Autophagy in Plants – What's New on the Menu?

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Review

Autophagy in Plants – What's New on the Menu? Simon Michaeli,1 Gad Galili,2 Pascal Genschik,3 Alisdair R. Fernie,4 and Tamar Avin-Wittenberg4,* Autophagy is a major cellular degradation pathway in eukaryotes. Recent studies have revealed the importance of autophagy in many aspects of plant life, including seedling establishment, plant development, stress resistance, metabolism, and reproduction. This is manifested by the dual ability of autophagy to execute bulk degradation under severe environmental conditions, while simultaneously to be highly selective in targeting specific compartments and protein complexes to regulate key cellular processes, even during favorable growth conditions. Delivery of cellular components to the vacuole enables their recycling, affecting the plant metabolome, especially under stress. Recent research in Arabidopsis has further unveiled fundamental mechanistic aspects in autophagy which may have relevance in non-plant systems. We review the most recent discoveries concerning autophagy in plants, touching upon all these aspects. Engulfing Plant Autophagy Research Macroautophagy (hereafter termed autophagy; see Glossary) is a common eukaryotic mechanism for the degradation of cellular components either in a housekeeping capacity or during stress [1]. Initially described in yeast (Saccharomyces cerevisiae) [2], autophagy was later shown to serve important roles in animal systems, ranging from development to involvement in neurodegenerative diseases [3,4]. Although characterized in plants (reviewed in [5–8]), autophagy has been relatively less studied in plant systems. However, in the past few years the study of autophagy in plants has flourished. First, it has expanded from researching the model plant Arabidopsis thaliana (Arabidopsis) to a multitude of photosynthetic organisms, including aquatic photosynthetic eukaryotes [9–12], gymnosperms [13], and angiosperms, including monocots [14–18] and dicots [18–20]. In addition, new evidence has emerged regarding the role of autophagy in cell survival [21–23] and cell death (recently reviewed by Minina et al. [24]. We describe here recent advances in plant autophagy, focusing on the mechanistic regulation of autophagy, selective autophagy, and autophagy function in nutrient availability and recycling.

Recent Insights into the Regulation and Mechanism of Autophagy in Plants Efforts to describe the components, assembly, and regulatory factors of the autophagy machinery in plants are ongoing. Several recent works have greatly increased our understanding of this multifaceted and important issue. The autophagy-related (ATG) 1/13 kinase complex has been characterized in Arabidopsis [25,26]. Plant homologs of ATG1, ATG13, ATG11, ATG17, and ATG101 have been described, with ATG1 being encoded by a four-member family and ATG13 encoded by a pair of genes [25]. ATG17 was not identified as a separate gene, but instead as an ATG17-like domain within the ATG11 protein [26]. ATG1 was shown to interact with ATG13 and ATG8, and ATG13 was demonstrated to interact with ATG11 and ATG101. ATG11, in turn, was shown to dimerize as well as interact with ATG101 and ATG8, strengthening the function of these various proteins as one complex. Both the atg13 double-knockout and

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Trends Autophagy is involved in almost every aspect of plant life, including germination, seedling establishment, development, reproduction, metabolism, and stress tolerance. Proteins that are involved in fundamental processes of autophagy, such as autophagosome biogenesis, were recently characterized in plants. Autophagy is intimately associated with other intracellular trafficking pathways. Several selective autophagy pathways were recently identified in Arabidopsis; most are common to all eukaryotes. Nevertheless, some pathways were initially discovered in plants and others are plant-specific. As an intracellular recycling system, autophagy is highly important for proper plant metabolism and nutrient allocation, both during stress and favorable growth conditions.

1 Biochimie et Physiologie Moléculaire des Plantes, Unité Mixte de Recherche 5004 Centre National de la Recherche Scientifique (CNRS)/Institut National de la Recherche Agronomique (INRA)/Université de Montpellier 2 (UM2)/SupAgro, 2 Place Viala, 34060 Montpellier, France 2 Weizmann Institute of Science, 234 Herzl Street, 7610001 Rehovot, Israel 3 Institut de Biologie Moléculaire des Plantes, CNRS Unité Propre de Recherche 2357, Conventionné avec l’Université de Strasbourg, 67084 Strasbourg, France 4 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam, Germany

*Correspondence: [email protected] (T. Avin-Wittenberg).

http://dx.doi.org/10.1016/j.tplants.2015.10.008 © 2015 Elsevier Ltd. All rights reserved.

