Qinwu Chen

Daiki Shinozaki

Jie Luo

Mathieu Pottier

Marien Havé

Anne Marmagne

Michèle Reisdorf-Cren

Fabien Chardon

Sébastien Thomine

Kohki Yoshimoto

Céline Masclaux-Daubresse
email: celine.masclaux-daubresse@inra.fr

Inbios

Autophagy and macro-and micro-nutrient management in plants

Nutrient recycling and mobilization from organ to organ all along the plant lifespan is essential for plant survival under changing environment. Nutrient remobilization to the seeds is also essential for good seed production. In this review we summarise the recent advances made to understand how plant manage nutrient remobilization from senescing organs to sink tissues, and what is the contribution of autophagy in this process. Plant engineering manipulating autophagy for better yield and plant tolerance to stresses will be presented.

Introduction

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 [START_REF] Dikic | Proteasomal and Autophagic Degradation Systems[END_REF][START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF]. The best studied and understood recycling system in plants is the ubiquitin-proteasome pathway in which proteins are ligated with a poly-ubiquitin 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, plants employ autophagy pathway for vacuolar bulk turnover of cytoplasmic components. Autophagy entails encapsulation of unwanted cytosolic materials within specialized autophagic vesicles, which are subsequently delivered to the vacuole for proteolysis or hydrolysis [START_REF] Li | Autophagy: a multifaceted intracellular system for bulk and selective recycling[END_REF].

Three distinct types of autophagy, micro-, macro-and mega-autophagy, have been reported in plant.

Micro-autophagy proceeds by the invagination of tonoplast to trap cytoplasmic material congregated at the vacuole surface to create autophagic bodies within the vacuole (Figure 1). Such micro-autophagy 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 micro-autophagy [START_REF] Pourcel | The Formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and Implications for the Sequestration of Anthocyanin Pigments[END_REF][START_REF] Chanoca | Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism[END_REF][START_REF] Masclaux-Daubresse | Stitching together the multiple dimensions of autophagy using metabolomic and transcriptomic analyses reveals new impacts of autophagy +15% (high N only) +3% (high N) AtATG8 OE atg5-KO -40% (low N) -10% (high N) Modification of N[END_REF]. Conversely, macro-autophagy is much better described. It involves double membrane vesicles, named autophagosomes, that sequester cytosolic components [START_REF] Thompson | Autophagic recycling: lessons from yeast help define the process in plants[END_REF]. 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. While micro-autophagy decreases tonoplast membrane area, macroautophagy provides new lipid material to the tonoplast. Thus, it is likely that these two types of autophagy may play opposite roles in tonoplast membrane homeostasis. 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] Kwon | The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis[END_REF].

Of these three types, macro-autophagy is the best characterized process and is considered as the major form and has received most attention [START_REF] Michaeli | Degradation of Organelles or Specific Organelle Components via Selective Autophagy in Plant Cells[END_REF]. During leaf senescence, expression of several AuTophaGy-related (ATG) genes encoding key components for autophagosome formation is increased [START_REF] Avila-Ospina | Identification of Barley (Hordeum vulgare L.) Autophagy Genes and Their Expression Levels during Leaf Senescence, Chronic Nitrogen Limitation and in Response to Dark Exposure[END_REF]. The suppression of these genes disrupts the normal development of autophagosomes and results in hypersensitivity to starvations as well as premature leaf senescence. This suggests that autophagy plays a key role in leaf senescence and nutrient recycling [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Xiong | AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana[END_REF].

Molecular machinery of macro-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 macro-autophagy (hereafter referred to as autophagy). Together with colleagues, he identified several ATG genes that participate in autophagic processes by yeast forward genetic experiments [START_REF] Ohsumi | Molecular dissection of autophagy: Two ubiquitin-like systems[END_REF]. To date, more than 40 ATG genes have been identified in yeast, 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 [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Xia | Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in Rice (Oryza sativa L.)[END_REF][START_REF] Pei | Identification of autophagyrelated genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses[END_REF][START_REF] Chung | The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability[END_REF][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][START_REF] Avila-Ospina | Identification of Barley (Hordeum vulgare L.) Autophagy Genes and Their Expression Levels during Leaf Senescence, Chronic Nitrogen Limitation and in Response to Dark Exposure[END_REF][START_REF] Li | Genome-wide analysis of autophagy-associated genes in foxtail millet (Setaria italica L.) and characterization of the function of SiATG8a in conferring tolerance to nitrogen starvation in rice[END_REF][START_REF] Wang | Characterization of an Autophagy-Related Gene MdATG8i from Apple[END_REF]. Functional analysis of these proteins reveals a canonical route for autophagy. Basically, the process of autophagy consists in 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 1) [START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF]. The ATG1and ATG13 proteins, together with two accessory proteins, ATG11 and ATG101, assemble into an active ATG1-ATG13 complex [START_REF] Li | Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy[END_REF] that promotes the nucleation and expansion of a cup-shaped double-membrane (phagophore), which is thought to originate from the endoplasmic reticulum (ER) [START_REF] Bars | ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants[END_REF][START_REF] Zhuang | ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis[END_REF][START_REF] Zhuang | Autophagosome Biogenesis and the Endoplasmic Reticulum: A Plant Perspective[END_REF]. The transmembrane protein ATG9 recruits lipids for phagophore elongation, ATG2 and ATG18 proteins facilitate ATG9 cycling [START_REF] Xiong | AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana[END_REF][START_REF] Zhuang | ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis[END_REF]. 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 [START_REF] Feng | The machinery of macroautophagy[END_REF][START_REF] Liu | Understanding and exploiting the roles of autophagy in plants through multi-omics approaches[END_REF]. 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 [START_REF] Yoshimoto | Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy[END_REF][START_REF] Woo | Differential processing of Arabidopsis ubiquitinlike Atg8 autophagy proteins by Atg4 cysteine proteases[END_REF], then conjugated to the lipid phosphatidyl ethanolamine (PE) by the conjugating enzyme ATG3 [START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF]. Another ubiquitin-fold protein ATG12 is conjugated to ATG5 by the conjugating enzyme ATG10 [START_REF] Phillips | The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana[END_REF]. Both of the conjugation systems share a single ATP-dependent activating enzyme ATG7 [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF]. The ATG12-ATG5 conjugate promotes the lipidation of ATG8 with PE and its anchorage into the phagophore membrane [START_REF] Chung | ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci[END_REF]. 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 [START_REF] Yoshimoto | Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy[END_REF], 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.

The TARGET OF RAPAMYCIN (TOR) kinase is a master player in sensing nutrient status of eukaryotic cells.

TOR orchestrates cell homeostasis in a fine crosstalk with several other players among which its LST8 and RapTOR partners and SnRK1 kinase [START_REF] Dobrenel | Regulation of plant growth and metabolism by the TOR kinase[END_REF]. TOR is a well-known positive regulator of ribosome protein synthesis and of translation and a negative post-translation regulator of autophagy under nutrient-rich conditions. It dampens autophagy at post-translational level by hyper-phosphorylating ATG13, which prevents its association with ATG1.

