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Regulation of nutrient recycling via autophagy
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ScienceDirect Regulation of nutrient recycling via autophagy
Ce´line Masclaux-Daubresse*, Qinwu Chen* and Marien Have´* Autophagy is a universal mechanism in eukaryotes that promotes cell longevity and nutrient recycling through the degradation of unwanted organelles, proteins and damaged cytoplasmic compounds. Autophagy is important in plant resistance to stresses and starvations and in remobilization. Autophagy facilitates bulk and selective degradations, through the delivery of cell material to the vacuole where hydrolases and proteases reside. Large metabolite modifications are observed in autophagy mutants showing the important role of autophagy in cell homeostasis. The control of autophagic activity by nutrients and energy status is supported by several studies in plant and animal. We review how autophagy contributes to nutrient management in plants and how nutrient status control this degradation pathway for adaptation to the environment. Address INRA-AgroParisTech, Institut Jean-Pierre Bourgin, UMR1318, ERL CNRS 3559, Saclay Plant Sciences, Versailles, France Corresponding author: Masclaux-Daubresse, Ce´line ([email protected]) * Institut Jean-Pierre Bourgin, INRA Route de Saint Cyr, 78000 Versailles, France. Current Opinion in Plant Biology 2017, 39:8–17 This review comes from a themed issue on Cell signalling and gene regulation Edited by Tzyy-Jen Chiou and Toru Fujiwara
http://dx.doi.org/10.1016/j.pbi.2017.05.001 1369-5266/ã 2017 Elsevier Ltd. All rights reserved.
Introduction Because plants are static, they cannot move to find the mineral nutrient they need, if the soil they explore is poor. Their survival then relies on their capacity to uptake the mineral nutrients available in their rhizosphere and to metabolize, recycle and conserve them efficiently all along their lifespan [1–3]. Recycling nutrients means to reuse the resources already present in the body, usually as constituents of macromolecules. Remobilization means that the nutrients released from recycling are translocated to the new growing organs and to sinks like seeds. The degradation of macromolecules for recycling and remobilization usually occurs in the senescing organs that are no more useful for the plant, except that they represent a source of nutrient. The unwanted, unneeded, Current Opinion in Plant Biology 2017, 39:8–17
useless macromolecules entering in the process, release sugars, amino acids (AA) and other small molecules useful for neo-syntheses. If plants are stressed or starved, the recycling processes are enhanced and exacerbated, leading occasionally to the sacrifice of healthy organs to feed and save the rest of the plant. In plant research, recycling and remobilization of nitrogen are of special interest for long [4] because of their strong impact on yield and grain quality, especially in cereals. Recycling and remobilization of micronutrients are also more and more studied due to their role in biofortification, which is important for food quality and human diet [5]. The recent advances on the physiological role of macroautophagy (then after termed autophagy) in plants show that autophagy machinery plays a crucial role in plant performance and resistance to stresses.
Autophagy definition and machinery in plants Autophagy (meaning self-eating) is a universal mechanism in eukaryotic cells that facilitates the vacuoledependent (in plant and yeast) or lysosome-dependent (in animal) degradation of unwanted cell constituents, and by the way, facilitates the recycling of cellular material [6]. Autophagy machinery consists in the formation in the cytosol of double membrane vesicles, termed autophagosomes that engulf and sequester cytoplasmic constituents (Figure 1). The substrates are diverse, for example damaged organelles or protein aggregates. They are not directly degraded inside the autophagosomes but driven thanks to the autophagosomes to the lytic vacuoles or lysosomes, where the proteases and hydrolases reside in. As cargos are not only proteins but also organelle fragments, autophagy is not only involved in the recycling of proteins or nitrogen sources, but also in the recycling of lipids and of any kind of micronutrients contained in the macromolecules [7,8]. When autophagosomes reach the lytic vacuoles, their outer membranes fuse to the vacuole membrane. The inner membrane and the cargo are then released into the lumen for degradation. At this step, the intra-vacuolar vesicle is named autophagic body. Autophagy thus also facilitates the degradation of a part of the membranes of its own machinery. Autophagy genes have been discovered by the pioneer work of Professor Yoshinori Ohsumi (Nobel Prize in medicine or physiology 2016) on yeast [9]. The screening of yeast mutants allowed him to identify the first autophagy gene APG1 (now ATG1). Further, more than 30 ATG genes were characterized by different groups, in Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha [10,6]. The characterization of the function of the www.sciencedirect.com
Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 9
Figure 1
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Schematic representation of the autophagy machinery. (A) More than 40 autophagy proteins are involved in the different steps of autophagy pathway in yeast. In plant, only few have been identified: www.sciencedirect.com
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10 Cell signalling and gene regulation
different ATG proteins (Figure 1) led to the identification of the ATG1–kinase complex involved (i) in the integration of environmental signals, including nutrient status sensed through the Target of Rapamycin (TOR) kinase, (ii) in the regulation of autophagy through the initiation of the pre-autophagosomal structure (PAS) formation, and (iii) with ATG9 in the elongation of the PAS membrane [6,11,12,13,14]. The ATG9 complex involves ATG9, ATG2 and ATG18. It participates to the recruitment of lipids. Membrane elongation also involves the PtdIns3K complex that is recruited to the PAS membrane to form the phagophore. Two conjugation systems ATG5– ATG12 and ATG8-PE contribute to the phagophore membrane expansion. Conjugating ATG8 to phosphatidylethanolamine facilitates its anchorage to the phagophore membrane. ATG8 abundance then regulates the size of the autophagosome. ATG8 is involved in cargobinding for selective autophagy [15,16]. Most of the proteins of the autophagy machinery, discovered in yeast, have been found in plant and animal. While each ATG protein is unique in yeast, multiple isoforms can exist in plant and animal. As such, nine AtATG8a-i isoforms are encoded by different genes in Arabidopsis. Autophagy has for long been considered as a nonselective bulk degradation of cytoplasm compounds under nutrient starvation. Selective autophagy, discovered more recently, involves ATG8, and adaptor proteins interacting with both ATG8 and cargos. In plant, the NBR1 (Neighbor of BRCA1 gene 1; [17]) and ATI1 (ATG8-interacting protein 1; [18]) proteins interact with AtATG8f. They are likely to be involved in selective autophagy, playing the role of adaptors. NBR1 targets ubiquitintagged proteins and ATI1 could participate in ER and thylakoid protein degradations. Although not fully understood, there are several lines of evidence that autophagy can degrade specifically mitochondria, peroxisomes, chloroplasts, proteasome, ribosomes, endoplasmic reticulum and other components in plant cells under specific
conditions [6,18,19,20,21,22,23,24,25,26–29,30,31] (Figure 2).
