (left,

Figure 5 .

 5 Figure 5. The hypothetical impact of an interrupted photorespiratory cycle on photosynthesis. Firstly, the interruption of photorespiratory carbon flux will lead to (i) a decrease of the 3PGA generation, which decreases RuBP regeneration via the Calvin-Benson Cycle; (ii) an accumulation of 2PG, which inhibits the activity of TPI and thus negatively impacts RuBP regeneration. (iii) an accumulation of glyoxylate, which inhibits the activity or activation state of RuBisCO; Secondly, accumulation of ATP and reducing power due to the decline of their consumption by the photorespiratory and Calvin-Benson cycles will slow down and modify photosynthetic electron transfer leading to a stimulation of ROS production

  Figure 6. The Krebs cycle and the respiratory Electron Transfer Chain in mitochondria. Abbreviation: I; II; III; and IV, complex I; complex II; complex III and complex IV; PDH, Pyruvate dehydrogenase; ICDH, isocitrate dehydrogenase; 2OGDH, 2-oxoglutarate dehydrogenase; SCoAS, succinyl-CoA synthase; SuDH, succinate dehydrogenase; MDH, malate dehydrogenase; 2OG, 2-oxoglutarate; OAA, oxaloacetate.

Figure 7 .

 7 Figure 7. The Krebs cycle and its interactions with the Calvin cycle, photorespiration, glycolysis and ammonia assimilation. Abbreviation: PDH, Pyruvate dehydrogenase; PDK,

Figure 8 .

 8 Figure 8. Ammonium assimilation and the interaction with photorespiratory and Krebs cycles in plants. Abbreviation: GS, plastidial glutamine synthetase; GOGAT, glutamate synthase; NRT, nitrate transporter; NR, nitrate reductase; NiR, nitrite reductase; AMT, ammonium transporter; NO 3 -, nitrate; NO 2 -, nitrite; NH 4 + , ammonium; N 2 , nitrogen.

Figure 9 .

 9 Figure 9. The role of tetrahydrofolate (THF) in C1 unit interconversions and transfers and their interaction with the photorespiratory cycle in mitochondria. (a) Chemical structure of THF and THF-related C1-substituted derivatives which comprises pteridine, paminobenzoate and glutamate, R and X are the sites where various C1 units oxidative states could be attached. (b) C1 unit interconversion and transfer between THF and its C1substituted derivatives. The carbon source is marked with blue color, and the carbon metabolite is marked with red color. (c) THF cycle interaction with SHMT1/GDC complex in the photorespiratory cycle. DHC, 5,10-methylene-THF dehydrogenase/5,10-methenyl-THF cyclohydrolase; FDH, formate dehydrogenase; FDF, 10-formyl THF deformylase; 5-FCL, 5-

  Transgenic rice (Oryza sativa L) with either a decreased RuBisCO content (RBCS-antisense plants) or an increased RuBisCO amount (RBCS-sense plants) has been generated. Under low-CO 2 ([CO 2 ] of 5 Pa) mimicking stomatal closure condition, the RBCS-antisense plants with a decreased RuBisCO content exhibited significantly decreased photorespiratory activity while his activity remained unchanged in RBCS-sense plants (with increased RuBisCO amount)

  photorespiratory enzymes were found to be involved in plant sensitivity to the Victoria blight fungus Cochliobolus victoriae which is associated with the LOV1 (Locus Orchestrating Victorin effect 1) protein. Silencing of GOX (but also GDC-T, GDC-P and GS2) brought about the suppression of LOV1-mediated Victorin-dependent cell death as well as the inhibition of cell death and disease resistance mediated by the PTO resistance gene[START_REF] Gilbert | Characterization of the LOV1-mediated, victorininduced, cell-death response with virus-induced gene silencing[END_REF]. More recently, a new study using Nicotiana benthamiana showed that when NbHAOX8, NbGOX1 and NbGOX4 were silenced by virus-induced gene silencing, plant resistance was impacted differently to various pathogens including Tobacco rattle virus, Xanthomonas oryzae pv. oryzae (Xoo) and Sclerotinia sclerotiorum. For example, after 21 days inoculation with Xanthomonas oryzae pv. oryzae (Xoo), NbGOX4, NbHAOX8 and NbGOX1 showed different degree of symptoms such as yellowing and/or retarded growth.

Figure 11 .

 11 Figure 11. Proposed mechanisms for THF-independent aldolase reaction. Only partial structure is shown for pyridoxal phosphate (PLP) and B1 is the base of protein that accepts the proton in the reaction[START_REF] Schirch | Serine hydroxymethyltransferase revisited[END_REF].

  AtSHMT4 and AtSHMT5 containing no signal peptides are located in the cytosol; AtSHMT6 and AtSHMT7 containing nuclear-targeting signals are located in the nucleus. Amongst the seven AtSHMT proteins, only SHMT1, SHMT2 and SHMT3 have been tested and shown to carry out a SHMT activity. The nuclear SHMT7 (name MSA1: More Sulphur Accumulation 1)

Figure 12 .

 12 Figure 12. The shm1 (shm1-2, SALK_083735) mutant shows long-term acclimation and survives if initially grown in high CO 2 . A: Phenotype of different mutants after two weeks in ambient air condition. The mutants firstly grew in high CO 2 condition for 8 weeks until developmental stage 5.1, then, transferred to ambient air for 2 weeks. The rosette leaves of glyk1 and pglp1 were completely bleached while shm1 mutant were not completely bleached

Figure

  Figure 1. Characterization of shm1-1 complemented lines Compl-SWT, Compl-S31A and Compl-S31D.

Figure

  Figure S1. Cleavage site analysis of SHMT1 in Compl-SWT-5, Compl-S31A-7, Compl-S31D-5 based on LC-MS/MS analysis.

Figure

  Figure S2. SHMT1 protein levels of high-CO2-grown Col-0 (wild-type), shm1-1 mutant and complemented lines (Compl-SWT, Compl-S31A, Compl-S31D) rosette leaves, related to Figure 2C.

Figure 2 .Figure S4 .