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atg11 knockout plants display the classical atg mutant phenotype, namely hypersensitivity to carbon and nitrogen starvation, albeit to lesser extent than the atg7 mutant [25,26]. These findings were surprising because the ATG1/13 complex was shown to function in phagophore assembly in yeast and animals [27,28], as well as to be involved in autophagosome biogenesis, unveiling an additional role in later stages of autophagy [29]. Nevertheless, in plants the complex was shown to be crucial for the existence of autophagic bodies in the vacuole, but not for ATG5 and ATG8 modification. These results, coupled with the milder knockout phenotype led to the hypothesis that, in plants, the ATG1/13 complex is involved solely in advanced steps of autophagy, namely autophagosome enclosure. Finally, rapid turnover of the complex was demonstrated under nutrient starvation. This turnover is autophagy-dependent and is mediated by ATG11, and thus has been suggested by the authors to serve as a feedback mechanism for autophagy induction during starvation [25,26]. The early steps of the autophagosome formation have not been well defined in plants. 3D visualization of autophagosome morphology in Arabidopsis root epidermal cells helped to define the role of ATG5 in structure determination of the growing autophagosome. The results led to a proposed model of autophagosome formation in plants [30]. Upon autophagy induction, ATG5 was shown to be localized to the external surface of the cortical endoplasmic reticulum (ER) and recruit ATG8 to the growing phagophore. As the phagophore expands, ATG5 continues to be localized to the edges of the growing autophagosome membrane and, when it becomes cupshaped, ATG5 forms a ring-like structure on the aperture of the phagophore. When the phagophore membrane is sealed, ATG5 leaves the structure and the newly formed autophagosome dislocates from the ER membrane [30]. In addition, a non-ATG protein, the SH3 domain-containing protein 2 (SH3P2) has recently been shown to regulate autophagosome formation in Arabidopsis. SH3P2 is recruited to the phagophore assembly site (PAS) upon autophagy induction and facilitates autophagosome formation via binding to phosphatidylinositol 3-phosphate (PIP), the PI3K complex, and ATG8. SH3P2 contains an N-terminal Bin– amphiphysin–Rvs (BAR) domain, implicated in membrane remodeling and the interaction between SH3P2 and ATG8, that is claimed to facilitate membrane deformation during autophagosome development [31]. Several studies point to a connection between autophagy and other trafficking mechanisms in the cell. The exocyst is a protein complex classically defined as mediating the targeting and fusion of vesicles generated in the Golgi apparatus and targeted to the plasma membrane (PM), though it has been shown to mediate other trafficking functions [32]. One version of the exocyst complex, containing EXO70B1 (EXOCYST COMPLEX COMPONENT EXO70B1), has been shown to regulate autophagy in Arabidopsis. exo70B1 mutants were hypersensitive to nitrogen starvation and displayed reduced numbers of autophagic bodies in their vacuoles compared to wild-type (WT) plants. Interestingly, they were not sensitive to carbon starvation, which might indicate the involvement of EXO70B1 in a more specific autophagic response [33]. In addition, all the Arabidopsis EXO70 homologs were shown to contain an ATG8 interacting motif (AIM), suggesting a possible interaction between EXO70 and ATG8 [34]. This report was preceded by evidence from mammalian systems, implicating other exocyst complex proteins in autophagosome initiation [35], begging the question of whether the interaction between autophagy and the exocyst complex is similar or different between the two kingdoms. Accumulating evidence has also implicated the endosomal complex required for transport (ESCRT) in the regulation of autophagy. This complex sorts ubiquitinated proteins in endosomes and targets them to intraluminal vesicles (ILVs) that compose the multivesicular body (MVB). The proteins are eventually transported to the vacuole for degradation [36] (Figure 1A). Studies in animal systems have shown that ESCRT function is necessary for autophagosomal degradation [37–39], and several recent works have also demonstrated this in Arabidopsis [40–43]. Surprisingly, the phenotypes of various ESCRT mutants are non-identical. Overexpression of the ESCRT-III