Autophagy for nutrient recycling in plants Autophagy, usually expressed at a basal level under normal conditions, constitutes housekeeping machinery that participates to cell homeostasis. During senescence and in response to stresses or starvations, autophagy is enhanced to facilitate the degradation of the increasing toxic and damaged components; the recycling of cell material is then used for nutrient remobilization. During leaf senescence autophagy is then likely to play two antagonistic roles, preserving cell longevity on one hand and participating to the methodic degradation of the cell constituents on the other hand. The tight balance between the two combined effects is likely to facilitate efficient recycling and remobilization as long as possible before cell death [32]. The first evidence that N remobilization from the leaves into the seeds is controlled by autophagy was provided in Arabidopsis by Masclaux-Daubresse group [33]. In their experiment, authors fed plants with 15NO3 during the vegetative growth period and evaluated 15N remobilization to the seeds in wild type (WT) and atg mutants. They showed that 15N remobilization was sharply decreased in all the atg mutants studied (atg18a RNAi, atg5 and atg9) compared to WT under N-limited conditions ([33]; Figure 3). Decrease was more moderate but significant in atg5 under nitrate-rich conditions. Salicylic acid (SA) was shown to accumulate in atg mutants and to be responsible of their early leaf senescence phenotype. Authors showed that the nitrogen remobilization defects were not related to the early leaf senescence phenotype of atg mutants and were maintained in the SA-free atg5.sid2 and atg5.NahG lines. Consistent with this, MasclauxDaubresse group showed in another publication that atg mutants accumulated more ammonium, amino acids (AA), and proteins in their rosette leaves than WT [34].
(Figure 1 Legend Continued) ATG1a-c, ATG11, ATG13a-b, ATG101 are involved in the induction of autophagy (a), ATG2, ATG9, ATG18a-h and the PtdIns3K (PI3K) complex ATG6–VPS15–VSP34 are involved in the elongation and the nucleation of the pre-autophagosomal structure (PAS) (b), and ATG3–ATG4ab–ATG7–ATG8a-i and ATG12ab–ATG5–ATG7–ATG10 are involved in the phagophore expansion and enclosure (c). Regarding fusion with tonoplast (d), digestion of cargos and recycling of nutrients (e), no gene has been clearly identified so far. Only genes identified in plant are presented in the figure. Genes in green colour are multigenic family genes, genes in black are single genes. (B) Detailed mechanism involved in the formation of autophagosome (core machinery). (a–c) Autophagy is induced by the dephosphorylation of the ATG1 kinase and ATG13 under nutrient-poor conditions. The activated ATG1–ATG13 complex promotes the nucleation of a cup-shaped doublemembrane involving the PI3K complex. The transmembrane protein ATG9 recruits lipids for membrane elongation, ATG2 and ATG18 proteins facilitate ATG9 recycling. (d–f) Expansion and closure of the phagophore membranes require two ubiquitination-like systems. ATG8 is initially processed by a cysteine protease ATG4, then conjugated to phosphatidyl ethanolamine (PE) by the conjugating enzyme ATG3. ATG12 is conjugated to ATG5 by the conjugating enzyme ATG10. Both of the conjugation systems share a single activating enzyme ATG7. The ATG12– ATG5 conjugate facilitates the lipidation of ATG8 with PE and its anchorage into the phagophore membrane. ATG8 membrane decoration facilitates the recruitment and enclosure of the cargos inside the autophagosome. ATG4 is also needed to remove and recycle ATG8 from ATG8PE. The autophagosome then transports the cargos to the vacuole for degradation by proteases and hydrolases. During the fusion process, the outer membrane of the autophagosome fuses with the tonoplast, and the remaining single-membrane structure (autophagic body) is delivered inside the vacuole. The digested products are then exported from the vacuole for recycling. Proteins in gray colour (ATG17, 29, 31, 14, and 16) participate to the autophagosome formation in yeast, but have not been identified in plant so far.
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Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 11
Figure 2
(j) TSPO-binding porphyrins degradation atg5
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Schematic representation of selective autophagy pathways reported in plants. (a) Degradation of chloroplasts (chlorophagy) through autophagy was observed by (i) engulfment of chloroplast and stroma fragments in Rubiscocontaining bodies (RCB) in individually darkened leaves, (ii) engulfment of entire chloroplasts in autophagosomal structures after UV-B treatment or (iii) formation of ATI bodies containing thylakoid proteins [18,24,25,27,31,60,61]. ATG8-Interacting Protein1 (ATI1) interact with both plastid proteins and ATG8 for autophagic plastid-to-vacuole trafficking. The ESCRT-III subunit paralogs Charged Multivesicular Body Protein1 CHMP1A and CHMP1B are required for autophagy-dependent plastid protein degradation in Arabidopsis. (b) Degradation of peroxisomes (pexophagy) has been reported in young seedlings submitted to hydrogen peroxide [21,22,62]. (c) Degradation of ribosomes (ribophagy). Ribosomal proteins selectively accumulated in autophagy mutants [34]. Autophagosomes and autophagic bodies contained ribosomes and RNA in rns2-2 mutants [63]. RNS2, an intracellular RNase T2 enzyme from Arabidopsis, is likely an essential component of ribophagy, for the decay and recycling of normal ribosomal RNA in plants. (d) Degradation of intracellular protein aggregates (aggrephagy) was shown in BY-2 tobacco cells after nitrogen starvation [64]. (e) Degradation of the protein Argonaute1 (AGO1) would participate to cellular reprograming [65]. The viral suppressor of RNA silencing protein P0 would trigger AGO1 degradation by the autophagy pathway. (f) Degradation of NBR1 (neighbour of BRCA1 gene) -targeted cargos was shown in Arabidopsis and tobacco [17,66]. The NBR1 cargo receptor targets ubiquitinated proteins for selective autophagy. (g) Degradation of mitochondria (mitophagy) is induced by senescence via a selective autophagic process requiring the ATG11 protein [20]. (h) Degradation of the 26S proteasome (proteaphagy) by autophagy involves the ubiquitin adaptor RPN10. RPN10 is essential for inhibitor-induced selective proteaphagy [26]. (i) Degradation of the endoplasmic reticulum (ER) by autophagy (reticulophagy/ER-phagy) has been shown in response to ER stress and depends on the ER stress sensor, Inositol-Requiring Enzyme-1b (IRE1b) [67]. (j) Degradation of TSPO-binding porphyrins [30]. TSPO is involved in binding and scavenging highly reactive porphyrins through autophagy by interacting with ATG8 proteins via a conserved AIM motif. Specific stress conditions and mutant backgrounds used to study each type of specific autophagy are mentioned.
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Current Opinion in Plant Biology 2017, 39:8–17
12 Cell signalling and gene regulation
Figure 3
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atg5, atg9, RNAi18a Current Opinion in Plant Biology
Schematic representation of the control of autophagy on nitrogen remobilization from leaves into the seeds and on protein degradation during leaf senescence. N remobilization from rosette leaves to the seeds was evaluated in wild type (A) and autophagy mutant plants (B) in Arabidopsis. (A, B) 15NO3 labeling pulse performed during vegetative stage, long time before flowering, facilitate 15N incorporation in the proteins of vegetative tissues. During plant growth and organ development, 15N remobilization into new sinks is performed through protein degradation, amino acid recycling and translocation to the phloem. At harvest, the amount of 15N determined by mass spectrometry in the seeds and the other plant organs facilitate the determination of the 15N partition to the seeds and thus nitrogen remobilization efficiency (NRE). N partitioning was affected in all the atg mutants which remobilized up to 60% less N to the seeds compared to the wild type under NO3 poor conditions and up to 40% less N under NO3 rich conditions revealing that N remobilization relies on autophagy machinery [33]. (C) In wild type autophagy machinery participates to the degradation of cytosolic components (a) chloroplasts (b), mitochondria (c) and peroxisomes (d). Cytoplasm components are engulfed in autophagosomes and delivered to the vacuole for degradation by resident proteases (e). Amino acids resulting from the protein degradation are then recycled in the cell or transferred to other organs such as developing seeds through the phloem (f). (D) atg mutants cannot deliver cargos to the vacuole and accumulate cytosolic protein aggregates (a), mitochondrial proteins (b) and peroxisomal proteins as catalase (c) and degradation products of chloroplastic proteins such as RuBisCo and GS2 (d) [34]. Chloroplast stromule buds increase (e) [24] and protein aggregate accumulation can become toxic, trapping transcription factors and signaling components [68]. The total proteolytic activities, which are mainly attributed to vacuolar proteases increase as well (g) [34] showing that protein accumulation in atg mutants is not due to a lack of proteolytic enzymes but rather to defects in substrate trafficking to the vacuole where they reside.