Under nutrient-limiting conditions, inactivation of TOR leads to rapid de-phosphorylation of ATG13, allowing it to bind ATG1. The TOR kinase also play a role in regulating the transcription of genes. It activates genes involved in anabolic processes that are essential for rapid growth like amino acid, lipid and nucleotide synthesis and the oxidative pentose phosphate pathway, and represses genes mediating the degradation of proteins, amino acids, lipids and xenobiotic, and autophagy regulation [START_REF] Xiong | Glucose-TOR signalling reprograms the transcriptome and activates meristems[END_REF]. On the contrary, SnRK1 is a positive regulator of autophagy in Arabidopsis. The KIN10 SnRK1 alpha catalytic subunit is necessary for the activation of autophagy under energy depleted conditions and in response to many other abiotic stresses. SnRK1 can control autophagy through both TOR-independent and TOR-dependent pathways depending on stresses [START_REF] Soto-Burgos | SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana[END_REF]. Autophagy genes and autophagic activity are then strongly induced by nitrogen and carbon limitation in several plant species, as well as by many other stresses as reviewed by Tang and Bassham [START_REF] Tang | Autophagy in crop plants: what's new beyond Arabidopsis?[END_REF].

Selective macro-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 cargoes by engaging a wide array of receptors or adaptor proteins that tether the cargoes and also interact with ATG8 [START_REF] Floyd | What to Eat: Evidence for Selective Autophagy in Plants[END_REF][START_REF] Li | Regulator and substrate Dual roles for the ATG1-ATG13 kinase complex during autophagic recycling in Arabidopsis[END_REF][START_REF] Farre | Mechanistic insights into selective autophagy pathways: lessons from yeast[END_REF]. The interaction between autophagic receptors and ATG8 is mediated by the presence of ATG8-interacting motif (AIM) in each receptor [START_REF] Noda | Atg8-family interacting motif crucial for selective autophagy[END_REF]. 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 [START_REF] Marshall | ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors[END_REF]. As ATG8 decorates and controls phagophore membrane expansion, its abundance determines the size of autophagosome [START_REF] Xie | Atg8 controls phagophore expansion during autophagosome formation[END_REF]. 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. Several forms of selective autophagy have been reported in plants as 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) and degradation of brassinosteroid-responsive transcription factor BES1 [START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF][START_REF] Marshall | Dynamic Regulation of the 26S Proteasome: From Synthesis to Degradation[END_REF][START_REF] Yoshimoto | Unveiling the Molecular Mechanisms of Plant Autophagy-From Autophagosomes to Vacuoles in Plants[END_REF]. Selective autophagy certainly allows fine organelle quality control and also to the removal of specific cellular waste. At the same time specific autophagy provides cargoes to degradation pathways performing hydrolysis and proteolysis inside the vacuole lumen, it facilitates the release of metabolites that contribute to nutrient recycling. Whether some specific autophagy pathways could be more related to the recycling of specific nutrient, macro-or micro-elements is an interesting question that remains to be investigated.

In plants, the first selective autophagy receptor, named Joka2, was identified in tobacco. This NBR1 (NEIGHBOR OF BRCA1 GENE 1) homologue was identified in yeast two-hybrid screen carried out to look for partners of the coiled-coil protein UP9C of unknown function that strongly over-accumulates under sulphur-deficiency [START_REF] Zientara-Rytter | Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors[END_REF]. Afterwards, the Arabidopsis NBR1 homologue was characterised and shown to target ubiquitinated protein aggregates formed under stress conditions through a C-terminal ubiquitin-associated (UBA) domain [START_REF] Zientara-Rytter | Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors[END_REF][START_REF] Svenning | Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1[END_REF]. Like ATG genes, it was shown that NBR1/Joka2 expression is enhanced under several nutrient starvations as C, N and S limitations. Functional analyses 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 [START_REF] Zhou | NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses[END_REF][START_REF] Jung | NBR1 Mediates Selective Autophagy of Defective Proteins in Arabidopsis[END_REF]. However, unlike atg5 and atg7 mutants, nbr1 is not sensitive to darkness stress or necrotrophic pathogen attack, suggesting that NBR1 is involved in the selective degradation of denatured or damaged non-native proteins generated under high temperature conditions, but not in other "bulk" autophagy. Therefore, autophagy operates through distinct cargo recognition and delivery systems according to biological processes. NBR1 is involved in the selective degradation of denatured or damaged non-native proteins generated under high temperature conditions, but is not involved in other "bulk" autophagy. Interestingly, it was recently reported that NBR1 also specifically binds viral capsid protein and particles of the cauliflower mosaic virus (CaMV) in xenophagy to mediate their autophagic degradation, and thereby restricting the establishment of CaMV infection [START_REF] Hafren | 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 (Maqbool et al., 2016). 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 hostpathogen 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].

Subsequently, the ATI1/ATI2 ATG8-binding proteins were also characterized as autophagy receptors. ATI1 is located in ER-bodied and plastid-associated bodies in dark induced leaves [START_REF] Honig | Selective autophagy in the aid of plant germination and response to nutrient starvation[END_REF][START_REF] Michaeli | Involvement of autophagy in the direct ER to vacuole protein trafficking route in plants[END_REF]. The plastid localised ATI1bodies were also detected in senescing cells and shown to contain stroma proteins. While they likely play a role in chlorophagy, their role in N remobilization during senescence has not been reported so far.

Another example of specific autophagy adaptor is RPN10. The proteasome subunit RPN10 was shown to mediate the autophagic degradation of the ubiquitinated 26S proteasomes, known as proteaphagy [START_REF] Marshall | Autophagic degradation of the 26S proteasome Is mediated by the dual ATG8/Ubiquitin receptor RPN10 in Arabidopsis[END_REF]. Upon stimulation by chemical or genetic inhibition of the proteasome, RPN10 simultaneously binds the ubiquitinated proteasome, via an ubiquitin-interacting motif (UIM), and to ATG8 through another UIM-related sequence that is distinct from the canonical AIM motif. In Arabidopsis, the inhibitor-induced proteaphagy was blocked in mutant expressing an RPN10 truncation that removed 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 [START_REF] Vanhee | Autophagy involvement in responses to abscisic acid by plant cells[END_REF]. 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 the tonoplast and the plasma membrane during abiotic stress conditions [START_REF] Hachez | The Arabidopsis Abiotic Stress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2;7 through Protein-Protein Interactions and Autophagic Degradation[END_REF].

Nutrient remobilization after organelle and protein degradations 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, plants senesce leaves 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 [START_REF] Avila-Ospina | Autophagy, plant senescence, and nutrient recycling[END_REF]. 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]. 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 progressively replaced by recycling from the catabolism of macromolecules such as proteins and nucleic acids. Up to 75% of the total mesophyll cellular nitrogen is localized in the chloroplasts [START_REF] Hortensteiner | Nitrogen metabolism and remobilization during senescence[END_REF]. The breakdown and recycling of these considerable nitrogen resources depend on three distinct chloroplast degradation pathways that rely on macroautophagy, senescence-associated-vacuoles and Chloroplast Vesiculation (CV) pathways (see [START_REF] Have | Nitrogen remobilization during leaf senescence: lessons from Arabidopsis to crops[END_REF] and [START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF], for reviews). Up to now, although detailed knowledge concerning interactions and relationships between these three chloroplast degradation pathways remains insufficient, the cysteine proteases localized in the vacuole appear to play a particularly important role in all these processes as they proceed during the last steps of the macromolecule break-down in the vacuole. Cysteine proteases as SAG12, Cathepsin B3 (CATHB3), Responsive-to-desiccation 21A (RD21A), Arabidopsis aleurain-like protease (AALP) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly overexpressed senescence-related proteases [START_REF] Lohman | Molecular analysis of natural leaf senescence in Arabidopsis thaliana[END_REF][START_REF] Pružinská | Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves[END_REF].

