Keywords: 15 N labelling, Nitrogen Use Efficiency, nutrient recycling, resource allocation, sink-source, yield ATG1a

Autophagy knock-out mutants in maize and in Arabidopsis are impaired for nitrogen recycling and exhibit reduced levels of nitrogen remobilization to their seeds. An important question is then to determine whether higher autophagy activity could at the reverse improve N remobilization efficiency and seed protein content, and under which circumstances.

As autophagy machinery involves many genes amongst which 18 are important for the core machinery, the choice of which ATG gene to manipulate to increase autophagy was examined. We choose ATG8 overexpressions since it has been shown in yeast that it could increase autophagosome size and autophagic activity. The results we report here are original as they show for the first time that increasing ATG8 gene expression in plant increases autophagosome number and promotes autophagy activity. More importantly our data demonstrate that, when cultivated under full nitrate conditions, known to repress N remobilization due to sufficient N uptake from the soil, N remobilization efficiency can be nevertheless sharply and significantly increased by overexpressing ATG8 genomic sequences under the control of ubiquitin promoter. We show that overexpressors have improved seed N% and at the same time reduced N waste in their dry remains. In addition, we show that overexpressing ATG8 does not modify vegetative biomass nor harvest index, thus does not affect plant development.

Leaf senescence is the final stage of leaf development. Leaf senescence is not a passive degeneration but an actively genetically controlled process. It involves highly coordinated cellular-, tissue-, organ-, and organism-level actions that trigger the recycling of energy and nutrients to other parts of plants like the developing seeds or storage organs (Figure 1) [START_REF] Buchanan-Wollaston | The molecular biology of leaf senescence[END_REF][START_REF] Lim | Leaf Senescence[END_REF]. The syndrome of leaf senescence involves morphological, physiological and molecular changes that include a number of distinguishing features such as the loss of the green color due to the dismantling of chloroplasts, membrane ion leakage, decline of photosystem II (PSII) photochemical efficiency, degradation of RNA, DNA and proteins, that ultimately lead to the death of the senescing leaves (Lohman et al., 1994;[START_REF] Bleecker | Last exit: Senescence, abscission, and meristem arrest in Arabidopsis[END_REF][START_REF] Woo | ORE9, an F-box protein that regulates leaf senescence in Arabidopsis[END_REF]Ougham et al., 2008). Proper initiation timing and progression rate of leaf senescence are critical for crop yield, biomass production, fruit ripening and postharvest storage [START_REF] Lim | Leaf Senescence[END_REF][START_REF] Wu | Regulation of Leaf Senescence and Crop Genetic Improvement[END_REF].

Transcriptomic studies performed during leaf senescence led to the identification of (i) many senescence-induced genes like the SAGs (Senescence-Associated Genes) amongst which SAG12, SAG13, SAG21 are well known, and the SEN (SENescence) genes and of (ii)

Senescence-Repressed Genes (SRG) that are for example involved in protein synthesis and photosynthesis like the CHLOROPHYLL A/B BINDING PROTEIN (CAB) and the RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN (RBCS) (Lohman et al., 1994;Park et al., 1998;[START_REF] Weaver | A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment[END_REF]Miller et al., 1999;[START_REF] Van Der Graaff | Transcription analysis of arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence[END_REF].

Leaf senescence program is controlled by both environmental and endogenous factors through different regulatory pathways. The different pathways activate distinct subsets of SAGs. Some of these SAGs are shared by several pathways, while others are unique to specific pathways. For example, the Arabidopsis SAG12 gene encoding a cysteine protease, is induced during senescence in an age-specific manner, and is not controlled by any other endogenous or environmental signals. SAG12 has thus been widely used as a developmental senescence marker gene in leaf senescence research. Arabidopsis. After leaf senescence is initiated, nutrients such as nitrogen, phosphorus, and metals are remobilized to younger plant parts such as developing seeds and leaves (c). Figure

from [START_REF] Lim | Leaf Senescence[END_REF].

Age/development-dependent senescence

As an integral part of plant development, the timing of leaf senescence is mainly governed by the developmental age of the leaf. When the plant is grown in optimal conditions with sufficient water and nutrition supply, free of various biotic and abiotic stresses, developmental leaf senescence onset theoretically only depends on leaf age. This supposes that there should be some kind of cellular mechanism to monitor the age of a cell, tissue, organ or the whole plant to initiate leaf senescence and control the progression after a certain developmental stage. However, there is very limited report regarding the genes that alter leaf senescence on the basis of developmental ageing. This may be because leaf senescence is a complex process that integrates the effects of various endogenous and external factors that participate to the developmental age-dependent senescence pathways, and to multiple regulatory pathways that respond to different factors that are possibly interconnected to each other to form regulatory networks [START_REF] Lim | Leaf Senescence[END_REF]. Regulatory factors only responding to leaf age have been investigated. The ore4-1 mutant presenting a T-DNA insertional mutation in the promoter region of the plastid ribosomal small subunit protein 17 (PRPS17) gene in Arabidopsis, exhibited delayed leaf senescence in an age-dependent manner, but had no special phenotype when leaf senescence was artificially induced by darkness or hormones [START_REF] Woo | Extended leaf longevity in the ore4-1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene[END_REF]. The delayed leaf senescence of ore4-1 is possibly due to the partial lesion in chloroplast functions and photosynthesis. The reduced metabolic rate in ore4-1 mutant may explain its earlier developmental senescence.

Stress/phytohormone-induced leaf senescence

Except for the inherent leaf developmental cues, leaf senescence process is also tightly regulated by various internal and external signals through specific pathways (Figure 2). The internal signals include various phytohormones and the occurrence of reproductive events.

The environmental cues that stimulate leaf senescence include a large number of stresses such as nutrient deficiency, extreme temperature, UV-B, high salinity, drought, pathogen infection, and darkness [START_REF] Lim | Leaf Senescence[END_REF].

Gibberellic acid (GA), auxin, and cytokinin were shown to induce delayed leaf senescence.

Abscisic acid (ABA), jasmonic acid (JA), ethylene, and salicylic acid (SA) were by contrast shown as leaf senescence triggering factors (van der [START_REF] Van Der Graaff | Transcription analysis of arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence[END_REF]. For example, based on the senescence-specific characteristics of SAG12, the P SAG12 -IPT autoregulatory senescence inhibition system has been widely explored by fusing the SAG12 promoter to a cytokinins-synthesizing enzyme, isopentenyl transferase (IPT), and successfully used in a variety of plant species including crops for inhibiting senescence [START_REF] Gan | Inhibition of leaf senescence by autoregulated production of cytokinin[END_REF][START_REF] Guo | Translational researches on leaf senescence for enhancing plant productivity and quality[END_REF].

Among the environmental cues, limited water and nutrient availability (especially nitrogen) are major factors that adversely affect the initiation and rate of leaf senescence [START_REF] Gan | Making Sense of Senescence[END_REF]. As the quantitatively most important plant mineral nutrient, nitrogen is very dominant in affecting senescence progress. Leaf senesces more slowly when nitrogen nutrition level is sufficient for plant growth (Ono et al., 2001). At the time of full leaf elongation, the rate of senescence was enhanced by the removal of nitrogen from the growth medium of wheat [START_REF] Crafts-Brandner | Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves[END_REF]. The early senescence phenotype under limited nitrogen condition is usually related to the enhanced breakdown of proteins and recycling of nitrogenous compounds. Multiple pathways that respond to various factors, including the genes involved in cell proliferation and growth, hormone perception and responses, ROS signaling, light signaling, and the circadian clock, are interconnected to form complex regulatory networks. Figure from [START_REF] Kim | New insights into the regulation of leaf senescence in Arabidopsis[END_REF].

Transcription factors controlling leaf senescence

From omics studies performed on senescing leaves and various mutant screens, master regulators of leaf senescence have been found. They mainly belong to the NAC and WRKY families and control each others while they integrate response to various signals (Kim et al. 2017https://doi.org/10.1093/jxb/erx287). Amongst them we can cite ORE1 (ANAC092) which is a positive regulator of the leaf senscence that responds to ethylene (Qiu et al. 2015). ORE1 is positively regulated by ATAF1 (ANAC2) that integrate ABA, dark and sugar signalling [START_REF] Garapati | Transcription factor A Activating Factor1 integrates carbon starvation responses with trehalose metabolism[END_REF]. In addition to stimulating the transcription of ORE1, it was shown that ATAF1 repress the expression of GLK1 that is involved in the chloroplast maintenance, and to induce autophagy genes. The AtNAP (ANAC029) transcription factor homologous of the wheat NAMB1 was shown to control stomata apperture in response to ABA and drought and its NAMB1 homologous gene to control N, Fe and Zn remobilization to the seeds and leaf senescence in wheat (Uay 2006 ;Guo and Gan 2006). The complex regulation network involving all these senescence regulators is likely connected to metabolic features and to the C and N status of the plant. It was for example shown that the NAC domain of ATAF1 interacts with the SNRK1 kinase which is a sensor of sugar and energy in the cell (Kleinow 2009).

Protein degradation during senescence

During senescence, in parallel with the decline of photosynthesis, leaf changes its metabolism from anabolism to catabolism. Given that proteins contain the largest fraction of organic nitrogen, which is potentially available for recycling to support the growth of younger plant parts in stress conditions, the major biochemical event occurring during leaf senescence is protein degradation (Guiboileau et al., 2010). The transcript levels or activity of various proteases are specifically induced in senescing leaves, and cysteine proteases, mainly accumulated in lytic vacuolar compartments, are amongst the most highly overexpressed.

Amongst them are SAG12, Cathepsin B3 (CATHB3), Responsive-to-desiccation 21A (RD21A), Arabidopsis aleurain-like protease (AALP) and Vacuolar Processing Enzymes (VPEs) (Lohman et al., 1994;[START_REF] Kinoshita | Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions[END_REF]Yamada et al., 2001;Guo et al., 2004;[START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]. Most of the leaf protein resource is present in chloroplasts, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) alone represents as much as 50%

of the total soluble leaf proteins [START_REF] Hörtensteiner | Nitrogen metabolism and remobilization during senescence[END_REF]. Therefore, efforts made to understand nitrogen remobilization during leaf senescence have mainly focused on the biochemistry of the degradation of plastidial proteins.

Chloroplasts contain large number of proteases suggesting that the degradation of plastidial proteins (such as Rubisco) might be at least initiated in the intact organelles (Thoenen and Feller, 1998;[START_REF] Hörtensteiner | Nitrogen metabolism and remobilization during senescence[END_REF]. A DNA-binding protease (CND41) identified in the tobacco chloroplast nucleoids, was shown to be involved in Rubisco degradation, as indicated by the retarded leaf senescence and persistent Rubisco protein levels in antisense suppression tobacco lines with a reduced amount of CND41 [START_REF] Kato | The DNA-binding protease, CND41, and the degradation of ribulose-1,5bisphosphate carboxylase/oxygenase in senescent leaves of tobacco[END_REF]. However, although chloroplasts contain a very large fraction of leaf proteins, most of the proteolytic activity in senescing leaf cells is located in other cell compartments and particularly in the lytic vacuole.

Originally observed in the cytoplasm and vacuole of naturally senescing wheat leaf cells by immuno-electron microscopy, Rubisco-containing bodies (RCBs) were proposed to be involved in the Rubisco degradation process outside of chloroplasts [START_REF] Chiba | Exclusion of Ribulose-1,5-bisphosphate Carboxylase/oxygenase from Chloroplasts by Specific Bodies in Naturally Senescing Leaves of Wheat[END_REF].

These RCBs contain the large and small subunit of Rubisco and another stromal protein, the chloroplastic glutamine synthetase, but lack chloroplast envelope or thylakoid components.

RCBs are surrounded by double membranes, which seem to be derived from the chloroplast envelope, and RCBs are further surrounded by other isolation membranes characteristic of the intermediate structures of autophagosomes (phagophores) in the cytoplasm [START_REF] Chiba | Exclusion of Ribulose-1,5-bisphosphate Carboxylase/oxygenase from Chloroplasts by Specific Bodies in Naturally Senescing Leaves of Wheat[END_REF][START_REF] Ishida | Roles of autophagy in chloroplast recycling[END_REF]. RCBs become frequently visible at the early stages of leaf senescence when Rubisco starts to decrease without prior chloroplast destruction nor chlorophyll degradation. It is now established that there are three vesicular pathways involved in chloroplast protein degradation: the Senescence-Associated Vacuoles (SAVs), macro-autophagy, and the Chloroplast Vesiculation (CV) pathway (Figure 3).

Senescence-associated vacuoles

SAVs are small, single membrane acidic proteolytic vacuolar compartments that accumulate in chloroplast-containing cells of senescing leaves [START_REF] Otegui | Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean[END_REF][START_REF] Carrión | In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescenceassociated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves[END_REF].

The cysteine protease SAG12 is localized to SAVs, and the expression of SAG12 coincides with the formation of SAVs. SAVs contain mostly cysteine protease activities, as suggested by the inhibited proteolytic activity in isolated SAVs upon incubation with a cysteine protease inhibitor. Interestingly, it was shown that isolated SAVs only contain chloroplast stromal proteins like Rubisco and glutamine synthetase, but lack the thylakoid-associated proteins [START_REF] Martínez | Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leaves[END_REF]. Although SAVs and RCBs both contain the same type of chloroplast stromal proteins (Rubisco and glutamine synthetase) and as thylakoid proteins are absent from these vesicle, whether they are distinct structures remains to be determined.

Despite the evidence that SAV pathway participates to chloroplast protein breakdown, detailed information regarding the origin of SAVs and how plastid proteins are relocated into the lumen of SAV is still very limited. The formations of SAVs, autophagosome and RCBs are all induced in senescent leaves. However, it was reported that SAVs formation is independent from the canonical autophagy machinery and from the synthesis of SAG12, as the autophagy atg7 and the sag12 Arabidopsis mutants develop SAVs during leaf senescence [START_REF] Otegui | Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean[END_REF]Otegui, 2018). It remains however unknown if SAVs fuse to the central vacuole, or to autophagosomes, and how they release nutrients to the cytosol.

Autophagy-dependent pathways

Efforts have been made toward the understanding of autophagy-dependent chloroplast degradation over the past decade. In the macro-autophagy (literally means 'self-eating' ;

thereafter named autophagy) process, unwanted cytoplasmic components are sequestered by a specialized double-membrane bound vesicle (termed autophagosome, see detailed molecular machinery in section 2) and are delivered to the vacuole for degradation (Thompson and Vierstra, 2005). Autophagy was shown to be involved in the mobilization of RCBs to the vacuole, as evidenced by the co-localization of the GFP-ATG8 autophagosome marker with RCBs labeled by a stroma-targeted DsRed fluorescent protein [START_REF] Ishida | Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process[END_REF][START_REF] Izumi | The autophagic degradation of chloroplasts via rubisco-containing bodies is specifically linked to leaf carbon status but not nitrogen status in Arabidopsis[END_REF]. Upon treatment with concanamycin A to suppress vacuolar lytic activity, the accumulation of RCBs in the vacuoles was disrupted in the autophagy-deficient atg5 and atg4a4b mutants exposed to darkness [START_REF] Ishida | Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process[END_REF][START_REF] Wada | Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves[END_REF]. These results confirm that RCBs are a type of autophagic body that specifically contain Rubisco and other stromal proteins.

It is still not well elucidated how RCBs form. The release of RCBs from the chloroplast seems to occur primarily by scission of stroma-filled tubules (stromules) or projections from plastids, using an Endosomal Sorting Complex Required for Transport (ESCRT)-type trafficking route (Spitzer et al., 2015). The ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN 1A and 1B (CHMP1A and CHMP1B) were found to be required for the efficient autophagy-dependent trafficking of RCBs to the vacuole by promoting the nucleation of phagophores (see below) on RCBs. The Arabidopsis mutant seedlings lacking the CHMP1A and CHMP1B display aberrant stromule morphology, accumulated RCBs in the cytoplasm and decreased degradation of chloroplast proteins.

Moreover, the release of RCBs from plastids depends on the autophagy machinery, as RCBs remain attached to stromules and are not released into the cytoplasm in the Arabidopsis autophagy atg5 and atg7 mutants.

In addition to the targeting of RCBs, the turnover of chloroplast proteins through autophagy pathway might also involve two specific adaptors that are the ATI1 and ATI2 plant-specific proteins ATG8-INTERACTING PROTEIN (ATI). ATI1 and ATI2 are localized at the membrane of a new type of compartments (named ATI1 bodies) that were first found associated with the endoplasmic reticulum (ER) membrane network upon exposure to carbon starvation and described as ER-associated ATI1 containing bodies (Honig et al., 2012). The ATI1 bodies harboring ATI1 protein were further found associated to the plastids and termed ATI1-PS bodies. These ATI1-PS bodies bud off directly from the surface of plastids into the cytoplasm in senescing cells, during carbon starvation or salt stress (Michaeli et al., 2014).

Like RCBs, a functional autophagy system is needed for the delivery of these ATI1-PS bodies to the vacuole, but the size of these bodies is much smaller than that of autophagosomes and their release into the cytoplasm does not depend on the autophagy machinery. In contrast to RCBs that exclusively contain stromal proteins, the ATI1-PS bodies also contain plastid membrane proteins from either the outer envelope of the chloroplasts or the thylakoids. As ATI1 was shown to interact with both plastid-localized proteins and the ATG8f protein, it was concluded that it may act as a specialized selective autophagy cargo receptor (Michaeli et al., 2014).

Entire chloroplast degradation through autophagy is named chlorophagy and corresponds to the encapsulation of entire damaged or abnormal chloroplasts into ATG8-decorated autophagosomes and their subsequent delivery to the vacuole through a route dependent on ATG8 lipidation [START_REF] Izumi | Entire photodamaged chloroplasts are transported to the central vacuole by autophagy[END_REF]. This process can be stimulated by strong photooxidative damage (UV-B or high-intensity visible light exposure) without prior activation of RCB production. The engulfed chloroplasts display a disorganized thylakoid structure and collapsed outer envelopes, and the size of these surrounding tubular shape autophagosome structures is much bigger than that of the autophagosomes observed in control conditions.

The Chloroplast Vesiculation pathway

Intriguingly, Wang and Blumwald (2014) found a third extraplastidic degradation pathway that is SAV-and autophagy-independent, and involves the budding of vesicle-like structures termed CV-containing vesicles (CCVs). CV (chloroplast vesiculation) is a plastid-targeted protein with a putative transmembrane domain. Work in Arabidopsis demonstrated that the expression of CV can be induced by senescence and abiotic stress (especially drought). After targeting to the chloroplast, CVs destabilize the chloroplast, which subsequently activate the formation of CCVs that promote chloroplast degradation and leaf senescence. Conversely, silencing of CV leads to delayed turnover of chloroplasts and senescence induced by abiotic stress. The CCVs bud from chloroplasts, carrying stromal, thylakoid membrane, and envelope membrane proteins, but apparently not plastoglobule components, are then mobilized to the vacuole for proteolysis via a pathway independent of both autophagy and SAVs. (2015).

Put another scheme with the CHMP1 ?

Nitrogen remobilization after protein degradation in senescing leaves

Nitrogen is quantitatively the most important mineral nutrient for plant growth. The use of nitrogen by plants involves several steps, including uptake, assimilation, translocation, recycling and remobilization [START_REF] Masclaux-Daubresse | Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture[END_REF]. Plants are static and cannot escape from the multitude of abiotic and biotic stress conditions occurring during their growth period. To deal with these environmental stresses and survive in the fluctuating environment conditions, plants use leaf senescence to massively remobilize phloem-mobile nutrients and energy from senescing leaves to developing tissues and storage organs. This way, plants can save and efficiently utilize the limited nutrients and energy for defence, growth, and reproduction (Avila-Ospina et al., 2014). Efficient nitrogen remobilization thus increases the competitiveness of plants especially under nitrogen limiting conditions. For agriculture, high nitrogen remobilization efficiency is interesting as it can reduce the need of nitrogen (N) fertilization, which represents a substantial cost of agricultural production and often causes environmental pollution. In crops, post-anthesis nitrogen remobilization during seed maturation is highly correlated to grain yield and quality [START_REF] Kichey | In winter wheat (Triticum aestivum L.), post-anthesis nitrogen uptake and remobilisation to the grain correlates with agronomic traits and nitrogen physiological markers[END_REF][START_REF] Masclaux-Daubresse | Leaf nitrogen remobilisation for plant development and grain filling[END_REF]. In small-grained cereals like wheat and rice, up to 90% of the grain nitrogen content is remobilized from the vegetative plant parts, while the proportion in maize is approximately 35-55% [START_REF] Hirel | The challenge of improving nitrogen use efficiency in crop plants: Towards a more central role for genetic variability and quantitative genetics within integrated approaches[END_REF].

Once senescence is initiated, carbon and nitrogen primary assimilations are replaced by recycling from the catabolism of macromolecules such as proteins and nucleic acids. As mentioned above, up to 75% of the total mesophyll cellular nitrogen is localized in the chloroplasts [START_REF] Hörtensteiner | Nitrogen metabolism and remobilization during senescence[END_REF]. The breakdown and recycling of these considerable nitrogen resources depend on the three distinct chloroplast degradation pathways cited above. Up to now, although detailed knowledge concerning interactions and relationships between these three chloroplast degradation pathways remains insufficient, cysteine proteases localized in the vacuole appear to play a particularly important role in all these processes. Nitrogenous macromolecules are hydrolyzed by vacuolar proteases and converted to amino acids, and amino acid interconversions facilitate transamination to glutamine, asparagine, glutamate, and aspartate that are likely the favorite amino acids used for phloem loading and transport to developing sinks [START_REF] Clément | Metabolomics of laminae and midvein during leaf senescence and source-sink metabolite management in Brassica napus L. leaves[END_REF].