 2S4 Figure 2. Salt sensitivity of air-grown complemented lines is reduced under high-CO2 growth conditions.

Figure 3 .

 3 Figure 3. SHMT1 protein stability is altered in Compl-S31D plants in response to salt stress.

  protein abundance in Compl-S31D lines in response to a NaCl treatment in ambient air condition, related to Figure 3C. Measurements were carried out on 7-day-old in vitro grown seedlings of Compl-SWT, Compl-S31D and Compl-S31A on ½MS plates and then transferred to new plates with or without 50 mM NaCl for 20 days. Leaf total soluble proteins were extracted and SHMT1 protein levels were determined by western-blot using potato SHMT1 antibodies. Actin was used as a loading control. Quantification of signal intensity was done using Image J software. SHMT1 protein levels were normalized to actin and SHMT1 protein levels without salt treatment were set to 100%, relative SHMT1 protein levels after salt treatment are given as the percentage of non-treated seedlings. Value are means ± SD (n=3 independent experiments). Statistical significance was determined by a Student's t test. Asterisks indicate significant difference compared with the initial value of the same genotype (*P < 0.05).

Figure 4 .

 4 Figure 4. Metabolic response to salt stress of Compl-S31D line differs from Col-0, Compl-SWT-5 and Compl-S31A-7 lines.

Figure 5 .(

 5 Figure 5. Salt stress induced H2O2 levels.

Figure

  Figure S6. Water content of air-grown Col-0, Compl-SWT, Compl-S31A, Compl-S31D lines, related to Figure 6.

Figure 6 .

 6 Figure 6. Compl-S31D lines are unable to recover after a long drought stress in ambient air conditions and exhibit a faster rate of water loss.

Figure 7 .

 7 Figure 7. Compl-S31D-5 stomata show an altered closure response to ABA and salt stress treatments.

a

  lower induction of P5CS1 in Compl-S31D-5 (2-fold) when compared to Col-0, Compl-SWT-5 and Compl-S31A-7 (10-fold) while RD29A expression in Compl-S31D-5 was induced more (8-fold) than in Col-0, Compl-SWT-5 and Compl-S31A-7 (5-fold) (Figure 8).

Figure 8 .

 8 Figure 8. Relative expression levels of several ABA-responsive genes in Compl-SWT-5, Compl-S31D-5 and Compl-S31A-7 in response to salt treatment.

  are shown with the Compl-SWT-5 line levels arbitrary set to1. Values are means ± SD (n=4 independant plants). Significantly different values from Col-0 under the same treatment condition are in bold (t-test, P<0.05). Significantly different values compared with the initial value of the same genotye are marked by an asterisk (t-test, P<0.05).



  Analyze stomatal starch content by lugol staining and microscopy or directly measure the starch content of isolated stomata. Detailed methods for GC-MS metabolite profiling and glycine quantification by HPLC for SHMT activity measurements All metabolomic analyzes were carried out in collaboration with the IPS2 Metabolism-Metabolome platform using their equipment and protocols. This included training by the referent engineers for each type of analysis I carried out during my PhD work.

  samples with calibration curves specific to glycine. The sample areas were normalized to the area of α-ABA (internal standard) before normalizing to the amount of the weight of the sample taken. HPLC was performed using a Waters Alliance instrument with a Waters 2475 multi-wavelength fluorescence detector set at λexc = 340, λem = 455. The reverse-phase 131 column was a Waters Symmetry C18 3.5 µm (150 mm• 4.6 mm i.d.) with a Sentry guard column (10 mm 2.1 mm i.d.) 133 CHAPTER II Additional results relative to the study of SHMT1 phosphoregulation PREAMBLE This chapter describes experiments carried out to strengthen certain observations presented in the SHMT1 manuscript that makes up chapter I of my PhD results. However, the obtained results were not included in the research paper either due to their preliminary nature, their coherence with leaf metabolism or because of their negative nature with respect to already published work. This chapter is divided into two parts:  Results related to the altered sensitivity of Compl-S31D lines to ABA.  Results related to the decreased level of SHMT1-S31D protein in salt-stressed Compl-S31D lines. The first part involves SHMT1 expression during seed germination and the effect of Ser31 phosphorylation on ABA-sensitivity to seed germination. The second part deals with saltinduced changes in SHMT1 stability by UBP16 linked to degradation by the 26S proteasome. I-ABA sensitivity: Compl-S31D lines show an altered ABA response during seed germination INTRODUCTION

Figure 1 .

 1 Figure 1. Public data for SHMT1 expression during seed germination.

Figure 2 .

 2 Figure 2. Seed germination of transgenic Ser31-phosphosite-modified SHMT1 lines and their sensitivity to ABA.



  photorespiration. Investigate seed germination salt-response to determine whether seed germination of Compl-SHMT1-S31D lines are specifically impacted. Measure seed SHMT1 activity in the different complemented lines during germination, with and without ABA or salt treatment to establish whether differences are correlated to SHMT1 activity. Investigate seed germination in the complemented lines without treatment to alleviate seed dormancy.These experiments should also include wild-type Col-0 seeds, and seeds from plants grown under the same conditions at the same time should be used.

Figure 3 .

 3 Figure 3. Cell-free degradation of Ser31 phosphosite mutated SHMT1 proteins.

Figure 4 .

 4 Figure 4. Yeast two-hybrid analysis of SHMT1-UBP16 interactions.

Figure 5 .

 5 Figure 5. Yeast two-hybrid analysis of SHMT1-SHMT1 interactions.

Figure 1 .

 1 Figure 1. Expression and purification of recombinant proteins of wild-type Arabidopsis HPR1 and mutated Arabidopsis HPR1 including HPR1-S229A, HPR1-S229D, HPR1-T335A and HPR1-T335D.

Figure 2 .

 2 Figure 2. Vmax and Km values of recombinant wild-type HPR1 and T335 site-mutated HPR1-T335A and HPR1-T335D proteins in the presence of different substrate-cofactor combinations.

Figure 3 .

 3 Figure 3. HPR1 protein content and growth phenotype of hpr1-complemented transgenic lines

Figure 4 .

