Autophagy maintains endosperm quality during seed storage to preserve germination ability in Arabidopsis.

To preserve germination ability, plant seeds must be protected from environmental stresses during the storage period. Here, we demonstrate that autophagy, an intracellular degradation system, maintains seed germination ability in Arabidopsis thaliana. The germination ability of long-term (>5 years) stored dry seeds of autophagy-defective (atg) mutant and wild-type (WT) plants was compared. Long-term stored (old) seeds of atg mutants showed lower germination ability than WT seeds, although short-term stored (new) seeds of atg mutants did not show such a phenotype. After removal of the seed coat and endosperm from old atg mutant seeds, the embryos developed into seedlings. Autophagic flux was maintained in endosperm cells during the storage period, and autophagy defect resulted in the accumulation of oxidized proteins and accelerated endosperm cell death. Consistent with these findings, the transcripts of genes, ENDO-ß-MANNANASE 7 and EXPANSIN 2, which are responsible for degradation/remodeling of the endosperm cell wall during germination, were reduced in old atg mutant seeds. We conclude that autophagy maintains endosperm quality during seed storage by suppressing aging-dependent oxidative damage and cell death, which allows the endosperm to perform optimal functions during germination, i.e., cell wall degradation/remodeling, even after long-term storage.

New insights into plant autophagy: molecular mechanisms and roles in development and stress responses.

Autophagy is an evolutionarily conserved eukaryotic intracellular degradation process. Although the molecular mechanisms of plant autophagy share similarities with those in yeast and mammals, certain unique mechanisms have been identified. Recent studies have highlighted the importance of autophagy during vegetative growth stages as well as in plant-specific developmental processes, such as seed development, germination, flowering, and somatic reprogramming. Autophagy enables plants to adapt to and manage severe environmental conditions, such as nutrient starvation, high-intensity light stress, and heat stress, leading to intracellular remodeling and physiological changes in response to stress. In the past, plant autophagy research lagged behind similar studies in yeast and mammals; however, recent advances have greatly expanded our understanding of plant-specific autophagy mechanisms and functions. This review summarizes current knowledge and latest research findings on the mechanisms and roles of plant autophagy with the objective of improving our understanding of this vital process in plants.

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light.

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.

Seed dormancy 4 like1 of Arabidopsis is a key regulator of phase transition from embryo to vegetative development.

Seed dormancy is an adaptive trait that enables plants to survive adverse conditions and restart growth in a season and location suitable for vegetative and reproductive growth. Control of seed dormancy is also important for crop production and food quality because it can help induce uniform germination and prevent preharvest sprouting. Rice preharvest sprouting quantitative trait locus analysis has identified Seed dormancy 4 (Sdr4) as a positive regulator of dormancy development. Here, we analyzed the loss-of-function mutant of the Arabidopsis ortholog, Sdr4 Like1 (SFL1), and found that the sfl1-1 seeds showed precocious germination at the mid- to late-maturation stage similar to rice sdr4 mutant, but converted to become more dormant than the wild type during maturation drying. Coordinated with the dormancy levels, expression levels of the seed maturation and dormancy master regulator genes, ABI3, FUS3, and DOG1 in sfl1-1 seeds were lower than in wild type at early- and mid-maturation stages, but higher at the late-maturation stage. In addition to the seed dormancy phenotype, sfl1-1 seedlings showed a growth arrest phenotype and heterochronic expression of LAFL (LEC1, ABI3, FUS3, LEC2) and DOG1 in the seedlings. These data suggest that SFL1 is a positive regulator of initiation and termination of the seed dormancy program. We also found genetic interaction between SFL1 and the SFL2, SFL3, and SFL4 paralogs of SFL1, which impacts on the timing of the phase transition from embryo maturation to seedling growth.

Autophagy triggered by iron-mediated ER stress is an important stress response to the early phase of Pi starvation in plants.

