Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

Global Analysis of the Role of Autophagy in Cellular Metabolism and Energy Homeostasis in Arabidopsis Seedlings under Carbon Starvation

Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.     

Autophagy Contributes to Leaf Starch Degradation

Transitory starch, a major photosynthetic product in the leaves of land plants, accumulates in chloroplasts during the day and is hydrolyzed to maltose and Glc at night to support respiration and metabolism. Previous studies in Arabidopsis thaliana indicated that the degradation of transitory starch only occurs in the chloroplasts. Here, we report that autophagy, a nonplastidial process, participates in leaf starch degradation. Excessive starch accumulation was observed in Nicotiana benthamiana seedlings treated with an autophagy inhibitor and in autophagy-related (ATG) gene-silenced N. benthamiana and in Arabidopsis atg mutants. Autophagic activity in the leaves responded to the dynamic starch contents during the night. Microscopy showed that a type of small starch granule-like structure (SSGL) was localized outside the chloroplast and was sequestered by autophagic bodies. Moreover, an increased number of SSGLs was observed during starch depletion, and disruption of autophagy reduced the number of vacuole-localized SSGLs. These data suggest that autophagy contributes to transitory starch degradation by sequestering SSGLs to the vacuole for their subsequent breakdown.     

Genes for plant Autophagy: Functions and interactions

Autophagy, or self-consuming of cytoplasmic constituents in a lytic compartment, plays a crucial role in nutrient recycling, development, cell homeostasis, and defense against pathogens and toxic products. Autophagy in plant cells uses a conserved machinery of core Autophagy-related (Atg) proteins. Recently, research on plant autophagy has been expanding and other components interacting with the core Atg proteins are being revealed. In addition, growing evidence suggests that autophagy communicates with other cellular pathways such as the ubiquitin-proteasome system, protein secretory pathway, and endocytic pathway. An increase in our understanding of plant autophagy will undoubtedly help test the hypothesized functions of plant autophagy in programmed cell death, vacuole biogenesis, and responses to biotic, abiotic, and nutritional stresses. In this review, we summarize recent progress on these topics and suggest topics for future research, after inspecting common phenotypes of current Arabidopsis atg mutants.     

Role of autophagy in triacylglycerol biosynthesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revealed by chemical inducer and inhibitors

Autophagy mediates degradation and recycling of cellular components and plays an important role in senescence and adaptive responses to biotic and abiotic stresses. Nutrient deprivation has been shown to trigger triacylglycerol (TAG) accumulation and also induces autophagy in various green algae. However, the functional relationship between TAG metabolism and autophagy remains unclear. To gain preliminary evidence supporting a role of autophagy in TAG synthesis, Chlamydomonas reinhardtii CC-2686 was grown in Tris-acetate phosphate medium with or without nitrogen and treated with an autophagy inducer (rapamycin) or inhibitors (wortmannin, 3-methyladenine, and bafilomycin A1). Fluorescence microscopic analysis of Nile red-stained cells following 72-h treatments showed that rapamycin induced accumulation of subcellular lipid droplets which are storage sites of TAG. Rapamycin treatment in combination with nitrogen starvation led to a greater abundance of lipid droplets. Wortmannin and bafilomycin A1, but not 3-methyladenine, inhibited lipid droplet accumulation in rapamycin-treated cells and to a less extent in nitrogen-depleted cells. These results suggested that autophagy contributes to TAG synthesis in C. reinhardtii, but is not a necessary process. Autophagy induction may also be used to further enhance TAG accumulation in microalgae under nutrient deprivation.     

The Arabidopsis thaliana ACBP3 regulates leaf senescence by modulating phospholipid metabolism and ATG8 stability

Bulk degradation and nutrient recycling are events associated with autophagy. The core components of the autophagy machinery have been elucidated recently using molecular and genetic approaches. In particular, two ubiquitin-like proteins, ATG8 and ATG12, which conjugate with phosphatidylethanolamine (PE) and ATG5, respectively, forming ATG8-PE and ATG12-ATG5 complexes, were shown to be essential in autophagosome formation. Our recent findings reveal that the Arabidopsis thaliana acyl-CoA-binding protein ACBP3 binds the phospholipid PE in vitro and that ACBP3 overexpression and downregulation correlate with PE composition in rosettes. Furthermore, ACBP3-overexpressors (ACBP3-OEs) display accelerated salicylic acid-dependent leaf senescence resembling the phenotype of Arabidopsis knockout (KO) mutants defective in autophagy-related (ATG) proteins. Consistently, downregulation of ACBP3 (ACBP3-KOs) delays dark-induced leaf senescence. By analysis of transgenic Arabidopsis expressing GFP-ATG8e as well as those co-expressing ACBP3-OE and GFP-ATG8e, we showed that ACBP3-overexpression disrupts autophagosome formation and enhanced degradation of ATG8 under starvation conditions, suggesting that ACBP3 is an important regulator of the ATG8-PE complex via its interaction with PE. Here, a working model for the role of ACBP3 in the regulation of autophagy-mediated leaf senescence is presented.     

ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.     

Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.     

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.     

The Role of Autophagy in Mitochondria Maintenance: Characterization of Mitochondrial Functions in Autophagy-Deficient S. cerevisiae Strains

Autophagy is a lysosome-dependent cellular degradation process. Organisms bearing deletions of the essential autophagy genes exhibit various pathological conditions, including cancer in mammals and shortened life span in C. elegans. The direct cause for these phenotypes is not clear. Here we used yeast as a model system to characterize the cellular consequence of ATG (autophagy-related) gene deletions. We found that the atg mutant strains, atg1?, atg6?, atg8? and atg12?, showed defects related to mitochondrial biology. These strains were unable to degrade mitochondria in stationary culture. In non-fermentable medium, which requires mitochondrial oxidative phosphorylation for survival, these atg strains showed a growth defect with an increased cell population at the G₁ phase of the cell cycle. The cells had lower oxygen consumption rates and reduced mitochondrial electron transport chain activities. Under these growth conditions, the atg strains had lower mitochondrial membrane potential. In addition, these mutants generated higher levels of reactive oxygen species (ROS) and they were prone to accumulate dysfunctional mitochondria. This study clearly indicates that an autophagy defect has a functional impact on various aspects of mitochondrial functions and suggests a critical role of autophagy in mitochondria maintenance.     

Self-eating to grow and kill: autophagy in filamentous ascomycetes

Autophagy is a tightly controlled degradation process in which eukaryotic cells digest their own cytoplasm containing protein complexes and organelles in the vacuole or lysosome. Two types of autophagy have been described: macroautophagy and microautophagy. Both types can be further divided into nonselective and selective processes. Molecular analysis of autophagy over the last two decades has mostly used the unicellular ascomycetes Saccharomyces cerevisiae and Pichia pastoris. Genetic analysis in these yeasts has identified 36 autophagy-related (atg) genes; many are conserved in all eukaryotes, including filamentous ascomycetes. However, the autophagic machinery also evolved significant differences in fungi, as a consequence of adaptation to diverse fungal lifestyles. Intensive studies on autophagy in the last few years have shown that autophagy in filamentous fungi is not only involved in nutrient homeostasis but in other cellular processes such as cell differentiation, pathogenicity and secondary metabolite production. This mini-review focuses on the specific roles of autophagy in filamentous fungi.