Autophagy in Development and Stress Responses of Plants

Autophagy ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20 Autophagy in Development and Stress Responses of Plants Diane C. Bassham, Marianne Laporte, Francis Marty, Yuji Moriyasu, Yoshinori Ohsumi, Laura J. Olsen & Kohki Yoshimoto To cite this article: Diane C. Bassham, Marianne Laporte, Francis Marty, Yuji Moriyasu, Yoshinori Ohsumi, Laura J. Olsen & Kohki Yoshimoto (2006) Autophagy in Development and Stress Responses of Plants, Autophagy, 2:1, 2-11, DOI: 10.4161/auto.2092 To link to this article: https://doi.org/10.4161/auto.2092 Published online: 09 Nov 2005. Submit your article to this journal Article views: 1075 View related articles Citing articles: 136 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=kaup20

[Autophagy 2:1, 2-11; January/February/March 2006]; 2006 Landes Bioscience Review Autophagy in Development and Stress Responses of Plants Diane C. Bassham 1, * Marianne Laporte 2 Francis Marty 3 Yuji Moriyasu 4 Yoshinori Ohsumi 5 Laura J. Olsen 6 Kohki Yoshimoto 5 1 Department of Genetics, Development and Cell Biology; Iowa State University; Ames, Iowa USA 2 Department of Biology; Eastern Michigan University; Ypsilanti, Michigan USA 3 Université de Bourgogne; Dijon, France 4 School of Food and Nutritional Sciences; University of Shizuoka; Shizuoka, Japan 5 National Institute for Basic Biology; Division of Molecular Cell Biology; Okazaki, Japan 6 Department of Molecular, Cellular and Developmental Biology; University of Michigan; Ann Arbor, Michigan USA *Correspondence to: Diane C. Bassham; Department of Genetics, Development and Cell Biology; Iowa State University; Ames, Iowa 50011 USA; Tel.: 515.294.7461; Fax: 515.294.1337; Email: bassham@iastate.edu Received 07/20/05; Accepted 08/09/05 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2092 KEY WORDS ATG genes, autophagosome, autophagy, nutrient starvation, senescence, vacuole formation ACKNOWLEDGEMENTS F.M. is supported by the Ministère de l'education Nationale, de l'enseignement Supérieur et de la Recherche. D.C.B. is supported by grants from the US Department of Agriculture and the National Science Foundation. ABSTRACT The uptake and degradation of cytoplasmic material by vacuolar autophagy in plants has been studied extensively by electron microscopy and shown to be involved in developmental processes such as vacuole formation, deposition of seed storage proteins and senescence, and in the response of plants to nutrient starvation and to pathogens. The isolation of genes required for autophagy in yeast has allowed the identification of many of the corresponding Arabidopsis genes based on sequence similarity. Knockout mutations in some of these Arabidopsis genes have revealed physiological roles for autophagy in nutrient recycling during nitrogen deficiency and in senescence. Recently, markers for monitoring autophagy in whole plants have been developed, opening the way for future studies to decipher the mechanisms and pathways of autophagy, and the function of these pathways in plant development and stress responses. Plants frequently encounter adverse environmental conditions, and their cellular and physiological responses to these conditions are critical for survival. Common stresses imposed upon plants include nitrogen deficiency, due to lack of available nitrogen in the soil, and carbon deficiency, caused by conditions that limit photosynthetic efficiency and thus carbon fixation. A key process by which eukaryotic cells respond to and survive such stresses is vacuolar autophagy. Autophagy, which etymologically means to eat oneself, is a catabolic process by which eukaryotic cells degrade portions of their own cytoplasm. This process is conserved between yeast, animal, and plant cells, and in plants has also been known for a number of years to be involved in cellular architectural (re)modeling that occurs during differentiation and development. The function of autophagy was revealed by morphogical observations with the electron microscope. The electron micrographs on which the concept of cellular autophagy rests show the bulk sequestration of cytoplasmic fragments and their subsequent digestion in the lytic vacuole. While many plant cells have at least two different types of vacuoles, lytic and storage, the lytic vacuole is thought to be functionally equivalent to the yeast vacuole or animal lysosome. Macroautophagy and microautophagy have been defined according to the size of the cytoplasmic material taken up for destruction (see Fig. 1) and the site of sequestration. Macroautophagy (hereafter referred to as autophagy) sequesters regions of cytoplasm, including organelles such as endoplasmic reticulum (ER), Golgi stacks, mitochondria, plastids and peroxisomes, into double-membrane bound structures called autophagosomes. The content of the autophagosome including the inner limiting membrane, which is defined as the autophagic body once it has been released into the vacuole lumen, is subsequently degraded. In contrast, in microautophagy the uptake was originally envisaged as occurring in small gulps in which a few cytosolic molecules were transferred to the vacuole interior within a membrane-delimited vesicle directly formed by invagination of the vacuolar surface, followed by disintegration of the membrane. The internalized vesicle formed after septation of the invaginated membrane usually looked empty. The reader is referred to previous reviews (refs. 1 5) for detailed information on earlier work on cellular autophagy in plants. In this review, we will attempt to integrate more recent molecular data on autophagy in plants cells with morphological and physiological information on the role of autophagy both during plant development and as a stress response. 2005 LANDES BIOSCIENCE. DO NOT DISTRIBUTE. 2 Autophagy 2006; Vol. 2 Issue 1