RNA contents were also higher in the atg rosettes whereas sugars and starch concentrations were lower [34]. The higher N/C ratio of atg rosette leaves was mainly due to the over accumulation of all sort of nitrogen compounds. While selective autophagy is usually considered for organelles, the western blots they performed showed that the accumulation of proteins in the atg mutants with ageing Current Opinion in Plant Biology 2017, 39:8–17
was selective and not related to the nature of the organelles that they reside in [34]. As such, degradationproducts of the chloroplast GS2 and of the Rubisco large subunit (LSU) accumulated in atg mutants, whereas the Fd-GOGAT, Thioredoxin-f and Rubisco small subunit (SSU) remained similar to WT [34,35]. The RPL13 and RPS6 ribosomal proteins (RP) also accumulated in atg www.sciencedirect.com
Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 13
mutants, while the S14 RP was unchanged. By contrast, all the mitochondrial proteins tested were likely to accumulate in atg mutants with ageing. Since the transcripts coding the accumulated proteins did not change, it was suggested that several proteins could be specifically targeted by autophagy [34]. Surprisingly, protease activities also increased in atg mutants with ageing. This then suggested that the overall protein accumulation observed was not due to a lack of protease activities, but to defects in the trafficking of these substrates to the vacuole where the proteases reside ([34,32]; Figure 3). Following the evidences obtained in Arabidopsis [33,34], N remobilization was studied in maize autophagy mutants by Vierstra group [36]. Authors showed that 15N remobilization to the kernels was affected in the maize atg12 autophagy-defective mutants [36]. The growth-rate of the atg12 maize mutants was sharply decreased under low-N conditions but not under high-N. Under high-N, seed yield was however much lower in atg12 than in WT, and 15N reallocation into the seeds was twice less in atg12. Surprisingly, the 15N partitioning in the new upper leaves produced after N labeling and located above the cob, was higher in atg12 compared to WT [36]. This suggests that N-remobilization from the old leaves to the new leaves was more efficient in atg12 while N-remobilization from the old leaves to the seeds was suppressed. This could indicate that, in maize, autophagy defects did not affect N recycling and mobilization from the source leaves, but rather modified N resource allocation during the formation of the new sinks that were the ear and kernels and upper leaves. Rice autophagy mutants were obtained by knocking down OsATG7 gene. Unfortunately the mutants were sterile and 15N remobilization could not be evaluated [28]. However investigations performed on vegetative tissues strongly suggested that autophagy also controls nutrient recycling in rice leaves [28]. While the accumulation of proteins in the leaves of Arabidopsis atg mutants described by Guiboileau et al. [34] appeared logical, the accumulation of AA and ammonium could not be explained unless considering that autophagy largely disturbs cell metabolism. The potential role of autophagy on ammonium or AA transporters still remains to be explored. As the abundance of ATG8 proteins at the cellular level could determine the size of autophagosomes, the overexpression of ATG8 genes was assayed in soybean, rice and Arabidopsis in order to determine whether increasing autophagic activity could benefit to the overall plant performance. Several reports demonstrate the positive effects of ATG8 overexpression on the tolerance to drought and to nitrate limitation [37–41]. However, the www.sciencedirect.com
impact on grain filling and N remobilization has not been determined so far.
Autophagy affects plant metabolism and controls C/N balance The function of autophagy in plant metabolism was examined by metabolomic analyses. Metabolite contents were measured by Izumi et al. [42] at the end of the night on 40d old plants grown under short days (SD), and by Masclaux-Daubresse et al. [43], 3 hours after the beginning of the light period, on 60 days plants grown under SD and both low or high nitrate conditions. Interestingly, these two independent studies showed similar features, for instance lower starch, sucrose and hexose contents and higher glutamate, aspartate and threonine contents in atg5 mutants compared to WT [42,43]. Comparing the atg5. sid2 and sid2 genotypes, Masclaux-Daubresse et al. showed that changes were SA-independent [34]. Autophagy, which is a recycling mechanism, essential for energy and nutrient supply under starvation, is especially required under SD conditions. Under SD, autophagy mutants are smaller than WT. The fact they do not store starch during the day and have low sugar contents makes them permanently carbon starved, and even more during the night [34,42,43]. The absence of carbon storage in autophagy mutants is certainly due to the fact that autophagy participates in chloroplast quality control. It also suggests that autophagy promotes growth during the night by delivering material for degradation, thus contributing to nutrient replenishment especially at the end of the night. Such hypotheses were supported by the close phenotypes observed in both the atg and starchless pgm1 mutants under SD [42]. Autophagy could provide AA for catabolism to compensate for sugar limitation in starchless mutants [42]. In addition to AA, atg mutants accumulated phospholipid-related intermediates. This suggested that autophagy mutants could not recycle membrane lipids that are source of energy through the b-oxidation pathway [42]. The accumulation of branched chain amino acids (BCCA) and aromatic amino acids (AAA) during leaf senescence is considered as a hallmark of enhanced protein degradation. The absence of modification in BCCA and AAA contents in atg5 is then consistent with the lower protein degradation suggested by the increase of protein concentrations [44,34]. The higher contents of glutamate, aspartate and threonine, if not arising from protein degradation, remain to be explained [11].
