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Autophagy degrades toxic materials and old organelles, and recycles nutrients in eukaryotic cells. Whereas the studies on autophagy have been reported in other eukaryotic cells, its functioning in plants has not been well elucidated. We analyzed the roles of OsATG10 genes, which are autophagy-related. Two rice ATG10 genes - OsATG10a and OsATG10b - share significant sequence homology (about 75%), and were ubiquitously expressed in all organs examined here. GUS assay indicated that OsATG10b was highly expressed in the mesophyll cells and vascular tissue of younger leaves, but its level of expression decreased in older leaves. We identified T-DNA insertional mutants in that gene. Those osatg10b mutants were sensitive to treatments with high salt and methyl viologen (MV). Monodansylcadaverine-staining experiments showed that the number of autophagosomes was significantly decreased in the mutants compared with the WT. Furthermore, the amount of oxidized proteins increased in MV-treated mutant seedlings. These results demonstrate that OsATG10b plays an important role in the survival of rice cells against oxidative stresses.

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H+-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.

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.

Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 A resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes ( ATG s) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein–ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4 s ( atg4a4b-1 ), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.

Autophagy is a fundamental process in the plant life story, playing a key role in immunity, senescence, nutrient recycling, and adaptation to the environment. Transcriptomics and metabolomics of the rosette leaves of Arabidopsis thaliana autophagy mutants (atg) show that autophagy is essential for cell homeostasis and stress responses and that several metabolic pathways are affected. Depletion of hexoses, quercetins, and anthocyanins parallel the overaccumulation of several amino acids and related compounds, such as glutamate, methionine, glutathione, pipecolate, and 2-aminoadipate. Transcriptomic data show that the pathways for glutathione, methionine, raffinose, galacturonate, and anthocyanin are perturbed. Anthocyanin depletion in atg mutants, which was previously reported as a possible defect in flavonoid trafficking to the vacuole, appears due to the downregulation of the master genes encoding the enzymes and regulatory proteins involved in flavonoid biosynthesis. Overexpression of the PRODUCTION OF ANTHOCYANIN PIGMENT1 transcription factor restores anthocyanin accumulation in vacuoles of atg mutants. Transcriptome analyses reveal connections between autophagy and (1) salicylic acid biosynthesis and response, (2) cytokinin perception, (3) oxidative stress and plant defense, and possible interactions between autophagy and the COP9 signalosome machinery. The metabolic and transcriptomic signatures identified for the autophagy mutants are discussed and show consistencies with the observed phenotypes.

Peroxisomes are essential organelles characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG) gene-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5 knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles by autophagy for degradation. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves for proper plant development under natural growth conditions.

Autophagy is a degradation pathway that recycles cell materials upon encountering stress conditions or during specific developmental processes. To better understand the physiological roles of autophagy, proper monitoring methods are very important. In mammals and yeast, monitoring of autophagy is often performed with a green fluorescent protein (GFP)-ATG8 fusion protein or with acidotropic dyes such as monodansylcadaverine (MDC) and LysoTracker Red (LTR). To evaluate these monitoring methods, here we examined these systems by inducing autophagy in Arabidopsis thaliana roots as a model for monitoring autophagy in planta. Under carbon- and nitrogen-starved conditions, the number and size of vesicles labeled by GFP-ATG8 was increased for several hours and then gradually decreased to a level higher than that observed before the start of the experiment. We also observed the disappearance of GFP-ATG8-labeled vesicles after treatment with wortmannin, a phosphatidylinositol 3-kinase inhibitor known as an autophagy inhibitor, showing that the GFP-ATG8 transgenic line constitutes an excellent method for monitoring autophagy. These data were compared with plants stained with MDC and LTR. There was no appreciable MDC/LTR staining of small organelles in the root under the induction of autophagy. Some vesicles were eventually observed in the root tip only, but co-localization experiments, as well as experiments with autophagy-deficient atg mutants, provided the evidence that these structures were located in the vacuole and were not manifestly autophagosomes and/or autolysosomes. Extreme caution should therefore be used when monitoring autophagy with the aid of MDC/LTR. Additionally, our observations strongly suggest that autophagosomes fuse directly to vacuoles in Arabidopsis roots.

The legend for Figure 1B has been corrected; The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; Autophagy is present at a basal level in all plant tissues and is induced during leaf ageing and in response to nitrogen (N) starvation. Nitrogen remobilization from the rosette to the seeds is impaired in autophagy mutants. This report focuses on the role of autophagy in leaf N management and proteolysis during plant ageing.Metabolites, enzyme activities and protein contents were monitored in several autophagy-defective (atg) Arabidopsis mutants grown under low and high nitrate conditions.Results showed that carbon (C) and N statuses were affected in atg mutants before any senescence symptoms appeared. atg mutants accumulated larger amounts of ammonium, amino acids and proteins than wild type, and were depleted in sugars. Over-accumulation of proteins in atg mutants was selective and occurred despite higher endopeptidase and carboxypeptidase activities. Specific over-accumulation of the ribosomal proteins S6 and L13 subunits, and of catalase and glutamate dehydrogenase proteins was observed. atg mutants also accumulated peptides putatively identified as degradation products of the Rubisco large subunit and glutamine synthetase 2 (GS2). Incomplete chloroplast protein degradation resulting from autophagy defects could explain the higher N concentrations measured in atg rosettes and defects in N remobilization.It is concluded that autophagy controls C:N status and protein content in leaves of Arabidopsis.