 4 Figure 4. Fresh weight, dry weight and water content of rosettes of Col-0, hpr1 complemented lines and the hpr1 mutant

Figure 5 .

 5 Figure 5. Predicted 3D-structure of Arabidopsis HPR1, showing the position of S229 and T335 in different domains.

  Figure 6. HPR1, HPR2 and HPR3 protein sequence alignment. Sequence alignment was done using Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/). Phosphorylated amino acids S229 and T335 of HPR1 are highlighted in red, D293 in HPR2 is highlighted in yellow and Q301 in HPR3 is highlighted in blue. HPR1 (Q93VM9), HPR2 (Q9CA90), HPR3 (Q9LE33).

  photorespiratory enzyme phosphorylation

  above neither the conditions (biotic or abiotic stresses, day/night cycle, nitrogen availability, etc) under which they occur, nor the enzymes (kinases, phosphatases, thioredoxins, etc) that are involved.Since seven out of the eight core enzymes of photorespiration are associated with phosphopeptides in the PhosPhAt database, protein phosphorylation is a good candidate to be a key regulatory component of the photorespiratory cycle. The MetaboActions team is currently deciphering the regulation of photorespiratory enzymes/cycle by phosphorylation and during my PhD, I was given the opportunity to study the phosphorylation of SHMT1 and HPR1. This mainly involved mutating the phosphorylation sites of SHMT1 (S31A/D) and HPR1 (S229A/D and T335A/D), to mimic a constitutive phosphorylation or to abolish the possibility of phosphorylation.

Table 1 . Overview of genes encoding photorespiratory enzymes in Arabidopsis thaliana.

 1

	Enzyme	Gene name	Location	References of the mutants identified in Arabidopsis thaliana
		RBCS1A	At1g67090
		RBCS1B	At5g38430
	RuBisCO	RBCS2B	At5g38420	Not known at present
		RBCS3B	At5g38410
		RBCL	AtCG00490
	Phosphoglycolate phosphatase	PGLP1	At5g36700	Sommerville and Ogren, 1979 Schwarte et Bauwe, 2007
	Plastidial glycolate/glycerate transporter	PLGG1	At1g32080	Pick et al., 2013
	Plastidial glycolate transporter	BASS6	At4g22840	South et al., 2017
	Glycolate oxidase	GOX1 GOX2	At3g14420 At3g14415	Rojas et al., 2012 Dellero et al., 2016
	Glutamate:glyoxylate aminotransferase	GGT1	At1g23310	Igarashi et al., 2003 Dellero et al., 2015b
		GDP1	At2g26080	Engel et al., 2007
		GDP2	At4g33010	Engel et al., 2007
		GDT	At1g11860	Timm et al., 2018
	Glycine decarboxylase complex	LPD2	At3g17240
		LPD1	At1g48030
		GDH1	At2g35370	Not known at present
		GDH2	At2g35120
		GDH3	At1g32470
	Serine hydroxymethyl transferase	SHMT1	At4g37930	Somerville and Ogren, 1981 Voll et al., 2006
		SHMT2	At5g26780	Engel et al., 2011
	Serine:glyoxylate aminotransferase	SGAT1	At2g13360	Somerville and Ogren, 1980
	Hydroxypyruvate reductase	HPR1 HPR2	At1g68010 At1g79870	Timm et al., 2008 Timm et al., 2011
		HPR3	At1g12550
	Glycerate kinase	GLYK1	At1g80380	Boldt et al., 2005
	Plastidial 2OG/malate transporter	DiT1	At5g12860	Taniguchi et al., 2002
	Plastidial glutamate-malate transporter	DiT2.1	At5g64290	Renné et al., 2003
	Glutamate synthase	Fd-GOGAT1	At5g04140	Coshigano et al., 1998
	Glutamine synthetase	GS2	At5g35630	Not known at present
	Catalase	CAT2	At4g35090	Queval et al., 2007

Core enzymes of the photorespiratory cycle Phosphoglycolate phosphatase (PGLP)

Table 2 . Classification of photorespiration mutants from Arabidopsis thaliana (Adapted from Timm and Bauwe, 2013) enzyme mutant subclass CO 2 recovery Reference of mutants in Arabidopsis thaliana

 2

	GDC-P	gldp1×gldp2	Class I	lethal	Engel et al., 2007
	GDC-T	gldt1-2	Class I	lethal	Timm et al., 2018
	SHMT1 and SHMT2	shm1-2xshm2-2	Class I	lethal	Engel et al., 2011
	PGLP1	pglp1	Class II	2%	Scharwtze et Bauwe, 2007
	SHMT1	shm1-1	Class II	0.3%	Somerville and Ogren, 1981
	SGAT1	sgat1	Class II	1%	Somerville et Ogren, 1980a
	GLYK1	glyk1	Class II	0.15-0.2%	Boldt et al., 2005
	HPR1 and HPR2	hpr1xhpr2	Class II	0.3-0.4%	Timm et al., 2011
	HPR1 and HPR2 and HPR3	hpr1xhpr2xhpr3	Class II	0.3-0.4%	Timm et al., 2011
	GOX1 and GOX2	amiRgox1/2	Class II	0.3%	Dellero et al., 2016a
	DiT2-1	dct1	Class II	1%	Renné et al., 2003
	Fd-GOGAT1	gls	Class II	1%	Somerville and Ogren, 1980b
	PLGG1	plgg1	Class II	0.3%	Pick et al., 2013
	GGT1	ggt1	Class III	0.3 %	Igarashi et al., 2003 Dellero et al., 2015a
	HPR1	hpr1	Class III	0.15-0.2%	Timm et al., 2011
	CAT2	cat2	Class III	0.3-0.4%	Queval et al., 2007
	BOU	bou-2	Class III	0.3%	Eisenhut et al., 2013
	BASS6	bass6	Class III	0.3%	South et al., 2017
	GOX	gox1	Class IV	0.038%	Rojas et al., 2012 Dellero et al., 2016a
	GOX	gox2	Class IV	0.038%	Rojas et al., 2012 Dellero et al., 2016a
	GOX	gox3	Class IV	0.038%	Rojas et al., 2012
	GDC-P	gldp1	Class IV	0.038%	Engel et al., 2007
	GDC-P	gldp2	Class IV	0.038%	Engel et al., 2007
	HPR2	hpr2	Class IV	0.038%	Timm et al.,2011
	HPR3	hpr3	Class IV	0.038%	Timm et al.,2011
	SHMT2	shm2	Class IV	0.038%	Engel et al., 2011
	DiT1	omt1	Class IV	0.038%	Taniguchi et al., 2002