Nutritional status controls autophagy recycling pathway In plant, like in yeast and animal, TOR kinase was shown to be a master player in the control of autophagy [45]. Under nutrient deficiency, the TOR kinase is inactivated and the lack of phosphorylation of ATG1 and ATG13 Current Opinion in Plant Biology 2017, 39:8–17
14 Cell signalling and gene regulation
activates the formation of the ATG1 regulatory complex and thus of autophagy machinery ([46]; Figure 4). However, it appears that TOR is not the sole level of regulation of autophagy. In mammals, it was recently shown that high acetyl-coenzymeA (AcetylCoA) cytosolic content represses autophagy and that the acetyltransferase EP300 is required for the AcetylCoA-mediated inhibition [47]. Indeed, several lines of evidence show that AcetylCoA donors activate the mice TOR kinase (mTORC1)
and that knockdown of EP300 inhibits the activation of mTORC1 by AcetylCoA. In addition, it was shown that histone acetylation could be involved in the epigenetic regulation of autophagy and that acetylation of ATG3 is required for autophagy while deacetylation of ATG7 by Sirtuin 1 is necessary to initiate the starvation-induced autophagy [48–50]. Major integrator of the nutritional status, AcetylCoA is at the crossroads of lipid, sugar, and protein catabolisms. The role of AcetylCoA on the
Figure 4
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Schematic representation of the regulation of autophagy by nutritional status. Checkpoint 1, described in plant, involves the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme, a key actor of glycolysis linking the energy-consuming to the energy-producing step of this pathway. In plant the cytosolic isoform of GAPDH is able to interact with ATG3. Interaction down-regulates autophagic activity preventing ATG3-dependent ATG8-PE conjugation (a) [51]. In animal, AMPK-dependent phosphorylation and nuclear translocation of GAPDH facilitate Sirt1 activation and subsequent autophagy initiation upon glucose starvation (a0 ) [53]. Checkpoint 2, described in yeast and animal, involves cytosolic AcetylCoA, which is a major integrator of the nutritional status, being at the crossroad of fatty acid, sugar and protein metabolisms [47]. AcetylCoA content links the cellular metabolic state to the regulation of autophagy via the acetylation of various proteins. (b) Hyperacetylation of histones might be involved in epigenetic control of autophagy by repressing the expression of several ATG genes including ATG7 [50]. (c) Under high cytosolic AcetylCoA content, acetyltransferase EP300 induces the acetylation of the nutrient-sensing TOR kinase which is activated and then represses autophagy via the phosphorylation of ATG1 and ATG13 [46,47]. (d) Acetylation of ATG3 is required for its interaction with ATG8 [49]. On the contrary, acetylation of ATG7 down-regulates autophagy and deacetylation of ATG7 by energy sensor NADH-dependent Sirtuin 1 (Sirt1) is necessary to enhance starvation-induced autophagy [48]. (e) ATAF1, a senescence-related transcription factor could also integrate carbon status and control the expression of autophagy genes [58] and constitute a third level of metabolic checkpoint. Finally the TOR kinase (f) represents the post-transcriptional control level certainly located downstream all the other checkpoint levels represented in the figure. Dashed line arrows indicate signal sensing. Plain line arrows and lines indicate positive and negative regulations. Thin arrows indicate proteins interactions or modifications. Green lines and arrows indicate pathways found in plant only, red found in animal and yeast only, blue found in all.
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Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 15
control of autophagy in plants has not yet been explored, however another key step in the energy metabolic pathway, controlled by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme was found to interfere with the regulation of autophagy in both animal and plant. GAPDH catalyzes the conversion of glyceraldehyde-3phosphate to 1,3-bisphosphoglycerate, linking the energy-consuming steps of glycolysis with its energyproducing steps. In tobacco, physical interactions between the cytosolic glyceraldehyde-3-phosphate dehydrogenases (cytosolic GAPDH: GAPCs) and ATG3 have been demonstrated in vitro and in vivo [51]. Authors showed that reactive oxygen species inhibited GAPC– ATG3 interaction and promoted ATG3–ATG8 interaction. Consistently, silencing of GAPCs activated autophagy while GAPCs overexpression limited autophagy activity by inhibiting ATG3–ATG8 interaction [51]. In Arabidopsis, it was confirmed that gapc1 KO mutant exhibited enhanced autophagy compared to WT [52]. In animal, the regulatory mechanism is different. GAPDH is phosphorylated by AMPK (AMP-activated protein kinase) under glucose starvation. Phosphorylated GAPDH is then translocated into the nucleus where it facilitates the displacing Sirt1’s repressor and the activation of Sirt1 (Sirtuin1). In turn, Sirt1 activates autophagy [53]. It is now assumed that the transcriptional up-regulation of autophagy genes is a prerequisite to enhance autophagic activity [54,55,56]. This is especially admitted in the case of the ATG8 genes, since ATG8 protein abundance determines the size of autophagosomes. Leaf senescence is a major metabolic cataclysm in plants. Several lines of evidence indicate that several ATG genes are up regulated during leaf ageing and that senescence-related transcription factors could regulate directly autophagy genes in plants [57–59].
environmental signals and how plants adapt and manage nutrient reallocation. Future perspectives of this subject should focus on the metabolic regulations of autophagy and on the characterization of the cargos and adaptors involved in specific autophagy under nutrient deficiency.
Acknowledgements We express thanks to the ANR (Agence Nationale pour la Recheche) AutoAdapt program (ANR-12- ADAPT-0010-0) for providing financial support and salary to Dr Marien Have´. We express thanks to the China Scholarship Council (CSC) for providing financial support as PhD grant to Mr Qinwu Chen.
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Conclusion Autophagy is essential for nutrient recycling in all eukaryotes. Its role in plant tolerance to mineral nutrient limitations has been demonstrated. Autophagy is important for nitrogen remobilization and for seed filling. Modulation of the autophagic activity is essentially controlled by abiotic stresses like drought, temperature and mineral nutrient deficiency and by ageing. Despite the progresses made in autophagy research field in plants, the nature of the cargos taken in charge by the autophagosomes depending on the environmental constraints remains unknown. The mechanisms involved in the specificity of autophagy, remain open questions. The transcriptional, epigenetic, and metabolic regulation levels controlling autophagy in plant, under stress or in response to nutrient limitations, are poorly understood. Increasing our knowledge about the factors controlling autophagy pathway under stress and in response to nutrient deficiency, should reveal how plants integrate all the www.sciencedirect.com
10. Klionsky DJ, Cregg JM, Dunn WA, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M et al.: A unified nomenclature for yeast autophagy-related genes. Dev. Cell 2003, 5:539-545. 11. Yang X, Bassham DC: New insight into the mechanism and function of autophagy in plant cells. In International Review of Cell and Molecular Biology, vol. 320. Edited by Jeon KW . Academic Press Elsevier Inc.; 2015:1-40. 12. Zientara-Rytter K, Sirko A: To deliver or to degrade—an interplay of the ubiquitin–proteasome system, autophagy and vesicular transport in plants. FEBS J. 2016, 283:3534-3555. 13. Le Bars R, Marion J, Le Borgne R, Satiat-Jeunemaitre B, Bianchi MW: ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants. Nat. Commun. 2014, 5. This paper proposes a model to explains how phagophore grows into a sealed autophagosome. ATG5 is present at the phagophore site from and defines a torus-shaped structure on a flat cisternal early phagophore. The underlying endoplasmic reticulum suggests a tight connection between phagophore and endoplasmic reticulum. Current Opinion in Plant Biology 2017, 39:8–17
16 Cell signalling and gene regulation