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Glossary ATG8-interacting motif (AIM): termed LIR (light-chain 3 interacting region) in animals, this motif is found in the amino acid sequence of most ATG8-interacting proteins and facilitates their interaction with ATG8 and subsequent incorporation into the growing autophagosome. Autophagic body: the consequence of an autophagosome fusing with a vacuole. Once the autophagosome reaches a vacuole, its outer membrane fuses with the vacuole limiting membrane (tonoplast) to enable the release of the autophagosome inner membrane and the cargo it harbors into the vacuole lumen. Inside the vacuole, this singlemembrane structure is termed the autophagic body. Although rapidly degraded within the vacuole, chemically elevating vacuolar pH enables the visualization of autophagic bodies. Autophagosome: a doublemembrane (or multi-membrane) structure engulfing cytosolic material that is destined for delivery to the vacuole or lysosome. Its biogenesis is mainly (but not exclusively) carried out by proteins annotated as autophagy-related (ATG). Autophagy: Greek for ‘self-eating’, a catabolic cellular process involving the delivery of cytosolic components (including entire organelles, macromolecules, and invading pathogens) to the vacuole for their degradation and recycling. The term autophagy encompasses several distinct pathways, including microautophagy (the engulfment of cytoplasm by vacuolar invagination), chaperone-mediated autophagy (delivery of proteins to the vacuole by chaperones), and macroautophagy, which is the focus of this review (and referred to throughout the text as ‘autophagy’). This type of autophagy is mediated by the generation of autophagosomes that engulf cytosolic cargo and deliver it to the lytic organelle (the vacuole in plants and yeast; lysosomes in animals). Autophagy-related (ATG) proteins: proteins that mainly function in the biogenesis of autophagosomes. Below is a list of some of these proteins divided into functional groups. Proteins participating in autophagosome trafficking to vacuole, its fusion with the tonoplast and release of the autophagic body,

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Figure 1. Autophagic Phenotypes of ESCRT Mutants. (A) In wild-type (WT) cells, endosomes are transported to the multivesicular body (MVB) for further degradation in the vacuole; autophagosomes engulf cargo (either from the chloroplast or from other compartments) and are then transported to the vacuole for degradation. (B) cells expressing the dominant negative 35S::VPS2.1–GFP display impaired endocytosis and an accumulation of autophagosomes in their cytosol stemming from lack of autophagosome fusion with the vacuole. (C) Cells lacking FREE1 display impaired endocytosis and autophagosome–vacuole fusion as well as defects in central vacuole morphogenesis. (D) cells lacking CHMP1 display diminished endocytosis as well as impaired autophagosome loading of plastid proteins and defective plastid division. Adapted from [40–43,93]. Abbreviations: CHMP1, CHARGED MULTIVESICULAR BODY PROTEIN 1; ESCRT, endosomal complex required for transport; FREE1, FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1, VPS2.1, VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 2 HOMOLOG 1; 35S::VPS2.1–GFP, VPS2-1–GFP fusion under the control of the cauliflower mosaic virus 35S promoter.

are not listed here. For further insight regarding ATG proteins mode of action we recommend the following reviews [7,8].ATG1 complex: an upstream regulator of autophagy that is itself negatively regulated by TARGET OF RAPAMYCIN (TOR) kinase which is sensitive to the nutritional status of the organism. When autophagy is required, TOR kinase is inhibited, allowing the ATG1 complex to assemble and induce autophagy in a so far unknown manner. The ATG1 complex includes ATG1, ATG13, ATG11, ATG17, ATG27, and ATG31 in yeast, and ATG1, ATG13, ATG101, and FIP200 (FAK family kinase-interacting protein 200 kDa) in mammals. No homolog of ATG 27 and ATG 31 (with respect to its amino acid sequence) was found in photosynthetic organisms, and an ATG17-like domain was found in ATG11.ATG9 cycling system: ATG9 is a transmembrane protein that apparently cycles between endomembranes and the phagophore assembly site (PAS) to supply membrane for the growing phagophore, where it interacts with ATG18 and ATG2.Class III phosphoinositide 3-kinase (PI3K) complex: generates phosphatidylinositol 3 phosphate (PI3P) in the PAS; seemingly to enable endomembrane machineries to distinguish between the phagophore and other endomembranes. This complex includes VPS34, ATG6, VPS15, and ATG14. No homolog of ATG 14 (with respect to its amino acid sequence) was found in photosynthetic organisms.Ubiquitin-like conjugation system: this system actually comprises two ubiquitin-like conjugation systems, ATG12 and of ATG8, that eventually coincide to conjugate ATG8 to phosphatidylethanolamine (forming the ATG8–PE adduct). ATG8–PE is required for autophagosome maturation and closure. ATG8–PE decorates the phagophore, and eventually autophagosome membranes, and is further delivered to the vacuole where it still decorates the autophagic body up to its degradation. Moreover, ATG8 proteins are able to bind a variety of proteins, some of which serving as cargo receptors, to enable cargo recognition during selective autophagy. The conjugation system