Efforts made to understand nitrogen remobilization during leaf senescence have mainly focused on the biochemistry of the degradation of plastidial proteins (Figure 2). Originally observed by immuno-electron microscopy in the cytoplasm and vacuole of naturally senescing wheat leaf cells, the RuBisCo-containing bodies (RCBs) were proposed to be involved in RuBisCo degradation process outside of the chloroplasts [START_REF] Chiba | Exclusion of ribulose-1,5biphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat[END_REF]. These RCBs contained the large and small subunit of RuBisCo and other stromal proteins as the chloroplastic glutamine synthetase. However, RCB lacked chloroplast envelope or thylakoid components. Sometime, RCBs were found to be surrounded by double membranes, which seem to be derived from the chloroplast envelope. Interestingly several observations presented RCBs in the cytoplasm closely bordered by kinds of bean shaped vesicles that might be isolation membranes characteristic of the intermediate structures of autophagosomes (phagophores) [START_REF] Chiba | Exclusion of ribulose-1,5biphosphate 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 were frequently visible at the early stages of leaf senescence when RuBisCo starts to decrease without prior chloroplast destruction or chlorophyll degradation and it was proposed that the budding of RCBs from chloroplast stromules was a way to bring chloroplast material out of the organelle [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][START_REF] Izumi | Entire photodamaged chloroplasts are transported to the central vacuole by autophagy[END_REF]. This material release may explain why during senescence the size of chloroplasts (c.a. 10 µm) decreases to form gerontoplasts (c.a. 4 µm).

The demonstration that autophagy plays a prominent role in RCB trafficking to the vacuole was provided using confocal microscopy to visualize stromal and ATG8 proteins tagged with different fluorescent probes. Authors showed that the release of RCBs inside the vacuole required functional autophagy and was absent in autophagy mutants such as atg5 and atg7. Co-localization of autophagosomes and RCBs was also demonstrated and moreover it was also shown that shrunk gerontoplasts could be released inside the vacuole in an ATG4-dependent microautophagy pathway [START_REF] Izumi | Establishment of Monitoring Methods for Autophagy in Rice Reveals Autophagic Recycling of Chloroplasts and Root Plastids during Energy Limitation[END_REF][START_REF] Izumi | Autophagic Turnover of Chloroplasts: Its Roles and Regulatory Mechanisms in Response to Sugar Starvation[END_REF]. The elimination of membrane damaged chloroplast via micro-autophagy was further confirmed and the role of macro-autophagy related membranes harbouring GFP-ATG8 decorations in this process suggested [START_REF] Nakamura | Selective Elimination of Membrane-Damaged Chloroplasts via Microautophagy[END_REF].

Role of autophagy in nitrogen recycling

As stated earlier, under normal conditions autophagy operates at a basal level that constitutes housekeeping machinery and participates in cell homeostasis. Under nutrient starvation and during leaf senescence autophagy activity is enhanced and its role in nutrient recycling and remobilization at the whole plant level was suspected.

The demonstration of the role of autophagy in nutrient recycling and mobilization from source to sinks was provided by Guiboileau et al. [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the wholeplant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF] (Figure 3). Monitoring 15 N fluxes to the seeds after labelling Arabidopsis rosettes at the vegetative stage, [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the wholeplant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF] showed that N remobilization was markedly decreased in atg mutants (atg18a RNAi, atg5, and atg9) compared to wild type plants (WT). The decrease was more moderate when plants were grown under high nitrate than under low nitrate conditions, but still significant. Accordingly, authors further found that atg mutants accumulated more ammonium, amino acids (AA), proteins, and RNA in their rosette leaves than WT [START_REF] Guiboileau | Physiological and metabolic consequences of autophagy defisciency for the management of nitrogen and protein resources in Arabidopsis leaves depending on nitrate availability[END_REF]. N remobilization was further evaluated using similar 15 N-labelling procedure in the atg12 maize mutants which revealed that N remobilization to the kernels was also impaired in autophagy deficient mutants [START_REF] Li | Autophagic Recycling Plays a Central Role in Maize Nitrogen Remobilization[END_REF].

The growth of atg12 mutants was most often arrested at seedling stage, and adult plants showed enhanced leaf senescence and stunted ear development under nitrogen-starved conditions but not under high-N. Under nutrientrich conditions, the seed yield of atg12 plants was much lower, and 15 N reallocation into the seeds was twice less in atg12 was half of that in WT. The investigation conducted during the vegetative growth period on the rice autophagy-deficient mutant Osatg7-1 suggested that N remobilization from senescent leaves to young leaves was suppressed [START_REF] Wada | Autophagy Supports Biomass Production and Nitrogen Use Efficiency at the Vegetative Stage in Rice[END_REF]. 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 sterile 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.

Recently over-expression of autophagy genes was assessed in several plant species. Overexpressing AtATG5 and AtATG7 in Arabidopsis delayed senescence, improved seed production and yield under certain conditions [START_REF] Minina | Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness[END_REF].

Similarly, several reports showed that overexpressing different ATG8 genes from soybean or millet in Arabidopsis or rice was beneficial to plant performances, increasing tolerance to nitrogen starvations and to drought [START_REF] Xia | Heterologous expression of ATG8c from Soybean confers tolerance to nitrogen deficiency and increases yield in Arabidopsis[END_REF][START_REF] Li | Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis[END_REF][START_REF] Luo | Autophagy Is Rapidly Induced by Salt Stress and Is Required for Salt Tolerance in Arabidopsis[END_REF][START_REF] Sun | MdATG18a overexpression improves tolerance to nitrogen deficiency and regulates anthocyanin accumulation through increased autophagy in transgenic apple[END_REF][START_REF] Sun | Improvement of drought tolerance by overexpressing MdATG18a is mediated by modified antioxidant system and activated autophagy in transgenic apple[END_REF].

Using the same 15 N labelling procedure as Guiboileau [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the wholeplant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF], Chen et al. [START_REF] Chen | Overexpression of ATG8 in Arabidopsis Stimulates Autophagic Activity and Increases Nitrogen Remobilization Efficiency and Grain Filling[END_REF] then showed that N remobilization of nitrogen from the rosette leaves to the seeds was improved in Arabidopsis plants overexpressing the AtATG8a or the AtATG8g gene. In these plants, N seed filling was increased and the amount of nitrogen lost in dry remains decreased. Interestingly the N-remobilization performances of the AtATG8a and AtATG8g over-expressors were improved only when plants were grown under abundant nitrate supply but not when grown under N limited conditions. This can be explained by the fact that under N-limitation, autophagy activity is already enhanced, which cancels the benefit of stimulating ATG8 expression through genetic transformation.