AUTOPHAGY PROCESSES DURING PLANT DEVELOPMENT Autophagy is a key process in the formation of the vegetative vacuole in plant cells. In nonvacuolated meristematic cells (the plant version of nondifferentiated stem cells), numerous autophagosomes resulting from the enwrapping of large portions of cytoplasm by double-membrane envelopes are made. The origin of the isolation membranes is still a subject of speculation. 3 Because the enveloping structure is a flattened sac possibly formed within minutes, it was considered likely, as still postulated in mammalian cells, that it arises directly from preexisting ER, which is also largely made of sacs. However, the landmark description of a complete high-resolution 3-D autophagic sequence in differentiating cells pointed to a totally different mechanism. 1,6 Provacuoles (similar to yeast preautophagosomal structures; see below) arise as vesicles at the trans-golgi network (TGN). Biochemical markers including phosphatidylinositol phosphate, GTPases and various phosphatases now support this structural model in plant and animal cells. 7,8 The provacuoles are a critical junction in post-golgi trafficking at which the endocytic and vacuolar pathways converge. 9,10 These vesicular structures elongate into tubules, which make cagelike traps around portions of cytoplasm. Autophagosomes are completed when the tubules fuse to completely seal the region in a double-membrane compartment. Tubules and derived autophagosomes contain acid hydrolases in an acidic environment. The lumen of the tubules contains microvesicles presumably derived from invagination of their membrane. It is speculated that this microautophagy might be responsible for the direct cytosol-to-provacuole transport of components required for the maturation of the content. The cytoplasm sequestered in the autophagosome is finally degraded by the digestive enzymes, which are released from the surrounding cavity, formed from the tubules, as the inner membrane of the autophagosome deteriorates. Autophagic-like pathways are used to deliver materials to the vacuole at numerous stages of plant development. During seed development, protein storage vacuoles (PSVs) replace the vegetative (lytic) vacuoles in the cells of maturing legume cotyledons (seed leaves). During this time, the vegetative vacuole is entrapped by a tubulo-cisternal envelope whose origin is not clearly understood. 11,12 The vegetative vacuole is progressively degraded as the novel PSV takes it over and fills up with reserve proteins. Degradative autophagy pathways are also operating when vegetative (lytic) vacuoles are substituted for PSVs in germinating legume seedlings. Fragments of cytoplasm are indeed engulfed by PSVs via local invaginations of their limiting membrane before they are pinched off and subsequently degraded. 13-15 Some protein precursors and other substances are deposited in the vacuole using an ER-to-vacuole targeting (ERvt) pathway, which is presented as a convergent analogue to the cytoplasm-to-vacuole targeting (Cvt) pathway described in yeast (see below). 16 In wheat grain, the endosperm storage protein prolamin accumulates transiently in ER-derived protein bodies, which enter the vacuoles by a process similar to microautophagy. The ER-derived limiting membrane is then degraded and the naked prolamin core combines with others to produce large storage protein aggregates that are resistant to vacuolar hydrolysis. 17 Similarly, in transgenic tobacco seeds expressing seed storage protein genes from maize, ER-derived protein bodies are engulfed into the vacuoles by autophagy. 18 A comparable process is responsible for the release of rubber particles into the vacuole. Unlike the ER-bodies with a ribosome-bearing membrane, rubber particles synthesized at the ER are coated by a Figure 1. Autophagy pathways in plant cells. Macroautophagy: (1) autophagosome formation from preautophagosomal structures (PAS or provacuoles); (2) completed autophagosome containing intact cytoplasm (class 1 autophagosome as described by Rose et al 2005); 58 (3) autophagosome in which the sequestered cytoplasm is being digested (class 2 autophagosome); (4) autophagosome fusing with the central vacuole, the digestion of the sequestered cytoplasm is almost complete (class 3 autophagosome); (5) the autophagic body is released into the lumen of the central vacuole. Microautophagy: (6) a small vesicle is formed directly by invagination of the vacuole membrane and released in the vacuolar lumen where it is degraded. Protrusion autophagy: (7) whole organelles (ERderived bodies, mitochondria, plastids, peroxisomes, rubber particles, etc.) and large portions of cytoplasm are delivered into the central vacuole by protrusion (8) and direct engulfment (9). Further processing is achieved by vacuolar hydrolases (10). single-unit membrane. After internalization into the vacuole the rubber membrane is digested and the stripped particles coalesce to form large aggregates. 19 Plant-specific, ER-derived vesicles containing precursors of cysteine proteinases (PPVs) have been described in seed-storage tissues and senescing tissues of vegetative organs. 