There is evidence that different plant species share common N remobilization mechanisms, and that N remobilization is mainly limited by the availability of the substrate in the vegetative source organs [START_REF] Bertheloot | Dynamics of Light and Nitrogen Distribution during Grain Filling within Wheat Canopy[END_REF][START_REF] Masclaux-Daubresse | Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture[END_REF]. Several key enzymes involved in nitrogen remobilization and specifically induced during leaf senescence have been identified, including the cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aminotransferases (Figure 4) [START_REF] Masclaux-Daubresse | Leaf nitrogen remobilisation for plant development and grain filling[END_REF], 2010). Based on the amino acid pool released from the chloroplast proteins proteolysis, a series of transamination reactions would enrich the glutamate pool, which could serve immediately as a substrate of GDH. Glutamate catabolism through GDH releases ammonia and alpha-ketoglutarate for mitochondria replenishment and energy production [START_REF] Masclaux-Daubresse | Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco[END_REF]Avila-Ospina et al., 2014). The released ammonia is in turn re-assimilated by the different GS1 to produce glutamine for export [START_REF] Clément | Metabolomics of laminae and midvein during leaf senescence and source-sink metabolite management in Brassica napus L. leaves[END_REF]. Besides, using glutamate and glutamine as substrates, aspartate aminotransferase and AS would help to synthesize another important N transport form, asparagine [START_REF] Masclaux-Daubresse | Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco[END_REF][START_REF] Gaufichon | Biological functions of asparagine synthetase in plants[END_REF]. The Arabidopsis genome contains 67 putative amino acid transporter genes, which belong to 11 gene families (Rentsch et al., 2007). However, our current knowledge regarding the biochemical, physiological and molecular mechanisms of transport processes needed for efficient N remobilization is still very limited. It can however be noticed that several amino acid transporters are induced during leaf senescence, which let us suspect they play a role in N translocation from source to sink during leaf senescence (Havé et al., 2017).

Autophagy

Intracellular recycling plays an essential role in the proper control of cellular events, such as modulating the levels of key regulators, and more importantly, as the main housekeeper that removes cellular debris and replenishes essential nutrients to support new growth (Dikic, 2017;Masclaux-Daubresse et al., 2017). The best studied and understood recycling system in plants is the ubiquitin-proteasome pathway in which proteins are ligated with a polyubiquitin chain to serve as effective substrates for cleavage by the 26S proteasome [START_REF] Hershko | THE UBIQUITIN SYSTEM[END_REF]. However, the selective degradation of this system is limited to some individual damaged or short-lived (regulatory) proteins, and seems insufficient in bulk protein degradation during leaf senescence.

Then, as explained above, plants employ autophagy pathway for vacuolar bulk turnover of cytoplasmic components (Figure 5). Autophagy entails encapsulation of unwanted cytosolic materials within specialized autophagic vesicles, which are subsequently delivered to the vacuole for proteolysis (Li and Vierstra, 2012). Three distinct types of autophagy, micro-, macro-and mega-autophagy, have been reported in plants [START_REF] Van Doorn | Ultrastructure of autophagy in plant cells: A review[END_REF].

Microautophagy proceed by the invagination of tonoplast to trap cytoplasmic material congregated at the vacuole surface to create autophagic bodies within the vacuole. Such microautophagy process is poorly described in plants. It was found that the transport of cytoplasmic anthocyanin aggregates into the vacuole is mediated by a process reminiscent of microautophagy, and the accumulation of anthocyanin is depressed in Arabidopsis autophagy mutants (Pourcel et al., 2010;[START_REF] Chanoca | Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism[END_REF].

Conversely, macro-autophagy is much well known. Autophagosomes consist in double membrane vesicles that sequester cytosolic components (Thompson and Vierstra, 2005).

After trafficking to the lytic vacuoles, their outer membrane fuses with tonoplast to release their contents (inner membrane plus cargoes) into the vacuolar lumen. Released bodies are then called autophagic bodies. Autophagic bodies containing luminal constituents are broken down by resident vacuolar hydrolases, and the products are exported back to the cytosol for reuse. Mega-autophagy involves the massive degradation of the cell at the final phase of developmental programmed cell death (PCD) [START_REF] Van Doorn | Ultrastructure of autophagy in plant cells: A review[END_REF]. During this process, the permeabilization or rupture of tonoplast results in the release of large amounts of hydrolases into the cytoplasm, which completely degrade the cytoplasm and even the cell walls, leading ultimately to cell death. Mega-autophagy has been mainly described in the case of xylem formation in plants [START_REF] Weir | Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells[END_REF][START_REF] Kwon | The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis[END_REF].

Of these three types, macro-autophagy (hereafter referred to as autophagy) is the best characterized process and is considered as the major form and has received most attention (Michaeli and Galili, 2014;[START_REF] Ishida | Impacts of autophagy on nitrogen use efficiency in plants[END_REF]. During leaf senescence, expression of several AuTophaGy-related (ATG) genes (ATG7, ATG5, ATG9, ATG18a) encoding key components for autophagosome formation is increased. The suppression of these genes disrupts the normal development of autophagosomes and results in hypersensitivity to nitrogen starvation as well as premature leaf senescence. This suggests that autophagy plays a key role in leaf senescence and nutrient recycling (Doelling et al., 2002;Hanaoka et al., 2002;Thompson et al., 2005;Xiong et al., 2005).

Molecular machinery of autophagy in plants

Professor Yoshinori Ohsumi was awarded the Nobel Prize for Physiology or Medicine in 2016 for his discovery of the molecular basis of autophagy. Together with colleagues, he identified several ATG genes that participate in autophagic processes by yeast forward genetic experiments (Ohsumi, 2001). To date, more than 40 ATG genes have been identified

in yeast [START_REF] Yao | Atg41/Icy2 regulates autophagosome formation[END_REF], and the orthologs for most of them have been found in different plant species such as Arabidopsis, rice, wheat, maize, tobacco, barley, foxtail millet, and apple (Doelling et al., 2002;Xia et al., 2011;Pei et al., 2014;Li et al., 2015a;[START_REF] Zhou | A comprehensive, genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues[END_REF]Avila-Ospina et al., 2016;Li et al., 2016;Wang et al., 2016;[START_REF] Yue | Genome-wide sequence and expressional analysis of autophagy Gene family in bread wheat (Triticum aestivum L.)[END_REF].

Functional analysis of these proteins reveals a canonical route for autophagy. Basically, autophagy machinery consists of the induction of the nucleation of pre-autophagosomal structures, membrane elongation, phagophore expansion and then closure, trafficking and delivery of the autophagosome to the vacuole, and finally breakdown of the autophagic membrane and its contents by hydrolases into the vacuole (Figure 6) (Masclaux-Daubresse et al., 2017).

The TARGET OF RAPAMYCIN (TOR) negative regulator of autophagy is active under nutrient-rich conditions and dampens autophagy by hyperphosphorylating ATG13, which prevents its association with ATG1 [START_REF] Suttangkakul | The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis[END_REF]Dobrenel et al., 2016). Under nutrient-limiting conditions, inactivation of TOR leads to rapid dephosphorylation of ATG13, permitting the binding of ATG1. Thus, ATG1, ATG13, together with two accessory proteins, ATG11 and ATG101, assemble into an active complex (Li et al., 2014). The activated ATG1-ATG13 complex then promotes the nucleation and expansion of a cup-shaped doublemembrane (phagophore), which is thought to originate from the endoplasmic reticulum (ER) (Le [START_REF] Bars | ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants[END_REF]Zhuang et al., 2017Zhuang et al., , 2018)). The transmembrane protein ATG9 recruits lipids for phagophore elongation, ATG2 and ATG18 proteins facilitate ATG9 cycling (Xiong et al., 2005;Zhuang et al., 2017). Another step involves phagophore decoration with phosphatidylinositol-3-phosphate (PI3P) generated by a class III complex containing the phosphatidylinositol-3-kinase (PI3K) encoded by VACUOLAR PROTEIN SORTING 34 (VPS34), along with three core accessory subunits, ATG6, VPS38 or ATG14, and VPS15 (Feng et al., 2014;Liu et al., 2018a).

Expansion and closure of the phagophore membranes require two ubiquitination-like systems. Ubiquitin-fold protein ATG8 is initially processed by a cysteine protease ATG4 to expose a C-terminal glycine (Yoshimoto et al., 2004;Woo et al., 2014), then conjugated to the lipid phosphatidyl ethanolamine (PE) by the conjugating enzyme ATG3 (Thompson et al., 2005). Another ubiquitin-fold protein ATG12 is conjugated to ATG5 by the conjugating enzyme ATG10 (Phillips et al., 2008). Both of the conjugation systems share a single ATPdependent activating enzyme ATG7 (Doelling et al., 2002). The ATG12-ATG5 conjugate promotes the lipidation of ATG8 with PE and its anchorage into the phagophore membrane (Chung et al., 2010). ATG8 decoration of the phagophore membrane facilitates the recruitment and seal of the cargoes inside the autophagosome. ATG4 is also needed to remove and recycle ATG8 from ATG8-PE lining the outer membrane (Yoshimoto et al., 2004), while the ATG8-PE adducts trapped on the autophagosome inner membrane are digested in the vacuole. The autophagosome then transports the cargoes to the vacuole by fusing the outer membrane with tonoplast, and the remaining single-membrane structure (autophagic body) is released inside the vacuole for degradation by proteases and hydrolases.

The digested products are then exported from the vacuole for recycling.

Selective autophagy

Although autophagy was originally considered as an unrestricted bulk degradation of cytoplasm compounds, recent studies reveal that various routes for selective autophagy exist.

Selective autophagy can specifically degrade appropriate cargo by engaging a wide array of receptors or adaptor proteins that tether the cargo and also interact with ATG8 for the recruitment of the cargo to the membranes of autophagosomes (Floyd et al., 2012;Li and Vierstra, 2012;Farré and Subramani, 2016;Wang et al., 2018). The interaction between autophagic receptors and ATG8 is mediated by the presence of ATG8-interacting motif (AIM) in each receptor (Noda et al., 2010). Recently, a new binding site for autophagy adaptors and receptors was discovered on ATG8. This site engages ubiquitin-interacting motif (UIM)-like sequences rather than the canonical AIM for high-affinity binding to a new class of ATG8 interactors (Marshall et al., 2019). As ATG8 decorates and controls phagophore membrane expansion, its abundance determines the size of autophagosome (Xie et al., 2008). In this way, autophagosomes may undergo drastic membrane expansion and develop into multiple sizes to efficiently and selectively sequester specific cargoes, including protein aggregates, mitochondria, peroxisomes, chloroplasts, proteasome, ribosomes, endoplasmic reticulum, invading pathogens, and other components in plant cells under specific conditions (Soto-Burgos et al., 2018).

Several forms of selective autophagy have been reported in plants (Figure 7), chlorophagy (degradation of chloroplasts), reticulophagy (degradation of endoplasmic reticulum), mitophagy (degradation of mitochondria), pexophagy (degradation of peroxisomes), proteaphagy (degradation of proteasomes), ribophagy (degradation of ribosomes), aggrephagy (degradation of intracellular protein aggregates), xenophagy (degradation of intracellular pathogens), degradation of the pre-autophagosomal structure, degradation of TRYPTOPHAN-RICH SENSORY PROTEIN (TSPO), degradation of brassinosteroidresponsive transcription factor BES1 (Marshall and Vierstra, 2018;[START_REF] Yoshimoto | Unveiling the molecular mechanisms of plant autophagyfrom autophagosomes to vacuoles in plants[END_REF].

In plants, the first selective autophagy receptor identified is Arabidopsis NBR1 (NEIGHBOR OF BRCA1 GENE 1) (also known as Joka2 in tobacco), which targets ubiquitinated protein aggregates formed under stress conditions through a C-terminal ubiquitin-associated (UBA)

domain (Svenning et al., 2011;Zientara-Rytter et al., 2011). Functional analysis using two nbr1 knockout mutants revealed that (i) NBR1 is important for plant tolerance to a large spectrum of abiotic stresses, like heat, oxidative, salt, and drought stresses, and (ii) there is an increased accumulation of ubiquitinated insoluble proteins in nbr1 mutants under heat stress (Zhou et al., 2013). However, unlike atg5 and atg7 mutants, nbr1 is not sensitive to darkness stress or necrotrophic pathogen attack, suggesting that autophagy involved in diverse biological processes operates through multiple cargo recognition and delivery systems and NBR1 is involved in the selective degradation of denatured or damaged non-native proteins generated under high temperature conditions, but is not involved in bulk autophagy.

Interestingly, it was recently reported that NBR1 also specifically binds viral capsid protein and particles of cauliflower mosaic virus (CaMV) in xenophagy to mediate their autophagic degradation, and thereby restricting the establishment of CaMV infection [START_REF] Hafrén | Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles[END_REF]. Similarly, Joka2/NBR1 mediated selective autophagy pathway contributes to defence against Phytophthora infestans. The Phytophthora infestans effector protein PexRD54 recognizes potato ATG8CL through an AIM [START_REF] Maqbool | Structural Basis of Host Autophagyrelated Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54[END_REF]. PexRD54 outcompetes binding of ATG8CL with the Joka2/NBR1 to counteract defence-related selective autophagy, thus possibly attenuating autophagic clearance for pathogen or plant proteins that negatively impact plant immunity [START_REF] Dagdas | An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor[END_REF][START_REF] Dagdas | Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen[END_REF]. Upon infection, ATG8CL/Joka2 labelled defence-related autophagosomes are diverted to the host-pathogen interface to focally restrict pathogen growth [START_REF] Dagdas | Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen[END_REF].

Other specific autophagy receptors/adaptors were found after NBR1. We already mentioned ATI1/ATI2 that play a role in chlorophagy in the Section 1.2 (Honig et al., 2012;Michaeli et al., 2014). Another example of specific autophagy adaptor is RPN10. The proteasome subunit RPN10 was shown to mediate the autophagic degradation of the ubiquitinated 26S proteasome [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF]. Stimulated by chemical or genetic inhibition of the proteasome, RPN10 simultaneously binds the ubiquitinated proteasome, via an ubiquitininteracting motif (UIM), and ATG8 through another UIM-related sequence that is distinct from the canonical AIM. In Arabidopsis, the inhibitor-induced proteaphagy was blocked in mutant expressing an RPN10 truncation without the C-terminal region containing these UIMs.

In addition to specifically eliminating macromolecular complexes, organelles, and pathogens, selective autophagy can also scavenge individual proteins. For example, TRYPTOPHAN-RICH SENSORY PROTEIN (TSPO) is involved in binding and eliminating highly reactive porphyrin molecules through autophagy by interacting with ATG8 proteins via a conserved AIM motif (Vanhee et al., 2011). A more recent study proposed another role for TSPO to control water transport activity by interacting with and facilitating the autophagic degradation of a variety of aquaporins present in tonoplast and plasma membrane, including plasma membrane intrinsic protein 2;7 (PIP2;7), during abiotic stress conditions [START_REF] Hachez | The Arabidopsis abiotic stress-induced TSPO-related protein reduces cellsurface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation[END_REF].

As introduced before, most of the autophagy machinery proteins discovered in yeast have been found in plants. Although each ATG protein is unique in yeast, higher eukaryotes can possess families with multiple isoforms. For example, unlike the single ATG8 gene in yeast, there are nine ATG8 genes (AtATG8a-i) in the model plant Arabidopsis thaliana (Doelling et al., 2002), implying that different ATG8 proteins may serve different functions, and involve in different types of selective autophagy. As such, the identification of more new selective autophagic cargoes and receptors could be achieved by some molecular or microscopy based techniques (such as Co-IP, Y2H, BiFC) using different ATG8 isoforms under specific stress conditions.

Autophagy in plant response to stress

Autophagy is known to play a crucial role in plant adaptation to the environment fluctuations (Masclaux-Daubresse et al., 2017). At basal levels, autophagy mainly serves as housekeeping functions in cellular homeostasis, whereas stimulated autophagy activity facilitates plant adaptation to environmental stress conditions including starvation, drought, and pathogen infection [START_REF] Hafrén | Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles[END_REF]. Under well-fed conditions, autophagy-defective mutants show very limited phenotype. They can easily complete their life cycle and produce viable seeds.

Remarkable phenotypes were observed when autophagy-defective plants are grown under nutrient deficient conditions such as nitrogen or fixed carbon starvation; in these conditions, mutants missing the core autophagy machinery components (e.g., ATG7, ATG5, ATG9, ATG18, ATG12) show slow growth, enhanced leaf senescence, lower fecundity, and reduced survival rate (Doelling et al., 2002;Hanaoka et al., 2002;Thompson et al., 2005;Xiong et al., 2005;Li et al., 2015a). It was also shown that these mutants are more sensitive to salt, drought, and oxidative stresses in both Arabidopsis and rice (Xiong et al., 2007;Liu et al., 2009;[START_REF] Shin | OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice[END_REF].

Conversely, overexpression of ATG5 or ATG7 in Arabidopsis increased plant resistance to oxidative stress and necrotrophic pathogens, delayed leaf ageing and enhanced plant growth, seed set, and seed oil content (Minina et al., 2018). Overexpression of MdATG18a in apple improved plant tolerance to drought, nitrogen starvation, and pathogen infection (Sun et al., 2018a[START_REF] Sun | MdATG18a overexpression improves tolerance to nitrogen deficiency and regulates anthocyanin accumulation through increased autophagy in transgenic apple[END_REF](Sun et al., , 2018c)). Although the Arabidopsis atg8 single mutants have no special phenotypes, the heterologous expression of ATG8 from other species (such as MdATG8i, SiATG8a, or GmATG8c) in Arabidopsis or rice conferred tolerance to nitrogen starvation, drought, and salt stresses and increased in some cases plant biomass or seed yield (Xia et al., 2012;Li et al., 2015bLi et al., , 2016;;Wang et al., 2016;Luo et al., 2017). Thus, increasing the expression of autophagy genes can provide benefits for plant performance and adaptation to nutrient limitation conditions probably through boosting nutrient mobilization via autophagy.

These findings implicate that genetic stimulation of autophagy might produce positive effects on the overall plant performance and environment adaptation in cereal crops, especially regarding the grain filling, plant productivity and N remobilization. However, this kind of hypothesis has not been tested.

Role of autophagy in nitrogen recycling

Under normal conditions, autophagy is expressed at a basal level that constitutes housekeeping machinery and participates to cell homeostasis. The selective and bulk removal of toxic and damaged components by autophagy is invoked when there is a need for rapid mobilization and resorption of nutrients as in senescence and stress responses. There is increasing evidence that autophagy is a primary mechanism of nutrient recycling during leaf senescence (Guiboileau et al., 2012;Masclaux-Daubresse et al., 2017). Autophagy then might have a dual role, preserving cell longevity by removing cell waste on one hand, triggering cell death with excessive autophagy activity via participating in the methodic degradation of the cell constituents on the other hand (Guiboileau et al., 2010). Depending on its fine-tuning, the tight balance between the two antagonistic effects is likely to facilitate efficient recycling and remobilization as long as possible before cell death (Havé et al., 2017). Guiboileau et al. (2012) firstly demonstrated the participation of autophagy in N remobilization from leaves into the seeds in Arabidopsis monitoring the fluxes of 15 N to the seeds after plant labelling at vegetative stage. Authors showed that N remobilization was sharply decreased in all the atg mutants studied (atg18a RNAi, atg5, and atg9) compared to wild type (WT) under N-limited conditions. Decrease was more moderate but still significant in atg5 under nitrate-rich conditions. Accordingly, authors further found that atg mutants accumulated more ammonium, amino acids (AA), proteins, and RNA contents, but less sugars and starch in the rosette leaves than WT (Guiboileau et al., 2013). The higher N/C ratio of atg rosette leaves was mainly due to the over accumulation of all sort of nitrogen compounds. Interestingly, protease activities also increased in atg mutants with ageing, suggesting that the overall protein accumulation observed was not due to a lack of proteolysis, but to defects in the trafficking of these substrates to the vacuole where the proteases reside (Guiboileau et al., 2013;Havé et al., 2017).

N remobilization in autophagy mutants was further evaluated by 15 N-labelling in crops. The study in maize revealed that N remobilization to the kernels was impaired in the maize atg12 mutants (Li et al., 2015a). The growth of atg12 mutants was severely arrested at seedling stage, and grown plants showed enhanced leaf senescence and stunted ear development under nitrogen-starved conditions but not under high-N. Under nutrient-rich conditions, seed yield of atg12 plants was much lower, and 15 N reallocation into the seeds was twice less in atg12 than WT. Surprisingly, 15 N partitioning in the new upper leaves produced after 15 N labeling was higher in atg12 compared to WT, which suggests that N-remobilization from the old leaves to the new leaves was more efficient while from the old leaves to the seeds was suppressed in atg12. This could indicate that, in maize, autophagy did not affect N recycling and mobilization from the source leaves but rather modified N resource allocation between different new sinks (the developing ear and kernels or the younger leaves).

The investigation conducted during the vegetative growth period on the rice autophagydisrupted Osatg7-1 mutant suggested that N remobilization from senescent leaves to young leaves was suppressed (Wada et al., 2015). Higher nitrogen content was retained in senescent leaves of Osatg7-1 mutants as soluble protein and Rubisco concentrations were higher than that of WT. The reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led to its reduced leaf area, tillers, and photosynthetic capacity. Unfortunately, the male sterility phenotype of Osatg7-1 mutants prevented authors from examining the contribution of autophagy-mediated nitrogen remobilization from leaves to seeds during the reproductive growth period. In this context, development of autophagy knock-down lines (OsATG antisense or RNA interference) in rice would be helpful in the future.

Recent advance shows that autophagy is not only involved in the recycling of nitrogen from proteins or other nitrogen sources, but also involved in the recycling and remobilization of micronutrients (Pottier et al., 2014). A more recent study of Arabidopsis found that the efficiency of iron (Fe) translocation from vegetative organs to the seeds was severely decreased in atg5-1 mutants compared with WT (Pottier et al., 2019). Authors further showed that the translocation of zinc (Zn) and manganese (Mn) to the seeds was also dependent on autophagy. These results are consistent with the observation that autophagy was induced under Zn limitation conditions, and that autophagy-deficient mutants (atg5-4, atg10-1)

showed an early senescence phenotype under Zn limitation and limited growth recovery when Zn resupplied (Eguchi et al., 2017).