This demonstration of the beneficial effect on NUE of over-expressing ATG8 was confirmed by Yu et al. [START_REF] Yu | Increased Autophagy of Rice Can Increase Yield and Nitrogen Use Efficiency (NUE)[END_REF].

The authors over-expressed the OsATG8a gene in rice and found that N% in seeds was increased while N% in dry remains was decreased attesting better N remobilization to the seeds. Interestingly, like in Arabidopsis, the positive effect on plant performances was only observed under sufficient N supply but not under N-limitation.

Cross-talk between autophagy and senescence-related cysteine proteases

Although both autophagy and cysteine proteases are key players during leaf senescence, protein proteolysis, and nutrient recycling as shown by recent publications from James et al. [START_REF] James | A New Role for SAG12 Cysteine Protease in Roots of Arabidopsis thaliana[END_REF][START_REF] James | SAG12, a Major Cysteine Protease Involved in Nitrogen Allocation during Senescence for Seed Production in Arabidopsis thaliana[END_REF], the relationship between them remains largely unknown. It is admitted that proteins are not degraded inside the autophagosomes but rather transported by them to the lytic vacuoles where proteases and hydrolases operate. As said before, autophagy mutants are impaired in N remobilization and they accumulate large amounts of proteins and amino acids in their rosette leaves.

They also present significantly higher protease activities in their rosette leaves than wild type, which supports the hypothesis that proteases and substrates cannot meet each other in autophagy mutants. In order to investigate the nature of the protease activities enhanced in autophagy mutants, Havé et al. [START_REF] Havé | Increase of proteasome and papainlike cysteine protease activities in autophagy mutants: backup compensatory effect or pro cell-death effect?[END_REF] used shotgun proteomics to identify these proteases and specific probes to monitor their activity. Results showed that cysteine proteases accounted for the largest proportion (38%) of the over-abundant proteases in autophagy-deficient lines. Activitybased protein profiling (ABPP) analysis with DCG-04 revealed that activities of papain like cysteine proteases (PLCPs) were higher in autophagy-defective plants grown under low-nitrate conditions. Further pull-down experiments using the DCG-04 biotinylated inhibitor of papain like cysteine protease (PLCP), showed that the active PLCPs accumulated in autophagy mutants in low-nitrate condition were mainly SAG12, RD21A, CATHB3, and AALP. The western blots using 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 low nitrate but not under high nitrate in autophagy mutants strongly suggested 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 double mutants. [START_REF] Havé | Increase of proteasome and papainlike cysteine protease activities in autophagy mutants: backup compensatory effect or pro cell-death effect?[END_REF] also found that the CND41-like aspartate protease AED1 (APOPLASTIC ENHANCED DISEASE SUSCEPTIBILITY-DEPENDENT 1) that have been described by Kato et al. [START_REF] Kato | The DNA-binding protease, CND41, and the degradation of ribulose-1,5bisphosphate carboxylase/oxygenase in senescent leaves of tobacco[END_REF] as one of the potential protease involved in RuBisCo degradation was also increased in autophagy mutant. Interestingly AED1 that was up-regulated in the senescing leaves of the sag12 mutants was also proposed to compensate the absence of SAG12 activity for N remobilization [START_REF] James | SAG12, a Major Cysteine Protease Involved in Nitrogen Allocation during Senescence for Seed Production in Arabidopsis thaliana[END_REF].

Autophagy and other nutrients

Autophagy is likeky involved in the recycling not only of proteins but also of membranes and other cell components that certainly contain micro-elements. It is well known that iron in the cell is mainly linked to ferritine and photosystem I, which are located in plants into the plastids in plants. In mammals, ferritine is degraded by NCOA4-mediated autophagy (ferritinophagy) which participates to control ferropoptosis and erythropoiesis [START_REF] Mancias | Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy[END_REF][START_REF] Santana-Codina | The Role of NCOA4-Mediated Ferritinophagy in Health and Disease[END_REF]. Although the role of autophagy in the degradation of ferritine has not been demonstrated in plant, it was found that the efficiency of iron (Fe) translocation from vegetative organs to the seeds is severely decreased in several autophagy mutants compared to wild type [START_REF] Pottier | Autophagy is essential for optimal translocation of iron to seeds in Arabidopsis[END_REF] (Figure 3). The authors confirmed the defect of iron translocation to the seeds in autophagy mutant using 57 Fe labelling and tracing experiment. This study also showed that not only iron but also manganese (Mn) and zinc (Zn) are sequestered into the rosette leaves. Consistently the lower amounts of Zn and Mn is the seeds of autophagy mutants also suggest that their translocation is dependent of autophagy. This observation is consistent with the study of Eguchi et al. [START_REF] Eguchi | Autophagy is induced under Zn limitation and contributes to Zn-limited stress tolerance in Arabidopsis (Arabidopsis thaliana)[END_REF] that showed that autophagy is induced under Zn limitation conditions, and that autophagy-deficient mutants (atg5-4, atg10-1) exhibit early senescence phenotype under Zn limitation and limited growth recovery after Zn resupply.

More recently, Shinozaki et al. (unpublished data) confirmed the hypersensitivity of autophagy mutants to zinc limitation. While Zinc limitation induced autophagy in wild-type it triggered accumulation of proteins in autophagy mutants as a mark of autophagy defect. Interestingly, Zn-deficiency symptoms in atg mutants recovered under low-light and iron-limited conditions, pointing out the role of Fenton-related oxidative stress in the response of plants to zinc deficiency. This also suggests that the induction of autophagy by zinc deficiency could be mediated by Fenton-generated hydroxy radicals. Inorganic phosphate like nitrogen is one of the major macro-elements needed for plant growth. The recent paper from Naumann et al. [START_REF] Naumann | The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy[END_REF] reveals that phosphate limitation stimulates autophagy in the root tips of Arabidopsis. Stimulation of autophagy by Pi deprivation was exacerbated in the pdr2 mutants which is hypersensitive to Pi deficiency. PDR2 protein is located at the endoplasmic reticulum and was hypothesized to play a role in ER-quality control. Blocking ER stress in pdr2 mutant introducing ire1a mutation, or providing ERstress inhibitors reduced autophagosome formation in response to Pi deprivation. This indicates that the ER-stress induced by low-Pi triggers autophagy in roots under low phosphate. Root growth of autophagy mutants was strongly reduced by Pi deprivation due to early root apical meristem differentiation that lowered meristem activity.

When suppressing locally Pi sensing using phosphite application, meristem activity was restored in autophagy mutants. Decreasing iron concentration in the low Pi culture medium also restored apical meristem activity in autophagy mutant, suggesting by the way that iron would also play a role in the ER stress response to Pi deficiency, possibly through the production of reactive oxygen species.

Conclusion

The results obtained from the studies of autophagy-defective mutants grown under various starvations clearly indicate the involvement of autophagy in the recycling and remobilization of nutrients at the whole plant level.