20-25 These ER-derived vesicles have the potential to accumulate a large repertoire of protease precursors stored in stable, inactive aggregates. They are delivered to the vacuole by a microautophagy-like protrusion autophagy process (see Fig. 1). Because the proteases would be activated only when autophagy occurs, it has been speculated that this Golgi-bypass trafficking could be useful when sudden and massive changes in proteolytic activities are required such as in storage protein mobilization and programmed cell death 16 (PCD). This is also a regulated cellular suicide strategy activated in the hypersensitive response (HR) to pathogens in plants. 21,26 HR is one strategy for resistance of plants to pathogens in which the rapid induction of PCD in infected cells restricts the growth and spread of the pathogen to adjacent cells. General cellular autolysis resulting from a sharp increase of vacuolar proteolytic activities followed by the permeabilization of the tonoplast (vacuolar membrane) is involved in the PCD of several plant tissues. 27 Distinctively, it has been recently demonstrated that autophagosome-mediated autophagy, i.e., macroautophagy, plays an important role during the innate immune response in plants. In order to prevent the HR from playing a pathologic rather than protective role, autophagy functions to restrict PCD to infection sites. 28 It is speculated that autophagy may eliminate death-promoting signals moving out of the pathogen-infected area or may clean up cellular damage www.landesbioscience.com Autophagy 3

caused during the defense response. A role for autophagy in host defense by limiting the growth of intracellular pathogens is also suggested. How this negative regulatory role for autophagy in PCD will be reconciled with the potential positive role described above remains to be seen. Autophagy driven by direct invagination of the tonoplast into the central vacuole (i.e., a form of microautophagy) in cells from senescing tissues has been reported. 29-32 The invagination is pinched off and the isolated body, containing cytoplasmic materials and membranes, is finally degraded in the vacuole. Interestingly, during the senescence of photosynthetic leaf cells from Arabidopsis and soybean, small specific senescence-associated vacuoles (SAVs) with intense proteolytic activity are formed in the peripheral cytoplasm. 33 They often contain dense aggregates, which may consist of partially degraded cellular material similar to the contents of late autophagic vacuoles. However, these SAVs are not derived from classical autophagosomes and their relationship to more typical autophagy processes remains to be determined. SELECTIVE AUTOPHAGY PATHWAYS The term pexophagy was introduced to describe the selective elimination of peroxisomes in different yeasts. 34 Similarly, it has been suggested that plants might also use selective sequestration to remove glyoxysomes when they are no longer needed in oil seeds. 35,36 Occasionally, vacuoles have been seen to engulf mitochondria, plastids or starch grains. 29,37-41 However, it is now known that autophagy does not play a major role in the degradation of chloroplast proteins, 2 but catabolites released from the senescing chloroplast are further metabolized in the vacuole. 42 Subregions of the tonoplast invaginate in round-shaped structures described as bulbs, inside the vacuole of cotyledonary cells and drought-acclimated cells. 43,44 They are highly dynamic structures, resistant to lysis and are thought to be reservoirs for tonoplast proteins. Whatever the mechanisms involved in the uptake of organelles and tonoplast, one may wonder whether these intriguing processes are essentially blind or selective. Several additional autophagy-related pathways have been shown to operate in other organisms. The Cvt pathway is biosynthetic and operates under nutrient-rich conditions. It involves sequestration by a double-membrane envelope, which appears morphologically to be a small version of the autophagosome. The existence of the Cvt pathway has only been demonstrated in the yeast S. cerevisiae. 45 Another degradative pathway, which operates in animal cells but is not found in yeast, is chaperone-mediated autophagy 46 (CMA), in which particular cytosolic proteins are delivered to lysosomes in a molecule-by-molecule fashion with the help of a chaperone complex in the cytosol and on the lumenal side of the lysosomal membrane. Finally, the vacuolar import and degradation (Vid) pathway is involved in the degradation of the gluconeogenic enzyme fructose- 1,6-bisphosphate in yeast. 47 None of these alternative autophagy pathways have been shown as yet to be present in plants. It is now clear that the vacuolar apparatus forms an intracellular digestive system and has evolved different autophagy pathways for recycling intracellular constituents. Because several types of vacuoles interplay in plant cells, 48-52 the question arises as to whether autophagy is a common function. MORPHOLOGICAL OBSERVATION OF AUTOPHAGY INDUCED BY NUTRIENT STARVATION The degradative autophagy pathway is induced in vacuolated cells to adapt to various environmental stresses, such as nutrient starvation. 37,53-60 In tobacco-cultured BY-2 cells, cytoplasmic degradation has been suggested to be confined to small lytic compartments, rather than the central vacuole, 57,61 although this remains controversial as other researchers report no differences between BY-2 cells and vacuolated cells from other species (Bonneau L, Marty F, unpublished). In sycamore and Arabidopsis-cultured cells autophagosomes are formed in the cytoplasm shortly after the cells are deprived of nutrient. 53,58 Unlike the situation in yeast cells, the encircled portion of cytoplasm is usually degraded before the outer membrane of the autophagosome fuses with the tonoplast. Upon fusion, the internal vesicle is released into the vacuole (the vesicle is now called an autophagic body) where the degradation of its membrane and internal remnants is completed. The degradation of the trapped cell components recycles intracellular substrates and makes resources sufficient for the cell to meet its vital needs and survive. Cellular autophagy thus plays an essential role in nutrient recycling. 