Class II: Lethal or very stunted in ambient air but rescued under high CO 2

	This class contains the mutants that have a rather classic "photorespiratory phenotype". These
	mutants are lethal or very stunted in ambient air but they grow normally under high CO 2
	concentrations, although the CO 2 concentration required to rescue the plants can differ
	according to the respective mutant (Timm and Bauwe, 2013). In addition, the mutants in this
	class show a very high accumulation of the respective substrates and a very large reduction of
	photosynthetic CO 2 assimilation rate after transfer from the elevated CO 2 condition to
	ambient air. This class includes the following mutants: phosphoglycolate phosphatase 1
	(pglp1; Scharwtze et Bauwe, 2007), serine hydroxymethyltransferase 1 (shm1; Somerville
	and Ogren, 1981), serine:glyoxylate aminotransferase1 (sgat1; Somerville et Ogren, 1980b),
	glycerate kinase 1 (glyk1; Boldt et al., 2005), double and triple hydroxypyruvate reductase
	lines (hpr1xhpr2 and hpr1xhpr2xhpr3; Timm et al., 2011), glycolate oxidase double knock-
	down line (amiRgox1/2;Dellero et al., 2016a), plastidial glutamate-malate transporter 1 (dct1;
	Renné et al., 2003), ferredoxin-dependent glutamate synthase 1 (gls; Somerville and Ogren,
	1980b), plastidial glycolate-glycerate transporter 1 (plgg1; Pick et al., 2013), mitochondrial
	transporter A BOUT DE SOUFFLE (BOU) (bou-2; Eisenhut et al., 2013). Remarkably,
	among the class II, phosphoglycolate phosphatase1 (pglp1; Scharwtze et Bauwe, 2007)
	knockout mutants are lethal in normal air, they even show only a partial recovery under 1%
	CO 2 and for a completely recovery, the mutants need to grow under 2% CO 2 condition
	(Scharwtze et Bauwe, 2007). Three mutants including the serine:glyoxylate aminotransferase1
	(sagt1; Somerville et Ogren, 1980b), ferredoxin-dependent glutamate synthase1 (glu;
	Coschigano, 1998), plastidial glutamate-malate transporter1 (dct1; Renné et al., 2003) are
	lethal in normal air, and could completely recover with 1% CO 2 concentration. The glycerate
	kinase 1 mutant (glyk1; Boldt et al., 2005) exhibited a classic photorespiratory phenotype
	thatcan be totally recovered under high-CO 2 concentration however, after one week of
	transfer from high-CO 2 to ambient air, the mutants were completely bleached (Timm et al.,
	2012) (Figure

Table 4 . SHMTs in Arabidopsis thaliana.

 4

	SHMT enzymes and gene localization	Enzyme localization	Molecular mass	Function	First references in Arabidopsis thaliana
	AtSHMT1 At4g37930	mitochondria	57.4	SHMT activity	Somerville and Ogren, 1981
	AtSHMT2 At5g26780	mitochondria	59.1	SHMT activity	Engel et al., 2011
	AtSHMT3 At4g32520	chloroplast	58.0	SHMT activity	Zhang et al., 2010
	AtSHMT4 At4g13930	cytosol	51.7	Not known at present	Zhang et al., 2010
	AtSHMT5 At4g13890	cytosol	52.2	Not known at present	Zhang et al., 2010
	AtSHMT6 At1g22020	nucleus	66.6	Not known at present	Zhang et al., 2010
	AtSHMT7 (MSA1) At1g36370	nucleus	66.3	Participate to SAM activity (More Sulphur Accumulation)	Huang et al., 2016

Table 5 . Phenotypes of Arabidopsis SHMT mutants.

 5

	SHM1 Enzymes	Mutants	Recovery	Phenotypic feature
	AtSHMT1 (At4g37930)	shm1-1 EMS mutation	0.3 % CO 2	Lethal in ambient air condition (Somerville and Ogren,1981)
	AtSHMT1 (At4g37930)	shm1-2 T-DNA insertion mutation (SALK_083735)	0.3 % CO 2	Lethal in ambient air condition (Moreno et al., 2005 ; Voll et al., 2006)
	AtSHMT1 (At4g37930)	shm1-3 EMS mutation	0.3 % CO 2	Exhibit chlorotic lesions but are viable in ambient conditions(Moreno et al., 2005)
	AtSHMT2 (At5g26780)	shm2-1 T-DNA insertion mutation (SALK _095881)	0.04% CO 2	no visible impairment in ambient air condition (Voll et al., 2006)
	AtSHMT2 (At5g26780)	shm2-2 T-DNA insertion mutation (SALK_096265)	0.04% CO 2	no visible impairment in ambient air condition (Engel et al., 2011)
		shm1-2 x shm2-2	1 % CO 2 +1 % Suc	Lethal in ambient air condition (Engel et al., 2011)

Table 6 . Phenotypic features of Arabidopsis HPR mutants (Timm et al., 2008, 2011).

 6 . The deletion of either HPR1, or HPR2 or HPR3 enzyme led to the reduction of extractable leaf HPR activity. Compared with WT plants, only 6% NADH/NADPH-

	dependent hydroxypyruvate activity was found in hpr1 mutants, and only 2%
	NADH/NADPH-dependent hydroxypyruvate activity remained in the hpr1xhpr2 double
	mutant.

compared with hpr1 Produces flowers but no seeds in ambient air hpr1xhpr3 0.15-0.2% Growth reduction as with hpr1 alone hpr2xhpr3 0.04% NO hpr1xhpr2xhpr3 0.3% Heavily stunted in growth, rarely flower

Table 7 . Phospho-peptides identified on the photorespiratory enzymes of Arabidopsis thaliana.