Taken together, results obtained using autophagy-defective mutants clearly indicate the involvement of autophagy in the remobilization of nutrients, especially under nutrient-limited conditions. It would be interesting to check whether the other key players in both bulk and selective autophagy play a role in nitrogen remobilization, such as ATG8 and other ATG genes involved in the core autophagy machinery, and also the recently identified selective autophagic receptors. Moreover, increasing the autophagic activity specifically during the seed filling stage by overexpressing some core autophagy genes may be a powerful approach to improve N remobilization from source tissues to the seeds, and more importantly, seed nutritional quality. However, this kind of investigation has not been reported so far.

Papain-like cysteine proteases (PLCPs)

Higher-plant genomes encode for hundreds of proteases, which include diverse families, according to their active site residue (e.g., cysteine, serine, aspartic, metallo, and threonine proteases), and play crucial roles in protein proteolysis for recycling [START_REF] Van Der Hoorn | Plant Proteases: From Phenotypes to Molecular Mechanisms[END_REF].

In Arabidopsis, more than 800 proteases encoded by the genome are subdivided into over almost 60 families (classified based on the sequence homology), which belong to 30 different clans (classified based on the ancestor; usually have similar protein folds) [START_REF] Van Der Hoorn | Plant Proteases: From Phenotypes to Molecular Mechanisms[END_REF]Rawlings et al., 2018). Despite the high number of proteases described and the importance of proteolytic processes during plant development (including germination, growth, senescence, and plant-environment interactions), no physiological substrates have been identified to date for the vast majority of the plant proteases. Hence, the molecular functions of most of these enzymes remain unknown.

Gene expression studies in different plant species have shown that cysteine proteases are the most abundant class of proteases upregulated in senescing leaves [START_REF] Bhalerao | Gene expression in autumn leaves[END_REF]Guo et al., 2004). Inhibition of cysteine proteases in tobacco by expressing the rice cystatin (an endogenous cysteine protease inhibitor gene) delayed the senescence-related decline of photosynthesis, and maintained the Rubisco protein content and activities under both optimal and stress conditions (Prins et al., 2008). Impairment in Rubisco degradation was also observed in wheat by using the cysteine protease chemical inhibitor E-64. This indicated the involvement of cysteine proteases in the turnover of Rubisco in wheat senescent leaves (Thoenen et al., 2007). Cysteine proteases use a catalytic Cys residue as nucleophile during proteolysis, activated by histidine (His) in the active site (van der Hoorn, 2008). Senescenceassociated cysteine proteases include papain-like cysteine proteases (PLCPs, family C1A in MEROPS protease Database), legumains or vacuolar processing enzymes (VPEs, family C13), metacaspases (family C14), calpains (family C2), and proteases related to the ubiquitin-proteasome-dependent pathway (families C12, C19, and C85) [START_REF] Grudkowska | Multifunctional role of plant cysteine proteinases[END_REF]Roberts et al., 2012;[START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]Liu et al., 2018b). Among them, PLCPs are the most abundant (Rawlings et al., 2018). In addition, the expression analysis of several senescence-related proteases in barley demonstrated that PLCPs had strong induction during developmental leaf senescence and correlated best with senescence markers like chlorophyll degradation under high C/N ratio conditions, suggesting that, among the proteases tested, PLCPs are most likely the major enzymes to participate in bulk protein degradation during leaf senescence (Parrott et al., 2010). Thus, the family of PLCPs is intensively investigated in plants among all the types of cysteine proteases.

The family of PLCPs

PLCPs are characterized by the occurrence in their structure of a papain-like fold that consists of two domains (lobes) delineating a substrate-binding groove which contains the catalytic residues in the order Cys, His, and Asn [START_REF] Van Der Hoorn | Plant Proteases: From Phenotypes to Molecular Mechanisms[END_REF]Rawlings et al., 2018). PLCPs are produced as inactive precursors, containing an N-terminal signal peptide for protein secretion and an auto-inhibitory pro-domain that folds back onto the catalytic site cleft and is removed during the protease activation, releasing a mature 25-35 kDa active protease (Taylor et al., 1995;Misas-Villamil et al., 2016). Some PLCPs also carry a C-terminal extension sequence with unknown function, consisting of a 2 kDa Pro-rich domain followed by a 10 kDa granulin domain, which shares homology to animal growth hormones, granulins, released upon wounding (Yamada et al., 2001;[START_REF] Bateman | The granulin gene family: from cancer to dementia[END_REF][START_REF] Richau | Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics[END_REF].

PLCPs genes belong to a large multigenic family. In plants, there are approximately 723

PLCPs, with 31 genes found in Arabidopsis, which are classified into 9 subfamilies based on phylogenetic analysis and conserved structural features [START_REF] Beers | The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis[END_REF][START_REF] Richau | Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics[END_REF]. Importantly, the Arabidopsis genome encodes PLCPs of all the 9 subfamilies, a detailed distribution of Arabidopsis PLCPs is summarized in Figure 8 according to [START_REF] Richau | Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics[END_REF]. Therefore, studies on Arabidopsis PLCPs may provide representative information to deduce the function of PLCPs from other species in the same subgroup. The authors then consequently refer to each of the nine subfamilies with a type member from Arabidopsis:

subfamily 1 (contains RD21A-like proteases; 9 members in Arabidopsis); subfamily 2 (CEP1-like; 3 members); subfamily 3 (XCP2-like; 2 members); subfamily 4 (XBCP3-like; 1 members); subfamily 5 (THI1-like; 1 members); subfamily 6 (SAG12-like; 6 members);

subfamily 7 (RD19A-like; 4 members); subfamily 8 (AALP-like; 2 members); and subfamily 9 (CTB3-like; 3 members).

PLCPs in leaf senescence, protein degradation, and cell death

In Arabidopsis, it has been described for a long time that many members of PLCPs are important participants in leaf senescence, including SAG12, RD21A, RD19A, AALP, CATHB1, and CATHB3 (Lohman et al., 1994;Yamada et al., 2001;Gepstein et al., 2003;Guo et al., 2004;Havé et al., 2017). SAG12 exhibits a strictly senescence-associated expression pattern and localizes specifically in the SAVs in senescing leaves (Lohman et al., 1994;[START_REF] Otegui | Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean[END_REF]. The sag12 Arabidopsis mutant did not exhibit any discernible phenotypical alteration during senescence under normal conditions. Whereas, under low nitrogen (LN) availability, decreases in both the seed N content and yield were observed in sag12 mutant, probably due to the fact that inducible proteolytic systems are not sufficient to cope with SAG12 depletion (James et al., 2018). These results suggesting that SAG12 could participate in Rubisco degradation and N remobilization during leaf senescence that sustain seed filling. The oilseed rape homologs of RD21A and SAG12 were also reported to play a crucial role in the efficient proteolysis associated with leaf senescence, especially in response to N limitation [START_REF] Desclos | A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation[END_REF][START_REF] Poret | Characterization of senescence-associated protease activities involved in the efficient protein remobilization during leaf senescence of winter oilseed rape[END_REF]. However, an opposite role was reported from the rice homologs of SAG12 that act as negative regulators of developmental leaf senescence and stress-induced cell death [START_REF] Singh | Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants[END_REF][START_REF] Singh | The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death[END_REF].

The investigation in individually darkened leaves of Arabidopsis revealed an increased activity of many PLCPs, among which RD21A and AALP were identified as the dominant active PLCPs in dark-induced senescing leaves [START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]. Authors used several knock-out mutants of senescence-induced PLCPs (rd21A-1, aalp-1, sag12-1, ctb3-1, rd21A-1/aalp-1, and ctb1/ctb2/ctb3) to determine the role of individual protease activities in senescence and only found a slight but significant delayed progression of the whole plant senescence in aalp-1 and rd21A-1/aalp-1 double mutants, suggesting a role for AALP in the process of plant senescence.

RD19A and RD21A are two important protein markers responsive to dehydration stress in Arabidopsis, and their transcription was strongly induced under drought and high salt conditions [START_REF] Koizumi | Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana[END_REF]. Interestingly, RD19A and RD21A were found recently to play a determinative role in regulating pathogen defence. Expression of RD19A was induced during infection by a bacterial pathogen Ralstonia solanacearum in Arabidopsis, and T-DNA insertional mutation of RD19A enhanced plant susceptibility to the Ralstonia, suggesting that RD19A provides immunity and is a positive regulator in plant resistance to Ralstonia [START_REF] Bernoux | RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector[END_REF]. In addition, Arabidopsis rd21A null mutants were significantly more susceptible to the necrotrophic fungal pathogen Botrytis cinerea [START_REF] Shindo | A Role in Immunity for Arabidopsis Cysteine Protease RD21, the Ortholog of the Tomato Immune Protease C14[END_REF]. The rd21A mutants exhibited significant increased cell death when treated with the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme. This indicated that RD21A behaves as a negative regulator of necrotrophic pathogen-induced cell death in Arabidopsis and may be rather involved in a protection mechanism to necrotrophic pathogen [START_REF] Ormancey | Sphingolipid-induced cell death in Arabidopsis is negatively regulated by the papain-like cysteine protease RD21[END_REF]. However, a pro-death function of RD21A was reported in another study, where detached leaves of rd21A Arabidopsis mutants and plants with AtSerpin1 (inhibitor of RD21A activity) over-expression were more resistant to the two necrotrophic fungi Botrytis cinerea and Sclerotina sclerotiorum as they displayed a significant reduction in cell death (Lampl et al., 2013). The discrepancies in these studies may arise from the use of different Botrytis cinerea isolates, from different plant culture conditions, or due to the fact that assays were performed on different pant parts (whole plant or detached leaves).

Like RD21A, the CATHB are also involved in plant immune response. It was discovered that silencing of the entire tobacco CATHB family prevented programmed cell death (PCD) and compromised disease resistance during the hypersensitive response (HR) triggered by two distinct non-host bacterial pathogens [START_REF] Gilroy | Involvement of cathepsin B in the plant disease resistance hypersensitive response[END_REF]. In Arabidopsis, the three CATHB homologues (AtCATHB1-3) act in a functionally redundant manner for full basal resistance against the virulent hemibiotrophic bacteria Pseudomonas syringae pv. tomato (Pst) DC3000.

They are crucial for the full development of PCD during the HR triggered by the avirulent strains expressing AvrB (McLellan et al., 2009). A novel function for CATHB was also demonstrated in promoting senescence. The cathB triple mutants indeed exhibited a delay in senescence and a seven-fold decrease in the accumulation of senescence-associated marker gene SAG12. Furthermore, the triple mutants of CATHB displayed a strong reduction in PCD induced by ultraviolet (UV), oxidative stress and ER stress in Arabidopsis [START_REF] Ge | Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis[END_REF][START_REF] Cai | Two proteases with caspase-3-like activity, cathepsin B and proteasome, antagonistically control ER-stress-induced programmed cell death in Arabidopsis[END_REF]. Thus, CATHB may represent a potential common element in the regulation of pathogen-induced, developmental, and abiotic stress-induced forms of senescence and PCD.

Altogether, these results indicate the important roles of PLCPs in the regulation of plant leaf senescence, protein degradation, and cell death. But whether they are involved in nitrogen remobilization remains to be characterized. A big challenge for the evaluation of their roles in plant physiology is the fact that many proteases are encoded by family genes that are presumed to contain other redundant proteases with compensatory effects. It may then be difficult to study their role in nitrogen remobilization using single knockout mutant.

Therefore, multiple mutations or employment of proteinaceous (Serpin1 or cystatin proteins)

or chemical (E-64) protease inhibitors would be helpful.

Cross-talk between autophagy and PLCPs

Although both autophagy and PLCPs are key players during leaf senescence, protein proteolysis, and nutrient recycling, the relationship between them remains largely unknown.

Proteins are probably not degraded directly inside the autophagosomes but rather transported by them to the lytic vacuoles where proteases and hydrolases operate. Previous results in our laboratory showed that protease activities were significantly increased in the leaves of atg mutants with increased protein accumulation and impaired N remobilization efficiency (NRE) compared to WT (Guiboileau et al., 2013). It was hypothesized that vacuole proteases and their cytoplasmic substrates cannot co-localize in the absence of autophagosome trafficking.

Then, Havé et al. (2018) used proteomics shotgun LC-MS/MS to identify the overaccumulated proteases in the leaves of autophagy mutants and specific probes to monitor their activity under both low-and high-nitrate (LN, HN) conditions (Figure 9). Results

showed that cysteine proteases accounted for the largest proportion (38%) of the 26 proteases that increased in autophagy-deficient lines. Activity-based protein profiling (ABPP) analysis with DCG-04 revealed that activities of PLCPs were higher in autophagy-defective plants grown under low-nitrate conditions. Further pull-down experiments using DCG-04 in lownitrate condition showed that the active PLCPs accumulated in autophagy mutants were mainly SAG12, RD21A, CATHB3, and AALP. The western blots performed using the RD21A, CATHB3, and SAG12 antibodies confirmed that both the mature and immature protease forms were accumulated in the mutant lines, suggesting that there was no defect in protease maturation or trafficking in the autophagy mutants. The specific over-accumulation of these PLCPs under LN but not under HN in autophagy impaired plants strongly suggests that they are involved in N remobilization, and possibly provide alternative remobilization pathways to autophagy. Such hypotheses need to be confirmed by further investigations and biochemical studies using protease and autophagy crossed mutants.

Recently, James et al. (2018James et al. ( , 2019) ) investigated the role of SAG12 in N remobilization for seed filling. They showed that the yield and N content of the seeds were decreased in the Arabidopsis sag12 mutants relative to the wild type under limited nitrogen nutrition.

Although leaf senescence was similar in both lines, there was an accumulation of 15 N in the roots while a lower partitioning of 15 N to the seeds in sag12 mutants compared to the wild type under LN conditions. This was accompanied by a significant decrease in root cysteine protease activity and an increase in root protein contents. Further investigation revealed that SAG12 is expressed in the root stele at the reproductive stage, particularly under LN condition. Taken together, these results suggest that SAG12 plays a crucial role in root protein degradation and N remobilization, which ensure seed filling and sustain yields under low nitrogen availability.

Interestingly, it was found that aspartate protease activity is significantly increased in sag12 mutant plants grown under optimal nitrogen conditions (James et al., 2018). Among the putative aspartate proteases involved, a CND41-like aspartate protease AED1

(APOPLASTIC ENHANCED DISEASE SUSCEPTIBILITY-DEPENDENT 1) was then identified and proposed to provide compensatory effects to cope with the absence of SAG12 activity in senescing leaves of sag12 mutants. The over-accumulation of AED1 was also observed in autophagy mutants (atg5) specifically under LN condition (Havé et al., 2018), suggesting its role in N remobilization.

Aims of the thesis

Leaf senescence program includes sequences of tightly regulated metabolic changes involving the degradation and recycling of many cytoplasmic components. N remobilization during this process is crucial for plant development and survival, especially in some stress conditions. The importance of autophagy in nutrient remobilization (N and other microelements) has been demonstrated using 15 N and 57 Fe tracing and elemental analyses on Arabidopsis atg5, atg9 and atg18a mutants (Guiboileau et al., 2012;Pottier et al., 2019). The role of specific autophagy in N remobilization remains to be established. The overexpression of autophagy genes in Arabidopsis enhanced plant tolerance to a variety of biotic and abiotic stresses (Xia et al., 2012;Li et al., 2015b;Wang et al., 2016;Luo et al., 2017;Minina et al., 2018), but whether overexpression could provide benefits in N remobilization remains unknown.

Several PLCPs and AED1 have been suggested as prominent participants in the process of leaf senescence and stromal protein degradation [START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]Havé et al., 2018;James et al., 2018). Except in the case of SAG12, there is no direct evidence to show that they are involved in N remobilization. Furthermore, our understanding of the relationship between PLCPs and autophagy in N remobilization is very limited.

The main objectives of this study were as follows:

1. To evaluate whether the genes involved in the autophagy core machinery or selective autophagy play a role in N remobilization in Arabidopsis.

2. To determine whether N remobilization could be improved through the overexpression of several individual AtATG8 genes in Arabidopsis, and the possible positive effect on seed quality.

3. To characterize the possible benefits in abiotic stresses tolerance and agronomic performance including the N remobilization and grain filling in barley plants by overexpressing the HvATG5 autophagy gene.

4. To investigate the contribution of PLCPs to N remobilization and the relationship with autophagy pathway using protease and autophagy crossed mutants.

Introduction

Agriculture is responsible of a large part of the greenhouse gas (GHG) emissions, in part due to the use of ammonio-nitrate fertilizers that have a strong negative impact on carbon balance. Therefore, today more than before, the objective of the sustainable agriculture is to reduce the use of fertilizers and to preserve environment.

Improving NRE in crops is a good way to achieve this goal. Nitrogen remobilization efficiency (NRE) is the capacity of plants to reuse their organic nitrogen through the recycling of macromolecules as proteins and nucleic acids in source organs to provide amino acids and other nitrogenous compounds, as small peptides, ammonium, allantoin or glucosinolates, to be translocated from source organs to the developing sinks as seeds [START_REF] Tegeder | Source and sink mechanisms of nitrogen transport and use[END_REF]. Plants with high NRE provide the advantage over low NRE plants to recycle and reuse several times their N resources and thus to resist more efficiently to nitrogen shortage [START_REF] Zhao | Foliar nutrient resorption patterns of four functional plants along a precipitation gradient on the Tibetan Changtang Plateau[END_REF]. For crops, high NRE results in less nitrate-demanding genotypes [START_REF] Avice | Leaf senescence and nitrogen remobilization efficiency in oilseed rape (Brassica napus L.)[END_REF].

Up to now several attempts to increase nitrogen use efficiency through plant engineering in model plants and crops have been reported [START_REF] Tegeder | Source and sink mechanisms of nitrogen transport and use[END_REF]; they mainly consisted in manipulating nitrate or ammonia transporters or glutamine synthesis through the overexpression of glutamine synthetases. No attempt to increase nitrogen remobilization from source organs to the sink has been described so far.

Autophagy (meaning self-eating) is a universal vesicular mechanism that facilitates the degradation of unwanted cell constituents in eukaryotic cells. Autophagy is essential for cell homeostasis and controls nitrogen recycling.

Autophagy is enhanced in senescing organs and contributes to the source to sink nutrient remobilization (Avila-Ospina et al. 2014). In Arabidopsis, Guiboileau et al, (2012) showed that autophagy is essential for N remobilization to the seeds and for seed filling. The same feature was demonstrated in maize by Li et al. (2015a).

The autophagy machinery consists of the formation of autophagosomes that are cytosolic double membrane vesicles that engulf and sequester unwanted cytoplasmic constituents to drive them to the central vacuole in plant or to lysosomes in animal. Unwanted material is then degraded in plant by the vacuole hydrolases and proteases that release amino acids and sugars to be recycled in the cell or exported to the sink organs [START_REF] Liu | Autophagy: pathways for self-eating in plant cells[END_REF]. Autophagosome formation incorporates the products of many AUTOPHAGY (ATG) genes. The ATG genes have been first discovered in yeast [START_REF] Tsukada | Isolation and characterization of autophagy defective-mutants of Saccharomyces cerevisiae[END_REF], and subsequently, homologous genes were found in plant and animal. Among the 50 ATG genes discovered in yeast, 18 are essential for the formation of autophagosomes and are part of the autophagy core-machinery [START_REF] Yang | New insight into the Mechanism and Function of Autophagy in Plant Cells[END_REF]. In Arabidopsis, all the core-machinery genes have been identified. They are either single genes or belong to gene families. For example, there are nine ATG8 genes in Arabidopsis (ATG8a-i). By contrast with the atg mutants defective in single genes, like atg5, atg7, or atg10 for example, it is likely that the atg8a-i single mutants do not display any specific phenotype relative to wild type (Phillips et al. 2008;[START_REF] Wang | ATG5 is required to limit cell death induced by Pseudomonas syringae in Arabidopsis and may be mediated by the salicylic acid pathway[END_REF][START_REF] Masclaux-Daubresse | Stitching together the multiple dimensions of autophagy using metabolomic and transcriptomic analyses reveals new impacts of autophagy defects on metabolism, development and plant response to environment[END_REF]Guiboileau et al., 2013;Guiboileau et al., 2012;[START_REF] Lenz | Autophagy differentially controls plant basal immunity to biotrophic and necrotrophic pathogens[END_REF][START_REF] Yoshimoto | The role of plant autophagy in nutrient starvation and aging[END_REF]Thompson et al. 2005).

The conjugation of the ATG8 proteins with phosphatidylethanolamine (PE) to form the ATG8-PE conjugate that decorates the autophagosome membrane, is however essential for the expansion of the membrane of the autophagosome and for the targeting of the cytoplasm components to be degraded (Noda et al. 2010;Masclaux-Daubresse et al. 2017). We then suppose that the different ATG8a-i proteins have redundant functions.

Redundancy may explain why single atg8 mutations have no effect on plant phenotype and autophagy activity, unlike mutations in the single ATG genes (as ATG5, ATG9, ATG7 and ATG2) that generate smaller rosettes, hypersensitivity to starvations and reduced yield (Thompson et al. 2005;Hanaoka et al. 2002;[START_REF] Yoshimoto | The role of plant autophagy in nutrient starvation and aging[END_REF]).

Although the Arabidopsis atg8 single mutants have no special phenotypes, the overexpression of ATG8 genes produced positive effects on plant adaptation to drought and nitrogen limitation. ATG8 overexpressors were reported to display higher yield than wild type (Xia et al. 2012;Li et al. 2015b;Li et al. 2016;Luo et al. 2017;Sun et al. 2018a;[START_REF] Sun | MdATG18a overexpression improves tolerance to nitrogen deficiency and regulates anthocyanin accumulation through increased autophagy in transgenic apple[END_REF]). Many reports then suggested that increasing the expression of autophagy genes can provide benefits for plant performance. Recently, Minina et al. (2018) showed that overexpressing ATG5 and ATG7 in Arabidopsis stimulates autophagosome formation, autophagic flux and increases resistance to necrotrophic pathogen, with delayed ageing and enhanced plant growth, seed set and seed oil content.