Inorganic phosphate (Pi) is essential for plant growth. However, Pi is often limiting in soil. Hence, plants have established several mechanisms of response to Pi starvation. One of the important mechanisms is Pi recycling, which includes membrane lipid remodeling and plastid DNA degradation via catabolic enzymes. However, the involvement of other degradation systems in Pi recycling remains unclear. Autophagy, a system for degradation of intracellular components, contributes to recycling of some nutrients, such as nitrogen, carbon, and zinc, under starvation. In the present study, we found that autophagy-deficient mutants depleted Pi early and exhibited severe leaf growth defects under Pi starvation. The main cargo of autophagy induced by early Pi depleted conditions was the endoplasmic reticulum (ER), indicating that ER-phagy, a type of autophagy that selectively degrades the ER, is involved in the response to the early phase of Pi starvation for contribution to Pi recycling. This ER-phagy was suppressed in an INOSITOL-REQUIRING ENZYME 1 double mutant, ire1a ire1b, in which ER stress responses are defective, suggesting that the early Pi starvation induced ER-phagy is induced by ER stress. Furthermore, iron limitation and inhibition of lipid-reactive oxygen species accumulation suppressed the ER-phagy. Interestingly, membrane lipid remodeling, a response to late Pi starvation, was accelerated in the ire1a ire1b under early Pi-depleted conditions. Our findings reveal the existence of two different phases of responses to Pi starvation (i.e. early and late) and indicate that ER stress-mediated ER-phagy is involved in Pi recycling in the early phase to suppress acceleration of the late phase.

Intracellular phosphate recycling systems for survival during phosphate starvation in plants.

Phosphorus (P) is an essential nutrient for plant growth and plants use inorganic phosphate (Pi) as their P source, but its bioavailable form, orthophosphate, is often limited in soils. Hence, plants have several mechanisms for adaptation to Pi starvation. One of the most common response strategies is "Pi recycling" in which catabolic enzymes degrade intracellular constituents, such as phosphoesters, nucleic acids and glycerophospholipids to salvage Pi. Recently, several other intracellular degradation systems have been discovered that salvage Pi from organelles. Also, one of sphingolipids has recently been identified as a degradation target for Pi recycling. So, in this mini-review we summarize the current state of knowledge, including research findings, about the targets and degradation processes for Pi recycling under Pi starvation, in order to further our knowledge of the whole mechanism of Pi recycling.

Autophagy balances the zinc-iron seesaw caused by Zn-stress.

Under zinc (Zn) deficiency, plants take up excess iron (Fe), but the uptake is inhibited under Zn excess. Coordination between intracellular recycling, transport, and sensing is essential for Zn-Fe homeostasis. A new study shows that autophagy resupplies Zn2+ and Fe2+ to correct intracellular Zn-Fe imbalances.

A proposed role for endomembrane trafficking processes in regulating tonoplast content and vacuole dynamics under ammonium stress conditions in Arabidopsis root cells.

Ammonium (NH4+) stress has multiple effects on plant physiology, therefore, plant responses are complex, and multiple mechanisms are involved in NH4+ sensitivity and tolerance in plants. Root growth inhibition is an important quantitative readout of the effects of NH4+ stress on plant physiology, and cell elongation appear as the principal growth inhibition target. We recently proposed autophagy as a relevant physiological mechanisms underlying NH4+ sensitivity response in Arabidopsis. In a brief overview, the impaired macro-autophagic flux observed under NH4+ stress conditions has a detrimental impact on the cellular energetic balance, and therefore on the energy-demanding plant growth. In contrast to its inhibitory effect on the autophagosomes flux to vacuole, NH4+ toxicity induced a micro-autophagy-like process. Consistent with the reduced membrane flux to the vacuole related to macro-autophagy inhibition and the increased tonoplast degradation due to enhanced micro-autophagy, the vacuoles of the root cells of the NH4+-stressed plants showed lower tonoplast content and a decreased perimeter/area ratio. As the endosome-to-vacuole trafficking is another important process that contributes to membrane flux toward the vacuole, we evaluated the effects of NH4+ stress on this process. This allows us to propose that autophagy could contribute to vacuole development as well as possible avenues to follow for future studies.

RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants.

Inorganic phosphate (Pi) and nitrogen (N) are essential nutrients for plant growth. We found that a five-fold oversupply of nitrate rescues Arabidopsis (Arabidopsis thaliana) plants from Pi-starvation stress. Analyses of transgenic plants that overexpressed GFP-AUTOPHAGY8 showed that an oversupply of nitrate induced autophagy flux under Pi-depleted conditions. Expression of DIN6 and DIN10, the carbon (C) starvation-responsive genes, was upregulated when nitrate was oversupplied under Pi starvation, which suggested that the plants recognized the oversupply of nitrate as C starvation stress because of the reduction in the C/N ratio. Indeed, formation of Rubisco-containing bodies (RCBs), which contain chloroplast stroma and are induced by C starvation, was enhanced when nitrate was oversupplied under Pi starvation. Moreover, autophagy-deficient mutants did not release Pi (unlike wild-type plants), exhibited no RCB accumulation inside vacuoles, and were hypersensitive to Pi starvation, indicating that RCB-mediated chlorophagy is involved in Pi starvation tolerance. Thus, our results showed that the Arabidopsis response to Pi starvation is closely linked with N and C availability and that autophagy is a key factor that controls plant growth under Pi starvation.