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subunit VPS2.1 (VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 2 HOMOLOG 1) conjugated to GFP renders it inactive, thus creating a dominant negative construct. Plants expressing VPS2.1–GFP under the control of the cauliflower mosaic virus 35S promoter (35S:: VPS2.1–GFP) displayed impaired endocytosis and also lacked autophagic degradation. These observations presumably stem not from impairment of autophagosome formation but rather from lack of autophagosome delivery to the vacuole, as demonstrated by the accumulation of ATG8 and NBR1 (NEIGHBOR OF BRCA1 GENE 1) proteins, and by the reduced abundance of monodansylcadaverine (MDC)-positive structures in the vacuoles of 35S::VPS2.1–GFP compared with WT plants [40]. Following these results, the authors went on to postulate in their model the accumulation of autophagosomes in the cytosol (although this was not demonstrated in [40]; Figure 1B). A similar, albeit milder, phenotype was observed in plants lacking the deubiquitinating enzyme AMSH1 (ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3) [40], an interactor of VPS2.1 [44]. Another ESCRT component, FREE1 (FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1), has been also shown to regulate autophagic degradation by facilitating the fusion of autophagosomes with the vacuole. In addition, free1 mutants displayed defects in central vacuole formation, presenting with many small vacuoles [41,42] (Figure 1C). Surprisingly, FREE1 was shown to directly interact with SH3P2 [41,42]. The authors claim that SH3P2 is at least transiently localized to MVBs [41,42], which raises the fascinating option of SH3P2 functioning in both autophagy initiation and autophagosome–vacuole fusion. Finally, an additional ESCRT protein, CHARGED MULTIVESICULAR BODY PROTEIN 1 (CHMP1) has also been implicated in autophagy regulation but also in phagophore loading of chloroplast proteins. chmp1 mutant plants are able to produce autophagosomes and transport them to the vacuole, but these autophagosomes are not able to take up chloroplast proteins for degradation, resulting in the accumulation of chloroplast proteins in these mutants. As a consequence of protein accumulation, chmp1 plastids are not able to divide properly, resulting in morphological defects [43] (Figure 1D). Two very recent publications demonstrated direct crosstalk between metabolic enzymes and autophagy. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway [45] and has been shown in animals to be involved in moonlighting activities such as DNA repair and membrane fusion and transport [46]. In Arabidopsis, GAPDH knockout mutants were shown to contain increased reactive oxygen species (ROS) levels and demonstrated enhanced disease resistance and constitutive autophagy [23], suggesting a role for GAPDH in restraining autophagy. Indeed, a parallel study in tobacco (Nicotiana benthamiana) revealed that GAPDH directly interacts with the ATG3 protein to negatively regulate its activity. When NbGAPDH1 and NbGAPDH2 are silenced, autophagy is induced whereas their overexpression resulted in autophagy inhibition. Notably, ROS disrupts GAPDH binding to NbATG3, thus allowing autophagy to escalate [19]. As discussed later in this review, autophagy is a significant contributor to plant metabolic homeostasis. Moreover, being a key component in glycolysis, GAPDH is believed to be expressed according to the metabolic state of the cell [47]. Thus, it is possible that GAPDH buffers the recycling contribution of autophagy according to the metabolic state of the cell by controlling ATG3 activity. However, when the plant is stressed and ROS accumulates, constitutive autophagy is required and autophagy inhibition via GAPDH is aborted. Further work to examine these speculations is required. Nevertheless, it is clear that a direct regulation of autophagy by a metabolic component has been revealed, which might represent yet another layer of regulation on top of the established target of rapamycin (TOR) kinase pathway [48] (see also Glossary under the term ATG1 complex).