However, there was no study on nutrient recycling and remobilization in autophagy overexpressor so far.

The use of GFP-ATG8 or RFP-ATG8 protein fusions to monitor autophagy fluxes in Arabidopsis and compare genotypes is very common [START_REF] Bassham | Methods for analysis of autophagy in plants[END_REF]. In the past, many groups have constructed such protein fusions for microscopy purpose. As such plant material consists in fact in ATG8 overexpressing lines that may be suitable for our study. As such we used the 35S::RFP-ATG8 and pActin::GFP-ATG8 lines previously selected in our laboratory for microscopy [START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF][START_REF] Di Berardino | Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds[END_REF]) and we constructed two new lines using the ubiquitin promoter and the ATG8a and ATG8g genomic sequences to run 15 N labelling experiments, as done previously by Guiboileau et al. (2012) to monitor N remobilization to the seeds in autophagy mutants.

Plants were labelled at vegetative stage and harvested at seed maturity. Our results showed that increasing ATG8 overexpression in Arabidopsis enhances N remobilization to the seeds, but only when plants are cultivated under full nitrate conditions. Under nitrate limiting conditions, no outperformance was observed in overexpressors, probably due to the fact that N limitation had already strongly stimulated autophagy activity and mobilized all the plant efforts in N remobilization.

Results

Overexpressing ATG8 enhances autophagy activity in Arabidopsis

In this study, two lines expressing the ATG8e or ATG8f cds (coding sequences) under the control of the 35S and actin promoters respectively and two lines expressing the ATG8a or ATG8g genomic sequences under ubiquitin promoter were used as ATG8 overexpressing lines. In all the constructs, the ATG8 were tagged in N-term with fluorescent proteins (FP; fluorescent protein; see Material and Methods). The overexpression of the different FP-ATG8 transgenes in these lines was verified using RT-qPCR to monitor FP overexpression (Supplementary Fig. S1AB). Accordingly, higher ATG8a, ATG8e, ATG8f, ATG8g mRNA levels were observed in the UbQ-ATG8a, 35S-ATG8e, Actin-ATG8f, UbQ-ATG8g lines than in the control (Supplementary Fig. S1C). The expression of ATG8a in the UbQ-ATG8a line was tremendously increased.

Although several publications state that autophagy flux and activity are increased in ATG8 overexpressing plants (Xia et al. 2012;Li et al. 2015b), such feature had never been verified so far. The reason is that monitoring autophagosome number and autophagic body accumulation to estimate autophagy flux necessitate to introduce same FP-ATG8 fusions in the genotypes to be compared. In the case of ATG8 overexpressing lines, which in our case consist in FP-ATG8 carrying lines, the control line cannot contain FP-ATG8; it only expresses free GFP in our experiments. Then it was then impossible to compare autophagosome and autophagic body fluxes between our ATG8 lines and control line using FP-ATG8 tool.

We then tried and estimated autophagy flux by monitoring (i) the NBR1 protein contents, (ii) the expression levels of autophagy genes and (iii) the number of autophagosomal structures in the different genotypes. We observed that, except few, all the transcripts of the ATG genes were overabundant in the ATG8 overexpressors in both seedlings (Supplementary Fig. S2) and in the leaves of old rosettes (Fig. 1; Supplementary Fig. S3). The sharp increase of the ATG transcripts in all the ATG8 overexpressors in the rosette leaves before was then likely to anticipate higher autophagy flux at later stages of development. The expressions levels of the ATG8 transcripts corresponding to the transgenes were especially high (Supplementary Fig. S3B) since primers used were able to quantify both native ATG8 and transgene FP-ATG8 mRNAs.

NBR1 is a protein adaptor involved in autophagy (Svenning et al. 2011). NBR1 targets cargoes and interacts physically with ATG8 proteins to facilitate the enclosure of cargos into the autophagosomes. The level of NBR1 neosynthesis then increases when autophagy is stimulated and the degradation of NBR1 is also stimulated when autophagy flux is increased. The higher NBR1 transcript levels in both 56 d old rosette leaves (Fig. 1) and seedlings (Fig. 2A) compared to control then also suggested higher autophagic activity in the ATG8 overexpressors. Despite the higher NBR1 transcript levels in all the ATG8 overexpressing lines, the fact that NBR1 protein content was slightly lower in overexpressors compared to control, especially in the UbQ-ATG8a

and UbQ-ATG8g seedlings, suggested that NBR1 degradation was stimulated in overexpressors (Fig. 2B). We then used treatments with the concanamycin-A (CA) drug to block the vacuolar ATPases and then inhibit the degradation of autophagic bodies and NBR1 into the vacuole. This treatment was performed on seedlings and we observed that the CA-treated seedlings accumulated NBR1 protein to a larger extent when they were ATG8 overexpressors. By contrast, there was no difference in NBR1 protein level between the ATG8 overexpressors and the control line in the absence of CA treatment (Fig. 2C).

It is possible to monitor the increase in autophagic bodies into the vacuole of young seedling roots using light microscopy [START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF]). This is however not feasible on leaves especially when getting old. We then submitted 7 d old seedlings to CA for 2-4 hours and observed autophagic bodies into the vacuole with a light microscope with DIC device. It was difficult to see any difference between the control and the ATG8 overexpressing lines even after short CA treatment, due to the high and fast accumulation of autophagic bodies in their vacuoles. However, in the absence of CA treatment we found a clear difference and more autophagic bodies in the ATG8 overexpressors than in the control line (Fig. 2D). This was statistically confirmed numbering the autophagosomal structures (Fig. 2E) and we verified that the observed bodies were autophagic bodies verifying they were fluorescent and thus decorated with FP-ATG8 (Supplementary Fig. S4).

ATG8 overexpression improves protein concentration in seeds and lower N waste in dry remains after harvest

The different genotypes were grown under low and full nitrate conditions and labelled with 15 N isotope as performed by Guiboileau et al. (2012) to monitor N use efficiency for grain filling and N remobilization efficiency. As done by Guiboileau et al. (2012) and in previous 15 N studies cited herein (Masclaux-Daubresse and Chardon 2011), we did not take into account the root compartments as the mass of the roots sampled at harvest, when plants are dry, is highly influenced by the plant manipulation, and that our estimation is that the collected root biomass never exceeded more than 2-3% of the total plant biomass, which is negligible especially considering the poor N concentration in these roots (1-2% in general under full nitrate nutrition). Then in our experiment only shoots were considered and separated as rosette, stem (flowering stem plus siliques) and seeds.

In their experiments, Guiboileau et al. (2012) showed that autophagy mutants were tremendously affected in N remobilization when nitrate supply was scarce and that under full nitrate nutritive conditions, the N remobilization capacity of mutants was much less affected even though still significantly. Therefore, we used both low and high nitrate conditions to test the N remobilization capacities of overexpressor lines.

Results presented in Fig. 3 show that there was globally no significant difference in dry weight (DW), C concentration (C%), N concentration (N%), 15 N content, DW partitioning and 15 N partitioning between genotypes when grown under nitrogen limiting conditions. When grown under full nitrate conditions, slight differences between genotypes were observed for DW and C% (Fig. 4AB), but these differences were not repeatable in other experiments. Nevertheless, the most striking result was that large and significant differences in N% and 15 N contents were observed between control and ATG8 overexpressors under full nitrate conditions (Fig. 4CD). Globally, N% was lower in the rosettes of the ATG8 overexpressors relative to the control while the N% of seeds was higher in the ATG8 overexpressors than in the control seeds. Higher N% in the seed of ATG8 overexpressors resulted in higher storage protein contents (Fig. 5). Like total N, 15 N content was lower in the rosette and higher in the seeds of the ATG8 overexpressors relative to the control (Fig. 4D). As there was no difference between ATG8 overexpressors and control plants for N uptake (Supplementary Fig. S5), we suspected that differences between them were linked to different capacities to allocate and remobilize nitrogen from the leaves to the seeds. Such assumption was confirmed comparing the biomass partitioning and the 15 N partitioning in rosette, stem and seeds between genotypes (Fig. 6). Indeed, while biomass allocation in the different organs of the plant was not different between genotypes (Fig. 6A), the 15 N partitioning was significantly higher in the seeds and lower in the rosettes of the ATG8 overexpressors relative to the control line (Fig. 6B). There was no difference in the 15 N partitioning in the stems between the ATG8 overexpressors and the control. This demonstrated that N remobilization efficiency was higher in the ATG8 overexpressors due to a better N flux from the rosettes to the seeds (Fig. 6C). The best performances were found in the UbQ-ATG8a and UBQ-ATG8g

plants. Although N remobilization to the seeds was higher in ATG8 overexpressors, there was no difference in total seed yield nor in individual seed weight (not shown).

Interestingly, regarding to the strong phenotype described by Minina et al. (2018) for the ATG5 and ATG7

overexpressors they produced, we did not observe major visual phenotypes on our ATG8 overexpressors but a slight early leaf senescence phenotype appearing on the rosettes of the UbQ-ATG8a and UBQ-ATG8g plants grown under high nitrate conditions (Supplementary Fig. S6). This weak senescence phenotype could be related to the higher N remobilization efficiency observed in these lines especially.

Altogether, results indicate that under full nitrate conditions, the ATG8 overexpressing lines outperformed in N remobilization from the rosette to the seed, thus improving protein content in the seeds and reducing N waste in the dry remains after harvest.

Discussion

Autophagy in plant is known to play an important role in plant adaptation to the environment and in nitrogen use efficiency (Masclaux-Daubresse et al. 2017;Pottier et al. 2014). Increasing the expression of autophagy genes in crops and model plants was shown to improve plant resistance to drought and N limitation and to increase in some cases plant biomass [START_REF] Bassham | Methods for analysis of autophagy in plants[END_REF]Xia et al. 2012;Sun et al. 2018a;[START_REF] Sun | MdATG18a overexpression improves tolerance to nitrogen deficiency and regulates anthocyanin accumulation through increased autophagy in transgenic apple[END_REF][START_REF] Wang | Ectopic expression of an autophagy-associated MdATG7b gene from apple alters growth and tolerance to nutrient stress in Arabidopsis thaliana[END_REF].

Based on transmission electron microscopy, in conjunction with other autophagy assays, and using several strains of yeast with various amounts of both ATG8 mRNA and its protein product, Klionsky's group found a positive correlation between the ScATG8 protein amount and autophagosome size/autophagy activity. As the PE is the cone-shaped lipid target of ATG8 conjugation, it was postulated that the presence of more ATG8-PE conjugates in the cell could modulate membrane curvature and regulate the size of the autophagosomes [START_REF] Jin | Regulation of autophagy: Modulation of the size and number of autophagosomes[END_REF]Xie et al. 2008;Minina et al. 2018;[START_REF] Avin-Wittenberg | Autophagy-related approaches for improving nutrient use efficiency and crop yield protection[END_REF]. We then decided to test the effect of ATG8 overexpression on plant yield and NRE in Arabidopsis. We choose four transgenic lines overexpressing ATG8a, ATG8e, ATG8f and ATG8g genes under the control of different promoters. These lines were chosen according to several criteria related to their expression levels in old leaves and in seeds (Guiboileau et al. 2013;Avila-Ospina et al. 2014;[START_REF] Di Berardino | Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds[END_REF]) and regarding their regulations by senescence related transcription factors as ATAF1 [START_REF] Garapati | Transcription factor A Activating Factor1 integrates carbon starvation responses with trehalose metabolism[END_REF]. They are in different clusters in the ATG8 phylogenic tree (Doelling et al. 2002).

We found in our Arabidopsis ATG8 overexpressors that all the autophagy genes were up-regulated and that there were more autophagosomal structures than in the control plants. This led us to consider that ATG8 overexpression could also lead to increased autophagosome number. While difficult to correlate, using our plant material that is far more complex than yeast to investigate, we did not observe obvious links between the levels of ATG8 transgene expression and the number of autophagosomal structures or the severity of the plant phenotypes. However, we observed that the effect of ATG8 overexpression was likely more performant when using the UbQ-ATG8a and UbQ-ATG8g than the Actin-ATG8f or 35S-ATG8e. Whether this feature was related to the nature of the promoter or to the nature of the ATG8 sequence cloned (genomic DNA or CDS; different ATG8 isoforms) in the construct remains to be determined.

Recently, Minina et al. (2018) showed that overexpressing ATG5 or ATG7 genes in Arabidopsis resulted in remarkable phenotypes on plant growth, seed production and ageing, although exclusively under low light intensity conditions. In our case, and maybe because we are in different growth conditions, we did not observe any remarkable macroscopic phenotype, just a weak early leaf senescence on UbQ-ATG8a and UbQ-ATG8g

rosettes. However, monitoring nitrogen fluxes at the whole plant level revealed very interesting phenotypes related to nitrogen management.

By monitoring NRE traits in nineteen Arabidopsis accessions, Masclaux-Daubresse and Chardon (2011) have

shown that N remobilization to the seeds is highly increased when plants are grown under low nitrate conditions.

Under low nitrate nutrition, the N remobilization capacity of the Col accession represents 50% to 60% of the 15 N absorbed by the plants at vegetative stage while only 20-25% under full nitrate conditions. Such discrepancy explains why the negative effect of the deleterious atg mutations on nitrogen remobilization was so high under low nitrate conditions and much moderate under full nitrate conditions (Guiboileau et al. 2012). Considering from these observations that the nitrogen remobilization capacity of the plant is optimal because it is highly stimulated under low nitrate conditions, it seems then difficult to be able to improve genetically the performance of wild type plants for N-remobilization under low nitrate conditions.

Like many enzymes involved in N remobilization, the expression of the autophagy genes is strongly increased by nitrate limitation [START_REF] Seay | Something Old, Something New: Plant Innate Immunity and Autophagy[END_REF]Xia et al. 2011;Avila-Ospina et al. 2016). For that reason, the overexpression of ATG8 in plants in which autophagy is already overstimulated by nitrate limitation, did not produce any remarkable effect on their performance to remobilize nitrogen. At the reverse, a strong effect was observed under full nitrate conditions in which autophagy is generally kept at a basal level and in which the progress margin is real. Improvement of NRE under full nitrate conditions was more especially observed in the new UbQ-ATG8a and UBQ-ATG8g transformants constructed for this study, and that also outperformed for seed N% and had less nitrogen leftover in their dry remains (Fig. 6C). It is then conceivable that the choice of promoters or of genomic sequences, that could include in intron regulatory sequences for plant engineering, is important. Such feature that is well known from breeders, is discussed in many reviews dealing with N use efficiency and could be the reason of the differences observed here between genotypes [START_REF] Li | Nitrogen use efficiency in crops: lessons from Arabidopsis and rice[END_REF][START_REF] Brauer | Nitrogen use efficiency: re-consideration of the bioengineering approach[END_REF].

The combination of traits as -higher NRE, higher seed N% and lower dry remain N%-is actually highly demanded by breeders for the cereal agriculture performances. Indeed, cereal grains need to be rich in proteins,

and their stubbles poor in N, especially when dedicated to biofuel [START_REF] Chardon | Manipulating NUE in Arabidopsis and crop plants to improve yield and seed quality[END_REF]. Decreasing N in the dry remains is highly recommended also for oilseed rape that is one of the most wasteful plant regarding N, due to the high N content left in dead leaves in the field [START_REF] Malagoli | Dynamics of nitrogen uptake and mobilization in fieldgrown winter oilseed rape (Brassica napus) from stem extension to harvest[END_REF].

In conclusion, our study shows that the overexpression of ATG8 genes provides beneficial effects on nitrogen management at the whole plant level without affecting yield or plant biomass. Selecting plants with higher ATG8 expression levels through plant engineering and/or marker-assisted breeding could be useful for cereals. The beneficial effects, obtained upon nitrate fertilization, are actually well adapted to the concrete agricultural practices used in many countries. Improving NRE as a prerequisite to mitigate fertilizer over use, may be achieved with ATG8 overexpressors.

Materials and Methods

Growth conditions

For N uptake and N remobilization assays, plants were grown on sand according to Masclaux-Daubresse and Chardon (2011) For RT-qPCR and western blots plants grown for 56 DAS under short days were harvested and stored at -80 °C.

For plant grown on plates, seeds were surface sterilized, chilled at 4 °C for 2 d, and then sown on solid MS/2 (Duchefa Biochemie B.V., Haarlem, The Netherlands) and grown for 7 or 18 days in cabinets under long day conditions (16-h-light/8-h-dark, 22/17 °C day/night).

For concanamycin A (CA; Sigma-Aldrich, Saint-Quentin Fallavier, France) treatment, 1-week-old seedlings grown on MS/2 plates were transferred to MES buffer (3 mM, pH 5.6) containing 1 µM CA (solved in DMSO)

or the same volume of DMSO (v/v = 1/100) as a solvent control and kept in the dark for 16 h. The seedlings were then collected in liquid nitrogen and stored at -80 °C for western blot analyses.

52

Plant material

The ATG8 overexpressors (35S-ATG8e, Actin-ATG8f, UbQ-ATG8a and UbQ-ATG8g) were compared to the control line (Prom35S::GFP). The Actin-ATG8f overexpressor corresponds to the transformant

RNA purification and RT-qPCR analysis

Total RNA was isolated from frozen plant tissue with Trizol reagent (Ambion) according to the manufacturer's instructions. cDNA synthesis and RT-qPCR were performed on 4 biological replicates as described by Di [START_REF] Di Berardino | Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds[END_REF]. Primers are listed in Supplementary Table S1. GAPDH and APC2 were used and combined as synthetic reference gene for the calculation of relative expressions.

N-labeling and tracing

For N remobilization assay, pulse 15 N-labeling was performed at the vegetative stage (42 DAS), a long time before flowering according to Masclaux-Daubresse and Chardon (2011). Plants were labelled with 15 N by watering for 24 h with nitrate solutions (10 mM or 2 mM nitrate) containing 10 atom% excess of 15 NO 3 -.

Afterwards, the pots containing sand were rinsed at least 5 times with deionized water to remove the remaining 15 N thoroughly. Unlabelled N solution was then applied regularly as before labelling for the rest of the culture cycle until harvest. At maturity, when plant is completely dry, shoots were harvested and separated into seeds, stem (including siliques and cauline leaves), and rosette to measure dry weight, nitrogen, carbon and 15 N concentrations. Roots of dry plants were not taken into account further as previous experiments performed in our laboratory have shown that the root system that can be collected at harvest represents very few biomass (2% of the total plant biomass) and does not contain more than 2% of nitrogen. For N uptake, plants were labelled at 56 DAS for 24 h precisely with solutions containing 10 atom% excess of 15 NO 3 -. Just after labelling, plants were harvested and fresh root and shoot harvested, dried and weighted, then samples were submitted to 15 N and N determination using IRMS as for remobilization.

Determination of total nitrogen, carbon, and 15 N abundance

Collected samples were dried and weighed to determine the dry weight (DW) of each part of the plant (seeds, stem and rosette). Then the samples were grounded into a fine powder. A subsample of each powder (0.800 to 1.200 mg) was assayed for total N and C contents and 15 N abundance using the FLASH 2000 Organic Elemental Analyzer coupled to Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific, Villebon, France). The N and C concentrations (N% and C%) of each sample were expressed as mg (of nitrogen or carbon) per 100 mg DW.

The 15 N abundance was calculated as atom% (percent of total N) and defined as Asample% = 100 × ( 15 N)/( 15 N + 14 N). The 15 N natural abundance value (Acontrol%) was 0.3684 in this study. 15 N enrichment (E%) was calculated as Asample% -Acontrol%. The absolute quantity of 15 N in each sample was defined as Q = (DW × N%) × E%. 15 N concentration was then calculated as the Q/DW and expressed as µg 15 N per g DW.

Indicators used to monitor NRE were calculated as described by Guiboileau et al. (2012). The dry matter partitioning in seeds, stem and rosette was calculated as % of whole plant (DW organ /DW whole plant ). The 15 N partitioning in seeds, stem and rosette was calculated as % of whole plant (µg of 15 N organ /µg of 15 N whole plant ).

Indicators for N uptake were calculated as described by [START_REF] Chardon | Natural variation of nitrate uptake and nitrogen use efficiency in Arabidopsis thaliana cultivated with limiting and ample nitrogen supply[END_REF].

Protein separation, gel electrophoresis, and Western blot

Total proteins extraction and the determination of protein concentrations were performed as described by Havé et al., (2018) for rosette material and as Di Berardino et al. ( 2018) for seeds. For NBR1, 5 µg of proteins were separated on 12% acrylamide SDS-PAGE. For 12S storage protein, proteins were extracted from same mass of seeds (2 mg DW in 80 µL of buffer) and same volume (2.25 µL in each lane) was separated on 12% acrylamide SDS-PAGE. Gels were electroblotted to polyvinylidene difluoride (PVDF) membranes using Trans-Blot Turbo transfer system (Bio-Rad, marne la Coquette, France) or stained using Coomassie Brilliant blue.

The anti-NBR1 antibody was kindly provided by T. Johansen (Svenning et al. 2011), and the 12S antibody was kindly provided by Dr. Sabine d'Andréa (IJPB, INRA Versailles, France). Antibodies were used at 1:1000 dilution in 3% milk PBS-T 0.1% buffer. Peroxidase-conjugated secondary antibody (1:10000 dilution) was used for chemiluminescence detection using an ImageQuant LAS 4000 (GE Healthcare Life Science, Velizy-Villacoublay, France). The membranes were then stained with Coomassie Brilliant Blue to verify that an equal amount of protein was loaded in each lane.

Microscopy conditions

The primary roots of 1-week-old vertically grown plants were mounted in water and then observed by fluorescence and DIC microscopy according to [START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF].

Statistical analysis

The significance of all the data was determined using Student's t-test (Excel software). XLSTAT ANOVA Newman-Keuls (SNK) comparisons were also performed for the phenotypic data.