55,56,60,62 Sucrose starvation in suspension cultured cells has proven to be an excellent model system for the analysis of plant autophagy. 37,53,57,58,63 When cells are transferred to sucrose-free medium, the transvacuolar strands, strands of cytoplasm that connect peripheral and perinuclear regions of the cytoplasm, degenerate, and 30 to 50% of the total protein is degraded over a two-day period, 61 concomitant with an increase in protease activity. Treatment with cysteine protease inhibitors such as E-64c rescues almost all polypeptides from degradation, showing that the decrease in the total protein derives from nonselective degradation of intracellular proteins and not from the degradation of specific proteins. 57 In BY-2 cells, cysteine protease inhibitors cause the accumulation of membrane vesicles around the nucleus, which contain electron-dense particles. 57 Among these particles, partially degraded mitochondria are occasionally seen (Moriyasu Y, Robinson DG, unpublished results), suggesting that the particles represent portions of the cytoplasm undergoing degradation. The vesicles are acidic compartments, containing cysteine protease activity and acid phosphatase, a marker enzyme of lysosomes. 57 Thus the membrane vesicles are thought to represent newly formed class 2 autophagosomes (autolysosomes; see Fig. 1). They are morphologically distinct from the central vacuole, in that they are smaller (1 to 6 µm in diameter) and the central vacuole accumulates very few electron- dense particles upon E-64c treatment. Without the addition of cysteine protease inhibitors, the accumulation of autophagosomes in tobacco cells is difficult to detect by light microscopy, 57 suggesting that the digestion of cytoplasm proceeds very rapidly during normal autophagy. In cells treated with E-64c, fusion profiles between autophagosomes and central vacuoles are observed. Thus, it is likely that some of the partially degraded cytoplasm in autophagosomes is released into the lumen of the central vacuole for further degradation during a typical autophagic process. On the other hand, it can be observed by electron microscopy that the number of empty vesicles increases significantly under sucrose starvation conditions in the absence of E-64c. 61 This suggests that, unlike in yeast, a significant proportion of autophagosomes finish the digestion of enclosed materials before their fusion with the central vacuole. Bafilomycin A 1 and concanamycin A are inhibitors of the vacuole-type H + -ATPase 64-66 and therefore prevent vacuolar hydrolase activity. Starvation-induced autophagy in BY-2 cells, as in 4 Autophagy 2006; Vol. 2 Issue 1

Arabidopsis 60 (see section on monitoring of plant autophagy, below), is perturbed by bafilomycin A 1 and concanamycin A. In these cells, autophagic bodies cannot be degraded but are still expelled into the central vacuole where they accumulate, resembling autophagic bodies in yeast vacuoles. 60 The pathway for maturation of autophagosomes is not fully understood in plant cells. In mammalian cells, newly formed autophagosomes fuse with preexisting lysosomes and/or endosomes, becoming autolysosomes. In yeast cells, autophagosomes fuse with the vacuole, resulting in the vacuole containing autophagic bodies. 67 In plant cells, acid hydrolases have been localized to the enwrapping autophagic cavities as well as to the precursor structures (provacuoles) from which they derive. 6 Some time after the sequestered portion of cytoplasm has been closed off, the hydrolases are released from the sequestering cavity into the autophagic body, which is subsequently degraded. It is therefore suggested that autophagosomes on their own are functionally self-sufficient to achieve the breakdown of the sequestered materials. 58 As a consequence, autophagosomes can finish the digestion of trapped components before their fusion with the central vacuole (see above section). When autophagosomes mature in BY-2 cells, there seem to be several pathways for transport of membrane vesicles to autophagosomes. The styryl dye FM 4-64 on the plasma membrane flows into autophagosomes, probably through an endocytic pathway. 10 Thus, it is probable that as in mammalian cells, the fusion of autophagosomes with endosomes contributes to the maturation of autophagosomes in tobacco BY-2 cells. 9,10 On the other hand, FM 4-64 residing on the membrane of central vacuoles, along with proteins existing in the membrane and lumen of central vacuoles, moves to autophagosomes under sucrose starvation conditions, suggesting that, upon fusion of autophagosomes with the central vacuole, there may be a flow of proteins and membrane lipids from the central vacuole to autophagosomes. 10 Thus it is possible that hydrolytic enzymes in the central vacuole also move back to autophagosomes and contribute to the maturation of autophagosomes. In contrast, in mammalian cells and yeast there is no evidence for the movement of membrane or proteins from the lysosome/vacuole to the autophagosome. IDENTIFICATION OF ARABIDOPSIS HOMOLOGUES OF YEAST AUTOPHAGY GENES More than 30 genes in yeast have been identified that are involved in various steps of autophagy (Table 1). Most of these genes also have mammalian homologues, and many have apparent homologues in plants. 