Optimal Distribution of Iron to Sink Organs via Autophagy Is Important for Tolerance to Excess Zinc in Arabidopsis.

Zinc (Zn) is nutritionally an essential metal element, but excess Zn in the environment is toxic to plants. Autophagy is a major pathway responsible for intracellular degradation. Here, we demonstrate the important role of autophagy in adaptation to excess Zn stress. We found that autophagy-defective Arabidopsis thaliana (atg2 and atg5) exhibited marked excess Zn-induced chlorosis and growth defects relative to wild-type (WT). Imaging and biochemical analyses revealed that autophagic activity was elevated under excess Zn. Interestingly, the excess Zn symptoms of atg5 were alleviated by supplementation of high levels of iron (Fe) to the media. Under excess Zn, in atg5, Fe starvation was especially severe in juvenile true leaves. Consistent with this, accumulation levels of Fe3+ near the shoot apical meristem remarkably reduced in atg5. Furthermore, excision of cotyledons induced severe excess Zn symptoms in WT, similar to those observed in atg5.Our data suggest that Fe3+ supplied from source leaves (cotyledons) via autophagy is distributed to sink leaves (true leaves) to promote healthy growth under excess Zn, revealing a new dimension, the importance of heavy-metal stress responses by the intracellular recycling.

Ammonium stress increases microautophagic activity while impairing macroautophagic flux in Arabidopsis roots.

Plant responses to NH4+ stress are complex, and multiple mechanisms underlying NH4+ sensitivity and tolerance in plants may be involved. Here, we demonstrate that macro- and microautophagic activities are oppositely affected in plants grown under NH4+ toxicity conditions. When grown under NH4+ stress conditions, macroautophagic activity was impaired in roots. Root cells accumulated autophagosomes in the cytoplasm, but showed less autophagic flux, indicating that late steps of the macroautophagy process are affected under NH4+ stress conditions. Under this scenario, we also found that the CCZ1-MON1 complex, a critical factor for vacuole delivery pathways, functions in the late step of the macroautophagic pathway in Arabidopsis. In contrast, an accumulation of tonoplast-derived vesicles was observed in vacuolar lumens of root cells of NH4+ -stressed plants, suggesting the induction of a microautophagy-like process. In this sense, some SYP22-, but mainly VAMP711-positive vesicles were observed inside vacuole in roots of NH4+ -stressed plants. Consistent with the increased tonoplast degradation and the reduced membrane flow to the vacuole due to the impaired macroautophagic flux, the vacuoles of root cells of NH4+ -stressed plants showed a simplified structure and lower tonoplast content. Taken together, this study presents evidence that postulates late steps of the macroautophagic process as a relevant physiological mechanism underlying the NH4+ sensitivity response in Arabidopsis, and additionally provides insights into the molecular tools for studying microautophagy in plants.

Thaumatin-like proteins and a cysteine protease inhibitor secreted by the pine wood nematode Bursaphelenchus xylophilus induce cell death in Nicotiana benthamiana.

Pine wilt disease (PWD) is an infectious disease of pines that typically kills affected trees. The causal pathogen of PWD is the pine wood nematode (PWN), Bursaphelenchus xylophilus. Understanding of the disease has advanced in recent years through the use of a highly sensitive proteomics procedure and whole genome sequence analysis; in combination, these approaches have enabled identification of proteins secreted by PWNs. However, the roles of these proteins during the onset of parasitism have not yet been elucidated. In this study, we used a leaf-disk assay based on transient overexpression in Nicotiana benthamiana to allow functional screening of 10 candidate pathogenic proteins secreted by PWNs. These proteins were selected based on previous secretome and RNA-seq analyses. We found that five molecules induced significant cell death in tobacco plants relative to a GFP-only control. Three of these proteins (Bx-TH1, Bx-TH2, and Bx-CPI) may have a role in molecular mimicry and likely make important contributions to inducing hypersensitive responses in host plants.

Importance of non-systemic leaf autophagy for suppression of zinc starvation induced-chlorosis.