14. Zhuang X, Chung KP, Cui Y, Lin W, Gao C, Kang B-H, Jiang L: ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 2017, 4:E426-E435. This study shows the role of ATG9 in autophagosome progression from the endoplasmic reticulum in plant cells, shedding new light on the membrane origins of autophagosome in plants. 15. Xie ZP, Nair U, Klionsky DJ: Atg8 controls phagophore expansion during autophagosome formation. Mol. Biol. Cell 2008, 19:3290-3298. 16. Noda NN, Ohsumi Y, Inagaki F: Atg8-family interacting motif crucial for selective autophagy. FEBS Lett. 2010, 584: 1379-1385. 17. Svenning S, Lamark T, Krause K, Johansen T: Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1. Autophagy 2011, 7:993-1010. 18. Michaeli S, Honig A, Levanony H, Peled-Zehavi H, Galili G: Arabidopsis ATG8-INTERACTING PROTEIN1 is involved in autophagy-dependent vesicular trafficking of plastid proteins to the vacuole. Plant Cell 2014, 26:4084-4101. 19. Floyd BE, Morriss SC, MacIntosh GC, Bassham DC: What to eat: evidence for selective autophagy in plants. J. Integr. Plant Biol. 2012, 54:907-920. 20. Li F, Vierstra RD: Arabidopsis ATG11, a scaffold that links the ATG1–ATG13 kinase complex to general autophagy and selective mitophagy. Autophagy 2014, 10:1466-1467. 21. Farmer LM, Rinaldi MA, Young PG, Danan CH, Burkhart SE, Bartel B: Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation. Plant Cell 2013, 25:4085-4100. 22. Shibata M, Oikawa K, Yoshimoto K, Kondo M, Mano S, Yamada K, Hayashi M, Sakamoto W, Ohsumi Y, Nishimura M: Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis. Plant Cell 2013, 25:4967-4983. 23. Goto-Yamada S, Mano S, Nakamori C, Kondo M, Yamawaki R, Kato A, Nishimura M: Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy. Plant Cell Physiol. 2014, 55:482496. 24. Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T: Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant Physiol. 2008, 148:142-155. 25. Spitzer C, Li F, Buono R, Roschzttardtz H, Chung T, Zhang M, Osteryoung KW, Vierstra RD, Otegui MS: The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis. The Plant Cell 2015, 27:391-402. This study shows that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1 B are required for the enclosure of autophagosomes and for the autophagic degradation of plastid proteins in Arabidopsis thaliana. 26. Marshall RS, Li FQ, Gemperline DC, Book AJ, Vierstra RD: Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol. Cell 2015, 58:1053-1066. 27. Michaeli S, Galili G, Genschik P, Fernie AR, Avin-Wittenberg T: Autophagy in plants—what’s new on the menu? Trends Plant Sci. 2016, 21:134-144. 28. Wada S, Hayashida Y, Izumi M, Kurusu T, Hanamata S, Kanno K, Kojima S, Yamaya T, Kuchitsu K, Makino A et al.: Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice. Plant Physiol. 2015, 168:60-U721. 29. Schreiber A, Peter M: Substrate recognition in selective autophagy and the ubiquitin–proteasome system. Biochim. Biophys. Acta 2014, 1843:163-181. Current Opinion in Plant Biology 2017, 39:8–17
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Current Opinion in Plant Biology 2017, 39:8–17
ScienceDirect Regulation of nutrient recycling via autophagy
Ce´line Masclaux-Daubresse*, Qinwu Chen* and Marien Have´* Autophagy is a universal mechanism in eukaryotes that promotes cell longevity and nutrient recycling through the degradation of unwanted organelles, proteins and damaged cytoplasmic compounds. Autophagy is important in plant resistance to stresses and starvations and in remobilization. Autophagy facilitates bulk and selective degradations, through the delivery of cell material to the vacuole where hydrolases and proteases reside. Large metabolite modifications are observed in autophagy mutants showing the important role of autophagy in cell homeostasis. The control of autophagic activity by nutrients and energy status is supported by several studies in plant and animal. We review how autophagy contributes to nutrient management in plants and how nutrient status control this degradation pathway for adaptation to the environment. Address INRA-AgroParisTech, Institut Jean-Pierre Bourgin, UMR1318, ERL CNRS 3559, Saclay Plant Sciences, Versailles, France Corresponding author: Masclaux-Daubresse, Ce´line ([email protected]) * Institut Jean-Pierre Bourgin, INRA Route de Saint Cyr, 78000 Versailles, France. Current Opinion in Plant Biology 2017, 39:8–17 This review comes from a themed issue on Cell signalling and gene regulation Edited by Tzyy-Jen Chiou and Toru Fujiwara
http://dx.doi.org/10.1016/j.pbi.2017.05.001 1369-5266/ã 2017 Elsevier Ltd. All rights reserved.
Introduction Because plants are static, they cannot move to find the mineral nutrient they need, if the soil they explore is poor. Their survival then relies on their capacity to uptake the mineral nutrients available in their rhizosphere and to metabolize, recycle and conserve them efficiently all along their lifespan [1–3]. Recycling nutrients means to reuse the resources already present in the body, usually as constituents of macromolecules. Remobilization means that the nutrients released from recycling are translocated to the new growing organs and to sinks like seeds. The degradation of macromolecules for recycling and remobilization usually occurs in the senescing organs that are no more useful for the plant, except that they represent a source of nutrient. The unwanted, unneeded, Current Opinion in Plant Biology 2017, 39:8–17
useless macromolecules entering in the process, release sugars, amino acids (AA) and other small molecules useful for neo-syntheses. If plants are stressed or starved, the recycling processes are enhanced and exacerbated, leading occasionally to the sacrifice of healthy organs to feed and save the rest of the plant. In plant research, recycling and remobilization of nitrogen are of special interest for long [4] because of their strong impact on yield and grain quality, especially in cereals. Recycling and remobilization of micronutrients are also more and more studied due to their role in biofortification, which is important for food quality and human diet [5]. The recent advances on the physiological role of macroautophagy (then after termed autophagy) in plants show that autophagy machinery plays a crucial role in plant performance and resistance to stresses.
Autophagy definition and machinery in plants Autophagy (meaning self-eating) is a universal mechanism in eukaryotic cells that facilitates the vacuoledependent (in plant and yeast) or lysosome-dependent (in animal) degradation of unwanted cell constituents, and by the way, facilitates the recycling of cellular material [6]. Autophagy machinery consists in the formation in the cytosol of double membrane vesicles, termed autophagosomes that engulf and sequester cytoplasmic constituents (Figure 1). The substrates are diverse, for example damaged organelles or protein aggregates. They are not directly degraded inside the autophagosomes but driven thanks to the autophagosomes to the lytic vacuoles or lysosomes, where the proteases and hydrolases reside in. As cargos are not only proteins but also organelle fragments, autophagy is not only involved in the recycling of proteins or nitrogen sources, but also in the recycling of lipids and of any kind of micronutrients contained in the macromolecules [7,8]. When autophagosomes reach the lytic vacuoles, their outer membranes fuse to the vacuole membrane. The inner membrane and the cargo are then released into the lumen for degradation. At this step, the intra-vacuolar vesicle is named autophagic body. Autophagy thus also facilitates the degradation of a part of the membranes of its own machinery. Autophagy genes have been discovered by the pioneer work of Professor Yoshinori Ohsumi (Nobel Prize in medicine or physiology 2016) on yeast [9]. The screening of yeast mutants allowed him to identify the first autophagy gene APG1 (now ATG1). Further, more than 30 ATG genes were characterized by different groups, in Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha [10,6]. The characterization of the function of the www.sciencedirect.com
Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 9
Figure 1
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Schematic representation of the autophagy machinery. (A) More than 40 autophagy proteins are involved in the different steps of autophagy pathway in yeast. In plant, only few have been identified: www.sciencedirect.com
Current Opinion in Plant Biology 2017, 39:8–17
10 Cell signalling and gene regulation
different ATG proteins (Figure 1) led to the identification of the ATG1–kinase complex involved (i) in the integration of environmental signals, including nutrient status sensed through the Target of Rapamycin (TOR) kinase, (ii) in the regulation of autophagy through the initiation of the pre-autophagosomal structure (PAS) formation, and (iii) with ATG9 in the elongation of the PAS membrane [6,11,12,13,14]. The ATG9 complex involves ATG9, ATG2 and ATG18. It participates to the recruitment of lipids. Membrane elongation also involves the PtdIns3K complex that is recruited to the PAS membrane to form the phagophore. Two conjugation systems ATG5– ATG12 and ATG8-PE contribute to the phagophore membrane expansion. Conjugating ATG8 to phosphatidylethanolamine facilitates its anchorage to the phagophore membrane. ATG8 abundance then regulates the size of the autophagosome. ATG8 is involved in cargobinding for selective autophagy [15,16]. Most of the proteins of the autophagy machinery, discovered in yeast, have been found in plant and animal. While each ATG protein is unique in yeast, multiple isoforms can exist in plant and animal. As such, nine AtATG8a-i isoforms are encoded by different genes in Arabidopsis. Autophagy has for long been considered as a nonselective bulk degradation of cytoplasm compounds under nutrient starvation. Selective autophagy, discovered more recently, involves ATG8, and adaptor proteins interacting with both ATG8 and cargos. In plant, the NBR1 (Neighbor of BRCA1 gene 1; [17]) and ATI1 (ATG8-interacting protein 1; [18]) proteins interact with AtATG8f. They are likely to be involved in selective autophagy, playing the role of adaptors. NBR1 targets ubiquitintagged proteins and ATI1 could participate in ER and thylakoid protein degradations. Although not fully understood, there are several lines of evidence that autophagy can degrade specifically mitochondria, peroxisomes, chloroplasts, proteasome, ribosomes, endoplasmic reticulum and other components in plant cells under specific
conditions [6,18,19,20,21,22,23,24,25,26–29,30,31] (Figure 2).