56 Though the functions of some ATG genes in Arabidopsis have been studied, their exact roles in autophagy are not known. It has been recently shown that the ATG genes linked to autophagosome formation are expressed in a coordinated manner at the onset of starvation, suggesting a role during starvation-induced autophagy. 58 An overview of the putative ATG homologues in Arabidopsis yields several interesting observations (summarized in Table 1). The induction of autophagy by starvation conditions or other stress signals is accomplished by the Tor kinase signaling cascade. 68 There is likely a single TOR gene in Arabidopsis. Tor ( Target of rapamycin ) 69 causes Atg13 to be phosphorylated, thereby decreasing its interaction with the kinase Atg1. 70 Atg13 is dephosphorylated under starvation conditions, concomitant with the induction of autophagy. Although two putative ATG13 genes are found in Arabidopsis, their similarity is restricted to a relatively small region of the protein and it is currently unclear if they are true Atg13 homologues. There are, however, multiple genes for ATG1 in Arabidopsis 56 nothing is known about their function, proteinprotein interactions or predicted localization. Many of the components required for the yeast-specific Cvt pathway are also required for autophagy. 67,71-73 Two genes, ATG11 and ATG19, are specifically required for cargo selection in the Cvt pathway. Arabidopsis does not have a homologue for either of these hydrolases. Not surprisingly therefore, Arabidopsis lacks an ATG19 homologue; Atg19 is the cargo receptor for the Cvt pathway. Based solely on computational analysis, it is not clear whether there are any ATG11 genes in Arabidopsis. In addition to the Cvt pathway, Atg11 is also required for pexophagy in Pichia pastoris. 74 Pexophagy has not yet been documented in plants, but it is possible that a putative AtATG11 may play a role in pexophagy or a pexophagy-related process in plants. Alternatively, it is possible that AtATG11, if it exists, functions in a unique way in plant autophagy. Most of the proteins required for autophagy function in formation of the autophagosome. This process, including vesicle nucleation, expansion and completion and targeting to the vacuolar membrane, is complex and poorly understood. 75 The regulation of vesicle induction and nucleation in yeast involves a class III phosphatidylinositol 3-kinase (PI3K), whose catalytic subunit is Vps34. There appears to be a single homologue in Arabidopsis of VPS34 and of VPS15, which is required for the kinase activity. Another subunit of the complex, ATG14, is apparently not found in Arabidopsis. Atg14 acts as a bridge between Atg6 and Vps15/Vps34. Thus it is likely that another protein, probably unrelated to Atg14, performs this function in Arabidopsis. Although a single gene for ATG6 is found in Arabidopsis, there appears to be 2 splice variants. Whether both variants are expressed at the same time, in the same place, and/or produce functional proteins is not known. There are two ubiquitin-like conjugation pathways that function during autophagosome membrane expansion and cargo engulfment. 35,75 The E1-like enzyme Atg7 activates Atg12 in one complex and Atg8 in the other complex. Atg12 forms a complex with Atg5 and Atg16. Atg10, an E2-like enzyme, is required for this complex formation in yeast, and is present as a single gene. 76 ATG5 and ATG7 are present as single genes, and there may be an Arabidopsis homologue of ATG16. There are two ATG12 genes in Arabidopsis. 77 The E2-like protein required for the formation of the Atg8 complex is Atg3. This protein is coded for by a single Arabidopsis gene. Two genes for ATG4 (both with distinct splice variants) are also found in Arabidopsis; Atg4 is a cysteine protease that removes residues from Atg8 after a conserved C-terminal glycine, and cleaves Atg8-phosphatidylethanolamine to remove the lipid moiety, and there are nine ATG8 genes in Arabidopsis. Many proteins are involved in regulating autophagy, but their function is currently defined mostly by their interacting partners. 75 Atg17 interacts with Atg1, Atg20, and Atg24 to regulate early steps in autophagosome formation. There are no apparent Arabidopsis homologues for ATG17, but the other three proteins are each produced by multigene families. The exact number and members of the gene families for ATG20 and ATG24 are difficult to distinguish using only sequence comparison and motif analysis to assign identity. Similarly, AtATG18 and AtATG21 gene families resemble each other in structure, making it hard to assign a given gene to a particular family. Atg18 is required for the localization of Atg2 to the preautophagosomal structure (PAS) in yeast. 78-80 Atg9 is an integral membrane protein that also localizes to the PAS, as well as to a non-pas structure, but is not present in the mature autophagosome. 81 Thus, Atg9 is www.landesbioscience.com Autophagy 5

