FYVE2, a phosphatidylinositol 3-phosphate effector, interacts with the COPII machinery to control autophagosome formation in Arabidopsis.

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase Secretion-associated Ras-related GTPase 1 (SAR1), which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.

Genetic Analyses of the Arabidopsis ATG1 Kinase Complex Reveal Both Kinase-Dependent and Independent Autophagic Routes during Fixed-Carbon Starvation.

Under nutrient and energy-limiting conditions, plants up-regulate sophisticated catabolic pathways such as autophagy to remobilize nutrients and restore energy homeostasis. Autophagic flux is tightly regulated under these circumstances through the AuTophaGy-related1 (ATG1) kinase complex, which relays upstream nutrient and energy signals to the downstream components that drive autophagy. Here, we investigated the role(s) of the Arabidopsis (Arabidopsis thaliana) ATG1 kinase during autophagy through an analysis of a quadruple mutant deficient in all four ATG1 isoforms. These isoforms appear to act redundantly, including the plant-specific, truncated ATG1t variant, and like other well-characterized atg mutants, homozygous atg1abct quadruple mutants display early leaf senescence and hypersensitivity to nitrogen and fixed-carbon starvations. Although ATG1 kinase is essential for up-regulating autophagy under nitrogen deprivation and short-term carbon starvation, it did not stimulate autophagy under prolonged carbon starvation. Instead, an ATG1-independent response arose requiring phosphatidylinositol-3-phosphate kinase (PI3K) and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), possibly through phosphorylation of the ATG6 subunit within the PI3K complex by the catalytic KIN10 subunit of SnRK1. Together, our data connect ATG1 kinase to autophagy and reveal that plants engage multiple pathways to activate autophagy during nutrient stress, which include the ATG1 route as well as an alternative route requiring SnRK1 and ATG6 signaling.plantcell;31/12/2973/FX1F1fx1.

Autophagy inducers lead to transient accumulation of autophagosomes in Arabidopsis roots.

KEY MESSAGE: This study reveals that plant roots show a rapid termination of autophagy induction, offering a plant model for studying how excessive autophagy is deterred. In eukaryotes, autophagy is an intracellular mechanism that is important for recycling nutrients by degrading various macromolecules and organelles in vacuoles and lysosomes. Autophagy is induced when the nutrient supply to plant cells is limited. The protein kinase target of rapamycin (TOR) complex negatively regulates autophagy when nutrients are present in adequate amounts. The TOR inhibitor AZD8055 is an autophagy inducer that is useful for studying starvation-induced autophagy in plant cells. The mechanism by which AZD8055 increases the autophagic flux in plant cells has not been studied in detail. Here, we show that AZD8055-induced autophagy requires phosphatidylinositol 3-kinase activity and canonical AUTOPHAGY-RELATED (ATG) genes in Arabidopsis thaliana. Autophagic flux rapidly increased in seedlings treated with AZD8055. Unexpectedly, autophagy induction was transient in root cells and terminated earlier than in cotyledon cells. Transient induction is partly caused by a temporary effect of AZD8055 on phagophore initiation. These findings indicate a TOR-independent mechanism for terminating autophagy induction, thereby paving the way for elucidating how excess autophagy is prevented in plant roots.

Arabidopsis cargo receptor NBR1 mediates selective autophagy of defective proteins.

Aggrephagy, a type of selective autophagy that sequesters protein aggregates for degradation in the vacuole, is an important protein quality control mechanism, particularly during cell stress. In mammalian cells, aggrephagy and several other forms of selective autophagy are mediated by dedicated cargo receptors such as NEIGHBOR OF BRCA1 (NBR1). Although plant NBR1 homologs have been linked to selective autophagy during biotic stress, it remains unclear how they impact selective autophagy under non-stressed and abiotic stress conditions. Through microscopic and biochemical analysis of nbr1 mutants expressing autophagy markers and an aggregation-prone reporter, we tested the connection between NBR1 and aggrephagy in Arabidopsis. Although NBR1 is not essential for general autophagy, or for the selective clearance of peroxisomes, mitochondria, or the ER, we found that NBR1 is required for the heat-induced formation of autophagic vesicles. Moreover, cytoplasmic puncta containing aggregation-prone proteins, which were rarely observed in wild-type plants, were found to accumulate in nbr1 mutants under both control and heat stress conditions. Given that NBR1 co-localizes with these cytoplasmic puncta, we propose that Arabidopsis NBR1 is a plant aggrephagy receptor essential for maintaining proteostasis under both heat stress and non-stress conditions.

Vacuolar Trafficking Protein VPS38 Is Dispensable for Autophagy.

Phosphatidylinositol 3-P (PI3P) is a signaling molecule that controls a variety of processes in endosomal, autophagic, and vacuolar/lysosomal trafficking in yeasts and mammals. Vacuolar protein sorting 34 (Vps34) is a conserved PI3K present in multiple complexes with specific functions and regulation. In yeast, the PI3K complex II consists of Vps34p, Vps15p, Vps30p/Atg6p, and Vps38p, and is essential for vacuolar protein sorting. Here, we describe the Arabidopsis (Arabidopsis thaliana) homolog of yeast Vps38p and human UV radiation resistance-associated gene protein. Arabidopsis VPS38 interacts with VPS30/ATG6 both in yeast and in planta. Although the level of PI3P in Arabidopsis vps38 mutants is similar to that in wild type, vps38 cells contain enlarged multivesicular endosomes and fewer organelles enriched in PI3P than the wild type. The vps38 mutants are defective in the trafficking of vacuolar cargo and its receptor VACUOLAR SORTING RECEPTOR2;1. The mutants also exhibit abnormal cytoplasmic distributions of endocytic cargo, such as auxin efflux carriers PINFORMED1 (PIN1) and PIN2. Constitutive autophagy is normal in the mutants but starvation-induced autophagy was slightly inhibited. We conclude that Arabidopsis VPS38 is dispensable for autophagy but essential for efficient targeting of biosynthetic and endocytic cargo to the vacuole.