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. Early morphological studies suggested that autophagy occurs in plant cells and predicted that autophagy has a variety of functions in plant growth and development. However, it is only since the identification of autophagy genes that the physiological roles of autophagy in plants have become apparent. Recent reverse genetic studies indicate that autophagy defects in higher plants result in early senescence and excessive immunity-related programmed cell death (PCD), irrespective of nutrient conditions, suggesting that plant autophagy has an important pro-survival function during these types of cell death. Further biochemical and pharmacological studies in combination with double mutant analyses revealed that excessive salicylic acid (SA) signaling is a major factor in autophagy-defective plant-dependent cell death and that the SA signal can induce autophagy. These results demonstrate a novel physiological function for plant autophagy that operates a negative feedback loop to modulate SA signaling.

We recently reported that autophagy plays a role in chloroplasts degradation in individually-darkened senescing leaves. Chloroplasts contain approximately 80% of total leaf nitrogen, mainly as photosynthetic proteins, predominantly ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco). During leaf senescence, chloroplast proteins are degraded as a major source of nitrogen for new growth. Concomitantly, while decreasing in size, chloroplasts undergo transformation to non-photosynthetic gerontoplasts. Likewise, over time the population of chloroplasts (gerontoplasts) in mesophyll cells also decreases. While bulk degradation of the cytosol and organelles is mediated by autophagy, the role of chloroplast degradation is still unclear. In our latest study, we darkened individual leaves to observe chloroplast autophagy during accelerated senescence. At the end of the treatment period chloroplasts were much smaller in wild-type than in the autophagy defective mutant, atg4a4b-1, with the number of chloroplasts decreasing only in wild-type. Visualizing the chloroplast fractions accumulated in the vacuole, we concluded that chloroplasts were degraded by two different pathways, one was partial degradation by small vesicles containing only stromal-component (Rubisco containing bodies; RCBs) and the other was whole chloroplast degradation. Together, these pathways may explain the morphological attenuation of chloroplasts during leaf senescence and describe the fate of chloroplasts.

Chloroplasts (chlorophyll-containing plastids) and other plastids are found in all plants and many animals. They are crucial to the survival of plants and most of the animals that harbour them. An example of a non-photosynthesizing plastid in animals is the apicoplast in the malaria-causing Plasmodium species, which is required for survival of the parasite. Many animals (such as sea slugs, sponges, reef corals, and clams) consume prey containing chloroplasts, or feed on algae. Some of these incorporate the chloroplasts from their food, or whole algal cells, into their own cells. Other species from these groups place algal cells between their own cells. Reef-building corals often lose their intracellular algae as a result of environmental changes, resulting in coral bleaching and death. The sensitivity of the chloroplast internal membranes to temperature stress is one of the reasons for coral death. Chloroplasts can also be a causal factor in the processes leading to whole-plant death, as the knockout of a gene encoding a chloroplast protein delayed the yellowing that proceeds death in tobacco plants. It is concluded that chloroplasts and other plastids are essential to individual survival in many species, including animals, and that they also play a role in triggering death in some plant and animal species.

During senescence and times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco). Previously by immunoelectron microscopy (IEM), we demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves (Chiba et al. 2003). In this study, we visualized RCBs in living cells of transgenic Arabidopsis plants containing stroma-targeted green fluorescent protein (GFP). When leaves of transgenic Arabidopsis plants were incubated under starvation conditions with a vacuolar-ATPase inhibitor, spherical bodies exhibiting GFP fluorescence without chlorophyll fluorescence were observed. Spherical bodies were not observed when leaves were provided with a sugar and nutrient solution. IEM of concanamycin-A-treated leaves with anti-Rubisco antibodies confirmed the existence of RCBs in the vacuolar compartment. These results suggest the hypothesis that stromal proteins can be mobilized to the vacuole via RCBs by possibly autophagy for the degradation.

Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b–8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.

Excluding the central vacuole, chloroplasts constitute the largest compartment within the leaf cells of plants and contain approximately 80 percent of the total leaf nitrogen, mainly as proteins. Much of this nitrogen is allocated to the carbon-fixing enzyme in photosynthesis, Rubisco. During senescence, plants can mobilize nitrogen from chloroplasts in older leaves to other organs, such as developing seeds. Whereas bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have recently demonstrated that stroma-targeted green fluorescent protein (GFP), DsRed, and GFP-labeled Rubisco can be mobilized to the vacuole of living cells via Rubisco-containing bodies, in an ATG gene-dependent manner. Our results indicate the presence of a specific autophagic pathway for chloroplast stromal proteins, which does not cause chloroplast lysis. Here, we also discuss the involvement of stroma-filled tubules, stromules, which are important for the structural flexibility of the organelle, on the autophagic transfer of stromal proteins to the vacuole.