Autophagy for nutrient recycling in plants Autophagy, usually expressed at a basal level under normal conditions, constitutes housekeeping machinery that participates to cell homeostasis. During senescence and in response to stresses or starvations, autophagy is enhanced to facilitate the degradation of the increasing toxic and damaged components; the recycling of cell material is then used for nutrient remobilization. During leaf senescence autophagy is then likely to play two antagonistic roles, preserving cell longevity on one hand and participating to the methodic degradation of the cell constituents on the other hand. The tight balance between the two combined effects is likely to facilitate efficient recycling and remobilization as long as possible before cell death [32]. The first evidence that N remobilization from the leaves into the seeds is controlled by autophagy was provided in Arabidopsis by Masclaux-Daubresse group [33]. In their experiment, authors fed plants with 15NO3 during the vegetative growth period and evaluated 15N remobilization to the seeds in wild type (WT) and atg mutants. They showed that 15N remobilization was sharply decreased in all the atg mutants studied (atg18a RNAi, atg5 and atg9) compared to WT under N-limited conditions ([33]; Figure 3). Decrease was more moderate but significant in atg5 under nitrate-rich conditions. Salicylic acid (SA) was shown to accumulate in atg mutants and to be responsible of their early leaf senescence phenotype. Authors showed that the nitrogen remobilization defects were not related to the early leaf senescence phenotype of atg mutants and were maintained in the SA-free atg5.sid2 and atg5.NahG lines. Consistent with this, MasclauxDaubresse group showed in another publication that atg mutants accumulated more ammonium, amino acids (AA), and proteins in their rosette leaves than WT [34].
(Figure 1 Legend Continued) ATG1a-c, ATG11, ATG13a-b, ATG101 are involved in the induction of autophagy (a), ATG2, ATG9, ATG18a-h and the PtdIns3K (PI3K) complex ATG6–VPS15–VSP34 are involved in the elongation and the nucleation of the pre-autophagosomal structure (PAS) (b), and ATG3–ATG4ab–ATG7–ATG8a-i and ATG12ab–ATG5–ATG7–ATG10 are involved in the phagophore expansion and enclosure (c). Regarding fusion with tonoplast (d), digestion of cargos and recycling of nutrients (e), no gene has been clearly identified so far. Only genes identified in plant are presented in the figure. Genes in green colour are multigenic family genes, genes in black are single genes. (B) Detailed mechanism involved in the formation of autophagosome (core machinery). (a–c) Autophagy is induced by the dephosphorylation of the ATG1 kinase and ATG13 under nutrient-poor conditions. The activated ATG1–ATG13 complex promotes the nucleation of a cup-shaped doublemembrane involving the PI3K complex. The transmembrane protein ATG9 recruits lipids for membrane elongation, ATG2 and ATG18 proteins facilitate ATG9 recycling. (d–f) Expansion and closure of the phagophore membranes require two ubiquitination-like systems. ATG8 is initially processed by a cysteine protease ATG4, then conjugated to phosphatidyl ethanolamine (PE) by the conjugating enzyme ATG3. ATG12 is conjugated to ATG5 by the conjugating enzyme ATG10. Both of the conjugation systems share a single activating enzyme ATG7. The ATG12– ATG5 conjugate facilitates the lipidation of ATG8 with PE and its anchorage into the phagophore membrane. ATG8 membrane decoration facilitates the recruitment and enclosure of the cargos inside the autophagosome. ATG4 is also needed to remove and recycle ATG8 from ATG8PE. The autophagosome then transports the cargos to the vacuole for degradation by proteases and hydrolases. During the fusion process, the outer membrane of the autophagosome fuses with the tonoplast, and the remaining single-membrane structure (autophagic body) is delivered inside the vacuole. The digested products are then exported from the vacuole for recycling. Proteins in gray colour (ATG17, 29, 31, 14, and 16) participate to the autophagosome formation in yeast, but have not been identified in plant so far.
Current Opinion in Plant Biology 2017, 39:8–17
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Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 11
Figure 2
(j) TSPO-binding porphyrins degradation atg5
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Schematic representation of selective autophagy pathways reported in plants. (a) Degradation of chloroplasts (chlorophagy) through autophagy was observed by (i) engulfment of chloroplast and stroma fragments in Rubiscocontaining bodies (RCB) in individually darkened leaves, (ii) engulfment of entire chloroplasts in autophagosomal structures after UV-B treatment or (iii) formation of ATI bodies containing thylakoid proteins [18,24,25,27,31,60,61]. ATG8-Interacting Protein1 (ATI1) interact with both plastid proteins and ATG8 for autophagic plastid-to-vacuole trafficking. The ESCRT-III subunit paralogs Charged Multivesicular Body Protein1 CHMP1A and CHMP1B are required for autophagy-dependent plastid protein degradation in Arabidopsis. (b) Degradation of peroxisomes (pexophagy) has been reported in young seedlings submitted to hydrogen peroxide [21,22,62]. (c) Degradation of ribosomes (ribophagy). Ribosomal proteins selectively accumulated in autophagy mutants [34]. Autophagosomes and autophagic bodies contained ribosomes and RNA in rns2-2 mutants [63]. RNS2, an intracellular RNase T2 enzyme from Arabidopsis, is likely an essential component of ribophagy, for the decay and recycling of normal ribosomal RNA in plants. (d) Degradation of intracellular protein aggregates (aggrephagy) was shown in BY-2 tobacco cells after nitrogen starvation [64]. (e) Degradation of the protein Argonaute1 (AGO1) would participate to cellular reprograming [65]. The viral suppressor of RNA silencing protein P0 would trigger AGO1 degradation by the autophagy pathway. (f) Degradation of NBR1 (neighbour of BRCA1 gene) -targeted cargos was shown in Arabidopsis and tobacco [17,66]. The NBR1 cargo receptor targets ubiquitinated proteins for selective autophagy. (g) Degradation of mitochondria (mitophagy) is induced by senescence via a selective autophagic process requiring the ATG11 protein [20]. (h) Degradation of the 26S proteasome (proteaphagy) by autophagy involves the ubiquitin adaptor RPN10. RPN10 is essential for inhibitor-induced selective proteaphagy [26]. (i) Degradation of the endoplasmic reticulum (ER) by autophagy (reticulophagy/ER-phagy) has been shown in response to ER stress and depends on the ER stress sensor, Inositol-Requiring Enzyme-1b (IRE1b) [67]. (j) Degradation of TSPO-binding porphyrins [30]. TSPO is involved in binding and scavenging highly reactive porphyrins through autophagy by interacting with ATG8 proteins via a conserved AIM motif. Specific stress conditions and mutant backgrounds used to study each type of specific autophagy are mentioned.