Figures

Fig. 1. ATG genes are overexpressed in the rosettes of ATG8 overexpressors grown under short days and high nitrate conditions for 56 days. Plants were grown on sand under short days (8-h-light/16-h-dark) under full N nutrition (10 mM nitrate) for 56 d after sowing and harvested then just before flowering. Transcript levels of the ATG genes involved in autophagosome formation and NBR1 were monitored using RT-qPCR and specific primers (Supplementary Table S1). All genes were normalized using GAPDH and APC2 as synthetic reference gene calculating Ct value as √(GAPDH × APC2). Data are the mean of the values of four biological replicates (histograms are presented as Supplementary Figure S3) and are presented as heat map fold change (log 2 ) according to the scale bar. Genes are grouped according to their functional roles, as indicated on the right. Table S1. Primers used for RT-qPCR analyses. Plants were grown on MS/2 plates with 1% sucrose for 18 days. Transcript levels of ATG genes were monitored using RT-qPCR. All genes were normalized using GAPDH and APC2 as synthetic reference gene calculating Ct value as √(GAPDH APC2). Relative expression level is represented as heat map fold change (log 2 ). Significant differences between control and ATG8 overexpressors are indicated according to Student's t-test, *P<0.05, **P<0.01, ***P<0.001.

Introduction

Recent advance shows that autophagy is not only involved in the recycling of nitrogen from cargo degradation, but also involved in the recycling and remobilization of micronutrients and of lipids (Pottier et al., 2014) Havé et al. 2019. A more recent study of Arabidopsis found that the efficiency of iron (Fe) translocation from vegetative organs to the seeds was severely decreased in the atg5-1 mutants compared with WT (Pottier et al., 2019). Authors further showed that the translocation of zinc (Zn) and manganese (Mn) to the seeds was also dependent on autophagy. These results are consistent with the observation that autophagy was induced under Zn limitating conditions, and that autophagy-deficient mutants (atg5-4, atg10-1) showed an early senescence phenotype under Zn limitation and limited growth recovery when Zn is resupplied (Eguchi et al., 2017).

In many areas of the world, undernutrition and micronutrient deficiencies (particularly for Fe and Zn) are widespread problems and account for 3 million child deaths each year [START_REF] Murgia | Biofortification for combating 'hidden hunger' for iron[END_REF]. The low quantity of micronutrients in most staple food crops is one of the main causes of micronutrient deficiencies, especially for the people living in poverty with a monotonous diet composed mainly of milled cereal grains [START_REF] Sperotto | Iron biofortification in rice: It's a long way to the top[END_REF]. Crop biofortification for essential micronutrients by conventional breeding or by genetic engineering has long been considered as a sustainable and low-cost way to provide micronutrients to people in developing countries to reduce the 'hidden undernutrition' caused by micronutrient deficiencies [START_REF] Murgia | Biofortification for combating 'hidden hunger' for iron[END_REF].

In this context, it would be interesting to investigate whether the overexpression of autophagy in Arabidopsis could improve other nutrients utilization and seed enrichment as nitrogen. We measured the concentration of other nutrients (with an emphasis on metal nutrients) using the same 15 N labelled materiel of the two ATG8 overexpressors UbQ-ATG8a and UbQ-ATG8g, and the control line described in the paper above, in which the UbQ-ATG8a and UbQ-ATG8g displayed stronger phenotype than the two other overexpressors. The analysis of calcium (Ca), magnesium (Mg), Fe, Mn, copper (Cu), and Zn concentrations was performed as described by Pottier et al. (2019). The content and partitioning of each nutrients were calculated as nitrogen in the paper above. Analyses were conducted on plants grown in the LN and HN conditions described before.

Results and discussion

Effect of autophagy overexpression on micronutrient partitioning and concentrations

Results showed that globally the overexpression of ATG8 had limited effect on micronutrients utilization. The partitioning of Cu was decreased in the stem and significantly overexpressors. Manipulation of autophagy may therefore represent a feasible transgenic approach to modify the seed nutritional value of micronutrients in addition to nitrogen, and for some toxic metal to decrease seed contamination.

HERE A SCHEME to summarize the most significant effects

Effect of N nutrition on micronutrient partitioning and concentrations (Table 1 of partition)

In addition, we found some interesting effects of N nutrition on the partition or absorption of other nutrients, irrespective of genotypes. In general, the partition pattern was similar between HN and LN for all the nutrients. Micronutrient partitioning was usually higher in the stem than in the rosette and the seeds. Micronutrient partitioning somehow followed dry weight partitioning (Fig. AB orange). This was verified for all the micronutrient monitored except for Zn under HN conditions whose partition was much higher in the seeds than in the stem and the rosette. Under HN conditions, the proportion of Zn in the seeds was almost 60%, whereas for other nutrient it did not exceeded more than 40%. Interestingly Zn partition in the rosette was similar under LN and HN accounting for about 20% of the total Zn content. This suggests that high N nutrition promoted the mobilization of Zn from the stem to the seeds. Increasing N supply may affect the abundance of Zn-chelating nitrogenous compounds or Zn transporters that facilitate the chelation and translocation of Zn in the phloem tissue.

The increased seed protein content under HN conditions may also facilitate Zn accumulation by enlarging the seed storage capacity for Zn. It was shown that N applications improved Zn concentration in wheat grains under adequate Zn condition, and a significant positive correlation was found between grain concentrations of Zn and N [START_REF] Kutman | Biofortification of Durum Wheat with Zinc Through Soil and Foliar Applications of Nitrogen[END_REF]. Further protein and Zn staining studies showed that Zn was co-localized with protein in the embryo and aleurone parts of grain. On the other hand, LN stress decreased total plant biomass by 2-fold (Table 2). While only the whole plant content of Fe and Cu was decreased for 2-fold as biomass in LN condition, suggesting that the concentration of these micronutrients in plant was unchanged by LN and HN conditions (Table 2). Plant Ca and Mn concentrations were much lower in LN condition, resulting in a much higher decrease of Ca and Mn contents (6-fold and 3-fold, respectively) than biomass. Interestingly, the content of Mg and Zn in plant was almost unchanged under low N stress, as their concentration was increased for 2-fold and counteracts the decrease of biomass. Thus, N nutrition plays a positive role for the absorption and assimilation of Ca and Mn while a negative role for Mg and Zn, and has no effect on Fe and Cu. The different responses to N nutrition indicate that the effect of N on other nutrient management seems to be specific and may involve distinct physiological and molecular mechanisms that remain to be elucidated by further investigations. If we consider only about the seed, under low N condition, the concentration of Ca and Mn was decreased, Mg was increased and unchanged in the case of Fe, Zn, and Cu (Table 2). This result suggests that the improvement of Ca and Mn concentrations in the seed could be achieved by the application of N fertilizer. modulating the levels of key regulators, removing cellular debris and replenishing essential nutrients to support new growth (Dikic, 2017;Masclaux-Daubresse et al., 2017). Autophagy entails encapsulation of unwanted cytosolic materials within double membrane vesicles named autophagosomes, which subsequently deliver their cargoes to the vacuole for hydrolysis and proteolysis (Li and Vierstra, 2012). During this process, more than 40

AuTophaGy-related (ATG) genes are involved. The TARGET OF RAPAMYCIN (TOR)

negative regulator of autophagy is active under nutrient-rich conditions and dampens autophagy by hyperphosphorylating ATG13, which prevents its association with ATG1 [START_REF] Suttangkakul | The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis[END_REF]Dobrenel et al., 2016). Under nutrient-limiting conditions, inactivation of TOR leads to rapid dephosphorylation of ATG13, permitting the binding of ATG1. Thus, ATG1, ATG13, together with two accessory proteins, ATG11 and ATG101, assemble into an active complex (Li et al., 2014). The activated ATG1-ATG13 complex then promotes the nucleation and expansion of a cup-shaped double-membrane (phagophore), which is thought to originate from the endoplasmic reticulum (ER) (Le [START_REF] Bars | ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants[END_REF]Zhuang et al., 2017Zhuang et al., , 2018)). The transmembrane protein ATG9 recruits lipids for phagophore elongation, ATG2 and ATG18 proteins facilitate ATG9 cycling (Xiong et al., 2005;Zhuang et al., 2017). Another step involves phagophore decoration with phosphatidylinositol-3phosphate (PI3P) generated by a class III complex containing the phosphatidylinositol-3kinase (PI3K) encoded by VACUOLAR PROTEIN SORTING 34 (VPS34), along with three core accessory subunits, ATG6, VPS38 or ATG14, and VPS15 (Feng et al., 2014;Liu et al., 2018).

Expansion and closure of the phagophore membranes require two ubiquitination-like systems. Ubiquitin-fold protein ATG8 is initially processed by a cysteine protease ATG4 to expose a C-terminal glycine (Yoshimoto et al., 2004;Woo et al., 2014), then conjugated to the lipid phosphatidyl ethanolamine (PE) by the conjugating enzyme ATG3 (Thompson et al., 2005). Another ubiquitin-fold protein ATG12 is conjugated to ATG5 by the conjugating enzyme ATG10 (Phillips et al., 2008). Both of the conjugation systems share a single ATPdependent activating enzyme ATG7 (Doelling et al., 2002). The ATG12-ATG5 conjugate promotes the lipidation of ATG8 with PE and its anchorage into the phagophore membrane (Chung et al., 2010). ATG8 decoration of the phagophore membrane facilitates the recruitment and seal of the cargoes inside the autophagosome. ATG4 is also needed to remove and recycle ATG8 from ATG8-PE lining the outer membrane (Yoshimoto et al., 2004), while the ATG8-PE adducts trapped on the autophagosome inner membrane are digested in the vacuole. Among all these genes, ATG1, ATG13, ATG18, ATG4, ATG12, and ATG8 are family genes, consist of multiple isoforms.

Although autophagy was originally considered as an unrestricted bulk degradation of cytoplasm compounds, recent studies reveal that various routes for selective autophagy exist.

Selective autophagy can specifically degrade appropriate cargo by engaging a wide array of receptors or adaptor proteins that tether the cargo and also interact with ATG8 for the recruitment of the cargo to autophagic membranes (Floyd et al., 2012;Li and Vierstra, 2012;Farré and Subramani, 2016;Wang et al., 2018). The interaction between autophagic receptors and ATG8 is mediated by the presence of ATG8-interacting motif (AIM) in each receptor (Noda et al., 2010). Recently, a new binding site for autophagy adaptors and receptors was discovered on ATG8. This site engages ubiquitin-interacting motif (UIM)-like sequences rather than the canonical AIM for high-affinity binding to a new class of ATG8 interactors (Marshall et al., 2019). In plants, some adaptor proteins have been identified. For example, the Arabidopsis NBR1 (NEIGHBOR OF BRCA1 GENE 1) (also known as Joka2 in tobacco) targets ubiquitinated protein aggregates formed under stress conditions through a C-terminal ubiquitin-associated (UBA) domain (Svenning et al., 2011;Zientara-Rytter et al., 2011).

ATG8-INTERACTING PROTEIN (ATI) 1/ATI2 play a role in the degradation of stromal and thylakoid proteins during carbon starvation or salt stress (Honig et al., 2012;Michaeli et al., 2014). Guiboileau et al. (2012) firstly demonstrated the participation of autophagy in N remobilization from leaves into the seeds in Arabidopsis monitoring the fluxes of 15 N to the seeds after plant labelling at vegetative stage. Authors showed that N remobilization was sharply decreased in all the atg mutants studied (atg18a RNAi, atg5, and atg9) compared to wild type (WT) under N-limited conditions. Decrease was more moderate but still significant in atg5 under nitrate-rich conditions. Accordingly, authors further found that atg mutants accumulated more ammonium, amino acids (AA), proteins, and RNA contents, but less sugars and starch in the rosette leaves than WT (Guiboileau et al., 2013). The higher N/C ratio of atg rosette leaves was mainly due to the over accumulation of all sort of nitrogen compounds. Similar defect of N remobilization was reported in maize and rice using atg12

and Osatg7-1 mutants, respectively (Li et al., 2015;Wada et al., 2015).

In order to identify other key ATG genes involved in the bulk or selective autophagy leading to nitrogen remobilization, we collected a large number of atg mutants. We paid a special attention to the atg8a-i mutants as they could reveal as specific role of ATG8 proteins in the targeting of cargoes especially dedicated to N remobilization. In addition, we investigated the roles of adaptors as NBR1 and ATI1/ATI2 in N-remobilization as they also participate in selective autophagy. The atg5-1 mutant previously studied by Guiboileau et al. (2012) was used as control.

The atg11, atg7, atg10, atg4a4b, atg12a12b, ati1, ati1/ati2, nbr1 mutants and those belonging to ATG8, ATG1, ATG13 and ATG18 families were collected and subjected to 15 N labelling experiments. We found that ATG5, ATG7, ATG10, ATG8h, ATG18a, ATG4, and ATG12 are key players in autophagy machinery, the disruption of these proteins impaired plant N remobilization process and caused hypersensitivity to low N stress. In addition, ATI1 contributes to N remobilization only under high N condition.

Materials and Methods

Growth conditions

For N remobilization assays, plants were grown on sand according to Masclaux-Daubresse and Chardon (2011) in growth chamber under low nitrogen conditions (2 mM nitrate) or full nitrogen conditions (10 mM nitrate). Plants were cultivated in short days (8-h-light/16-hdark; 21/17 °C day/night; 160 µmol.m -2 .s -1 ) and labelled with 15 N for 24 h at 42 days after sowing (DAS). After two more weeks in short day condition, plants were transferred to long days (16-h-light/8-h-dark; 21/17 °C day/night; 160 µmol.m -2 .s -1 ) at 56 DAS to induce flowering. Plants were kept in long days until seed maturity.

Indicators used to evaluate NRE were calculated as described by Guiboileau et al. (2012).

The partitioning of dry matter in stem, rosette, and seeds was calculated as % of whole plant (DW organ /DW whole plant ). In the same manner, the partitioning of C, N, and 15 N in stem, rosette, and seeds was calculated as % of whole plant. Indicators for nitrogen utilization efficiency (NUtE) include grain N utilization efficiency (grainNUtE) defined as seed DW/total aboveground N and total N utilization efficiency (plantNUtE) defined as total above-ground DW/total above-ground N [START_REF] Le Gouis | Genetic differences for nitrogen uptake and nitrogen utilisation efficiencies in winter wheat[END_REF].

Statistical analysis

The significance of all the data was determined using Student's t-test (Excel software).

Results

Selection of homozygous atg8a-i T-DNA insertion mutants

All the atg mutants we obtained from ABRC (Arabidopsis Biological Resource Center, Table 1) have been reported as homozygous mutant by the resource center. As we pay special attention to the atg8 mutants, we decided to genotype and verify they are homozygous and knock out mutants. The Arabidopsis ATG8 proteins are encoded by nine genes (ATG8a-i) located on the chromosomes 1, 2, 3, and 4. We identified eight atg8 T-DNA insertion mutants from ABRC stock (Figure 1A). There is one allele for ATG8a, ATG8c, ATG8d, ATG8f, ATG8h, ATG8i, and two alleles for ATG8g, which were designated as atg8a, atg8c, atg8d, atg8f, atg8h, atg8i, atg8g-1, and atg8g-2, respectively. No mutant could be found for ATG8b and ATG8e. PCR analyses confirmed the T-DNA insertion in the exon or intron for all of them.

Sequencing ??? To further determine whether all native ATG8 transcripts were absent in the insertion mutants, RT-PCR analyses were performed using gene-specific primers that span the T-DNA insertion to amplify the full-length ORFs. Results confirmed the absence of fulllength ATG8 mRNA in all of the respective atg8 mutants (Figure 1B). We conclude that these alleles represent knock out mutations. by the sequencing of the T-DNA borders…..

Genotyping of NBR1 (Figure 1C,D)

Phenotypes of autophagy mutants, nbr1 mutants and ati1/2 mutants We screened all the mutants in low N condition to elucidate their role in leaf senescence and N remobilization (Figure 2). We confirmed that atg5-1, atg7, atg10, atg4a4b, atg12a12b, and atg18a plants show strong early senescence phenotype, and that their rosette size is smaller compared to Col. Interestingly, in this experiment, among all the atg8 mutants, the atg8h exhibited slightly early senescence phenotype, but had similar rosette size with Col. The phenotype of atg8h mutants was reproduced at least three times under low nitrate condition in our laboratory.

N remobilization and plant performance of atg mutants

In agriculture, crop performance is evaluated measuring grain yield (g of grains per ha or per plant) and harvest index ratio (HI; g of grain .g -1 of the whole plant) and nitrogen use efficiency can be evaluated monitoring nitrogen harvest index ratio (NHI; mg N in seeds. mg - 1 N in the whole plant). Grain quality evaluates the protein, lipid and starch concentrations in seeds.

In our study we have chosen as plant biomass indicators to consider the dry weight of rosettes, stems and seeds and the harvest index (DW partition in seeds). For nitrogen use efficiency we choose to evaluate NHI, the N remobilization efficiency ( 15 N partition in seeds)

and the nitrogen utilization efficiency (grainNUtE and plantNUtE). We estimated grain quality measuring N% and C% (mg C or N .(100 mg) -1 DW).

It has been reported that atg5, atg9 and atg18a mutants and RNAi lines are highly impaired in N remobilization to the seeds (Guiboileau et al., 2012). We measured N remobilization in atg1, atg11, atg13, atg5-1, atg7, atg10, atg4a4b, atg12a12b and atg8 mutants to confirm such phenotype in other mutants from the autophagy core machinery and to explore the behaviour of mutants in genes belonging to multigenic families. In addition, we separated the dry remains into stems and rosettes in order to determine better where nitrogen flux to the seeds is blocked. The remarkable difference in N remobilization found in atg5-1 relative to WT was observed in the atg7, atg10 single and atg4a4b, atg12a12b double mutants in which we also observed early yellowing phenotypes (Figures 2). Globally, all these mutants had lower biomass but higher N concentration in the different plant parts, and for some lower C% in their rosettes and stems. Compared to WT, the partitioning of DW, C, N, and 15 N was significantly higher in their rosettes and lower in their seeds, while not different in their stems (Figure 3). This indicated that the defect of mobilization was originating from the management of the protein sources in rosettes but not from the stem tissues mainly in charge of nutrient mobilization in phloem saps. Due to the bad N remobilization efficiency and to the higher N% and lower DW, the NUtE of these five mutants was largely decreased compared to the WT.

Generally, the phenotypes of the single mutants from the atg1, atg11, and atg13 families were not different from WT, suggesting that these genes could compensate each other without impacting rosette phenotype nor N-remobilization efficiency.

In the case of the atg18 mutants that also belong to large gene family that includes eight members (ATG18a-h), only the atg18a mutant displayed the same strong phenotypes that characteristic of the atg single mutants presented above. This included the lower biomass, the higher N% in stem, rosette, and seeds (Figure 4) and the lower N and 15 N in seeds that show defects in N remobilization to the seeds. Accordingly, the NUtE was also largely reduced in atg18a plants.

No phenotype has been reported so far for the Arabidopsis atg8 mutants in literature although ATG8 protein has been widely studied as it represents the best marker to monitor autophagy activity. In this study, we found that only atg8h displayed some phenotype as the early senescence phenotype observed in low N although less severe as in other well-known mutants like atg5-1 and atg7 (Figure 5). Our investigations using 15 N tracing then revealed that atg8h had a lower rosette DW and higher N% (Figure 5). The partitioning of DW and C was lower in rosette and higher in the seeds for atg8h compared with Col (Figures 5).

Although no difference was found for N partitioning in atg8h plants, their 15 N partitioning was significantly increased in rosette and decreased in the seeds. Moreover, atg8h mutants had a lower plant NUtE. Therefore, although less dramatic than in atg single gene mutants, atg8h presented all the characteristics of autophagy defective lines.

N remobilization of nbr1 and ati mutants

In addition to the autophagy mutants, we also screened mutants affected in the autophagy receptors as the nbr1 in low N condition, ati1/2 mutants in both low and high N conditions.

There was absolutely no phenotype for both leaf senescence and N utilization in any of the nbr1 mutants (Figure 6).

The ati1 single and ati1/ati2 double mutants did not exhibit any interesting phenotype under low N conditions (Figures 7; Supplemental Figure S1). Under high N conditions, both ati1 and ati1/ati2 had lower C% and higher N% in stem, rosette, and seeds (Figure 8). NHI and 15 N partitioning in seeds were lower in ati1 and ati1/ati2 suggesting defects in N allocation to the seeds. Due to their high N% compared to WT, both grainNUtE and plantNUtE was lower in ati1 and ati1/ati2 mutants. The lower NUE observed in ati1 and ati1/ati2 under high N suggest that these mutants suffer from other limiting factors. As their C% is lower than WT, we hypothesize that these mutants have lower photosynthesis capacity that WT. This could explain why, although not significant, the DW of ati1 and ati1/ati2 tend to be lower than that of WT. Overall, results show that phenotypes of ati1 and ati1/ati2 that are mainly observed under high N are different from those observed in atg5.

Discussion

In Arabidopsis, remarkable phenotypes were observed when autophagy-defective plants are grown under nutrient deficient conditions such as nitrogen or fixed carbon starvation; in these conditions, mutants missing the core autophagy machinery components (e.g., ATG7, ATG5, ATG9, ATG18a) show slow growth, enhanced leaf senescence, lower fecundity, and reduced survival rate (Doelling et al., 2002;Hanaoka et al., 2002;Thompson et al., 2005;Xiong et al., 2005). Similarly, we also observed that atg5-1, atg7, atg4a4b, and atg18a mutants are hypersensitive to low N stress, with accelerated leaf senescence and smaller rosette size (Figure 2). These phenotypes were also observed in the atg12a12b and atg10 mutants.

Interestingly, we confirmed that all the mutants that display the typical senescence phenotypes were also defective in N remobilization.