The studies that increased autophagic activity through the over-expression of some ATG genes demonstrated that it could be a powerful approach to improve plant tolerance to starvations and nutrient remobilization from source to sinks. However, recent results enlighten the strong link between nutrient deprivation (N, S, Zn and Pi), and oxidative stress and ER-stress [START_REF] Havé | Proteomic and lipidomic analyses of the Arabidopsis atg5 autophagy mutant reveal major changes in ER and peroxisome metabolisms and in lipid composition[END_REF][START_REF] Naumann | The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy[END_REF]. This questions whether the ER, which is the source of lipid for autophagosome formation [START_REF] Zhuang | Autophagosome Biogenesis and the Endoplasmic Reticulum: A Plant Perspective[END_REF], could be a sensor of plant environmental stresses and an intermediate in autophagy induction.

Figure 1: Schematic representation of macro and micro-autophagy pathways in plants.

Nutrient availability controls the TOR kinase activity that in turn regulates posttranscriptionally maro-autophagy through the phosphorylation of ATG1 and ATG13. After nucleation of the pre-autophagosomal structures, the ATG9, ATG18 and ATG2 proteins (in blue) are involved in the expansion of the membrane of the autophagosome. Several ATG proteins (in orange) involved in the conjugation of ATG8 to phosphatidyl-ethanolamine, facilitate ATG8 anchorage to the membrane of the pre-autophagosome and per se autophagosome formation and enclosure. The ATG8 interacting motifs facilitate the capture of cargoes to be driven to the central vacuole for degradation. Micro-autophagy consists in the invagination of the tonoplast and participates to the formation of anthocyanin vacuole inclusions (AVI). Chloroplast material and unwanted cytoplasmic material are driven to the central vacuole for degradation through the macro-autophagy pathway. Once delivered to the vacuole lumen, autophagic bodies (inner membrane of autophagosome and cargoes) are degraded by the resident proteases and hydrolases. The nutrients released are exported to the cytosol and using transporters or canals. Once inside the cytosol, nutrients are either used for cell metabolism or released out of the cell for source to sink translocation. Interconversions of amino-acids occur in the cytosol to produce the glutamine and asparagine forms that are preferentially used for long distance transport in the phloem. Many black boxes remain to be explored, especially regarding the docking of autophagosomes to the tonoplast and the transport of nutrients out of the vacuole and further out of the cell (question marks).

Organic acids

Fatty acids Glycerides

Fe 2+ Zn 2+ Mn 2+ Source to sink remobilization

Figure 2 :

 2 Figure 2: Schematic representation of the different steps of nutrient recycling in plant cells.Chloroplast material and unwanted cytoplasmic material are driven to the central vacuole for degradation through the macro-autophagy pathway. Once delivered to the vacuole lumen, autophagic bodies (inner membrane of autophagosome and cargoes) are degraded by the resident proteases and hydrolases. The nutrients released are exported to the cytosol and using transporters or canals. Once inside the cytosol, nutrients are either used for cell metabolism or released out of the cell for source to sink translocation. Interconversions of amino-acids occur in the cytosol to produce the glutamine and asparagine forms that are preferentially used for long distance transport in the phloem. Many black boxes remain to be explored, especially regarding the docking of autophagosomes to the tonoplast and the transport of nutrients out of the vacuole and further out of the cell (question marks).

Figure 3 :

 3 Figure 3: Modification of macro-and micro-nutrient fluxes in autophagy mutants and over-expressors in Arabidopsis. The green and blue arrows indicate the lack (as percentages) of micro and macro-nutrient allocation to the seeds in the atg5-KO mutant relative to wild type [74,92]. The red arrows indicate the extra nitrogen remobilization measured in ATG8 Arabidopsis over-expressors by comparison to control line under plethoric nitrate conditions [84].

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Funding information

This work was supported the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS) for IJPB, the China Scholarship Council for Qinwu Chen and the Agreenskills+ program for Jie Luo. Marien Havé was supported by ANR-12-ADAPT-0010-0.


Corentin Anex

Ewen Touzé

Léonard Curet

Frédéric Gohier

Charles Cougnon
email: charles.cougnon@univ-angers.fr

Base-Assisted Electrografting of Aromatic Amines

Contrary to aliphatic amines, aromatic amines have received less attention for the surface modification by oxidation due to the low acidity of their cation radicals. To overcome this limitation, bases are often added in solution to facilitate or make possible the grafting. However, there has only been a modest focus on their impact on the grafting efficiency. Here, the anodic behavior of a series of para-substituted anilines was investigated in the presence of different bases to address this issue. From complete sets of Brønsted acidity/basicity data and H-bond acidity/basicity factors for both parasubstituted anilines and bases, a grafting efficiency index was proposed as a practical tool to choose the most suitable base for the grafting. As a proof-of-concept for base-assisted grafting of aromatic amines, two aniline/base systems were tested to modify carbon surfaces at lower potential, compared to the modification achieved from pure aniline solutions.

Introduction

Since the 1990s, interest in the oxidation of amines was renewed by the possibility for metallic and carbon surfaces to be covalently modified when attacked by an aminyl radical produced by deprotonation of the amino cation radical. [1] In this electrografting procedure, the stability of the cation radical (envisioned as the proton loss rate from the cation radical) governs the grafting efficiency of amines. Because of the exergonic deprotonation of the primary aliphatic amine cation radicals by the neutral amines, the resulting cation radicals are only short-lived intermediates in amine oxidations and the primary aliphatic aminyl radicals, produced near to the electrode surface, can be efficiently attached to the surface. [2] In contrast, when the charge density at nitrogen is decreased by delocalization, the lifetime of amine cation radicals becomes appreciable, so that the electrochemical oxidation of such amines mostly involves cation radicals. An example is the acidity of the aniline cation radical lower than that of the protonated aniline in water and DMSO, [3] which makes endergonic the proton-transfer reaction from the cation radical to the neutral aniline, favoring the formation of coupling products and, in certain conditions, highly condensed coupling products such as polyanilines. [4] It is the reason why the covalent attachment of aromatic primary amines to the electrode surface upon oxidation has received less attention and was not reported for a long time. For this last class of amines, an excess of organic base, such as collidine or 3,5-lutidine, is often added in solution to facilitate or make possible the grafting. [5] While the effect of a base on the electrochemical oxidation of substituted anilines was widely studied, [6] results are discussed on a case by case basis, depending on the electronic effect of substituents expected to exert a strong influence on both the acido-basic properties of the neutral amine and the amine cation radical. [7] Although it is well recognized in literature that the base aids in the deprotonation of the amine cation radical, their role in the mechanism of the oxidation of aromatic amine compounds is still being debated. [8] Here, in order to clarify the role of the base in the oxidation of aromatic amines, the anodic behavior of a series of para-substituted anilines was investigated in the presence of different nitrogen bases.

Results and discussion

Complete sets of Brønsted acidity/basicity data and H-bond acidity/basicity factors for both the bases, identified by Roman numerals, and the para-substituted anilines, identified by Greek numerals, permit to rationalize the effect of a base on the oxidation of aniline compounds (see Tables 1 and2). A salient point of this study is an efficiency parameter to help in choosing the most suitable base for the grafting and proof-of-concept for base-assisted grafting of aromatic amines shall be provided.