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Current Opinion in Plant Biology 2017, 39:8–17
12 Cell signalling and gene regulation
Figure 3
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atg5, atg9, RNAi18a Current Opinion in Plant Biology
Schematic representation of the control of autophagy on nitrogen remobilization from leaves into the seeds and on protein degradation during leaf senescence. N remobilization from rosette leaves to the seeds was evaluated in wild type (A) and autophagy mutant plants (B) in Arabidopsis. (A, B) 15NO3 labeling pulse performed during vegetative stage, long time before flowering, facilitate 15N incorporation in the proteins of vegetative tissues. During plant growth and organ development, 15N remobilization into new sinks is performed through protein degradation, amino acid recycling and translocation to the phloem. At harvest, the amount of 15N determined by mass spectrometry in the seeds and the other plant organs facilitate the determination of the 15N partition to the seeds and thus nitrogen remobilization efficiency (NRE). N partitioning was affected in all the atg mutants which remobilized up to 60% less N to the seeds compared to the wild type under NO3 poor conditions and up to 40% less N under NO3 rich conditions revealing that N remobilization relies on autophagy machinery [33]. (C) In wild type autophagy machinery participates to the degradation of cytosolic components (a) chloroplasts (b), mitochondria (c) and peroxisomes (d). Cytoplasm components are engulfed in autophagosomes and delivered to the vacuole for degradation by resident proteases (e). Amino acids resulting from the protein degradation are then recycled in the cell or transferred to other organs such as developing seeds through the phloem (f). (D) atg mutants cannot deliver cargos to the vacuole and accumulate cytosolic protein aggregates (a), mitochondrial proteins (b) and peroxisomal proteins as catalase (c) and degradation products of chloroplastic proteins such as RuBisCo and GS2 (d) [34]. Chloroplast stromule buds increase (e) [24] and protein aggregate accumulation can become toxic, trapping transcription factors and signaling components [68]. The total proteolytic activities, which are mainly attributed to vacuolar proteases increase as well (g) [34] showing that protein accumulation in atg mutants is not due to a lack of proteolytic enzymes but rather to defects in substrate trafficking to the vacuole where they reside.
RNA contents were also higher in the atg rosettes whereas sugars and starch concentrations were lower [34]. The higher N/C ratio of atg rosette leaves was mainly due to the over accumulation of all sort of nitrogen compounds. While selective autophagy is usually considered for organelles, the western blots they performed showed that the accumulation of proteins in the atg mutants with ageing Current Opinion in Plant Biology 2017, 39:8–17
was selective and not related to the nature of the organelles that they reside in [34]. As such, degradationproducts of the chloroplast GS2 and of the Rubisco large subunit (LSU) accumulated in atg mutants, whereas the Fd-GOGAT, Thioredoxin-f and Rubisco small subunit (SSU) remained similar to WT [34,35]. The RPL13 and RPS6 ribosomal proteins (RP) also accumulated in atg www.sciencedirect.com
Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 13
mutants, while the S14 RP was unchanged. By contrast, all the mitochondrial proteins tested were likely to accumulate in atg mutants with ageing. Since the transcripts coding the accumulated proteins did not change, it was suggested that several proteins could be specifically targeted by autophagy [34]. Surprisingly, protease activities also increased in atg mutants with ageing. This then suggested that the overall protein accumulation observed was not due to a lack of protease activities, but to defects in the trafficking of these substrates to the vacuole where the proteases reside ([34,32]; Figure 3). Following the evidences obtained in Arabidopsis [33,34], N remobilization was studied in maize autophagy mutants by Vierstra group [36]. Authors showed that 15N remobilization to the kernels was affected in the maize atg12 autophagy-defective mutants [36]. The growth-rate of the atg12 maize mutants was sharply decreased under low-N conditions but not under high-N. Under high-N, seed yield was however much lower in atg12 than in WT, and 15N reallocation into the seeds was twice less in atg12. Surprisingly, the 15N partitioning in the new upper leaves produced after N labeling and located above the cob, was higher in atg12 compared to WT [36]. This suggests that N-remobilization from the old leaves to the new leaves was more efficient in atg12 while N-remobilization from the old leaves to the seeds was suppressed. This could indicate that, in maize, autophagy defects did not affect N recycling and mobilization from the source leaves, but rather modified N resource allocation during the formation of the new sinks that were the ear and kernels and upper leaves. Rice autophagy mutants were obtained by knocking down OsATG7 gene. Unfortunately the mutants were sterile and 15N remobilization could not be evaluated [28]. However investigations performed on vegetative tissues strongly suggested that autophagy also controls nutrient recycling in rice leaves [28]. While the accumulation of proteins in the leaves of Arabidopsis atg mutants described by Guiboileau et al. [34] appeared logical, the accumulation of AA and ammonium could not be explained unless considering that autophagy largely disturbs cell metabolism. The potential role of autophagy on ammonium or AA transporters still remains to be explored. As the abundance of ATG8 proteins at the cellular level could determine the size of autophagosomes, the overexpression of ATG8 genes was assayed in soybean, rice and Arabidopsis in order to determine whether increasing autophagic activity could benefit to the overall plant performance. Several reports demonstrate the positive effects of ATG8 overexpression on the tolerance to drought and to nitrate limitation [37–41]. However, the www.sciencedirect.com
impact on grain filling and N remobilization has not been determined so far.
Autophagy affects plant metabolism and controls C/N balance The function of autophagy in plant metabolism was examined by metabolomic analyses. Metabolite contents were measured by Izumi et al. [42] at the end of the night on 40d old plants grown under short days (SD), and by Masclaux-Daubresse et al. [43], 3 hours after the beginning of the light period, on 60 days plants grown under SD and both low or high nitrate conditions. Interestingly, these two independent studies showed similar features, for instance lower starch, sucrose and hexose contents and higher glutamate, aspartate and threonine contents in atg5 mutants compared to WT [42,43]. Comparing the atg5. sid2 and sid2 genotypes, Masclaux-Daubresse et al. showed that changes were SA-independent [34]. Autophagy, which is a recycling mechanism, essential for energy and nutrient supply under starvation, is especially required under SD conditions. Under SD, autophagy mutants are smaller than WT. The fact they do not store starch during the day and have low sugar contents makes them permanently carbon starved, and even more during the night [34,42,43]. The absence of carbon storage in autophagy mutants is certainly due to the fact that autophagy participates in chloroplast quality control. It also suggests that autophagy promotes growth during the night by delivering material for degradation, thus contributing to nutrient replenishment especially at the end of the night. Such hypotheses were supported by the close phenotypes observed in both the atg and starchless pgm1 mutants under SD [42]. Autophagy could provide AA for catabolism to compensate for sugar limitation in starchless mutants [42]. In addition to AA, atg mutants accumulated phospholipid-related intermediates. This suggested that autophagy mutants could not recycle membrane lipids that are source of energy through the b-oxidation pathway [42]. The accumulation of branched chain amino acids (BCCA) and aromatic amino acids (AAA) during leaf senescence is considered as a hallmark of enhanced protein degradation. The absence of modification in BCCA and AAA contents in atg5 is then consistent with the lower protein degradation suggested by the increase of protein concentrations [44,34]. The higher contents of glutamate, aspartate and threonine, if not arising from protein degradation, remain to be explained [11].