We performed 15 N labelling experiments, as conducted previously in our laboratory by Guiboileau et al. (2012), to monitor N remobilization to the seeds. Consistent with the early senescence phenotype, atg5-1, atg7, atg10, atg8h, atg18a single and atg4a4b, atg12a12b

double mutants were all impaired in N remobilization. Although a higher N concentration in different plant parts including the seeds in these mutants, their 15 N partitioning was significantly increased in rosette and decreased in the seeds, resulted in a lower NUE. These results are consistent with previous findings reported by Guiboileau et al. (2012), in which N remobilization was sharply decreased in all the atg mutants studied (atg18a RNAi, atg5, and atg9) compared to wild type (WT) under N-limited conditions. In fact, we used the same atg5-1 mutant allele as them for a positive control.

These strong phenotypes in mutants confirmed they are key autophagy genes that were shown to be essential for the formation of autophagosomes and the function of the whole autophagy process. For instance, it was found for a long time that autophagosomes could not be observed in atg5, atg7 and atg4a4b, while few were found in in the roots of atg18a RNAi lines and atg9 mutants that displayed less severe phenotypes than that of atg5 for example (Yoshimoto et al., 2004;Thompson et al., 2005;Xiong et al., 2005). By contrast, the absence of phenotype for the mutation of other autophagy genes may due to the fact that they are either less important or family genes encoded by multiple isoforms with a compensatory effect. Therefore, screening the phenotypes of mutants in multigenic families under Nlimitation should led to the identification of the genes involved in autophagy and essential for N remobilization.

We confirmed here the results from Guiboileau et al. (2012), showing the participation of ATG18a in N remobilization by using another mutant allele in our study. The absence of phenotype in the other atg18 show that the other members of the ATG18 family are not essential for N-remobilization. Whether these genes, that have been identified through sequence homologies with the yeast ScATG18, are actually involved in autophagy machinery remains an open question.

Regarding genes from multigenic families, the fact that the atg8h mutant displayed slight early senescence phenotype and defects in N remobilization indicate that ATG8h may have a more specific and important role than the other ATG8 in targeting substrates for Nremobilization during leaf senescence and under low N conditions. As we only had one KO mutant for ATG8h, complementation of this mutant will be carried on in order to confirm our hypothesis. The use of ATG8h as a key gene to improve NRE as we did previously (Chen et al. 2018) might be considered.

Overall, our results indicate the important role of ATG5, ATG7, ATG10, ATG4, ATG18a and ATG12 in N remobilization process and reveal a specific role of ATG8h in the same process.

Previous functional analysis using two nbr1 knockout mutants revealed that (i) NBR1 is important for plant tolerance to a large spectrum of abiotic stresses, like heat, oxidative, salt, and drought stresses, and (ii) there is an increased accumulation of ubiquitinated insoluble proteins in nbr1 mutants under heat stress (Zhou et al., 2013). However, unlike atg5 and atg7 mutants, nbr1 is not sensitive to darkness stress or necrotrophic pathogen attack. In the present study, the phenotype of the three homozygous nbr1 mutants we isolated was not different from Col in limited N condition, suggesting that NBR1 is not essential for Nremobilization.

Although no difference of leaf senescence was observed in both ati1 single and ati1/ati2

double mutants compared to Col, we found that the rosette size of ati1/ati2 double mutants was smaller than Col under both low and high nitrate conditions during vegetative growth period (data not shown). This phenotype was recovered along with time and there was no difference in biomass at the end of plant cycle. In high N condition, ati1 and ati1/ati2 mutants had lower C% and higher N% in stem, rosette, and seeds. It seems then that ati1 and ati1/ati2

mutants displayed the same phenotype as autophagy mutants but specifically under high nitrate. The N and 15 N partitioning was higher in the vegetative tissues but less in the seeds, suggesting that these mutants were impaired for N remobilization but under high nitrate especially. Such defects resulted in a lower plantNUtE and grainNUtE in ati1 and ati1/ati2

mutants. These results suggest the role of ATI1 in N remobilization under N favourable conditions. As there was no independent ati2 null mutant allele and the double mutant developed symptoms similar to but not more severe than the single mutant, we were unable to verify whether ATI2 contributes to N remobilization. Since the discovery of ATI1 and ATI2 by (Michaeli et al., 2014), other ATI has been found.

Conclusion

In this study, we screened a large number of mutants involved in the key components in both bulk and selective autophagy in low N condition to investigate their role in environment adaptation and N remobilization. Our results showed that ATG5, ATG7, ATG10, ATG8h, ATG18a, ATG4, and ATG12 are key players in autophagy machinery, the disruption of these proteins impaired plant N remobilization process and caused hypersensitivity to low N stress.

In addition, ATI1 contributes to N remobilization only under high N condition. The investigation in individually darkened leaves of Arabidopsis revealed an increased activity of many PLCPs, among which RD21A and AALP were identified as the dominant active PLCPs in dark-induced senescing leaves [START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]. Authors used several knock-out mutants of senescence-induced PLCPs (rd21A-1, aalp-1, sag12-1, ctb3-1, rd21A-1/aalp-1, and ctb1/ctb2/ctb3) to determine the role of individual protease activities in senescence and only found a slight but significant delayed progression of the whole plant senescence in aalp-1 and rd21A-1/aalp-1 double mutants, suggesting a role for AALP in the process of plant senescence.

RD19A, RD21A, and CATHB were found to play a determinative role in regulating pathogen defense, either a pro-survival or a pro-death function upon the infection by various pathogens. For example, Arabidopsis rd21A null mutants were significantly more susceptible to the necrotrophic fungal pathogen Botrytis cinerea [START_REF] Shindo | A Role in Immunity for Arabidopsis Cysteine Protease RD21, the Ortholog of the Tomato Immune Protease C14[END_REF]. The rd21A mutants exhibited significant increased cell death when treated with the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme. This indicated that RD21A behaves as a negative regulator of necrotrophic pathogen-induced cell death in Arabidopsis and may be rather involved in a protection mechanism to necrotrophic pathogen [START_REF] Ormancey | Sphingolipid-induced cell death in Arabidopsis is negatively regulated by the papain-like cysteine protease RD21[END_REF]. However, a pro-death function of RD21A was reported in another study, where detached leaves of rd21A Arabidopsis mutants and plants with AtSerpin1

(inhibitor of RD21A activity) over-expression were more resistant to the two necrotrophic fungi Botrytis cinerea and Sclerotina sclerotiorum as they displayed a significant reduction in cell death (Lampl et al., 2013).

The importance of autophagy in nutrient remobilization (N and other microelements) has been demonstrated using 15 N and 57 Fe tracing and elemental analyses on Arabidopsis atg5, atg9 and atg18a mutants (Guiboileau et al., 2012;Pottier et al., 2019). Although both autophagy and PLCPs are key players during leaf senescence, protein proteolysis, and nutrient recycling, the relationship between them remains largely unknown. Proteins are probably not degraded directly inside the autophagosomes but rather transported by them to the lytic vacuoles where proteases and hydrolases operate. Previous results in our laboratory showed that protease activities were significantly increased in the leaves of atg mutants with increased protein accumulation and impaired N remobilization efficiency (NRE) compared to WT (Guiboileau et al., 2013). It was hypothesized that vacuole proteases and their cytoplasmic substrates cannot co-localize in the absence of autophagosome trafficking.

Argentina). The homozygous double mutant plants (sag12/atg5-2, cathB3-1/atg5-2, and rd21A-1/atg5-2) were selected by PCR in F2 progeny after F1 self-pollination.

Plant growth conditions, methods for 15 N labelling and nutrients determination were same as the paper 1. Except that the double mutant plants were grown on compost, and labelled by pipetting 2 ml nitrate solutions (2 mM nitrate) containing 10 atom% excess of 15 NO 3 -per plant at the plant-soil interface. Pots were not rinsed but supplied normal N solution regularly after labelling until harvest.

Statistical analysis

Statistical analysis was performed by comparing the single protease mutants with Col, and the double mutants with atg5-2 using Student's t-test (Excel software).

Results

N remobilization of sag12 and sag12/atg5-2 mutants

When grown under low N condition, the rosette size and fresh weight measured at vegetative stage were higher in the sag12 single mutant and the sag12/atg5-2 double mutants by comparison to Col and atg5-2, respectively (HN). When measured at harvest, we also found that the rosette and the stem biomasses were higher in sag12 compared to Col. This was observed under both low and high N conditions (Figures 2A and3A). Irrespective of the N conditions, sag12 and sag12/atg5-2 mutants did not exhibit any special phenotypes regarding N utilization compared to Col and atg5-2, respectively (Figures 1 and2). Only the 15 N partitioning was significantly increased in the rosette of sag12 mutants under low N condition compared with Col, and the partitioning in the seeds was slightly but not significantly decreased (Figure 2E).

N remobilization in cathB3, rd21A, serpin1 single mutants and Serpin1-HA overexpressor

Before investigating the cooperation of autophagy and proteases for N remobilization, the phenotype of proteases single mutants was investigated. At vegetative stage, the rosette size and fresh weight of cathB3-1 and rd21A-1 mutants were increased compared with Col under low N condition (HN, Figure 1). At harvest, the rosette biomass was significantly increased in cathB3-1 mutants compared to Col under low N condition; the rosette and stem biomasses of cathB3-1, rd21A-1 mutants and Serpin1-HA overexpressor lines were also increased under high N condition (Figures 4A and5A). Thus like for the sag12 mutant it seems that suppressing PLCP activities could increase plant growth.

The seed dry weight of rd21A-2 under low N and cathB3-2 under high N was lower than that of Col (Figures 4A and5A). The rd21A-2 had higher N% in the rosette, and the partitioning of N and 15 N was higher in the vegetative tissues and lower in the seeds compared with Col under low N condition (Figures 4B, 4C, and4D). As a result, the grain N utilization efficiency was significantly reduced in rd21A-2 mutants under low N condition (Figure 4E).

Under high N condition, difference was only found for the serpin1 mutant, in which its N% was lower in the stem (Figure 5B). The partitioning of N in the seeds was significantly increased in serpin1 mutants, resulted in an increased N utilization efficiency (Figures 5C and5E). These results suggested that RD21A is involved in N remobilization. However differences between single mutants and WT were weak. It can also be noticed that the different mutant alleles of CATHB3 did not give similar results and that results were also different for the two lines of the rd21A mutant originating from ABRC and Pr. R. Fluhr laboratory.

N remobilization in cathB3-1/atg5-2 and rd21A-1/atg5-2 double mutants

To further investigate the relationship between protease and autophagy in N remobilization, we monitored N remobilization in double mutants under low N condition. We found that the rosette size and fresh weight were increased in cathB3/atg5-2 mutants compared to atg5-2 mutants at the vegetative stage. From 15 N tracing and mobilization, different phenotype from atg mutant was only found in rd21A-1/atg5-2. The biomass of stem, rosette and seeds was increased in rd21A-1/atg5-2 compared with atg5-2 single mutant (Figure 6A). The N% was higher in the seeds of rd21A-1/atg5-2 (Figure 6B). The partitioning of DW and N was similar between rd21A-1/atg5-2 and atg5-2 mutants, however the 15 N partitioning was much lower in the seeds of rd21A-1/atg5-2 mutant than in atg5-2 (Figures 6C, 6D, and6E). Decrease in plant N utilization efficiency was observed in rd21A-1/atg5-2 relative to atg5-2 (Figure 6F).

Therefore, mutation in RD21A is likely to increase autophagy mutant defects for N remobilization and NUtE suggesting that RD21A is involved in N-remobilization in an autophagy-independent way.

Discussion

In our study, the lack of SAG12 protein did not lead to significant differences in seed yield.

However we noticed a significant increase of rosette weight irrespective of the nitrogen regime (low and high nitrate) that has not been reported before but no noticeable differences in leaf senescence, which was in agreement with previous studies [START_REF] Otegui | Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean[END_REF][START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]James et al., 2018). Our 15 N remobilization assay did not show any difference between sag12 and Col, and between sag12/atg5-2 and atg5-2. The absence of 15 N remobilization from the shoot to the seeds confirmed the results obtained by James et al. (2018James et al. ( , 2019) ) that demonstrated the predominant role of SAG12 in N-remobilization from the roots while the role of SAG12 in N mobilization from the shoot was likely compensated by alternative proteolytic activities. Using a different labelling procedure than us that consisted in transferring plants grown under hydroponics from high to low nitrogen conditions, they showed that the yield and N content of the seeds were decreased in the Arabidopsis sag12 mutants relative to the wild type under nitrogen starvation. The accumulation of 15 N in the roots and the lower partitioning of 15 N to the seeds in sag12 mutants under LN conditions were consistent with the significant decrease of cysteine protease activity in root and the presence of SAG12 in the root stele at the reproductive stage, particularly under LN condition.

In our study, bigger rosettes were also observed in the cathB3-1 and rd21A-1 mutants. Only rd21A-2 presented interesting phenotype that suggested that RD21A was involved in N utilization efficiency and N-remobilization to the seeds. Such assumption was consistent with results obtained on the RD21A inhibitor serpin1 mutant, in which the partitioning of N in the seeds and N utilization efficiency were by contrast significantly increased, although only under high N condition. The role of RD21A in N remobilization might however be revisited as the rd21A-1 did not show similar phenotype as rd21A-2. Also different labelling method as used by James et al. ( 2019) might be assayed to try and reveal higher difference compared to wild type.

Using double mutants, we found that the 15 N partitioning in the seeds and total N utilization efficiency were significantly decreased in rd21A-1/atg5-2 mutant compared with atg5-2 single mutant. This then strengthened our previous assumption about the role of RD21A in N-remobilization and led us to conclude that RD21A could play a role to compensate the lack of N remobilization in autophagy mutants by providing alternative degradation pathway.

As suggested by James et al. (2018), the absence of phenotype in sag12 and cathB3 mutants could be due to compensatory effect by other protease activities. PLCPs genes belong to a large multigenic family. In plants, there are approximately 723 PLCPs, with 31 genes found in Arabidopsis, which are classified into 9 subfamilies based on phylogenetic analysis and conserved structural features [START_REF] Beers | The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis[END_REF][START_REF] Richau | Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics[END_REF]. [START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF] demonstrated that cysteine proteases play redundant roles during leaf senescence and are able to substitute for each other. For instance, it was reported that the three Arabidopsis CATHB homologues (AtCATHB1-3) act in a functionally redundant manner for full basal resistance against the virulent hemibiotrophic bacteria Pseudomonas syringae pv. tomato (Pst) DC3000

( McLellan et al., 2009). Furthermore, only the triple cathB mutants exhibited a significant delay in dark-induced senescence, which confirmed that the three CATHB genes act redundantly in leaf senescence. Interestingly, it was found that aspartate protease activity is significantly increased in sag12 mutant plants grown under optimal nitrogen conditions (James et al., 2018). Among the putative aspartate proteases involved, a CND41-like aspartate protease AED1 (APOPLASTIC ENHANCED DISEASE SUSCEPTIBILITY-DEPENDENT 1) was then identified and proposed to provide compensatory effects to cope with the absence of SAG12 activity in senescing leaves of sag12 mutants.

Conclusion and perspectives

Although this study suggests a role of RD21A in N remobilization that co-operates with autophagy pathway, the effect of RD21A mutation is weak. It is then possible that the depletion of the proteolytic activity in the single sag12, cathB3, and rd21A mutants are compensated by other protease activities. Such hypotheses need to be confirmed by further protease activity screening, especially in cathB3 and rd21A mutants and by monitoring N remobilization using multiple mutations and proteinaceous (cystatin protein) or chemical (E-64) protease inhibitors. overexpression in abiotic stresses tolerance and agronomic performance including the N remobilization and grain filling in barley.

Materials and Methods

Plant material and treatments

The barley (Hordeum vulgare L.) cultivar 'Golden Promise' was used as the recipient of the HvATG5-overexpressing transformation in this study. Three independent lines (L20, L10, and L18) of HvATG5 overexpressor were compared with two different control lines (C1 and C2), which were null segregants lacking the transgene.

For N remobilization assays, barley plants were grown in greenhouse conditions and supplemented with artificial illumination when daylight was below 10 klx to ensure long day photoperiod (day/night cycle of 16/8 h). Plants were cultivated in small pots of compost and supplied a standard nutrient solution (10 mM nitrate) for 35 days until 15 N labelling. After labelling, plants were supplied with 2 mM nitrate solution until harvest, which represent a low nitrate supply. Temperature and humidity were controlled by passive ventilation.

For phenotyping experiments under stress at seedling stage, plants were grown in small pots (7 x 7 x 6.5 cm) filled with medium particle size sand in a growth chamber with long day

Construction of plant-overexpressing binary vector for HvATG5

The NIAS Hv2006J04 clone containing the complete CDS of HvATG5 was kindly provided by Pr. Fuminori Kobayashi (NIAS Plant Genome Research Unit, Ibaraki, Japan). HvATG5

CDS was amplified by PCR and recombined into the pDONR207 (Invitrogen) entry vector using Gateway technology (Thermo Fisher Scientific). The insert in pDONR-HvATG5 was then introduced into the destination binary vector pIPKb002 (kindly provided by Dr B. Leps, IPK, Germany; [START_REF] Himmelbach | A Set of Modular Binary Vectors for Transformation of Cereals[END_REF]) by LR recombination. CDS in this vector was driven by the maize ubiquitin (Ubi-1) promoter and the hygromycin phosphotransferase (hpt)

gene was carried as a selectable marker in plants. Sequencing-verified plasmid was transformed to Agrobacterium tumefaciens (strain AGL0) by electroporation for subsequent barley transformation.

Genetic transformation of barley and homozygote selection

The Agrobacterium-mediated transformation was performed by inoculating barley 'Golden Promise' immature embryos [START_REF] Holme | Cisgenic barley with improved phytase activity[END_REF]. Regenerated lines were screened by real time qPCR on genomic DNA for the presence and the copy number of the transgene.

Genomic DNA was isolated from barley leaf samples using the procedure of Edwards et al.

( 1991). The presence of the transgene was detected by qPCR using specific forward primer

(5'-GGAAGGAGGGAG/GTAGAAGC-3') distributing in the end of the 7 th exon for the first part and in the beginning of the 8 th exon for the second part (separated by an intron) that can specifically amplify the transgene but not the native HvATG5. The reverse primer (5'-GGGCTCGCTGGAGGTGGTTT-3') was normal and distributed totally in the middle of 8 th exon of HvATG5. The reference gene HvGAPDH was used for normalization and determination of the transgene copy numbers. The qPCR was performed same as RT-qPCR described below except that the template was 50 ng genomic DNA instead of the cDNA. The null segregants from the transgenic lines that were negative in the screening tests of the transgene were designated null-transgenic and used as controls in the following experiments.

RNA extraction and RT-qPCR analysis

Total RNA was extracted from frozen leaves using Trizol reagent (Ambion) according to the manufacturer's instructions. cDNA synthesis and RT-qPCR were performed with four biological replicates as described by Chen et al. (2019). HvGAPDH and HvActin were used and combined as synthetic reference gene to calculate the relative expression level (Avila-Ospina et al., 2016). Primers are listed in Supplementary Table S1.

Phenotyping of transgenic barley lines under stress conditions 123

Before harvesting the plant materials, chlorophyll contents (SPAD) were measured on the 1 st and 2 nd leaves in all the stress conditions except dark recovery, and on the 3 rd leaf for the dark and LS treatments. Measurements were made on three independent leaf groups corresponding to 12 plants per genotype. SPAD values were recorded at the tip, mid-point, and base of the leaf, and values were averaged for each leaf of each group. The plant height (for all the stress conditions) and the 3 rd leaf length (only for HN and LS) were also measured to monitor plant development.

At each harvesting time, three independent groups containing 12 plant shoots in total were harvested and separated into old leaf blades (1 st and 2 nd leaves), young leaf blades (the remaining leaves), and leaf sheaths. Fresh weight of each plant part was determined, then samples were stored at -80 ℃ for further experiments. For the dark recovery experiment, shoot fresh weight and plant height were determined, then shoots were harvested without separating in different parts.

Statistical analysis

The significance of all the data was determined by comparing the three independent lines with the two control lines (combined together) using Student's t-test (Excel software).

Results

recovered much better than the control, and exhibited significantly higher plant height and shoot fresh weight (Figure 4C).

Overexpression of HvATG5 improves N remobilization and plant productivity

In this experiment, plants were grown under N limiting conditions and labelled at vegetative stage to follow N-remobilization to their seeds. Interestingly, while no difference in plant vegetative biomass could be observed at maturity, the seed yield (seed DW; Figure 5A) of the HvATG5 overexpressors (L10 and L18) was significantly increased compared to the control plants. Accordingly, DW partitions in leaves and stem were significantly decreased and harvest index of HvATG5 overexpressors was significantly improved (Figure 5B). Nitrogen concetration (N%) was not significantly modified in the HvATG5 overexpressors relative to control, except a weak and significant increase in L18 stem (Figure 5C). Then N partitioning was slightly different in overexpressors relative to controls and the L10 and L18 lines presented significantly higher N harvest index than controls (NHI; Figure 5D). The partitioning of 15 N in leaves, stem and seeds showed a significant decrease of 15 N allocation in the leaves and stem and a significant increase of the allocation of 15 N in the seeds of the three HvATG5 overexpressing lines (Figure 5E). Consequently, the seed N utilization efficiency (NUtE), calculated as the ratio of seed DW/total N in plant, was increased in HvATG5 overexpressing lines (Figure 5F). Furthermore, the better seed yield in HvATG5 overexpressors was attributed to the increase of seed number per tiller and not to the tiller number (Figure 6A). Single seed DW was not changed in HvATG5 overexpressors, except weakly higher in L20 (Figure 6B). Altogether, these results indicate that overexpressing HvATG5 improves N remobilization and plant productivity in barley.

Discussion

It was recently shown that the overexpression of autophagy AtATG5 in Arabidopsis enhances plant tolerance to biotic and abiotic stresses (Minina et al., 2018). Our study demonstrates the role of HvATG5 in barley tolerance to N deficiency, S deficiency and dark stress. In addition we show that the overexpression of HvATG5 promotes plant development and increases vegetative biomass under high nitrate supply. Plant recovery after dark stress treatment at seedling stage is also remarkably improved in overexpressors. Finally the higher N-remobilization efficiency in HvATG5 overexpressors that is paralleled by higher seed yield and higher harvest index demonstrates the role of autophagy in nutrient recycling and mobilization in crops.