Characterization of Selective Autophagy in Plants Research regarding plant selective autophagy is quickly advancing. Most of the selective autophagy processes previously described in metazoans have recently been found to act in plants as well [7,8,49,50]. Furthermore, autophagy seems to be highly involved in the clearance

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also comprises ATG3, ATG4, ATG5, ATG7, ATG10, and ATG16. Phagophore: also known as isolation membrane, a membrane structure serving as the precursor for autophagosomes. The phagophore (whose membrane origin is still under debate) typically associates with the degradation-prone cargo to ultimately enable its engulfment by the autophagosome. Phagophore assembly site (PAS): also known as pre-autophagosomal structure, the site where several complexes consisting of ATG proteins are gathered for the initiation of the phagophore and its maturation to form an autophagosome. Selective autophagy: refers to processes which are disassociated from bulk- and nutrient starvationrelated autophagy and which seem to be selective for a particular type of cargo. Although selective autophagy can be induced under stressful conditions, this type of autophagy takes place constitutively at basal levels. Types of selective autophagy include: aggrephagy, the degradation of protein aggregates; chlorophagy, degradation of chloroplasts; mitophagy, degradation of mitochondria; nucleophagy, degradation of nuclear components; pexophagy, degradation of peroxisomes; reticulophagy, degradation of the endoplasmic reticulum; ribophagy, degradation of ribosomes; and xenophagy, degradation of pathogens. Ubiquitination: the process of conjugating ubiquitin molecules to a target. This is classically assigned as labeling the protein for degradation via the 26S proteasome. However, there are several types of ubiquitination events, and these are classified according to the number and arrangement of the molecules (poly-, multi- or mono-ubiquitination) and the position of the lysine that is conjugated (K11, K48, K63, etc.). Thus ubiquitination may represent a variety of signals, including for intracellular trafficking and autophagy. Several cargo receptors for selective autophagy, such as NBR1 and RPN10, recognize ubiquitinated substrates prior to their sequestering by autophagosomes.

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of plant-specific compartments, such as chloroplasts [51], via rubisco-containing bodies (RCBs [52]) and ATI1 (ATG8 INTERACTING 1)-containing plastid bodies [53]. The ESCRT component CHMP1 addressed earlier is more probably involved in the RCB pathway [43]. Although selective autophagy was initially considered to be a housekeeping process, recent studies demonstrate its importance in stress tolerance and proper developmental transitions. This has been nicely shown by the clearance of peroxisomes during seedling growth [54]. Moreover, highly oxidized peroxisomes and also those lacking an active LON2 (LON PROTEASE HOMOLOG 2, PEROXISOMAL) protease were shown to be degraded via pexophagy [54–56]. Mitophagy in Arabidopsis has only recently been identified following the functional characterization of AtATG11. Although it appears that ATG11 is not specific for mitophagy, its key role in this process was demonstrated. Nevertheless, its mode of action is not yet clear, and it is not known whether it is involved in the recognition of mitochondria destined for degradation [26]. Autophagy of ER membranes [57] was also demonstrated to take place in Arabidopsis. Looking at selective autophagy of specific proteins, autophagic degradation of the RNA-silencing component ARGONAUTE 1 (AGO1) [58] was first reported in Arabidopsis. [9_TD$IF]Later, the autophagic degradation of RNA-silencing components was also reported in humans and C. elegans, suggesting that the interaction between [10_TD$IF]Protein [1_TD$IF]and [12_TD$IF]RNA degradation is evolutionarily conserved [59]. Most of the components participating in each selective autophagy process have been identified in metazoans and unicellular fungi [60]. However, the identification of such components, namely cargo receptors, in plants is in its infancy. NBR1 ()and p62 were the first two selective-autophagy cargo receptors to be identified (in human cells) and were shown to act in aggrephagy[13_TD$IF] (see Glossary under the term Selective autophagy) by their ability to bind to ubiquitinated protein aggregates and ATG8 [61,62]. An Arabidopsis homolog of human NBR1 (AtNBR1) was the first non-metazoan (potential) aggrephagy cargo-receptor to be described in detail. Its ability to homopolymerize suggested that AtNBR1 is a functional hybrid of the human NBR1 and p62 [63]. Indeed, AtNBR1 and its tobacco homolog (JOKA2) bind to both ATG8 and ubiquitin and are delivered to the vacuole [63,64] (Figure 2C). Relocation to the vacuole depended on a functioning ATG7, homopolymerization of NBR1, and the integrity of its AIM domain [63]. NBR1 has been implicated in abiotic stress tolerance while, in contrast to core autophagy components, no effect of NBR1 deficiency had been observed on plant resistance to a necrotrophic fungal pathogen [65]. Evidently, there is a tight relationship between NBR1 and catalase[14_TD$IF] (CAT) proteins in Arabidopsis. Accumulation of several [15_TD$IF]CAT[4_TD$IF] isoforms was detected in an nbr1 mutant following heat stress [66]. Reciprocally, plants deficient in CATALASE 2 or NO CATALASE ACTIVITY 1 (cat2 or nca1) accumulated NBR1 and ubiquitinated substrates upon treatment with the avrRpm1 effector from Pseudomonas syringae that elicits bacterial pathogenicity as well as the plant defensive hypersensitive response (HR) that includes localized programmed cell death (PCD) [67]. NBR1 may well act in the selective clearance of catalases either directly or as part of pexophagy[16_TD$IF] (see Glossary under the term Selective autophagy), a function that was already demonstrated for this protein in animals [68]. Accumulation of CAT has been demonstrated in Arabidopsis atg mutants, further strengthening this hypothesis [56]. In parallel, it seems that CATs may modulate the levels of NBR1 by their positioning upstream of autophagy, as was recently suggested [67]. Such a mechanism, whereby an autophagy regulator is also consumed by autophagy, is reminiscent of the fate of ATG1/ATG13 [25], suggesting tight feedback regulation of autophagy intensity and duration. In an attempt to discover additional plant cargo receptors, Honig et al. screened for potential ATG8f interacting proteins from Arabidopsis and recovered two homologous, previously uncharacterized proteins, termed ATG8 INTERACTING 1 AND 2 (ATI1 and ATI2). Both proteins are similar in domain architecture, namely, a predicted transmembrane domain and two predicted AIMs. Moreover, they seem to be plant-specific, suggesting their involvement in unique plant functions. ATI1 and ATI2 were found to be associated with the ER and, upon carbon starvation, translocate to newly formed spherical structures (ATI bodies) that move along