Table 1. Electrochemical and basicity data in acetonitrile for the nitrogen bases studied.

Base ∆Eonset (mV) [a] pKa(BH + )ACN [b] β [c] (1) Quinoline -34 12.03 0.645

( [a] Shift in the onset oxidation potential of the p-bromoaniline (1 mM) measured by cyclic voltammetry at 100 mV.s -1 in acetonitrile after addition of 5 equivalents of the base B. [b] pKa values of the conjugate acid HB + of the base B in acetonitrile. [9] [c] Basicity factor proposed by Abraham as a scale of solute hydrogen-bond basicity. [10] Table 2. Electrochemical and basicity data in acetonitrile for the para-substituted anilines studied.

Substituent σp [a] α [b] ∆Eonset (mV) [c] pKa(XPhNH3+) [d] pKa(XPhNH2•+) [e] (I) OMe -0. [a] Hammett coefficients. [11] [b] Acidity factor proposed by Abraham as a scale of solute hydrogen-bond acidity; [12] α values in parenthesis are deduced to their dependence with the Hammett coefficients, taking α = 0.264 for the aniline. [13] [c] Shift in the onset oxidation potential of para-substituted anilines (1 mM) measured by cyclic voltammetry at 100 mV.s - 1 in acetonitrile after addition of 5 equivalents of collidine. [d] pK a values for the protonated forms of para-substituted anilines in acetonitrile; [14] data in parenthesis are estimated from a linear Hammett correlation of pK a with σ p . [e] Since the pK a values of the para-substituted anilines cation radicals are unknown in acetonitrile, they were calculated from the N-H bond dissociation enthalpies (BDEs) for para-substituted anilines; [15] the pK a values in parenthesis are deduced to the dependence of the N-H BDE with the Brown σ p + constants of the substituents. [16] Note that similar values can be deduced to the linear correlation of pK a with E pa . [17] As a starting-point, evidence for the existence of hydrogen bonding between the neutral pbromoaniline and the collidine has come from cyclic voltammetry and proton NMR experiments (Figure 1). For the 1 H NMR characterization of the H-bonded complex, equimolar (50 mM) benzene-d6 solutions of p-bromoaniline and collidine were mixed at constant volume (500 µL) by varying the molar fraction of p-bromoaniline, χp-BrPhNH2, from 0.1 to 0.9. As it is evidenced by partial proton NMR spectra presented in the Figure S1, the peak associated with the NH2 protons of p-bromoaniline shifts downfield with addition of aliquots of collidine, indicating the formation of a non-covalent hydrogen bond between p-bromoaniline and collidine. The Job plot analysis presented in the inset of the Figure 1 shows a peak at a molar fraction of p-bromoaniline corresponding to the the formation of a complex of 1:1 stoichiometry. [18] Noted that NMR experiments were achieved in a non-polar solvent such as benzene, because the changes in the chemical shift of the amino protons are very small in acetonitrile, which is a competitive hydrogen-bonding solvent. [19] The p-bromoaniline (1 mM) was also titrated with collidine by cyclic voltammetry in acetonitrile (Figure 1). In pure pbromoaniline solution, the irreversible anodic peak obtained indicates that the amine cation radical is unstable in this time scale and underwent an overall deprotonation/dimerization sequence, yielding an electroactive head-to-tail dimer responsible to the reversible system appearing at less positive potential as demonstrated by Bacon and Adams in aqueous media and Farsang in organic ones. [20] When collidine is present, an oxidation prepeak (better visible at sub-stoichiometric amounts of the base) appears at a less positive potential than for the pure pbromoaniline solution and the reduction peak of the oxidized form of the dimer product shifts towards the negative potential due to the more basic medium (not visible in Figure 1). Noted that studies have demonstrated that the dimerization remains the main pathway in the presence of a non-nucleophilic base. [21] Further increase in the base concentration produced an increase in the current peak intensity to almost twice because the base replaces the parent p-bromoaniline in the deprotonation step, avoiding the formation of an equivalent amount of the electroinactive p-bromoanilinium ion, [22] but no further significant shift in potential is noticed. Such cyclic voltammogram (CV) features were already reported in literature and were attributed to the oxidation of a hydrogen-bonding complex. [23] Figure 1. Cyclic voltammograms of p-bromoaniline (1 mM) in acetonitrile solutions containing 0.1 M Bu4NPF6 recorded on a glassy carbon electrode at 0.1 V.s -1 with 0, 0.6, 1, 2 and 5 equivalents of collidine. The inset shows a Job plot analysis for the complexation of p-bromoaniline and collidine using 1 H NMR spectroscopy.

-0.3 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 E (V) vs. Ag/AgNO 3 10 µA 0.6 eq 1 eq 2 eq 0 eq 5 eq Br NH 2

N

+ n e q 0 0.5 1 χ

Figure 2 shows the changes in the anodic peak potential and the current peak intensity obtained for different aniline/base pairs in acetonitrile. It should be noted that none of the bases was electroactive in the potential windows studied. In all cases, CVs were recorded first in the absence of the base (solid lines in Figure 2), and then in the presence of 5 equivalents of the base (dotted lines in Figure 2) to make sure that there is a maximum change. In this study, the change in potential was defined as the shift in the onset of the oxidation peak rather than the change in the half-peak potential or the peak potential, because of the changes in the peak shape obtained with some bases. The method used for the determination of the onset potential is described in the Supporting Information and illustrated with the p-bromoaniline/collidine in the Figure S2. Based on Figure 2 and E onset values presented in Tables 1 and2 for all p-substituted aniline/base pairs studied, some important conclusions can be drawn: i) the onset of oxidation of substituted anilines shifts towards less positive potentials either when the basicity of the base increases or when the acidity of the substituted aniline is exalted by an electronwithdrawing para substituent, suggesting that the amine cation radical of substituted anilines is deprotonated (this assumption is reinforced by the large shift obtained in the presence of DBU); ii) the energetics of the proton-transfer step, estimated from the difference between the pK a of the protonated form of the base and the pK a of the p-substituted aniline cation radicals (see Tables 1 and2), gives an overestimated value of the negative shift in potential (indicated by the length of arrows in Figure 2), proving that deprotonation alone is clearly insufficient to account for the potential shifts observed, so that an interaction between aromatic amines and bases is supposed to exist, the energy cost to break it justifying the lower values of the shift in potential. On the basis of NMR results, the postulated interaction is treated as being mainly a hydrogen bond interaction, so that the thermochemical cycle presented in Scheme 1 provides a simple conceptual framework for thinking about the role of the base on the oxidation of aromatic amines. Noted that all other predominantly electrostatic interactions can be treated by the same conceptual framework. According to these thermochemical concerns, the H-bond formation and the deprotonation have opposite effects on the potential, since a positive shift in potential is required to break the hydrogen bond interaction, while a negative shift results to the energetic deprotonation (see Equation (1) in Scheme 1). p-substituted aniline (1 mM) + 5 eq of the base

Scheme 1. Thermochemical cycle showing the effect of the hydrogen bonding and deprotonation steps on the oxidation potential of aniline compounds.