The enhanced tolerance to abiotic stress conditions in HvATG5 overexpressing lines at seedling stage is probably due to the improved cleavage and recycling of accumulated toxic and damaged macromolecules rather than to higher nutrient recycling. At this stage, the role conditions. The reason of this could be that in plants, the expression level of ATG5 genes is invariably very low by comparison to that of the various ATG8 isoforms. Then the ATG5 expression level might remain limiting for autophagy activity even under low N conditions that induces the expressionof all the autophagy genes including that of ATG5. While the simple stimulation of ATG8 expression under low N is enough to reach the best autophagy performance that ensure the optimal N recycling and N mobilization capacities of the plant, we can suppose that increasing the expression level of HvATG5 by 18-30 fold is not still enough to obtain the best N remobilization efficiency in barley. Further experiments are ongoing to explore whether improved N remobilization in HvATG5 overexpressors can also be reached under high N conditions.

Conclusion and perspectives

Measuring autophagic activity in plants is very complex as it necessitate the use of Physiology in Germany involving several restriction enzymes (Yan et al., 2016). I performed the Crispr-Cas9 construction in the laboratory of another collaborator, Dr Salma Balazadeh, as they have all the related high fidelity restriction enzymes from same company and more importantly these enzymes work efficiently in a single buffer, which will greatly facilitate the completion of the work. With the help from one of their PhD student, Venkatesh Periyakavanam Thirumalai Kumar, I introduced all the nine gRNAs in one construct in three weeks to mutate all the ATG8 family genes.

As mentioned above, the Arabidopsis atg8 single mutants have no special phenotypes except atg8h. The screening of all the atg8 single mutants in different stress conditions is actually a lot of work. Pr Pyung Ok Lim, another collaborator at Daegu Gyeongbuk Institute of Science and Technology in Korea, developed a phenomic approach to perform high-throughput and computational study of leaf senescence (Lyu et al., 2017). I went to her laboratory for one month last year to screen the phenotype of all the atg8 single mutants and ATG8 overexpressors in hormone-induced leaf senescence using the high-throughput phenotyping In addition, I participated in three international conferences during the last four years, including the 8th and 9th International Symposium on Plant Senescence and the workshop on Plant Autophagy: Improving Crop and Energy Production. These conferences gave me

Figure 1 .

 1 Figure 1. Whole-plant senescence (a) and leaf senescence (b) characteristics in

Figure 2 .

 2 Figure 2. A model summarized the potential regulatory factors during leaf senescence.

Figure 3 .

 3 Figure 3. A working model summarized the degradation pathways of chloroplast proteins during leaf senescence. Basically, chloroplast proteins can be degraded through three different pathways: (A), senescence-associated vacuoles (SAVs); (B), autophagydependent pathways; (C), the CV-containing vesicles (CCVs) pathway. Figure from Xie et al.

Figure 4 .

 4 Figure 4. A working model summarized the key enzymes involved in nitrogen remobilization in senescing leaves. Senescence-associated vacuole (SAV); Glutamate dehydrogenase (GDH); Cytosolic glutamine synthetase (GS1); Asparagine synthetase (AS); Asparagine (ASN); Glutamine (GLN); Glutamate (GLU). Figure adapted from Masclaux-Daubresse et al. (2010).

Figure 5 .

 5 Figure 5. Morphological steps during microautophagy and macroautophagy in plants. ER, endoplasmic reticulum; PAS, pre-autophagosomal structure; A, protein aggregate; C, chloroplast; M, mitochondrion; P, peroxisome; VP, vacuole protein. Figure from Marshall and Vierstra (2018).

Figure 6 .

 6 Figure 6. Schematic representation of the core autophagy machinery in plants. (A) Genes in green color are family genes, genes in black are single genes. (B) Proteins in gray color (ATG17, ATG29, ATG32, ATG14, and ATG16) participate to the autophagosome formation in yeast, but have not been identified in plants. Figure from Masclaux-Daubresse et al. (2017).

Figure 7 .

 7 Figure 7. Selective autophagy pathways reported in plants. Specific stress conditions and mutants used to study each type of specific autophagy are noted. Figure from Masclaux-Daubresse et al. (2017).

Figure 8 .

 8 Figure 8. Nomenclature and subclassification of Arabidopsis PLCPs. The 9 subfamilies of PLCPs are marked in the left column, followed by the gene accession codes and the putative PLCPs encoded. The domain structure consists of an N-terminal signal peptide (sp), pro-domain (pro), protease domain with catalytic Cys (c), and in some cases a C-terminal Pro-rich domain (p) and a granulin domain. Figure adapted from Richau et al. (2012).

Figure 9 .

 9 Figure 9. Schematic representation of the proteases and protease-related proteins differentially accumulated in autophagy mutants. The predicted cellular localization of

  PromActin::GFP::ATG8f generated by Di[START_REF] Di Berardino | Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds[END_REF]. The 35S-ATG8e overexpressor corresponds to the Prom35S::RFP::ATG8e plants generated by[START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF]. The UbQ-ATG8a and UbQ-ATG8g GFP::ATG8g constructs in Col Arabidopsis wild type. The PromUBQ10::GFP::ATG8a and PromUBQ10::GFP::ATG8g were generated by cloning the ATG8a and ATG8g coding DNA plus introns (from ATG to STOP) in the pDONR207 (Invitrogen, Carlsbad, California, USA) entry vector and transferred in the pUBN-Dest GFP[START_REF] Grefen | A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies[END_REF] using the Gateway ® technology (ThermoFisher Scientific, Waltham, Massachusetts, USA). Plant transformations were performed via Agrobacterium tumefaciens (GV3101::pMP90) using the floral dipping. The Prom35S::GFP correspond to plants transformed by the same pGWB6 empty vector (GATEWAY binary vector, Invitrogen, Carlsbad, California, USA) as used by[START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF] to generate the Prom35S::RFP::ATG8e constructs.

Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 2345 Fig. 2. Overexpression of ATG8 stimulates the formation of autophagosomes and increases autophagic flux in seedlings.A-B: NBR1 transcript levels (A) and NBR1 protein contents (B) were estimated in the ATG8e, ATG8f, ATG8a and ATG8g overexpressor lines by comparison to control on seedlings grown in vitro for 18 d on MS/2 medium. RT-qPCR were performed and expression levels were normalized using GAPDH and APC2 as synthetic reference gene calculating Ct value as √(GAPDH × APC2) and represented as 2 -ΔΔCt . Significant differences between control and ATG8 overexpressors are indicated according to Student's t-test, *P<0.05, **P<0.01, ***P<0.001. For western blot, 5 µg of total proteins were separated on 12% SDS-PAGE gels and detected using NBR1-specific antibody. Rubisco large sub-unit (RBCL) bands identified on Coomassie Brilliant Blue-stained blot provide loading and blotting controls. C: Autophagic flux in ATG8-overexpressing lines was estimated by monitoring NBR1 protein level in 7 d old seedlings grown on MS/2 medium in vitro and transferred to MES buffer containing 1 µM concanamycin A (CA) dissolved in DMSO, or only DMSO as solvent control, for 16 h in the dark. Western blots were performed as indicated for B. D: Autophagic flux was also estimated in seedling roots by monitoring the number of autophagic bodies by DIC microscopy on the different genotypes. Representative images of the epidermal cells of the primary roots of control and UbQ-ATG8a lines are presented. Seedlings were grown on vertical plates containing MS/2 medium for 7d. Roots were mounted in water and then observed by DIC microscopy. Note that no CA was used in this experiment. Insets show enlargement of indicated boxes. Bars = 10 µm for main figure and 5 µm for insets. E: The number of autophagic bodies as observed in D was quantified in the epidermal root cells of each genotype. The number of autophagosomal structures per image was converted per µm 2 . Five roots were imaged per genotype and three non-consecutive optical sections were examined for each root. Error bars indicate standard deviations. The means of each genotype were compared with the control using Student's t-test, *P<0.05, **P<0.01, ***P<0.001.

Fig. 6 .

 6 Fig. 6. Nitrogen remobilization efficiency (NRE) is increased in the ATG8 overexpressor lines. Plants were grown on sand under full nitrogen nutrition as described for Figure 4. The partitioning of dry weight (DW; A) and the partitioning of 15 N (B) in the rosette, stem, and seeds were computed as in Figure 3. Means and standard deviations of six biological replicates are shown. Both Student's t-test (*P<0.05, **P<0.01, ***P<0.001) and XLSTAT ANOVA Newman-Keuls (SNK; P<0.05) comparison were performed and statistically significant differences are shown. Two independent experiments were performed showing similar results. (C) The

  Fig. S1. Expression level of GFP, RFP and of ATG8 mRNA in Arabidopsis overexpressors.Plants were grown on MS/2 1% sucrose solid medium for 18 days. A-B. Transcript levels of GFP (A) and RFP (B) were monitored using RT-qPCR. The control line only expresses free GFP. The 35S-ATG8e line carries RFP-ATG8 fusion while all the other lines have GFP-ATG8 constructs. Relative expression level is represented as 2 -ΔCt . C. Transcript levels of ATG8e, ATG8f, ATG8a, and ATG8g were also monitored using RT-qPCR. They cumulate the expression of the native ATG8 genes and of transgenes. Relative expression level is represented as 2 -ΔΔCt . Means and standard deviations of four biological replicates are shown. Significant differences between control and ATG8 overexpressors are indicated according to Student's t-test, *P<0.05, **P<0.01, ***P<0.001. Genes were normalized using GAPDH and APC2 as synthetic reference gene calculating Ct value as √(GAPDH APC2).

Fig. S3 .

 S3 Fig. S3. Histograms of ATG gene expressions in the rosette of ATG8 overexpressors at 56 DAS. The same data set was presented in Figure 1 using heat map. (A) Expression level of ATG genes other than ATG8 and NBR1. (B) Expression level of ATG8 genes/transgenes in the ATG8 overexpressors at 56 days after sowing (DAS). All genes were normalized using GAPDH and APC2 as synthetic reference gene calculating Ct value as √(GAPDH APC2) and representing relative expression level as 2 -ΔΔCt . Means and standard deviations of four biological replicates are shown. Significant differences between control and ATG8 overexpressors are indicated according to Student's ttest, *P<0.05, **P<0.01, ***P<0.001.

Fig. S6 .

 S6 Fig. S6. Slight early senescence phenotype of UbQ-ATG8a and UbQ-ATG8g lines. Rosettes of control and ATG8 overexpressors grown under short day and high nitrate conditions were harvested 56 days after sowing. Slight early leaf senescence was observed on the UbQ-ATG8a and UbQ-ATG8g lines. Phenotype was observed on several (n>3) independent plant cultures.

  increased in the seeds in ATG8 overexpressors compared with control under both low and high nitrate conditions (Figure A,B). This impacted on the Cu concentration in seeds that was significantly increased in the ATG8 overexpressing lines but only under LN condition (Figure C,D). Positive effects were also found for Zn under LN condition only, in which the Zn partitioning was decreased in rosette and stem, while increased in the seeds of the ATG8 overexpressors (Figure E,F). Zn concentration was accordingly decreased in the stem and increased in the seeds of ATG8 overexpressing plants under LN (Figures G,H). Although there was no difference for the partitioning of other nutrients between ATG8 overexpressors and control (Figure I,J,M,N,Q,R,U,V), the concentration of Fe in seeds was significantly increased under LN (Figure I), and Mn concentration was slightly decreased in the stem and the seeds of the ATG8 overexpressors under LN condition (Figure P). There was no modification of the Mg and Ca concentrations in the seeds of any of the two ATG8

  referred to as autophagy) is essential for intracellular recycling and plays an essential role in the control of cellular homeostasis and reprograming, by

Figure 1 .Figure 2 .Figure 5 .

 125 Figure 1. Gen etic descr iption of Ar abidopsis atg8 T-DNA insertion mutants. (A) Gene diagram of the Arabi dopsis ATG 8 loc us showing the ex on/i ntron or ganization and the positions of at g8 T-DNA inserti on mutations generat ed with GSDS2.0 (Gene Struc ture Displ ay Server; http://gsds.c bi.pk u.edu.cn/). Lines represent i ntrons and the r ectangl es filled with or without black c olor indic ate c odi ng and untranslated regions , r espectively. (B) RT-PCR analysis of the ex pression of ATG 8 genes i n wild type (Col) and T-DNA insertional m utants. Total cellular RNA was isolated from wild type, atg8a, atg8c, at g8d, at g8f, at g8g-1, at g8g-2, atg8h, and atg8i plants and s ubjec ted to RT-PCR. RT-PCR was perf ormed using gene-specific primers to amplify the full-length ORFs with two biological replicates . (C) Gene di agram of the Arabidopsis NBR1 loc us showing the exon/intron organiz ation and the positions of nbr1 T-DNA insertion mutations. (D) Western blots performed usi ng the NBR1 antibody on the three nbr1 mutants , atg5 mutant and Col wild type.

Figure 6 .Figure 8 .

 68 Figure 6. Nitrogen use efficiency of nbr1 mutants.

  Figure 2. N remobilization in sag12 single and sag12/atg5-2 double mutants under low nitrate condition.

  photoperiod and controlled humidity and temperature (25 ℃/17 ℃, day/night). Day light intensity was 200 µE m -2 s -1 . Plants were supplied with 5 mM nitrate solution for the high nitrate (HN) and dark treatments, 0.5 mM nitrate solution for the low nitrate (LN) treatment, and 5 mM nitrate solution lacking sulphur for the low sulphur (LS) treatment. Plants were harvested at 17 days after sowing (DAS) for HN and LN treatments, and 23 DAS for LS treatment. For the dark treatment, 14 d old plants were completely deprived of light for 4 days and harvested, another group of plants were kept in dark for 5 more days and then transferred back to the normal condition with light for a 16 d recovery period (the dark recovery treatment). During the dark treatment, nutrient solution was supplied regularly as before. There were 12 plants per genotype in three independent groups for each treatment.

  15 N labelling and samples collectionPulse 15 N labelling was performed at the vegetative stage (35 DAS), before the emergence of spikes. Each plant was watered 8 mL of 10 mM nitrate solutions containing 10 atom% excess of15 NO 3 -at the plant-soil interface for 6 times during 2 weeks. Watering of plants was completed using deionized water instead of nutrient solutions when needed to avoid drought during these 2 weeks. After labelling, plants were kept in nitrate-starved condition until harvest by providing 2 mM nitrate solution to ensure all the 15 N will be absorbed before flowering and to induce the process of N remobilization. At maturity, when plants were completely dry, plant height, tiller number, and seed number per tiller were recorded. Then shoots were harvested and separated into seeds, leaves, and stem (including leaf sheath, peduncle, and awn) to measure dry weight (DW), C, N, and 15 N concentrations. The methods for the measurements and calculations were same as in Chen et al. 2019.

  of autophagy is mainly preserving cell longevity by removing cell waste. It cannot be excluded that N acquisition could be improved by overexpressing HvATG5 in response to N deficiency. Determination of nutrient concentrations in the harvested samples of seedlings could clarify this point. The fact that plant recovery after dark stress treatment is faster in overexpressors is also certainly due to the positive effect of autophagy activity on organelle quality control during stress treatment and also to faster nutrient recycling during recovery period.At the late reproductive stage, the effect of HvATG5 overexpression on 15 N partitioning demonstrates the role of autophagy at whole plant level in nutrient recycling and remobilization from senescent source organs to sink. As a result of their higher N remobilization capacities under N-limiting conditions, barley transformants produced more seeds per tiller without modifying seed quality. If we compare this result with recent findings of Chen et al. (2019) in Arabidopsis and of[START_REF] Yu | Increased Autophagy of Rice Can Increase Yield and Nitrogen Use Efficiency (NUE)[END_REF] in rice, we can conclude that our barley transformant behave differently. While these reports showed a positive effect of the overexpression of ATG8 on plant performances under normal N conditions only, we show here in barley that the positive effect of the HvATG5 overexpression is obtained under low N

Figure 1 .Figure 4 .

 14 Figure 1. HvATG5 is highly expressed in the transgenic lines.The HvATG5 expression level was measured using qRT-PCR and specific primers on ???? Of the L20, L10 and L18 transfgenic lines and on the null-transgenic segregating control lines. Expression was normalized ... Mean and SD of n=? are shown ....

  platform. With a lot of help from a post-doc, Su Jin Park, I detached the 3 weeks old 3 rd /4 th leaf of atg8 mutants and incubated them in different senescence inducer solutions (Mock, H 2 O 2 , jasmonic acid, and salicylic acid). Then I transferred the leaves to the platform to monitor senescence progression by 4 different types of cameras (fluorescence, RGB, infrared, and hyperspectral imaging) during one week. I also learned how to convert the images to numeric data to facilitate the following analysis. I learned a lot of techniques that are essential to finish my PhD thesis work from the visiting of different laboratories. During the stay, I attended their group meetings, department or institute scientific meetings and talked with their PhD students and post-docs. I learned different types of laboratory organization and supervision of the students. The professors and their students are always very kind and accommodating both in my experiments and daily life. The discussion about what they are working on also enriched my scientific knowledge background that may helpful in the future. I harvested sincere friendship with many people from different countries and knew more about their different cultures in the world.

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RESULTS 1

Overexpression of ATG8 in Arabidopsis stimulates autophagic activity and increases nitrogen remobilization efficiency and grain filling Running Title:

  Increasing autophagy for better recyclingQinwu Chen 1 , Fabienne Soulay 1 , Baptiste Saudemont 1 , Taline Elmayan 1 , Anne Marmagne 1 and Céline Masclaux-Daubresse 1*

  For N uptake, plants were harvested at 56 days after 24 h of 15 N labelling. For N remobilization, plants were labelled earlier (42 days after sowing; DAS) and transferred to long days (16-h-light/8-h-dark; 21/17 °C day/night; 160 µmol.m -2 .s -1 ) at 56 DAS to induce flowering. Plants were kept in long days until seed maturity.

in growth chamber under full nitrogen conditions (10 mM nitrate) and low nitrogen conditions (2 mM nitrate). Plants were cultivated in short days (8-h-light/16-h-dark; 21/17 °C day/night; 160 µmol.m -2 .s -1 ) for 56 days.

uptake at vegetative stage is not different from control line in ATG8 overexpressors

	Control	35S-ATG8e	Actin-ATG8f
	Control	UbQ-ATG8a	UbQ-ATG8g
					. 15 N labelling
			was performed over 24 hours
			precisely to monitor N uptake. Roots
			and shoots were collected and N and
			15 N content determined. The DW (A),
			total N concentration (B) and 15 N
			concentration (C) were not different
			between	control	and	ATG8
			overexpressors according to XLSTAT
			ANOVA	Newman-Keuls	(SNK;
			P<0.05) comparison.

Table 1 :

 1 Partitioning of micronutrients and DW in rosette, stem and seeds in control plants (wild type) grown under high (10 mM) and low (2 mM) nitrate. Mean and SD of 4-6 plants are shown.

		High N		Low N		High N		Low N		High N		Low N
		Rosette/WP		Rosette/WP		Stem/WP		Stem/WP		Seed/WP		Seed/WP
		mean	SD	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
	Ca	37.2	± 3.0 37.0	± 5.4 56.1	± 2.6 51.4	± 4.1 6.7	± 1.1 11.5	±1.8
	Cu	15.5	± 1.7 15.1	± 3.1 62.3	± 1.0 68.5	± 3.3 22.2	± 2.2 20.8	± 1.1
	Fe	20.7	± 1.9 18.4	± 2.1 51.5	± 4.5 55.3	± 8.3 25.1	± 4.5 23.0	± 4.7
	Mg	16.9	± 2.5 25.8	± 4.7 51.1	± 3.5 42.2	± 3.6 32.0	± 4.8 32.0	± 1.2
	Mn	35.7	± 3.5 26.3	± 4.1 42.0	± 1.9 59.9	± 3.8 22.2	± 2.1 13.8	± 3.4
	Zn	16.9	± 2.8 21.0	± 2.7 23.9	± 2.7 54.8	± 2.6 59.2	± 2.8 26.3	± 3.6
	DW

Table 2 :

 2 Micronutrient concentrations in rosette, stem and seeds of the control plants grown under high and low nitrate conditions. Means and SD of 4-6 plants are shown. Significant differences between HN and LN are indicated by * Overall, overexpressing ATG8 in Arabidopsis improves the partition of Zn and Cu to the seeds and increases seed concentration of Zn, Cu, and Fe under low nitrate condition. N nutrition enhances Zn partition from stem to seeds and provides benefits for the absorption of Ca and Mn.Conclusion

		Rosette				Stem				Seeds
		High N		Low N		High N		Low N		High N	Low N
		mean SD	Mean SD	mean SD	mean SD	mean SD	mean SD
	Ca (mg.g -1 ) 50.1	± 3.7 14.2	± 3.1	23.3	± 2.1	5.4	± 0.7 4.9	± 0.4 2.8	± 0.3
	Cu (µg.g -1 )	8.0	± 0.6 6.7	± 1.7	9.9	± 1.1	8.1	± 1.3 6.2	± 0.5 5.0	± 1.0
	Fe (µg.g -1 )	98.9	± 8.2 76.2	± 15.1 78.9	± 15.3 65.1	± 9.5 67.1	± 7.7 61.2	± 5.4
	Mg (mg.g -1 ) 1.6	± 0.2 4.2	± 0.8	1.5	± 0.2	2.1	± 0.2 1.7	± 0.1 3.3	± 0.1
	Mn (µg.g -1 ) 129.5 ± 8.4 84.6	± 21.9 46.8	± 1.5	52.1	± 3.3 44.0	± 2.1 27.1	± 2.8
	Zn (µg.g -1 )	30.4	± 1.7 67.9	± 12.6 13.4	± 2.1	48.1	± 2.0 58.9	± 2.7 51.6	± 0.9

Table 1 :

 1 List of mutants used in this study.

	original name	Accession No.	Mutant	Mutant ID	Reference	Genotyping
	At Cath B3 KO	At4g01610	cathB3-1	SALK_019630	(McLellan et al., 2009)	from Hazel McLellan
	Cath B3 Salk	At4g01610	cathB3-2	SALK_132724	from ABRC	from ABRC
	SERPIN 1 KO	At1g47710	serpin1	SALK_075994	(Lampl et al., 2010)	from Robert Fluhr
	SERPIN 1 HA ox	At1g47710	Serpin1-HA	over-expressor	(Lampl et al., 2010)	from Robert Fluhr
	RD 21 KO	At1g47128	rd21A-1	SALK_090550	(Lampl et al., 2010)	from Robert Fluhr
	RD 21 Homo	At1g47128	rd21A-2	SALK_090550	from ABRC	certified homozygous
	sag12	At5g45890	sag12	SALK_124030	Homozygous	from Juan Guiamet
	Atg 5.2	At5g17290	atg5-2	SAIL_129B07	(Guiboileau et al., 2012)
	CathB3 x atg5.2		cathB3-1/atg5-2		This work
	RD21 x atg 5.2		rd21A-1/atg5-2		This work
	atg5/sag12		sag12/atg5-2		from Juan Guiamet

Figure 1.