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(A) TSPO pathway Plasma membrane

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Figure 2. Plant Cargo Receptors and the Pathways in which They Act. (A) PIP[5_TD$IF]2;7 is recycled between the plasma membrane and endomembranes via recycling endosomes. Following abiotic stresses, TSPO interacts with PIP[5_TD$IF]2;7 and with ATG8 to facilitate their engulfment by autophagosomes and delivery to the vacuole. (B) During natural or stress induced senescence, ATI1-PS bodies that contain ATI1 are generated in plastids. ATI1 further interacts with plastid proteins, including those associated with thylakoid membranes (such as PSBS) and with ATG8 to enable the delivery of the ATI-PS bodies to the vacuole via autophagy. (C) Soluble proteins are prone to aggregate during stressful conditions. To enable the clearance and recycling of the aggregates, they are labeled with ubiquitin molecules, enabling their recognition by NBR1 which further interacts with ATG8 for autophagic delivery to the vacuole. (D) Inhibition of the 26S proteasome leads to the ubiquitination of several of its subunits. This event increases the association of the RPN10 subunit with the complex thereby facilitating its interaction with ATG8 to enable the clearance of the entire complex to the vacuole. Abbreviations: ATG8, AUTOPHAGY-RELATED 8; ATI1, ATG8 INTERACTING 1; NBR1, NEIGHBOR OF BRCA1 GENE 1; [6_TD$IF]PS body; plastidassociated body; PIP[7_TD$IF]2;7, PLASMA MEMBRANE INTRINSIC PROTEIN [8_TD$IF]2;7; RPN10, REGULATORY PARTICLE NONATPASE SUBUNIT; TSPO, TRYPTOPHAN-RICH SENSORY PROTEIN.