 7

			LIEGVPE(pT)LDMLR	96-108/362	Cauline leaf, 2 months old	Aryal et al. (2012)
	phosphoglycolat e phosphatase	PGLP1 At5g36700	TLLVLSGV(pT)SISMLESPENK ISDFL(pS)PK	322-341/362 351-358/362	Cauline leaf, 2 months old Rosette, 22 days old	Aryal et al. (2012) Reiland et al. (2009)b
			(pT)LLVLSGVTSISMLESPK IQPDFYTSK	322-341/362	Cell culture (Nitrogen starvation)	PhosPhAt
			MEI(pT)NVTEYDAIAK	1-14/367	Leaves (oxygen depletion)	PhosPhAt
			AIAL(pT)VDTPRL	151-161/367	Rosette	Choudhary et al. (2015)
		GOX1			Rosette (irradiation treatment)	Roitinger et al. (2015)
		At3g14420	AIALTVD(pT)PRL	151-161/367	Seedling (ABA and dehydration treatment)	Umezawa et al. (2013)
					Rosette	Choudhary et al. (2015)
	glycolate		NHITTEWD(pT)PR	347-357/367	Rosette	Choudhary et al. (2015)
	oxidase		MEI(pT)NVTEYDAIAK	1-14/367	Leaves (oxygen depletion)	PhosPhAt
					Rosette (irradiation treatment)	Roitinger et al. (2015)
		GOX2	AIALTVD(pT)PRL	151-161/367	Seedling (ABA and dehydration treatment)	Umezawa et al. (2013)
		At3g14415			Rosette	Choudhary et al. (2015)
			NHITTEWD(pT)PR	347-357/367	Rosette	Choudhary et al. (2015)
			TL(pS)WK	210-214/367	Seedling (ABA and dehydration treatment)	Umezawa et al. (2013)
			AL(pS)LPTGLGIVCASPK	202-217/401	Cauline leaf, 2 months old	Aryal et al. (2012)
		SGAT	ALSLPTGLGIVCA(pS)PK	202-217/401	Rosette, 22 days old	Reiland et al. (2009)b
		At2g13360			Rosette (irradiation treatment)	Roitinger et al. (2015)
			NNEDYR(pS)PAIPALTK	30-45/401
	aminotransferas				flowering plants for 3 weeks	Yang et al. (2013)
	es		ALD(pY)DTLNENVK	5-16/481	Cell culture (Nitrogen starvation)	PhosPhAt
		GGAT1	KVLMEMG(pS)PFSK	268-279/481
		At1g23310	VLEMG(pS)PFSK	269-279/481	Rosette (irradiation treatment)	Roitinger et al. (2015)
			LP(pT)GALQAAK	397-406/481
			EY(pT)KFLEEEDAAH	153-165/165	Cauline leaf, 2 months old	Aryal et al. (2012)
					Cauline leaf, 2 months old	Aryal et al. (2012)
			VKP(pS)SPAELESLMGPK	137-152/165	Seedling (ABA and dehydration treatment)	Umezawa et al. (2013)
		GDH1 At2g35370			Seedlings (ABA treatment)	Wang et al. (2013)
					Seedling (ABA and dehydration treatment)	Umezawa et al. (2013)
			VKPS (pS) PAELESLMGPK	137-152/165	Rosette	Choudhary et al. (2015)
					Seedlings (ABA treatment)	Wang et al. (2013)
		GDH2 At2g35120	GF(s)(s)VVLKDLK-	23-33/156	Seedling Isoxaben-treated	PhosPhAt
			EY(pT)KFLEEEDAAH	154-166/166	Cauline leaf, 2 months old	Aryal et al. (2012)
	glycine		(s)HLLPAF(s)I(s)R	32-42/166	Rosette, 25 days old	Reiland et al. (2009)b
	decarboxylase complex	GDH3 At1g32470	VKP(pS)SPAELEALMGPK	138-153/166	Seedlings (ABA treatment)	Wang et al., 2013
					Seedling (ABA and dehydration treatment)	Umezawa et al. (2013)
			VKPS(pS)PAELEALMGPK	138-153/166	Seedlings (ABA treatment	Wang et al. (2013)
			R(pY)IHKLQSK	556-564/1037	Seed, 7 days after flowering	Meyer et al. (2012)
		GPD1 At4G33010	ADGFDLKVVT(pS) DLK	257-270/1037	Cell culture Nitrogen starvation/Nitrate resupply Cell culture Nitrogen starvation/KCl supply	PhosPhAt PhosPhAt
			S(pS)KFWPTTGR	1001-1010/1037	Rosette (irradiation treatment)	Roitinger et al. (2015)
		LPD1 At1g48030	TPFT(pS)GLDLEK	315-325/507

Table 1 . V max and K m values of recombinant wild-type and phosphorylation site mutated HPR1 in the presence of NADH and hydroxypyruvate

 1

	and threonine 335 (Aryal

. Values shown are mean values of Vmax and Km ± SD from three independent recombinant protein purifications. Statistical significance was determined by a Student's t-test (*p ≤ 0.05, ** p ≤ 0.01).

Table 2 .

 2 Primers for PCR and cloning.

REFERENCE

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my supervisor, Dr. Mathieu Jossier, for his patience, continuous support and encouragement during my PhD. I greatly appreciate not only

ACKNOWLEDGEMENTS

Deep appreciation is also given to all the colleagues at IPS2 for their help in technology. I would like to thank the technical services of the institute, especially the help from Jean-Paul Bares and Holger Ornstrup, which allowed me to carry out the experiments in good conditions. I would like to thank Andreas Weber (University of Dusseldorf, Germany) and Stefan Timm (University of Rostock, Germany) for kindly providing us the shm1-1 and hpr1-1 mutants, the anti-SHMT1 and antiHPR1 antibodies and the pBAD-HPR1 plasmid, all of which are so important for finishing my project.