Biomass and rosette size of protease single and double mutants under low nitrate condition.

Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000, Versailles, France * corresponding author Abbreviations: Nitrogen remobilization efficiency (NRE); Autophagy gene (ATG)

Acknowledgements

Authors thank Pr. Kohki Yoshimoto for advices and for providing plant material. Authors thank Joël Talbotec and Philippe Maréchal for taking care of plants.

Funding information

This work was supported by the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS) for IJPB, the China Scholarship Council for Qinwu Chen and the ANR-10-BLAN-1707-01 program for Dr. Baptiste Saudemont.

Accession numbers

At4g21980; At2g45170; At3g60640; At4g16520

original name Accession No. Mutant Mutant ID Reference atg8a1 At4g21980 atg8a SALK_045344C this work atg8c K At1g62040 atg8c SALK_003706 (O) this work atg8d At2g05630 atg8d SALK_131853(BW) this work atg8f1 At4g16520 atg8f SALK_052510.43.10.X this work atg8g At3g60640 atg8g-1 SALK_205553 this work atg8g2 At3g60640 atg8g-2 GK-735H09 this work atg8h2 At3g06420 atg8h SALK_136493.30.25.X this work atg8i1 At3g15580 atg8i SALK_123914.53.50.N this work atg18ak At3g62770 atg18a GK-651D08 atg18b1 At4g30510 atg18b-1 SALK_039939 atg18b2 At4g30510 atg18b-2 SALK_039947.52.50.X atg18c At2g40810 atg18c SALK_088893.49.85.X atg18d At3g56440 atg18d SALK_205978 atg18e At5g05150 atg18e ARR11-2 SALK_013832 atg18f1 At5g54730 atg18f SALK_129486.25.25.X atg18g2 At1g03380 atg18g SALK_000928 atg18h At1g54710 atg18h-1 SALK_005379 atg18hk At1g54710 atg18h-2 SAIL_818_H04 atg1a1 At2g37840 atg1a WISCDSLOX506B07 atg1b1 At3g53930 atg1b-1 SALK_114104.24.40.N atg1b2 At3g53930 atg1b-2 SALK_130294.47.25.X atg1c At3g61960 atg1c SALK_054351.43.45.X atg11.1k At4g30790 atg11 SAIL_1166_G10 atg13a At3g49590 atg13a SALK_065240.50.45.X atg13bprom At3g18770 atg13b SALK_129206 atg5-1 At5g17290 atg5-1 SALK_020601 (Guiboileau et al., 2012) atg7 K At5g45900 atg7 GK-655B06 atg10k At3g07525 atg10 SALK_084434 atg4a4b At2g44140/At3g59950 atg4a4b SAIL_740_H03/? (Yoshimoto et al., 2004) atg12a12bk At1g54210/At3g13970 atg12a12b SAIL_1287_A08 salk_003192 (Q) (CA) nbr1 Gabi1 At4g24690 nbr1-1 GK-246H08 (From Anne) nbr1 Gabi2 At4g24690 nbr1-2 GK-281A10 (exon) nbr1 Salk At4g24690 nbr1-3 SALK 144852C (exon) Ati1KO At2g45980 ati1 SAIL_404_D09 (Honig et al., 2012) Ati1/Ati2KO At2g45980/At4g00355 ati1/ati2 SAIL_404_D09/ATI2-RNAi (Honig et al., 2012) B1, B2, B4 At2g25625 cv Artificial microRNA silencing (Wang and Blumwald, 2014)

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RESULTS 2

Both autophagy and nitrogen nutrition modify micronutrient remobilization and allocation to the seeds in Arabidopsis

RNA extraction and semiquantitative RT-PCR analysis

Total RNA was isolated from frozen seedlings with Trizol reagent (Ambion) according to the manufacturer's instructions and cDNA was synthesized as described by Di Berardino et al.

(2018). RT-PCR was performed as described by XXX (REF) using gene-specific primers to amplify the full-length ORFs with two biological replicates. Primers are listed in Supplementary Table S2.

Western blot

Total protein extraction, protein concentration determination, protein separation and Western blot for NBR1 were performed as described by Chen et al. ( 2019).

N-labeling and tracing

Pulse 15 N-labeling was carried out at the vegetative stage (42 DAS), a long time before flowering according to Masclaux-Daubresse and Chardon (2011). Briefly, plants were watered for 24 h with nitrate solutions (10 mM or 2 mM nitrate) containing 10 atom% excess of 15 NO 3 -. After removing the 15 N solution, the pots were rinsed thoroughly with deionized water (at least 5 times). Normal N solution was then resupplied regularly for the rest of the culture cycle until harvest. At maturity, when plant is completely dry, shoots were harvested and separated into rosette, stem (including siliques and cauline leaves), and seeds to determine the dry weight (DW), nitrogen, carbon, and 15 N concentrations.

Determination of total nitrogen, carbon, and 15 N abundance

Determination of total nitrogen, carbon, and 15 N abundance was performed as described by Chen et al. (2019). Collected samples were dried in oven and weighed to determine the DW.

Then the samples were grounded into fine powder, which was subsampled (0.800 to 1.200 mg) and submitted to the determination of total N and C concentrations and 15 N abundance using the FLASH 2000 Organic Elemental Analyzer coupled to Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific, Villebon, France). The N and C concentrations (N% and C%) were defined as mg (of nitrogen or carbon) per 100 mg DW. The quantity of N and C was then calculated as DW × N% (C%). The 15 N abundance was calculated as atom% (percentage of total N) and defined as Asample% = 100 × ( 15 N)/( 15 N + 14 N). The 15 N natural abundance value (Acontrol%) was 0.3684 in this study. 15 N enrichment (E%) was calculated as Asample% -Acontrol%. The absolute quantity of 15 N in each sample was defined as Q = (DW × N%) × E%.

RESULTS 4

Role of papain-like cysteine proteases in nitrogen remobilization and the relationship with autophagy

Introduction

During leaf senescence, in parallel with the decline of photosynthesis, leaf changes its metabolism from anabolism to catabolism. Given that proteins contain the largest fraction of organic nitrogen, which is potentially available for recycling to support the growth of younger plant parts in stress conditions, the major biochemical event occurring during leaf senescence is protein degradation (Guiboileau et al., 2010). The transcript levels or activity of various proteases are specifically induced in senescing leaves, and papain-like cysteine proteases (PLCPs), mainly accumulated in lytic vacuolar compartments, are amongst the most highly overexpressed. Amongst them are senescence-associated gene 12 (SAG12), Cathepsin B3 (CATHB3), Cathepsin B1, Responsive-to-desiccation 21A (RD21A), RD19A, and Arabidopsis aleurain-like protease (AALP) (Lohman et al., 1994;Yamada et al., 2001;Gepstein et al., 2003;Guo et al., 2004;Havé et al., 2017;[START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF]Havé et al., 2018).

SAG12 exhibits a strictly senescence-associated expression pattern and localizes specifically in the SAVs in senescing leaves (Lohman et al., 1994;[START_REF] Otegui | Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean[END_REF]. The sag12 Arabidopsis mutant did not exhibit any discernible phenotypical alteration during senescence under normal conditions. Whereas, under low nitrogen (LN) availability, decreases in both the seed N content and yield were observed in sag12 mutant, probably due to the fact that inducible proteolytic systems are not sufficient to cope with SAG12 depletion (James et al., 2018). These results suggested that SAG12 could participate in Rubisco degradation and N remobilization during leaf senescence that sustain seed filling. The oilseed rape homologs of RD21A and SAG12 were also reported to play a crucial role in the efficient proteolysis associated with leaf senescence, especially in response to N limitation [START_REF] Desclos | A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation[END_REF][START_REF] Poret | Characterization of senescence-associated protease activities involved in the efficient protein remobilization during leaf senescence of winter oilseed rape[END_REF]. However, an opposite role was reported from the rice homologs of SAG12 that acted as negative regulators of developmental leaf senescence and stress-induced cell death [START_REF] Singh | Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants[END_REF][START_REF] Singh | The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death[END_REF].

Then, Havé et al. (2018) used proteomics shotgun LC-MS/MS to identify the overaccumulated proteases in the leaves of autophagy mutants and specific probes to monitor their activity under both low-and high-nitrate (LN, HN) conditions. Results showed that cysteine proteases accounted for the largest proportion (38%) of the 26 proteases that increased in autophagy-deficient lines. Activity-based protein profiling (ABPP) analysis with DCG-04 revealed that activities of PLCPs were higher in autophagy-defective plants grown under low-nitrate conditions. Further pull-down experiments using DCG-04 in low-nitrate condition showed that the active PLCPs accumulated in autophagy mutants were mainly SAG12, RD21A, CATHB3, and AALP. The western blots performed using the RD21A, CATHB3, and SAG12 antibodies confirmed that both the mature and immature protease forms were accumulated in the mutant lines, suggesting that there was no defect in protease maturation or trafficking in the autophagy mutants. The specific over-accumulation of these PLCPs under LN but not under HN in autophagy impaired plants strongly suggests that they are involved in N remobilization, and possibly provide alternative remobilization pathways to autophagy.

All these studies highlight the ambiguous role of PLCPs in the regulation of plant leaf senescence, protein degradation, and cell death. But whether they are involved in nitrogen remobilization remains to be characterized. In this context, our aim was to clarify their contribution to N remobilization by performing 15 N labelling experiments using several mutants of RD21A, CATHB3, and the Serpin1 inhibitor of RD21A, as well as the Serpin1-HA overexpressing line. Furthermore, these mutants were crossed with the well-known autophagy-defective mutant atg5-2 to evaluate the possible relationship with autophagy pathway.

Materials and Methods

Plant material

In this study, we used one mutant for SAG12 (sag12), two mutants for CATHB3 (cathB3-1, cathB3-2), two different lines of the same RD21A mutant (rd21A-1, rd21A-2), one mutant for Serpin1 (serpin1) and one Serpin1 overexpression line (Serpin1-HA), detailed information were listed in Table 1. To evaluate the relationship between autophagy and protease in N remobilization, we crossed the autophagy-defective mutants atg5-2 with cathB3-1 and rd21A-1; crossed lines with sag12 were provided by Pr. Juan Guiamet (La Plata University, favorable conditions, autophagy occurs at a basal level that mainly serves as housekeeping functions in cellular homeostasis, whereas stimulated autophagy activity facilitates the selective and bulk removal of toxic and damaged components in response to environmental stress conditions including starvation, drought, and pathogen infection [START_REF] Hafrén | Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles[END_REF]Marshall and Vierstra, 2018). Autophagy-defective mutants show very limited phenotype under well-fed conditions. They can easily complete their life cycle and produce viable seeds.

Remarkable phenotypes were observed when autophagy-defective plants are grown under nutrient deficient conditions such as nitrogen or fixed carbon starvation; in these conditions, mutants missing the core autophagy machinery components (e.g., ATG7, ATG5, ATG9, ATG18, ATG12) show slow growth, enhanced leaf senescence, lower fecundity, and reduced survival rate (Doelling et al., 2002;Hanaoka et al., 2002;Thompson et al., 2005;Xiong et al., 2005;Li et al., 2015a). It was also shown that these mutants are more sensitive to salt, drought, and oxidative stresses in both Arabidopsis and rice (Xiong et al., 2007;Liu et al., 2009;[START_REF] Shin | OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice[END_REF].

Conversely, overexpression of AtATG5 or AtATG7 in Arabidopsis increased plant resistance to oxidative stress and necrotrophic pathogens, delayed leaf ageing and enhanced plant growth, seed set, and seed oil content (Minina et al., 2018). Overexpression of MdATG18a in apple improved plant tolerance to drought, nitrogen starvation, and pathogen infection (Sun et al., 2018a[START_REF] Sun | MdATG18a overexpression improves tolerance to nitrogen deficiency and regulates anthocyanin accumulation through increased autophagy in transgenic apple[END_REF](Sun et al., , 2018c)). Although the Arabidopsis atg8 single mutants have no special phenotype, the heterologous expression of ATG8 from other species (such as MdATG8i, SiATG8a, or GmATG8c) in Arabidopsis or rice conferred tolerance to nitrogen starvation, drought, and salt stresses and increased in some cases plant biomass or seed yield (Xia et al., 2012;Li et al., 2015bLi et al., , 2016;;Wang et al., 2016;Luo et al., 2017).

After Guiboileau et al. (2012) firstly demonstrated the participation of autophagy in N remobilization from leaves into the seeds in Arabidopsis using atg mutants (atg18a RNAi, atg5, and atg9), similar investigations were conducted in crops like maize and rice. The study in maize revealed that the growth of atg12 mutants was severely arrested at seedling stage, and plants showed enhanced leaf senescence and stunted ear development under nitrogenstarved conditions but not under high-N (Li et al., 2015a). Under nutrient-rich conditions, seed yield of atg12 plants was much lower, and 15 N reallocation into the seeds was twice less in atg12 than WT, suggesting that N remobilization to the kernels was impaired. The investigation conducted during the vegetative growth period on the rice autophagy-disrupted

Osatg7-1 mutant also suggested that N remobilization from senescent leaves to young leaves was largely decreased (Wada et al., 2015).

Recently, Chen et al. ( 2019) demonstrated that overexpressing AtATG8 genes in Arabidopsis significantly improves nitrogen remobilization efficiency to the seeds under normal N conditions, resulting in an increase of protein concentrations in the seeds and a decrease of the amount of nitrogen lost in the dry remains after harvest. Accordingly, the rice OsATG8a overexpressors presented better yield and increased N use efficiency compared to wild type under normal N conditions [START_REF] Yu | Increased Autophagy of Rice Can Increase Yield and Nitrogen Use Efficiency (NUE)[END_REF].

Taken together, all these studies demonstrate that increasing the expression of autophagy genes can provide benefits for plant performance and adaptation to stress conditions in Arabidopsis probably through boosting autophagic flux. In addition, results obtained using autophagy-defective mutants and overexpressors clearly indicate that autophagy controls N remobilization in both Arabidopsis and crops.

Barley is an important cereal crop for forage and beverage industry, and is a useful model system for wheat. To date a total of 24 autophagy genes have been identified in barley, and the HvATG transcript levels were shown to be increased globally by leaf senescence, nitrogen deficiency and dark stress (Avila-Ospina et al., 2016). The function of these genes still remains to be characterized. In this paper, we selected the HvATG5 as a candidate for functional analysis, as the important role of AtATG5 in N remobilization and adaptation to environment in Arabidopsis is well documented, and because the expression level measured in various plant species for atg5 was always estimated very low compared to any other atg gene. The aim of this study was to characterize the possible benefits of HvATG5

HvATG5 is highly expressed in the transgenic lines Before barley transformation, the functionality of the HvATG5 CDS sequence provided by NIAS was verified in Arabidopsis through the complementation of the atg5 Arabidopsis mutant (Avila-Ospina et al., 2016). In the barley transformants selected, the expression level of HvATG5 was increased for 30-fold in L20 and L10, and 16-fold for the L18 (Figure 1).

The qPCR genotyping performed on their DNA confirmed that these lines had only one T-DNA insertion. The absence of T-DNA and the low level of HvATG5 expression, similar to WT, in the segregating null-transgenic control plants were verified.

Overexpression of HvATG5 increases tolerance to N deficiency, S deficiency and to darkstress

Under LN condition, the HvATG5 overexpressing lines exhibited alleviative senescence symptoms on their 1 st and 2 nd leaves. Accordingly, chlorophyll contents were significantly increased compared to the control lines under LN (Figure 2A). Under HN condition, the senescence phenotype was not different in the HvATG5 overexpressing and control lines (Figure 2A). However the overexpression of HvATG5 promoted plant development under HN, as indicated by the increased plant height (Figure 2B) that is mainly due to the longer 3 rd leaves observed on the HvATG5 overexpressors as shown in Fig. 2A. Differences in the fresh weight of the different parts of HvATG5 overexpressors and control plants under both LN and HN conditions were mostly undetectable, except for L18 under HN (Figure 2C).

Like in LN condition, the HvATG5 overexpressors were less senescent upon LS treatment.

The yellowing area located at the tip of the 1 st leaf twas smaller in overexpressors and higher chlorophyll content was recorded (Figure 3A). It indicates that overexpression of HvATG5 increases sulphur deficiency tolerance, although the phenotype is weaker than that observed under LN. Like under LN, there was almost no difference for the shoot fresh weight, plant height and leaf length between HvATG5 overexpressors and control (Figure 3B).

The leaf-yellowing and chlorophyll-loss under dark treatment were attenuated in the HvATG5 overexpressors in comparison to the control (Figure 4A). The shoot fresh weight and plant height were also higher in the HvATG5 overexpressors than in control (Figure 4B), suggesting the higher tolerance of overexpressors to dark treatment. Interestingly, after a long In the first year, we tried barley transformation for several times but the transformation efficiency was quite low, we could not get enough transformants. Then Céline decided to send me to the laboratory of Dr Per L. Gregersen at Aarhus University to learn how did they exactly do. Although they gave us very detailed protocol, sometimes some important but tiny manipulations may be not included but will greatly affect the result. Dr Per L. Gregersen is working primarily with barley and wheat using molecular biology tools to study quality characters in the crops. Together with the technician Dr Inger B. Holme, they showed me how to select the optimum immature embryos (1.5-2 mm in diameter) with a ruler, which is quite important for the development of callus in the later stage but we did not perform properly in our own laboratory before. They also showed a new method for Agrobacterium inoculation of embryos by dipping the scalpel into a 1.5 ml Eppendorf tube containing Agrobacterium before using it to remove the embryonic axis, so the embryo will touch with the Agrobacterium much better. I visited and recorded all the growth conditions of different stages, including the donor barley plants, the embryos on different medium, the seedlings on root induction medium and the big transgenic barley plants in greenhouse. I used three different constructs (overexpressed HvATG5, down regulated HvATG7 and a GFP construct provided by Per's lab for test), and around 75 embryos were infected for each of them. Résumé : L'autophagie est importante pour le recyclage et la mobilisation des éléments nutritifs dans la plante. Plusieurs lignées surexprimant les gènes AtATG8a-i d'Arabidopsis ont été sélectionnées afin de déterminer l'effet de l'augmentation de l'activité autophagique sur l'efficacité de la remobilisation de l'azote. Les lignées sur-expresseur ont présenté une remobilisation de l'azote des feuilles de la rosette jusqu'aux graines supérieure à celle du sauvage, mais cela uniquement dans des conditions de culture en nitrate pléthorique. Une vaste collection de mutants atg, comprenant les notament les mutants atg8a-i, a ensuite été utilisée pour mesurer la remobilisation de l'azote vers les graines, dans le but de déterminer si un membre de la famille ATG8 pourrait être plus spécifiquement dédié au recyclage de l'azote lors de la sénescence des feuilles pour la remobilisation.

Bien que les divers mutants atg8 n'aient pas présenté de différence majeure par rapport au sauvage, l'un d'entre eux a montré un léger phénotype de sénescence précoce, suggérant que cette isoforme pourrait être plus spécialisée dans la remobilisation de l'azote. Afin d'améliorer l'activité de l'autophagie chez l'orge, nous avons surexprimé HvATG5 chez l'orge et testé la sensibilité des sur-expresseurs à plusieurs conditions de stress. Nous avons constaté que les sur-expresseurs étaient plus tolérants à une faible alimentation en nitrate, à une faible teneur en soufre, et surtout moins sensibles aux effets de l'obscurité prolongée. Afin d'estimer les rôles respectifs des protéases induites par la sénescence et de l'autophagie, plusieurs mutants de protéase (sag12, rd21A, cathB3) ont été croisés avec des mutants d'autophagie (atg5, atg7). Leur contribution à la remobilisation de l'azote a été mesurée par marquage 15 N. Title : Role of autophagy in nitrogen remobilization and adaptation to environment in plants Keywords : Nutrient recycling, nitrogenuse efficeincy, leaf senescence Abstract : Autophagy is important for nutrient recycling and mobilization in plants. Several Arabidopsis AtATG8 overexpressing lines were selected in order to determine the effect of increasing autophagy on nitrogen remobilization efficiency. The overexpressing lines remobilized more nitrogen from the rosette leaves to the seeds but only when cultivated under high nitrate conditions. A large collection of atg mutants including the atg8a-i mutants was then used to monitor N-remobilization from the rosettes to the stems and seeds in order to determine which ATG genes are essential for N remobilization.

Although the various atg8 mutants were not different from wild-type, one of them presented slight early senescence phenotype, suggesting this isoform could be more specialized in N remobilization. In order to enhance autophagy activity in barley, the HvATG5 was overexpressed.

Barley HvATG5 over-expressors were more tolerant to low nitrate, to low sulfur, and especially less sensitive to dark-stress. Finally, the respective roles of autophagy and senescence induced proteases was investigated by crossing protease mutants (sag12,rd21A, cathB3) with autophagy mutants (atg5, atg7) in order to monitor their respective contributions to nitrogen remobilization using 15 N.