In parallel to this stepwise mechanism for the electron-transfer-deprotonation process, there is also a possibility for the protonic motion to be concerted to the electronic transfer with a unique transition state. [24] Eventually, as in other dissociative electron transfer reactions, a transition between a stepwise and a concerted mechanism may occur, since a large variation of the anodic peak potential and of the peak width is obtained over the extended series of aniline/base pairs studied. [25] Nevertheless, the determination of a possible passage from the stepwise to the concerted mechanism requires a detailed examination of the evolution of the anodic peak as a function of the scan rate and this issue will be discussed in a future article. In any case, whatever the mechanism (concerted or stepwise), the increase in the peak width when the base becomes stronger indicates that the electron-transfer step participates more and more significantly in the mixed control of the reaction kinetics as the driving force of the deprotonation step increases, implying that the main factor is the increase of the deprotonation rate constant. Noted that with the strong base DBU the large peak width, uncorrected from ohmic drop, suggests that complete kinetic control by the electron-transfer step is reached and the fact that the ensuing onset peak potential is out of the correlations derived from the scheme 1 (vide infra), may be an indication of a possible transition between stepwise and concerted mechanisms. Since such a transition cannot be excluded, the scheme 1 must be just regarded as a comprehensive thermochemical description of the effect of the hydrogen bonding on a proton-coupled electron transfer redox potential without any regard as the mechanism for the one-electron oxidation of the hydrogen-bonded aniline compounds occurs. [26] Theoretically, quantitative information such as the H-bond strength can be obtained by the Nernst equation when the H-bond donor or acceptor site is electroactive as long as the redox kinetics is fast and reversible. Therefore, the main reservation here is about the irreversibility of the amine oxidation and for this reason, it is of interest for the following discussion, to estimated how the anodic peak potential of substituted anilines deviated from their formal potentials. In seminal work on the mechanism of electrohydrodimerization and its impact on the shape of cyclic voltammograms, the Savéant groups have demonstrated that the peak potential deviate from its formal potential by only 20 mV and 30 mV per decade of scan rate or of their apparent dimerization rate constants for a DIM1 and DIM2 mechanisms under pure kinetic conditions (i.e. in a range of scan rate where no cathodic trace in cyclic voltammetry is observed). [27] Furthermore, in a previous study, Amatore et al. explored the impact of a base on the kinetics of the overall dimerization reaction of p-halogenoanilines in DMF. [21] Although the dimerization remains the major pathway in the presence of a base, authors have demonstrated that the kinetic of the dimerization reaction can be accelerated and even become irreversible, causing a shift in the anodic peak potential of 30 mV per decade of base concentration. It follows from such kinetic concerns and the above discussions on a possible transition from a stepwise to a concerted mechanism for the electron-transfer and deprotonation steps, that kinetic or mechanism changes may have an impact both on the shape of the irreversible anodic wave and the shift of the peak potential. These effects are assumed to be more and more significant as the base becomes stronger. In the following discussion, we assume that the impact of a base on the dimerization mechanism accounts for a small part of the shift in potential in comparison to the H-bonding interaction or the deprotonation of the amino cation radical, since the apparent dimerization rate constant, typically lower by 5 orders of magnitude than the diffusion limit, [21,22,28] implies a maximum potential shift of around 0.15 V. We also assume that there is no passage from stepwise to concerted mechanisms (or vice versa). It is also important to note that while such kinetic concerns complicate the use of absolute potentials, only the change in potential is important in equation ( 1), which is assumed to be close to the change in the formal potential between the H-bonded and non-H-bonded forms of the substituted anilines, pursuant to the above assumptions. It is clear that these simplifying assumptions are increasingly irrelevant the base becomes stronger, as attested by the system with the strong base DBU, which is out of the correlations (vide infra). Figure 3 depicts the shift in the onset potential of the oxidation of p-substituted anilines in the presence of nitrogen bases of different strengths with the driving force of the deprotonation step. One important observation is that, with the bases covering the pK a range 12.03 -24.13 in acetonitrile, the variation of the onset potential of the p-bromoaniline with the deprotonation reaction exergonicity is close to a Nernstian behavior (open symbols in Figure 3). This becomes even more apparent when the DBU is excluded from the linear fit of data. Conversely, the magnitude of the shift in the onset potential of the oxidation of the p-substituted anilines when collidine is present, strongly depends on the nature of the para substituent (closed symbols in Figure 3). While the oxidation peak of the p-methoxyaniline undergoes a very small shift because of the low acidity of its cation radical, p-substituted anilines are becoming more sensitive to the deprotonation as the electron-withdrawing nature of the para-substituent is increasing. For the strongest ones, the onset potential change parallels what is obtained for the pbromoaniline in the presence of bases, approaching a Nernstian behavior. These results can be partly rationalized by regarding the change in the H-bond acidity  and basicity  par eries of solutes studied in this work (see the inset in Figure 3). For clarity, a and b parameters are normalized to their minimum values (a min is for p-methoxyaniline and b min is for pyridine).

Figure 3. Variation of the onset potential of the oxidation of para-substituted anilines (1 mM) in acetonitrile + 0.1 M Bu4NPF6 in the presence of nitrogen bases (5 equivalents) with the driving force of the deprotonation of the anilinium radicals. Open symbols are for the p-bromoaniline/bases pairs and closed symbols are for the para-substituted anilines/collidine pairs (Roman and Greek numerals refer to the Tables 1 and2). Note that the solid curve serves just as a guideline for the eye. The inset shows the evolution of the H-bond acidity α and basicity β parameters with ∆pKa.

In a pioneering work, Abraham et al. have demonstrated that the Gibbs energy of a H-bond interaction (as log K HB ) can be expressed by the general equation ( 2), where m and c only depend on the solvent. [29,30] log K HB = mαβ + c

(2) In this αβ formalism, it appears that the energies of the H-bond interactions between the pbromoaniline and the bases used in this work are nearly the same, since except for DBU, the β parameter for the other bases is comprised within a very narrow range. It is the reason why the increase in electrochemical potential to make the H-bond interaction broken can be assumed constant, so that the apparent anodic potential shifts in a Nernstian manner with the exergonicity of the deprotonation step pursuant to equation (1). Conversely, because p-substituted anilines studied cover a wide H-bond acidity range, deviation to the Nernstian behavior is obtained. On plotting these log K HB values versus the product αβ for all the H-bond donor:acceptor (i.e. substituted-aniline/base pairs) combinations, there is some linear relationship (Figure 4), that is a good experimental evidence for the dominant H-bonding character in the molecular interaction between aniline and bases. Noted that the deviation obtained with the DBU (see the system V:8 in Figure 4) is presumably due to a different mechanism for the oxidation of the p-bromoaniline in the presence of a very strong base, as it was evoked above. It can also be due to a singular stoichiometry for the complex V:8, since the equation ( 2) is only applicable to 1:1 hydrogen bonded complexes, or to a deprotonation of the p-bromoaniline becoming energetically accessible with DBU. [31] Figure 4. Equilibrium constants (as log K values) vs. the αβ product for the formation of H-bonded complexes between para-substituted anilines (identified by Greek numerals) and bases (identified by Roman numerals).