Table 1 Genes involved in autophagy in Arabidopsis and yeast Yeast Predicted Number of Homologues Functions/Characteristics of Yeast Proteins a Protein in Arabidopsis Thaliana Induction of Autophagy, Signal Transduction, Cargo Selection TOR Single gene Protein kinase; negative regulator of autophagy ATG11 None Peripheral membrane protein; interacts with ATG1; required for Cvt pathway, but not for macroautophagy in S. cerevisiae ATG19 None Cargo receptor; required for Cvt pathway, but not for macroautophagy in S. cerevisiae Autophagosome Formation Phosphatidylinositol-3-kinase complex: ATG6 Single gene;two predicted splice variants Interacts with ATG14, VPS34, VPS15 ATG14 None Subunit of PI3K complex VPS15 Single gene Protein kinase required for Vps34 VPS34 Single gene PI-3-K catalytic subunit Ubiquitin-like conjugation pathways: Pathway 1: ATG5 Single gene Forms complex with ATG12 ATG7 Single gene E1-like enzyme; activates ATG12 and ATG8 ATG10 Single gene; two predicted splice variants E2-like enzyme ATG12 Two genes Interacts with ATG5 ATG16 Single gene with restricted similarity Interacts with ATG5 Pathway 2: ATG3 Single gene E2 protein for ATG8 complex ATG4 Two genes; both with predicted splice variants Cysteine protease; processes ATG8 and ATG8-PE; ATG7 Single gene E1-like enzyme; activates ATG12 and ATG8 ATG8 Small gene family; two loci with predicted Ubiquitin-like modifier; regulates autophagosome size splice variants Regulatory machinery: ATG1 Small gene family Serine/threonine kinase; interacts with ATG13 ATG13 Two possible genes with restricted similarity Phosphoprotein; dephosphorylated under starvation conditions; not required for Cvt pathway ATG17 None Interacts with ATG1, ATG20, ATG24; not required for Cvt pathway ATG18 Small gene family with multiple splice variants Needed for ATG2 localization and ATG9 recycling from PAS ATG20 Small gene family Interacts with ATG24 and ATG17; required for Cvt pathway, but not for macroautophagy in S. cerevisiae ATG21 Difficult to distinguish from ATG18 Required for recruitment of ATG8 to PAS; required for Cvt pathway, but not for macroautophagy in S. cerevisiae ATG24 Difficult to distinguish from ATG20 Interacts with ATG1, ATG17, and ATG20; required for Cvt pathway, but not for macroautophagy in S. cerevisiae ATG27 None PtdIns3P-binding protein; membrane protein; required for Cvt pathway, but not for macroautophagy in S. cerevisiae VAC8 N.D. b Armadillo repeat protein; interacts with ATG13; required for Cvt pathway, but not essential for macroautophagy in S. cerevisiae ATG9 COMPLEX ATG2 Single gene Interacts with ATG9 ATG9 Single gene Interacts with ATG2 and ATG23; integral membrane protein ATG23 None Recycles ATG9 from PAS; required for Cvt pathway, but not essential for macroautophagy in S. cerevisiae Breakdown of Autophagic and Cvt Vesicles in Vacuole ATG15 None Putative lipase; integral membrane protein ATG22 Single gene Putative permease homologue; integral membrane protein; not required for Cvt pathway PEP4 Multigene family Vacuolar proteinase A PRB1 Small gene family Vacuolar proteinase B VPE N.D. Plant specific; see Thompson and Vierstra, 2005 Homology was predicted based upon database annotation, sequence similarity, and the presence of conserved domains and residues. a Partially based on Farré and Subramani, 2004; b N.D., not determined. 6 Autophagy 2006; Vol. 2 Issue 1

retrieved from the PAS prior to autophagosome maturation. Atg23 localization in yeast is similar to Atg9, 82 but its retrieval from the PAS is different. 80 The retrieval of Atg9 and/or Atg23 from the PAS requires Atg18, Atg2, and Atg1. It is not yet known whether plants possess a PAS in the same way as yeast, but there is a single homologue in Arabidopsis of ATG9. 56 Atg23 is not required for autophagy in yeast it might be Cvt specific; 82 there is no apparent Arabidopsis homologue of ATG23. Ultimately, autophagosomes (and Cvt vesicles) fuse with the vacuole, releasing single-membrane vesicles, which are degraded by vacuolar enzymes. 35,75 Vacuolar proteinase A (Pep4), vacuolar proteinase B (Prb1), and the acidic lumen of the vacuole may act directly or indirectly to break down the autophagic vesicles. 83,84 Arabidopsis has many homologues of these vacuolar proteases. In addition, the putative lipase Atg15 and a potential permease, Atg22, also may be involved in this step. 85,86,87 Although there seems to be a single gene for ATG22 in Arabidopsis, there is not an obvious ATG15 homologue. In summary, of the 31 genes shown in Table 1 to be involved in autophagy in yeast, at least 24 have homologues in Arabidopsis. Several are present as multigene families e.g., ATG8, ATG18, VAC8. Some of the genes are predicted to express splice variants (e.g., ATG4, ATG6, ATG8), although the significance of this is unknown. Finally, some genes that appear to be homologues of yeast genes, based on sequence comparisons, may ultimately be shown to play a different role in plant autophagy or to be involved in different processes altogether. REVERSE GENETIC APPROACH FOR STUDYING PLANT AUTOPHAGY Sequence homologies between Arabidopsis ATG genes and those of other species are not particularly high, and only AtATG4, AtATG6 and AtATG8s have been shown to complement the corresponding yeast atg mutant. 28,56,60 Most of the essential residues in the yeast Atg proteins are conserved in these AtATG proteins, suggesting that most of the autophagic machinery is well conserved in plants. This prediction has been further supported by experiments using Arabidopsis T-DNA insertion mutants of ATG genes. For example, in a T-DNA mutant of AtATG7 (Atatg7-1), wild-type AtATG7 could complement the mutant phenotype, but AtATG7C/S, which contains a substitution in a catalytic active cysteine residue, could not, suggesting that AtATG7 also functions as an E1 enzyme in two Atg conjugation reactions. 55 Recently, a double knockout mutant (Atatg4a4b-1) of the functionally-redundant proteins AtATG4a and AtATG4b was obtained, corresponding to the genes encoding the processing and deconjugation enzyme of AtATG8. It has been clearly shown that the putative phosphatidylethanolamine (PE)-conjugated AtATG8s and AtATG8- AtATG3 intermediates were detected in wild-type plants but not in the Atatg4a4b-1 double mutant. 