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the ER network and are distinct from ‘classical’ autophagosomes [69]. These findings raised the hypothesis that ATI bodies are involved in direct ER-to-vacuole trafficking, a route repeatedly suggested to function in plant cells [7,70]. Because plants deficient in both ATI1 and ATI2 exhibit retarded germination following treatment with abscisic acid (ABA), it was further suggested that ATI bodies mediate selective degradation of ABA, or ABA-related proteins. Surprisingly, ATI1 was also shown to associate with plastids, resulting in the identification of selective delivery of plastid proteins to the vacuole, mostly following senescence inducing conditions. Under these circumstances ATI1 incorporated onto special plastid-associated bodies (ATI-PS bodies), which are distinct from the earlier described ATI bodies (that associate with the ER) and the plastidderived RCBs that are apparently larger and contain predominantly stroma-derived proteins such as rubisco [52,53,69]. ATI1 interacts with chloroplast-localized proteins, some of which associated with thylakoid membranes, and its delivery to the vacuole requires its interaction with ATG8f and a functional ATG5 (Figure 2B). These findings suggest that ATI1 acts as a plantspecific autophagy cargo receptor [53]. Another potential Arabidopsis cargo receptor is TSPO (TRYPTOPHAN-RICH SENSORY PROTEIN). This protein was initially described as a heme-binding protein whose interaction with ATG8 enables the degradation of porphyrins via autophagy [71]. Recently, TSPO was shown to interact with the aquaporin PIP[5_TD$IF]2;7 (PLASMA MEMBRANE INTRINSIC PROTEIN [5_TD$IF]2;7) and mediate its delivery from the plasma membrane to the vacuole (Figure 2A). Notably, this autophagy-mediated reduction in cell surface quantity of PIP[5_TD$IF]2;7 seems to regulate water permeability, an important function under heat and drought stresses [72]. A surprising type of selective autophagy has recently been discovered in Arabidopsis, involving the degradation of the 26S proteasome (termed proteaphagy). Evaluation of the proteasome complex fate following nutrient starvation revealed its delivery to the vacuole, apparently via bulk autophagy. Marshal et al. [73] examined additional conditions which may induce this type of degradation and revealed that proteasome inhibition, either chemically or via mutated subunits, also induces proteaphagy. Notably, inhibitor-induced proteaphagy was coupled to ubiquitination of several proteasome subunits, including RPN10 (REGULATORY PARTICLE NONATPASE SUBUNIT). This subunit, commonly disassociated from the 26S proteasome complex, showed increased association when it was ubiquitinated (Figure 2D). Importantly, rpn10 mutants could not carry out inhibitor-induced proteaphagy. Finally, RPN10 was shown to interact with ATG8, confirming its role as a cargo receptor mediating the delivery of the entire complex to the vacuole (Figure 2D). RPN10 is conserved among different plant species, and RPN10 orthologs from Physcomitrella patens, Brachypodium distachyon, and Zea mays were shown to interact with their ATG8 counterparts [73]. This report highlighted an interesting interplay between the two major degradation pathways in plants. It will be interesting to examine potential restriction of autophagy by the 26S proteasome, perhaps by proteasomal degradation of key autophagy proteins. Such type of counter-regulation may ensure proper level of cellular degradation and recycling. Further identification of cargo receptors is mandatory to classify the different types of selective autophagy which will able to determine the relative contribution of each of the processes for the different aspects of plant life.

The Role of Autophagy in Plant Nutrient Availability Hypersensitivity to carbon and nitrogen starvations are hallmark phenotypes of atg mutants [74–78]. Furthermore, ATG genes were shown to be transcriptionally upregulated under these conditions [78–83]. Recently, several studies aimed to decipher the role of autophagy in nutrient recycling and remobilization. Plants normally use carbohydrates as the major carbon source for respiration, and these are stored as starch in the chloroplast for nighttime consumption [84]. The autophagy mechanism