These log K HB values correspond to Gibbs energies of the hydrogen bonding interactions comprised between 6 kcal.mol -1 to 16 kcal.mol -1 at 293 K. Here, it is important to bear in mind that the amino group can forms three H-bonds, in two of them NH 2 is a proton donor and in the third one nitrogen acts as a proton acceptor, so that the log K HB values (and the corresponding Gibbs energies) are for the overall H-bonding interactions. Noted also that, in the αβ formalism used in Figure 4, α values for individual hydrogen atoms were used and the contribution of the nitrogen acceptor sites to the H-bonding interaction was neglected. That being so, the results obtained agree well with the relevant literature, since classical hydrogen bonds (i.e., noncovalent interactions) span energies between 0.2 and 15 kcal.mol -1 . [32] The correlation in Figure 4 provides an important clue how to treat the impact of a base on the oxidation of aromatic amines, since, in most cases, solvatochromic parameters α and β provide a reasonable prediction about their initial interactions. So that this study provide us with a unique opportunity to explore the possibility that the overall H-bonding/deprotonation sequence influences the grafting efficiency of aromatic amines, which could help to establish guiding principles to rationalize the choice of the most suitable base for the grafting. Because the base must have both a high pK a value for increasing the driving force of the deprotonation step and, at the same time, a weak β parameter to make easier the breaking of the hydrogen bond, it is possible to obtain an efficiency parameter for the base in the grafting process that we express simply as ∆pK a /(αβ). Noted that for a base makes possible the grafting of an amine, its efficiency parameter must be positive. Figure 5a shows the evolution of this parameter with the Hammett coefficient σ p of the para substituent (for clarity, only the evolution of the efficiency index for the pyridine, collidine and DBU were showed). Remarkably, this efficiency index asymptotically tends towards the same value whatever the base considered. This constant value is assumed to be characteristic to the solvent as it can be obtained from equations (1) and (2). Figure 5b shows the evolution of the onset potential with the efficiency index of bases in the oxidation of the pbromoaniline. Importantly, up to an efficiency index of ca. 30 -40, the bases have practically no effect on the anodic potential, because, for this class of compounds, a certain increase in the driving force of the deprotonation step is required to compensate the energy to provide to break the H-bond interaction. In acetonitrile, conditions making the oxidation of the p-bromoaniline easier are obtained with the DBU. Therefore, as a proof-of-concept for base-assisted grafting of aromatic amines, these conditions were used to modify a carbon surface (Figure 5c). Importantly, when the modification of a glassy carbon surface was achieved in the presence of DBU, the very exergonic deprotonation of the amino cation radical makes possible the grafting of the pbromoaniline at very low potential, as evidenced by a strong Br 3d XPS signal for the carbon surface modified at 0.3 V vs. AgIAgNO 3 for 5 min with DBU in solution, compared to a blank experiment achieved in a pure p-bromoaniline solution (Figure 5c). For information, the survey XPS spectrum for the carbon surface modified with the p-bromoaniline/DBU system is presented in Figure S3. In order to generalize this approach, the p-nitroaniline/collidine system was also tested for the electrografting. This system leads to a situation very similar to that of the oxidation of the p-bromoaniline in the presence of DBU, since in both cases a very exergonic deprotonation step is obtained either by using a very strong base or a very acidic amino cation radical, as it is evidenced in Figure 2 and in the inset of Figure S4. In this system, the collidine is responsible of shifting the peak potential of the oxidation of the pnitroaniline by around 120 mV towards the less positive potentials, because of the exergonic deprotonation of the p-nitroaniline cation radical. But with the p-nitroaniline/collidine mixture, the Hbond interaction between the NH 2 group and the collidine has a strong antagonist effect on the potential compared to the deprotonation step and a moderate apparent potential shift is obtained. Nevertheless, the net increase in the driving force is sufficient to make possible the grafting of the p-nitroaniline at 1 V vs. Ag/AgNO 3 in the presence of collidine, while this is not the case in the absence of collidine, as it was evidenced by the presence or the absence of a photoelectron peak at ca. 406 eV ascribed to the nitro group in the N 1s XPS spectrum of carbon surfaces polarized in the presence or absence of collidine (see Figure S5).

In conclusion, this study shows the existence of an interaction between neutral aniline compounds and nitrogen bases that go against the driving force of the deprotonation of the amine cation radicals in the oxidation process of aromatic amines. Prediction of this interaction from H-bond acidity and basicity parameters provides a simple conceptual framework to study how the electrografting of aromatic amines is impacted by a base. An factor for the base in the oxidation process was proposed as a practical tool to choose the mostly suitable base for the grafting.

Experimental Section

Electrochemical measurements were made at 293 K in a three-electrode cell containing distilled acetonitrile + 0.1 M nBu 4 NPF 6 . The working electrode was a glassy carbon electrode mirror polished between each measurement. All potential values were referred to the Ag/AgNO 3 system. 1 H NMR experiments were conducted in benzene-d6. In the Job diagram, solutions of p-bromoaniline and collidine were prepared with equal concentration (50 mM) and various aliquots of each solution were mixed at constant volume (500 µL). XPS measurements were performed with a Kratos Axis Ultra spectrometer using a Al Kα monochromatic beam working at 1486.6 eV. All spectra were recorded in the constant energy mode at a pass energy of 20 eV.

Figure 2 .

 2 Figure 2. Cyclic voltammograms of different p-substituted anilines (1 mM) / bases (5 equivalents) pairs in acetonitrile + 0.1 M Bu4NPF6 recorded on a glassy carbon electrode at 0.1 V.s -1 .

Figure 5 .

 5 Figure 5. Base-assisted grafting of primary aromatic amines. (a) Variation of the efficiency parameter of the pyridine, collidine and DBU bases in the oxidation of para-substituted anilines with the Hammett coefficient σp of the para substituent. (b) Evolution of the onset potential with the efficiency parameter of the collidine in the oxidation of p-substituted anilines (closed symbols) and of the other bases studied in the oxidation of the pbromoaniline (open symbols). (c) Br 3d core level XPS spectra of glassy carbon surfaces polarized at 0.3 V vs. Ag/AgNO3 for 5 min in a pbromoaniline (1 mM) acetonitrile solution containing 0.1 M Bu4NPF6 in the absence and presence of 5 equivalents of DBU. Note that the dotted curves serve just as a guideline for the eye.

) vs. Ag/AgNO 3 base effect

	p-substituted aniline (1 mM)	Quinoline/p-BrPhNH 2
			(∆pK a = 7.84)
		Collidine/p-BrPhNH 2
		(∆pK a = 10.58)
	-0.4 substituent effect -0.2 20 µA	0 a 19.94) 0.2 (∆pK a = 2.91) E (VDBU/p-BrPhNH P-MeOAPhNH 2 0.4 0.6 Collidine/p-O 2 NPhNH 2 0.8 1 P-O 2 NPhNH 2 1.2 P-BrPhNH 2 Collidine/p-MeOPhNH 2 (∆pK a = 17.67)	1.4

Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (CNRS-France). We thanks the French Ministry for the PhD grant of E. Touzé.