I am so lucky to have many friends in the IPS2, especially for the friendship from Younes

ACKNOWLEDGEMENTS

This work is supported by a public grant overseen by the French National Research Agency (ANR) as part of the « Investissement d'Avenir» program, through the "Lidex-3P" project and a French State grant (ANR-10-LABX-0040-SPS) funded by the IDEX Paris-Saclay, ANR-11-IDEX-0003-02. This work was also supported by REGUL3P ANR-14-CE19-0015 awarded to MH. YL was supported by a PhD grant from the China Scholarship Council. We would like to thank Françoise Gilard, Florence Guerard, Marlène Lamothe-Sibold of the Institute of Plant Sciences Paris-Saclay Métabolisme-Métabolome facility, analyses and for their help with the GC-TOF-MS and HPLC analyses. We thank Stefan Timm and Andreas Weber for kindly providing us the shm1-1 mutant and anti-SHM antibodies. We thank Sophie Filleur and Julie Hodin for helping us in the stomata aperture experiments. The present work has benefited from the light microscopy facility of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02). We thank Marlène Davanture and Michel Zivy from Plateforme PAPPSO, (http://pappso.inra.fr) for their help in N-teminal sequencing of SHMT1.

ABSTRACT

The photorespiratory cycle is a crucial pathway in photosynthetic organisms since it removes toxic 2-phosphoglycolate made by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and retrieves its carbon as 3-phosphoglycerate. Mitochondrial serine hydroxymethyltransferase 1 (SHMT1) is an essential photorespiratory enzyme converting glycine to serine. SHMT1 regulation remains poorly understood although it could involve the phosphorylation of serine 31. Here we report the complementation of Arabidopsis shm1-1 by SHMT1 wild-type, phosphorylation mimetic (S31D) or non-phophorylatable (S31A) forms.

All SHMT1 forms could almost fully complement the photorespiratory growth phenotype of shm1-1, however each transgenic line only had 50% of normal SHMT activity. In response to either a salt or drought stress, Compl-S31D lines showed a more severe growth deficiency compared to the other transgenic lines. This sensitivity to salt appeared to reflect reduced SHMT1-S31D protein amounts and a lower activity that impacted leaf metabolism leading to proline under-accumulation and over-accumulation of polyamines. The S31D mutation in SHMT1 also led to a reduction in salt-induced and ABA-induced stomatal closure. Taken together, our results highlight the importance of maintaining photorespiratory SHMT1 activity in salt and drought stress conditions and indicate that SHMT1 S31 phosphorylation could be involved in modulating SHMT1 protein stability.

Seed germination ABA-sensitivity of the SHMT1 transgenic lines

Such observations convinced us to test whether our different SHMT1 lines exhibited altered germination properties and whether, as with stomata, the phospho-mimetic line was less sensitive to an ABA-induced inhibition of germination. Seeds of the different complemented lines (Compl-SWT-5/1, Compl-S31D-5/4 and Compl-S31A-7/6) were sown on ½ MS medium plates containing or not 5µM ABA and the germination rate was determined. When germinated on ½ MS medium lacking ABA, all lines showed similar germination rates.

However, with 5µM ABA, the germination of all lines was inhibited, but the inhibition in Compl-SWT1/5 and Compl-S31A6/7 lines was greater than Compl-S31D4/5 lines (Figure 2a).

Perhaps, the Compl-S31A6/7 lines were slightly less inhibited than Compl-SWT1/5 lines. The major difference, observed after 3 days, concerned the Compl-S31D4/5 lines that presented a germination rate twice that of the Compl-SWT1/5 lines (Figure 2a). Moreover, after two weeks on ½ MS 5µM ABA, the Compl-S31D4/5 lines plants had reached the stage of two cotyledons while this was not the case for the Compl-S31A6/7 and Compl-SWT1/5 lines (Figure 2b). These observations were consistent with the ABA-induced stomatal closure assays (Figure 7 in Chapter 1) indicating that Compl-S31D5 was less sensitive to ABA.

CONCLUSIONS & PERSPECTIVES

The experiments described above were carried out to determine whether the lower SHMT1-S31D protein levels in response to a salt stress could be attributed to a modified interaction with UBP16 and thus to an increased susceptibility to 26S proteasome degradation.

Unfortunately, in our hands, the yeast two-hybrid assay was negative since UBP16 was not found to interact with SHMT1, even though we used the same vectors/constructs and the same experimental methods described in Zhou et al (2012). However, in our cell-free assay, SHMT1-S31D appeared to exhibit significant 26S proteasome-dependent degradation when compared to SHMT1 from the other transgenic lines, and this might reflect the lower SHMT1-S31D protein levels in salt stressed Compl-S31D lines. However, the ubiquitinproteasome system (UPS) is a major cytosolic protein degradation system providing both quality control and turnover regulation of numerous cellular proteins involved in essential cellular processes [START_REF] Li | A divide-conquer quasi-physical and quasihuman algorithm for the circle and rectangle orthogonal packing problem with equilibrium constraints[END_REF][START_REF] Bragoszewski | Control of mitochondrial biogenesis and function by the ubiquitin -Proteasome system[END_REF]. Therefore, how can mitochondrial SHMT1 be degraded by this mechanism? Although the cytosolic UPS system and internal mitochondrial proteins are separated, recent evidence has suggested that UPS can be involved in mitochondrial proteins degradation. It is possible that mitochondrial proteins are ubiquitin-conjugated during shuttling to the mitochondria or following retro-translocation to the intermembrane space or outer membrane of mitochondria [START_REF] Bragoszewski | Control of mitochondrial biogenesis and function by the ubiquitin -Proteasome system[END_REF].

For example, the mitochondrial uncoupling protein 2 (UCP2) [START_REF] Azzu | Degradation of an intramitochondrial protein by the cytosolic proteasome[END_REF] and uncoupling protein 3 (UCP3) [START_REF] Mookerjee | Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria[END_REF] were shown to undergo 26Sproteasome-dependent degradation following retro-translocation, but whether the ubiquitination occurred in the mitochondria remains unclear. Moreover [START_REF] Bragoszewski | The Ubiquitin-Proteasome System Regulates Mitochondrial Intermembrane Space Proteins[END_REF] reported that some mitochondrial intermembrane space proteins were ubiquitinated prior to their importation into mitochondria and a recent study demonstrated that ubiquitinated proteins exist in the mitochondrial matrix (Lehmann et al., 2016a;Lehmann et al., 2016b).