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was shown to be active during the night, and autophagy-deficient Arabidopsis and tobacco (N. Benthamiana) leaves accumulated starch. In addition, small starch granule-like structures (SSGLs) microscopically colocalized with autophagosomes. Autophagy was therefore suggested to possibly function in starch breakdown. However, the authors claim that this is a minor starch degradation pathway compared to starch degradation in the chloroplast [85]. When the carbohydrate pool is depleted, proteins and lipids can be used as an alternative carbon source [86]. When crossed with starch-deficient mutants, Arabidopsis atg mutants displayed a severe growth phenotype under both long and short day conditions. This phenotype was alleviated under continuous light, suggesting a role for autophagy in carbon availability [87]. Furthermore, the double mutants displayed increased signs of starvation during the night compared to single mutants. Metabolic profiling revealed that autophagy was responsible for the supply of free amino acids during the night, possibly utilized as an alternative carbon source [87]. The metabolic profile of single atg mutants was also different from that of WT plants. Although there was no decrease in free amino acids, presumably because the sugar resources have not been completely exhausted, an increase in lipid degradation products was observed, which could also be used as alternative energy sources [88]. An altered lipid profile has also been observed in the anthers of atg mutant rice [15], suggesting an impact of autophagy on lipid metabolism. Reduction in free amino acids has also been reported in atg mutant etiolated seedlings grown without sucrose, strengthening the role of autophagy as a supplier of free amino acids for usage under carbon starvation [22]. Isotope labeling experiments revealed that, indeed, carbon-starved atg mutants use amino acids as respiratory substrates in the tricarboxylic acid (TCA) cycle, while displaying decreased flux into protein synthesis. Altered lipid composition has also been observed in this system [22]. Nitrogen is another element crucial for plant growth and survival [17]. Under nitrogen starvation atg mutants are smaller and exhibit early senescence [17,74,75,89]. Metabolic analysis revealed an accumulation of free amino acids in atg mutants compared to WT plants [89,90]. This finding is contradictory to the reduction in free amino acids in atg mutants observed in carbon starvation [22,87]. One possible explanation is the age of the plants. Accumulation of free amino acids was observed 60 days after sowing [89,90], while a decrease in free amino acids was observed in either seedlings or samples collected 40 days after sowing [22,87]. Masclaux-Daubresse et al. [90] suggest the accumulation of free amino acids stems from an attempt to regulate oxidative stress. Because it has recently been shown that autophagy regulates the degradation of oxidized peroxisomes [56], it is possible that, as plants age, and especially when under stress, atg mutants are exposed to higher oxidative stress than are younger plants. Inducible atg mutants would allow us to answer this question. Nitrogen remobilization from source tissues, such as senescing leaves, to sink tissues, such as developing seeds, is crucial for plant productivity, especially under nitrogen starvation [91]. Several studies have examined nitrogen remobilization into developing seeds of atg mutants [16,17,89–91]. In pulse-chase experiments using 15[1_TD$IF]N, atg mutants accumulated more labeled nitrogen in leaves and less in seeds, suggesting an impairment of nitrogen remobilization [16,17,91]. Nitrogen starvation further exacerbated this phenotype [91]. An important control arising from both nitrogen and carbon starvation is the incorporation of stay-green atg mutants into the analysis [22,90]. atg mutants accumulate salicylic acid (SA) as they age, resulting in an early senescence phenotype. Crossing atg mutants with plants which degrade or do not produce SA resulted in a stay-green phenotype [92]. atg mutants and stay-green lines do not display an identical metabolic profile, necessitating the use of these lines as a control for metabolic effects resulting from SA accumulation [22,90].

Concluding Remarks and Future Perspectives Despite the tremendous progress made in autophagy research in plants, several key questions remain open (see Outstanding Questions). All these questions, and others, may be answered only if the ongoing identification of new ATG genes and focused research on the already known

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genes is maintained. It is essential to further identify selective autophagy pathways and characterize the components and mechanism of their action. Although research in the model organisms Arabidopsis and Chlamydomonas is ongoing, it is of great importance to continue to develop the study of autophagy in crop plants. As discussed here, autophagy research in plants can strongly contribute to the characterization of novel and fundamental molecular mechanisms governing autophagy, and future work must position plants as an important eukaryotic system for autophagy research alongside animal and yeast systems.

Outstanding Questions Are all autophagy processes in plants similar? For example, are the core autophagy components that act during nitrogen starvation identical to those that act during carbon starvation or mitophagy (excluding the unique selective components)? Is there an alternative ATG7/ATG5-independent autophagy in plants as shown in mammals?

Acknowledgments We apologize to researchers whose work has not been included in this manuscript [17_TD$IF]owing to lack of space. The work of S.M. and P.G. has received funding from the European Research Council under the European Commission Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement 338904. G.G. is the incumbent of the Bronfman Chair in Plant Sciences. The work of A.R.F. is supported by the Max-Planck Society, and that of T.A.W. by Minerva, Alexander von Humboldt, and European Molecular Biology Organization (EMBO) fellowships.

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