60 In addition, the C-terminus of the nascent form of AtATG8s was cleaved in an AtATG4-dependent manner, and Ala substitution of the C-terminal Gly of AtATG8s results in a defect in the normal behavior of AtATG8s. These results indicated that in plant autophagy an ubiquitination-like Atg8 lipidation system functions as it does in yeast. A C Figure 2. Monitoring of the autophagic process in plant roots. Localization of GFP-AtATG8a in wild-type and Atatg4a4b-1 roots. Under nutrient-rich conditions (MS), many ring-shaped structures, which are thought to be autophagosomes, are observed in wild-type roots (A, inset), but not in Atatg4a4b-1 roots (C). When the plants are transferred to nitrogen-starved conditions (MS-N), GFP-AtATG8a is delivered to the vacuolar lumen in wild-type roots (B), but not in Atatg4a4b-1roots (D). Larger GFP-labeled structures are nuclei, into which most likely the GFP fusion can diffuse due to its small size. Bar=1 µm (A) and 20 µm (D). [modified from Yoshimoto K, et al. Plant Cell 2004; 16:2967-83]. MONITORING OF PLANT AUTOPHAGY The lack of a convenient assay for autophagy in plants has hampered previous efforts to study this process. It was predicted that an ubiquitin-like protein, Atg8, would be suitable as a marker for monitoring plant autophagy, 60,63 as it has proven useful in other organisms. Atg8 is modified with a lipid molecule PE by ubiquitination-like reactions; consequently, Atg8-PE behaves like an intrinsic membrane protein, and the PE-conjugated form is involved in autophagosome formation. The Atg8 conjugated to PE is localized to autophagosomes and their intermediates, and finally a portion of it is transferred to the vacuole during autophagy. Therefore, in yeast, Atg8 is a useful molecular marker for monitoring the autophagic process. 88 In mammalian cells, an Atg8 homologue, LC3, is used as a molecular marker for monitoring the autophagic process. 89 In order to address this need for a marker protein, transgenic plants expressing N-terminally GFP-fused AtATG8 protein (GFP-AtATG8) were generated, and were observed by confocal laser scanning microscopy. 60 In wild-type roots expressing GFP-AtATG8, under nutrient-rich conditions, GFP-AtATG8 is observed as many ring-shaped and punctate structures, which correspond to autophagosomes and their intermediates, respectively (Fig. 2A). In contrast, these structures are not observed in roots of Atatg4a4b-1 plants expressing GFP-AtATG8, although some small dot structures, possibly aggregates, are observed (Fig. 2C). Under nitrogen-starved conditions, GFP-AtATG8 is delivered to vacuolar lumens in wild-type roots (Fig. 2B). On the other hand, in the Atatg4a4b-1 mutant, none of the GFP-AtATG8s, even GFP-AtATG8i, which has no C-terminal extension after the Gly residue, are delivered to the B D www.landesbioscience.com Autophagy 7

A B C Figure 3. Plant autophagic bodies. In wild-type Arabidopsis roots treated with concanamycin A, a V-ATPase inhibitor, many autophagic bodies accumulating in the vacuolar lumens of root cells are observed (A), but not in Atatg4a4b-1 mutant roots (C). The autophagic bodies contain cytoplasmic structures, such as mitochondria, endoplasmic reticulum, and Golgi bodies (B). Bar=10 µm (A and C) and 500 nm (B). [modified from Yoshimoto K, et al. Plant Cell 2004; 16:2967-83]. vacuole (Fig. 2D and data not shown), suggesting that AtATG4s are essential for autophagosome formation not only as processing enzymes but presumably also deconjugation enzymes of putative PE-conjugated AtATG8s. These results suggest that AtATG8s are suitable for monitoring the autophagic process in plants. The general utility of this approach has been demonstrated by transient transfection of protoplasts with GFP-AtATG8. Whereas starved protoplasts from wild-type plants contained many GFP-labeled autophagosomes, 63 those derived from transgenic lines with reduced expression of the autophagy gene AtATG18a lack these structures, 90 indicating that transient transfection provides a rapid assay for identifying autophagy defects. When roots are treated with concanamycin A, a V-ATPase inhibitor, many spherical bodies are observed in vacuolar lumens of wild-type roots, but not in those of Atatg4a4b-1 roots using conventional light microscopy (data not shown). Electron microscopy revealed that these spherical bodies contain cytoplasmic structures, such as mitochondria, endoplasmic reticulum, and Golgi bodies (Fig. 3A and B), suggesting that these structures correspond to autophagic bodies in plants. Concanamycin A is known to raise the ph of the vacuolar lumen by inhibiting the V-type ATPase when exogenously added to plant cells. Consequently, under such high ph conditions, vacuolar hydrolases cannot act, resulting in accumulation of autophagic bodies in the vacuole. Similarly, when roots are treated with the cysteine protease inhibitor E-64d, aggregates, presumably consisting of cytoplasmic degradation products, are observed in the vacuolar lumen in wild-type roots, but not Atatg4a4b-1 roots (data not shown). Treatment with Concanamycin A or E-64d are therefore very easy ways to detect whether autophagy occurs in plants. Using these monitoring systems, it is now clear that AtATG proteins are responsible for autophagy. Recently, it has been proven that AtATG12-AtATG5 conjugates are also essential for plant autophagy by using mutants in the responsible genes. 