To continue to study the mechanisms involved in regulating SHMT1-S31D levels, it will be necessary to:

 Carry out cell-free degradation assays with total soluble proteins extracted from saltstressed plants to determine whether 26S proteasome degradation is involved in the observed reduction of SHMT-S31D protein levels.

 Carry out bimolecular fluorescence complementation (BiFC) analyses in Arabidopsis protoplast as an alternative method to analyze the interaction between UBP16 and different versions of SHMT1.

CHAPTER III

Phospho-mimetic HPR1-T335D alters HPR1 enzymatic kinetic parameters in vitro and is unable to fully complement the photorespiratory growth phenotype of hpr1 in short-day air-grown conditions

RESULTS

Recombinant phosphorylation-mimetic HPR1 T335D exhibits altered kinetic parameters

In order to increase our knowledge of the phosphoregulation of the photorespiratory cycle by peroxisomal HPR1, it was decided to evaluate the effect of S229 and T335 phosphorylationmimetic mutations on the kinetics parameters of purified recombinant HPR1. To do this, both phosphorylation sites S229 and T335 were altered by site-directed mutagenesis using pBAD-HPR1 (that was kindly provided by H. Bauwe, University de Rostock, Germany) to generate pBAD-HPR1-S229A / D and pBAD-HPR1-T335A / D (Figure 1a and1b) that were used to produce his-tagged phosphorylation-mimetic (S229D and T335D) HPR1 and nonphosphorylatable (S229A and T335A) HPR1 in E.coli (Figure 1c). The original pBAD-HPR1 was used to produce control, WT HPR1 recombinant protein (Figure 1c).

The in vitro activities of the recombinant HPR1 proteins were measured by spectrophotometry following the change in absorbance at 340 nm due to the oxidation of either NADH or NADPH. Initial analyses of NADH-dependent HPR1 activity showed that in the presence of hydroxypyruvate (HP) and NADH, the V max of recombinant HPR1-S229A and HPR1-T335D proteins were reduced, by 25% and 50%, respectively, when compared to the V max of recombinant wild-type HPR1 (Table 1). On the other hand, the V max of recombinant HPR1-T335A and HPR1-S229D were not significantly different to wild-type HPR1. In comparison with V max , the K m for hydroxypyruvate (of between 149 and 183 M) was not significantly affected by the different phosphorylation-site mutations (Table 1). These results suggested that T335 phosphorylation in planta could modulate HPR1 activity while S229 phosphorylation did not appear to alter HPR1 kinetic properties.

Short day grown HPR1-T335D complemented plants show reduced growth in ambient air

To evaluate the impact of phosphorylation-mimetic HPR1 and non-phosphorylatable HPR1 on plant development, hpr1 was transformed with constructs to express the different mutated forms of AtHPR1. To achieve this, the WT and mutated phosphorylation site HPR1 cDNAs under the control of an SHMT1 promoter region (Voll et al., 2006) were used to carry out agrobacterium-mediated transformation of hpr1 by floral dip (Clough and Bent, 1998). The different constructs used (and their resulting HPR1 proteins) are as follows: Compl-hpr1-pSHMT1:HPR1-S229D (HPR1-S229D), Compl-hpr1-pSHMT1:HPR1-T335D (HPR1-T335D),

Compl-hpr1-pSHMT1:HPR1-S229A (HPR1-S229A), Compl-hpr1-pSHMT1:HPR1-T335A (HPR1-T335A) and Compl-hpr1-pSHMT1:HPR1 (HPR1) (see Figure 3a). Homozygous lines expressing the different mutated versions of HPR1 protein were selected for analysis.

Western-blot analyses showed that HPR1 protein was successfully produced in all hpr1 complemented lines although at various levels (Figure 3b). All constructs complemented the photorespiratory growth phenotype of hpr1 in our short day ambient air conditions (Figure 3c and 3d). Compl-HPR1-S229A and Compl-HPR1-S229D lines exhibited no differences in growth compared to Col-0 and WT-HPR1-transformed lines (Figure 3c). On the other hand, Compl-HPR1-T335D lines displayed an obvious reduced growth compared with Compl-HPR1-S335A and Compl-HPR1 lines (Figure 3d). This suggested that the in planta HPR1 activity was reduced in a similar manner to that observed in vitro with recombinant HPR1-T335D protein (Table 1), The differences in plant growth between the different plant lines was confirmed by the analysis of rosette fresh and dry weights (Figure 4a and4b). In conclusion, Compl-HPR1-T335D plants had a reduced (about 20%) growth when compared to all other lines while their relative water contents were unchanged (Figure 4c).

Taken together, our limited data suggest that phosphorylation of Thr335 could lead to an inhibition of HPR1 activity in planta and this becomes limiting for normal plant growth in standard short-day air conditions. With respect to S229 phosphorylation, our data suggest that this modification does not affect HPR1 activity and therefore it must have another effect.