Nutritional status controls autophagy recycling pathway In plant, like in yeast and animal, TOR kinase was shown to be a master player in the control of autophagy [45]. Under nutrient deficiency, the TOR kinase is inactivated and the lack of phosphorylation of ATG1 and ATG13 Current Opinion in Plant Biology 2017, 39:8–17
14 Cell signalling and gene regulation
activates the formation of the ATG1 regulatory complex and thus of autophagy machinery ([46]; Figure 4). However, it appears that TOR is not the sole level of regulation of autophagy. In mammals, it was recently shown that high acetyl-coenzymeA (AcetylCoA) cytosolic content represses autophagy and that the acetyltransferase EP300 is required for the AcetylCoA-mediated inhibition [47]. Indeed, several lines of evidence show that AcetylCoA donors activate the mice TOR kinase (mTORC1)
and that knockdown of EP300 inhibits the activation of mTORC1 by AcetylCoA. In addition, it was shown that histone acetylation could be involved in the epigenetic regulation of autophagy and that acetylation of ATG3 is required for autophagy while deacetylation of ATG7 by Sirtuin 1 is necessary to initiate the starvation-induced autophagy [48–50]. Major integrator of the nutritional status, AcetylCoA is at the crossroads of lipid, sugar, and protein catabolisms. The role of AcetylCoA on the
Figure 4
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Schematic representation of the regulation of autophagy by nutritional status. Checkpoint 1, described in plant, involves the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme, a key actor of glycolysis linking the energy-consuming to the energy-producing step of this pathway. In plant the cytosolic isoform of GAPDH is able to interact with ATG3. Interaction down-regulates autophagic activity preventing ATG3-dependent ATG8-PE conjugation (a) [51]. In animal, AMPK-dependent phosphorylation and nuclear translocation of GAPDH facilitate Sirt1 activation and subsequent autophagy initiation upon glucose starvation (a0 ) [53]. Checkpoint 2, described in yeast and animal, involves cytosolic AcetylCoA, which is a major integrator of the nutritional status, being at the crossroad of fatty acid, sugar and protein metabolisms [47]. AcetylCoA content links the cellular metabolic state to the regulation of autophagy via the acetylation of various proteins. (b) Hyperacetylation of histones might be involved in epigenetic control of autophagy by repressing the expression of several ATG genes including ATG7 [50]. (c) Under high cytosolic AcetylCoA content, acetyltransferase EP300 induces the acetylation of the nutrient-sensing TOR kinase which is activated and then represses autophagy via the phosphorylation of ATG1 and ATG13 [46,47]. (d) Acetylation of ATG3 is required for its interaction with ATG8 [49]. On the contrary, acetylation of ATG7 down-regulates autophagy and deacetylation of ATG7 by energy sensor NADH-dependent Sirtuin 1 (Sirt1) is necessary to enhance starvation-induced autophagy [48]. (e) ATAF1, a senescence-related transcription factor could also integrate carbon status and control the expression of autophagy genes [58] and constitute a third level of metabolic checkpoint. Finally the TOR kinase (f) represents the post-transcriptional control level certainly located downstream all the other checkpoint levels represented in the figure. Dashed line arrows indicate signal sensing. Plain line arrows and lines indicate positive and negative regulations. Thin arrows indicate proteins interactions or modifications. Green lines and arrows indicate pathways found in plant only, red found in animal and yeast only, blue found in all.
Current Opinion in Plant Biology 2017, 39:8–17
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Regulation of nutrient recycling via autophagy Masclaux-Daubresse, Chen and Have´ 15
control of autophagy in plants has not yet been explored, however another key step in the energy metabolic pathway, controlled by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme was found to interfere with the regulation of autophagy in both animal and plant. GAPDH catalyzes the conversion of glyceraldehyde-3phosphate to 1,3-bisphosphoglycerate, linking the energy-consuming steps of glycolysis with its energyproducing steps. In tobacco, physical interactions between the cytosolic glyceraldehyde-3-phosphate dehydrogenases (cytosolic GAPDH: GAPCs) and ATG3 have been demonstrated in vitro and in vivo [51]. Authors showed that reactive oxygen species inhibited GAPC– ATG3 interaction and promoted ATG3–ATG8 interaction. Consistently, silencing of GAPCs activated autophagy while GAPCs overexpression limited autophagy activity by inhibiting ATG3–ATG8 interaction [51]. In Arabidopsis, it was confirmed that gapc1 KO mutant exhibited enhanced autophagy compared to WT [52]. In animal, the regulatory mechanism is different. GAPDH is phosphorylated by AMPK (AMP-activated protein kinase) under glucose starvation. Phosphorylated GAPDH is then translocated into the nucleus where it facilitates the displacing Sirt1’s repressor and the activation of Sirt1 (Sirtuin1). In turn, Sirt1 activates autophagy [53]. It is now assumed that the transcriptional up-regulation of autophagy genes is a prerequisite to enhance autophagic activity [54,55,56]. This is especially admitted in the case of the ATG8 genes, since ATG8 protein abundance determines the size of autophagosomes. Leaf senescence is a major metabolic cataclysm in plants. Several lines of evidence indicate that several ATG genes are up regulated during leaf ageing and that senescence-related transcription factors could regulate directly autophagy genes in plants [57–59].
environmental signals and how plants adapt and manage nutrient reallocation. Future perspectives of this subject should focus on the metabolic regulations of autophagy and on the characterization of the cargos and adaptors involved in specific autophagy under nutrient deficiency.
Acknowledgements We express thanks to the ANR (Agence Nationale pour la Recheche) AutoAdapt program (ANR-12- ADAPT-0010-0) for providing financial support and salary to Dr Marien Have´. We express thanks to the China Scholarship Council (CSC) for providing financial support as PhD grant to Mr Qinwu Chen.
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Conclusion Autophagy is essential for nutrient recycling in all eukaryotes. Its role in plant tolerance to mineral nutrient limitations has been demonstrated. Autophagy is important for nitrogen remobilization and for seed filling. Modulation of the autophagic activity is essentially controlled by abiotic stresses like drought, temperature and mineral nutrient deficiency and by ageing. Despite the progresses made in autophagy research field in plants, the nature of the cargos taken in charge by the autophagosomes depending on the environmental constraints remains unknown. The mechanisms involved in the specificity of autophagy, remain open questions. The transcriptional, epigenetic, and metabolic regulation levels controlling autophagy in plant, under stress or in response to nutrient limitations, are poorly understood. Increasing our knowledge about the factors controlling autophagy pathway under stress and in response to nutrient deficiency, should reveal how plants integrate all the www.sciencedirect.com
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16 Cell signalling and gene regulation
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