76,77 Using anti-atatg12b antibodies that recognize both AtATG12a and AtATG12b, AtATG12-AtATG5 conjugates can be detected in wild-type and AtATG10/Atatg10-1 (heterozygous) plants. As expected, in Atatg4a4b-1, whose mutated genes are known not to be involved in Atg12-Atg5 conjugation, AtATG12-AtATG5 conjugates are also detected. In contrast, in Atatg5-1 and Atatg10-1 plants, which are deficient mutants of the target and E2-like enzymes of AtATG12, respectively, AtATG12-AtATG5 conjugates are not detected. Instead, unconjugated AtATG12s increase in these mutants compared with plants that can form AtATG12-AtATG5 conjugates. It was further examined whether these mutants have a defect in autophagy by concanamycin A treatment. In wild-type and AtATG10/Atatg10-1 (heterozygous) roots treated with concanamycin A, many autophagic bodies are detected in the vacuoles, but not in those of Atatg5-1 and Atatg10-1 roots. The result shows that Atatg5-1 and Atatg10-1 plants are defective in autophagy, suggesting that AtATG12-AtATG5 conjugates are essential for plant autophagy. In mammalian cells, it is known that the Atg12-Atg5 conjugate dissociates from autophagosomes when their formation is complete; therefore this is a good marker for monitoring autophagosome formation. 91 As in mammalian cells, it is expected that AtATG5 will provide a useful marker for monitoring the process of plant autophagosome formation. PHYSIOLOGICAL ROLES OF AUTOPHAGY IN WHOLE PLANTS The Atatg mutant plants that have been described can achieve normal embryogenesis, germination, cotyledon development, shoot and root elongation, flowering, and seed production under normal nutrient-rich conditions. However, careful phenotypic analyses revealed some differences between wild-type and Atatg mutants. 55,56,60,77,90 In Atatg mutants, leaf senescence is accelerated even in nutrient-rich conditions (Fig. 4A and B). In addition, bolting, the production of inflorescence (floral) stems, of Atatg9-1 is accelerated under normal conditions. 56 These results suggest that autophagy plays some roles in normal developmental processes even in nutrient-rich conditions. When Atatg mutants are grown under nitrogen depleted or limiting conditions, they exhibit drastic acceleration of starvation-induced yellowing of the leaves. When leaves are artificially starved by detachment and dark-treatment, the mrna levels of senescence marker genes such as SEN1 increase at earlier times in the mutants than wild-type, resulting from acceleration of artificially induced senescence. 55,56,90 An Arabidopsis mutant of the v-snare VTI12, whose yeast homologue has been shown to be involved in docking and fusion of autophagosomes with the vacuole, also exhibited a similar acceleration of senescence in detached leaves, suggesting a function in autophagy. 62 It is currently unclear why Atatg mutants show an early senescence phenotype. In Atatg mutants, chlorophyll degradation is accelerated compared with wild-type plants; therefore, it seems that autophagy is not necessary for degradation of chloroplast proteins. This result is consistent with the idea that the degradation of chloroplast proteins during the initial stages of leaf senescence occurs within the chloroplast itself. 2,42,92 Thus, the accelerated senescence seen in Atatg mutants may reflect the need for vacuolar degradation of components that reside outside of the chloroplast. Atatg mutants also show some subtle growth defects. They develop fewer inflorescence branches because of early senescence, resulting in lower yield of seeds. In addition, root elongation of Atatg mutants is inhibited under nutrient-depleted conditions compared with those 8 Autophagy 2006; Vol. 2 Issue 1

of wild-type (Fig. 4C). It is thought that when the nutrient supply from the environment is limited, autophagy is required for efficient recycling of protein in the cells, and so its defect may cause a less efficient supply of nutrients; consequently cell growth is inhibited. Further phenotypic analysis of Atatg mutants also provides novel information on potential differences in autophagy in plants compared with other organisms. The phenotypes seen in Atatg4a4b-1 are more severe than those of Atatg9-1. In Atatg9-1 roots, although autophagic bodies accumulate more slowly compared with wild-type, they finally accumulate in the vacuolar lumen in the presence of concanamycin A, despite the presence of a null mutation. This is also seen in another mutant allele, Atatg9-2. Atatg9 mutants are leaky for unknown reasons, even though autophagy is completely abolished in the yeast atg9 mutant. These results suggest that higher plants are in some ways distinct from yeast and may have further plant-specific autophagy pathways. A C B PERSPECTIVES Research on plant autophagy is still in its infancy, and many important questions remain to be answered. One issue that is still unresolved is the possible variation in the degradative compartment. It has been proposed that the main lytic compartments of autophagy in plant cells are autophagosomes and not the central vacuole. 53,57,58 However, in vacuolated plant cells, autophagosomes appear to fuse with the central lytic vacuole as they do in yeast cells. 53,93 These observations suggest that plant cells may use two types of lytic compartments in autophagy and that the contributions of these organelles are controlled differently in different cells. It is also possible that plant species may differ in the extent of degradation of cytoplasmic components within autophagosomes, before fusion with the vacuole, rather than in the mechanism of autophagy itself. How the contribution of these two pathways to autophagy is regulated will be one of the future issues to be addressed in autophagy of plant cells. Autophagy monitoring systems established recently in whole plants allow the determination of when and where autophagy occurs, and under which conditions autophagy is induced. These systems will contribute greatly to the further understanding of the physiological roles of plant autophagy. 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