Autophagy, which is one of the self-degradation systems, promotes intracellular zinc (Zn) recycling under Zn deficiency (-Zn) in plants. Therefore, autophagy defective plants show severe chlorosis under -Zn. Root is the plant organ which directly exposed to Zn deficient environment, however, in our recent study, -Zn symptom was prominently observed in leaves as chlorosis. Here, we conducted micrografting to determine which organ's autophagic activities are important to suppress the -Zn induced chlorosis. Grafted plants that have autophagic activities only in roots or leaves were grown under -Zn and then compared chlorotic phenotypes among them. As a result, regardless of the autophagic activities in rootstocks, -Zn induced-chlorosis in leaves was occurred only when autophagy in scion was defective. This data indicates that Zn resupplied by autophagic degradation in root cells could not contribute to suppress the chlorosis in leaves. Thus, autophagy in the aerial part is critical for controlling -Zn induced-chlorosis in leaves. Taken together, along with our recently reported data, we conclude that the mechanism of Zn resupply by autophagic degradation is not systemic throughout the plant but rather a local system.

Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated Reactive Oxygen Species Production under Zinc Deficiency.

Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency (-Zn) in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during -Zn in Arabidopsis (Arabidopsis thaliana). Autophagy-defective plants (atg mutants) failed to grow and developed accelerated chlorosis under -Zn. As expected, -Zn induced autophagy in wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn2+ was lower in atg mutants than in control plants. Interestingly, -Zn symptoms in atg mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in -Zn atg mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of -Zn, is accelerated in atg mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain reactive oxygen species homeostasis under -Zn in plants.

Autophagy and Nutrients Management in Plants.

Nutrient recycling and mobilization from organ to organ all along the plant lifespan is essential for plant survival under changing environments. Nutrient remobilization to the seeds is also essential for good seed production. In this review, we summarize the recent advances made to understand how plants manage nutrient remobilization from senescing organs to sink tissues and what is the contribution of autophagy in this process. Plant engineering manipulating autophagy for better yield and plant tolerance to stresses will be presented.

Unveiling the Molecular Mechanisms of Plant Autophagy-From Autophagosomes to Vacuoles in Plants.

Autophagy is an evolutionarily conserved intracellular vacuolar process. Since Christian de Duve first coined the term 'autophagy' in 1963, it had not been well understood at the molecular level until much later, due to limitations in biochemical approaches and/or morphological approaches posed by electron microscopy. An important milestone was achieved with the isolation and identification of autophagy-related (ATG) genes by genetic screening using yeast Saccharomyces cerevisiae. ATG genes are well conserved in most eukaryotic organisms, which allowed the subsequent isolation of ATG gene-knockouts in plants. From the phenotypic analyses of the autophagy-defective plants, the physiological roles of autophagy have been predicted. However, in some cases, all the phenotypes cannot be simply explained by defects in autophagy. Therefore, in order to fully understand the physiological implications of plant autophagy, it is quite important to elucidate the molecular mechanisms involved in each process in macro-/micro-autophagy. Although, until recently, our understanding of the molecular mechanisms of plant autophagy was lagging compared to similar research in yeast and animals, current studies have made many great advances in the plant research field. In this review, we discuss current knowledge of the molecular mechanisms of plant autophagy, from autophagy-induction/autophagosome-formation to vacuolar degradation, comparing these to processes in yeast and mammals. We also review aspects of plant autophagy research that require further investigation in the future.

Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds.

Autophagy is essential for nutrient recycling and plays a fundamental role in seed production and grain filling in plants. Autophagy participates in nitrogen remobilization at the whole-plant level, and the seeds of autophagy mutants present abnormal C and N contents relative to wild-type (WT) plants. It is well known that autophagy (ATG) genes are induced in leaves during senescence; however, expression of such genes in seeds has not yet been reported. In this study we show that most of the ATG genes are induced during seed maturation in Arabidopsis siliques. Promoter-ATG8f::UIDA and promoter-ATG8f::GFP fusions showed the strong expression of ATG8f in the phloem companion cells of pericarps and the funiculus, and in the embryo. Expression was especially strong at the late stages of development. The presence of many GFP-ATG8 pre-autophagosomal structures and autophagosomes confirmed the presence of autophagic activity in WT seed embryos. Seeds of atg5 and WT plants grown under low- or high-nitrate conditions were analysed. Nitrate-independent phenotypes were found with higher seed abortion in atg5 and early browing, higher total protein concentrations in the viable seeds of this mutant as compared to the WT. The higher total protein accumulation in atg5 viable seeds was significant from early developmental stages onwards. In addition, relatively low and early accumulation of 12S globulins were found in atg5 seeds. These features led us to the conclusion that atg5 seed development is accelerated and that the protein storage deposition pathway is somehow abnormal or incomplete.

Stitching together the Multiple Dimensions of Autophagy Using Metabolomics and Transcriptomics Reveals Impacts on Metabolism, Development, and Plant Responses to the Environment in Arabidopsis.

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.

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality.

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.