DISCUSSION

Protein phosphorylation is a major regulatory mechanism, and phosphoproteomic studies have shown that photorespiratory HPR1 can be phosphorylated at two sites [START_REF] Aryal | Identification of phosphoproteins in arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metalion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry[END_REF][START_REF] Umezawa | Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in arabidopsis thaliana[END_REF][START_REF] Roitinger | Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana[END_REF]. In this study we began to determine the role of HPR1 phosphorylation at Ser229 and Thr335 by analyzing the enzymatic activities of phosphorylation-mimetic recombinant proteins and their capacity to complement the photorespiratory growth phenotype of hpr1. Since different HPR isoforms appear to be able to use differentially two substrates (hydroxypyruvate and glyoxylate) and two cofactors (NADH and NADPH) (see [START_REF] Kleczkowski | Purification and characterization of a novel NADPH ( NADH ) -dependent glyoxylate reductase from spinach leaves[END_REF][START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF][START_REF] Timm | The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end[END_REF], different substrate/cofactor combinations were also tested for the Thr335 phosphorylation site since only this phospho-mimetic HPR1 form exhibited an inhibition of NADH-dependent HPR activity (Table 1). Indeed, our initial activity assays in the presence of hydroxypyruvate and NADH revealed that the Vmax of HPR1-S229A was inhibited by 25 % compared to wild-type HPR1 (Table 1) while the activity of HPR1-S229D remained unaltered. For the Km for hydroxypyruvate, no differences were detected between all recombinant proteins (Table 1). A structural model, based on the crystallized Pyrococcus HPR1 protein, suggested that neither phosphorylation site had the potential to alter HPR1 dimerization (Figure 5d), however Ser229 was located within the NAD(P)H-binding domain (Figure 5) [START_REF] Yoshikawa | Structure of archaeal glyoxylate reductase from Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate[END_REF] (http://www.ebi.ac.uk) (http://zhanglab.ccmb.med.umich.edu/) [START_REF] Yang | I-TASSER server: New development for protein structure and function predictions[END_REF].

Therefore, phosphorylation of a residue in this domain could affect enzymatic activity by altering NADH binding however, this was not observed for phospho-mimetic S229D HPR1. Surprisingly, the non-phosphorylatable S229A HPR1 did exhibit a reduced maximal activity, thus indicating that Ser229 has a certain importance with respect to enzymatic activity. On the other hand, the HPR1-T335D recombinant protein showed a 50% inhibition of NADHdependent HRP1 activity compared to the activity of both wild-type HPR1 and HPR1-T335A. Indeed, the activities of HPR1-T335A and wild-type HPR1 were very similar (Table 1).

Based on the structural model, Thr335 is located in the substrate-binding domain of HPR1 (Figure 5) however the Km for hydroxypyruvate was not modified in the phospho-mimetic protein (Table 1). Therefore, it could be hypothesized that the loss of activity was due to a structural change that affected interactions between the two domains that somehow altered the co-factor domain. Indeed, it was found that HPR1-T335D had a higher activity in the

MATERIALS AND METHODS

Plants material and growth conditions

Arabidopsis thaliana Columbia (Col-0) was used as wild-type control plants in all experiments. The hpr1-1 mutant [START_REF] Timm | A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[END_REF] was kindly provided by Professor Herman Bauwe (University of Rostock, Plant Physiology Department, Germany).

Arabidopsis seeds were germinated on potting soil for 10 days in short-day growth conditions (8h light / 16h dark, 200 μmol of photons.m -2 .s -1 , 65% humidity, 20°C day / 18°C night).

Subsequently, the seedlings were transplanted to individual pots and continued to grow.

For the fresh weight and dry weight, rosettes of 4-week-old individual plants were detached and weighed to give the fresh weight, then they were dryed at 65°C for 48 h in a dessicator MMM Medcenter™ (MC001812) and weighed to give the dry weight.

Plasmid constructions, site-directed mutagenesis, and generation of complemented lines

To produce recombinant proteins, the pBAD-AtHPR1 plasmid (kindly provided by Professor Herman Bauwe of the University of Rostock, Plant Physiology Department, Germany) was used as a template to introduce S229A, S229D, T335A and T335D point mutations using primers pairs (Table 2) using the QuikChange® II XL Site-Directed Mutagenesis Kit (Agilent®), according to the manufacturer's instructions. In this way, pBAD-AtHPR1-S229A, pBAD-AtHPR1-S229D, pBAD-AtHPR1-T335A and pBAD-AtHPR1-T335D were obtained. All constructions were subsequently verified by DNA sequencing using the primers pBAD-seq-F and pBAD-seq-R (see Table 2).

To generate hpr1 complemented lines, pBAD-AtHPR1, pBAD-AtHPR1-S229A, pBAD-AtHPR1-S229D, pBAD-AtHPR1-T335A and pBAD-AtHPR1-T335D were used as templates for two consecutive PCR reactions (using primers GW-HPR1-F / GW-HPR1-R then GateWay-F/GateWay-R, see Table 2) to add attB Gateway® recombination sites to the 5' and 3' ends, respectively. The resulting DNAs were further cloned into pDONR207 using the Gateway® cloning system to generate pDONR207-AtHPR1, pDONR207-AtHPR1-S229A, pDONR207-AtHPR1-229D, pDONR207-AtHPR1-T335A and pDONR207-AtHPR1-T335D. After sequencing, the different constructs were sub-cloned into pK7WG-promSHMT1 (Dellero et al., 2016) using the Gateway® cloning system to generate pH7WG-promSHMT1::HPR1, Keywords : Photorespiration, Phosphorylation, SHMT1, HPR1, Metabolism, Abiotic stress Abstract Photorespiration is an essential process in oxygenic photosynthetic organisms, that is triggered by the oxygenase activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) leading to the production of 3-phosphoglycerate and 2phosphoglycolate (2PG). Toxic 2PG is recycled by the photorespiratory cycle which involves eight core enzymes and takes place in chloroplasts, peroxisomes, mitochondria and the cytosol. Photorespiration leads to a reduction of photosynthetic CO 2 assimilation and the photorespiratory cycle has a high energetic cost, so it has been considered as a wasteful process. However, the severe growth phenotypes of photorespiratory mutants reflect the importance of this process in air-grown conditions.

Current data indicate that seven of the eight core photorespiratory enzymes can be phosphorylated, thus suggesting that phosphorylation could be a critical regulatory component of the photorespiratory cycle. In order to understand the regulation of the photorespiratory cycle, we explored the role of mitochondrial serine hydroxymethyltransferase 1 (SHMT1) and peroxisomal hydroxypyruvate reductase 1 (HPR1) phosphorylation on enzymatic activities, plant metabolism and abiotic stress resistance. This was achieved by producing phospho-mimetic (serine/threonine mutated to aspartate) or non-phophorylatable (serine/threonine mutated to alanine) recombinant enzymes either in E. coli or in relevant Arabidopsis thaliana mutants (shm1-1 and hpr1).