BPH1, a novel substrate receptor of CRL3, plays a repressive role in ABA signal transduction.

KEY MESSAGE: BPH1 acts as a substrate receptor of CRL3 complex and negatively regulates ABA-mediated cellular responses. The study on its function provides information that helps further understand the relationship between ABA signaling and UPS. Abscisic acid (ABA) plays a crucial role in a variety of cellular processes, including seed dormancy, inhibition of seedling growth, and drought resistance in plants. Cullin3-RING E3 ligase (CRL3) complex is a type of multi-subunit E3 ligase, and BTB/POZ protein, a component of CRL3 complex, functions as a receptor to determine a specific substrate. To elucidate the CRL3 complex that participates in ABA-mediated cellular processes, we first investigated ABA-inducible BTB/POZ genes based on data from the AtGenExpress Visualization Tool (AVT). We then isolated an ABA-inducible gene encoding a potential CRL3 substrate receptor in Arabidopsis, BPH1 (BTB/POZ protein hypersensitive to ABA 1). The isolate gene has a BTB/POZ domain and a NPH3 domain within its N-terminal and C-terminal region, respectively. Yeast two-hybrid and co-immunoprecipitation assays showed that BPH1 physically interacted with cullin3a, a scaffold protein of CRL3, suggesting that it functions as an Arabidopsis CRL3 substrate receptor. The functional mutation of BPH1 caused delayed seed germination in response to ABA and enhanced sensitivity by NaCl and mannitol treatments as ABA-related stresses. Moreover, bph1 mutants exhibited enhanced stomatal closure under ABA application and reduced water loss when compared with wild-type, implying their enhanced tolerance to drought stress. Based on the information from microarray/AVT data and expression analysis of various ABA-inducible genes between wild-type and bph1 plants following ABA treatments, we concluded loss of BPH1 resulted in hyper-induction of a large portion of ABA-inducible genes in response to ABA. Taken together, these results show that BPH1 is negatively involved in ABA-mediated cellular events.

Autophagic recycling plays a central role in maize nitrogen remobilization.

Autophagy is a primary route for nutrient recycling in plants by which superfluous or damaged cytoplasmic material and organelles are encapsulated and delivered to the vacuole for breakdown. Central to autophagy is a conjugation pathway that attaches AUTOPHAGY-RELATED8 (ATG8) to phosphatidylethanolamine, which then coats emerging autophagic membranes and helps with cargo recruitment, vesicle enclosure, and subsequent vesicle docking with the tonoplast. A key component in ATG8 function is ATG12, which promotes lipidation upon its attachment to ATG5. Here, we fully defined the maize (Zea mays) ATG system transcriptionally and characterized it genetically through atg12 mutants that block ATG8 modification. atg12 plants have compromised autophagic transport as determined by localization of a YFP-ATG8 reporter and its vacuolar cleavage during nitrogen or fixed-carbon starvation. Phenotypic analyses showed that atg12 plants are phenotypically normal and fertile when grown under nutrient-rich conditions. However, when nitrogen-starved, seedling growth is severely arrested, and as the plants mature, they show enhanced leaf senescence and stunted ear development. Nitrogen partitioning studies revealed that remobilization is impaired in atg12 plants, which significantly decreases seed yield and nitrogen-harvest index. Together, our studies demonstrate that autophagy, while nonessential, becomes critical during nitrogen stress and severely impacts maize productivity under suboptimal field conditions.

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.

Autophagy-related (ATG) 11, ATG9 and the phosphatidylinositol 3-kinase control ATG2-mediated formation of autophagosomes in Arabidopsis.

KEY MESSAGE: Using quantitative assays for autophagy, we analyzed 4 classes of atg mutants, discovered new atg2 phenotypes and ATG gene interactions, and proposed a model of autophagosome formation in plants. Plant and other eukaryotic cells use autophagy to target cytoplasmic constituents for degradation in the vacuole. Autophagy is regulated and executed by a conserved set of proteins called autophagy-related (ATG). In Arabidopsis, several groups of ATG proteins have been characterized using genetic approaches. However, the genetic interactions between ATG genes have not been established and the relationship between different ATG groups in plants remains unclear. Here we analyzed atg2, atg7, atg9, and atg11 mutants and their double mutants at the physiological, biochemical, and subcellular levels. Involvement of phosphatidylinositol 3-kinase (PI3K) in autophagy was also tested using wortmannin, a PI3K inhibitor. Our mutant analysis using autophagy markers showed that atg7 and atg2 phenotypes are more severe than those of atg11 and atg9. Unlike other mutants, atg2 cells accumulated several autophagic vesicles that could not be delivered to the vacuole. Analysis of atg double mutants, combined with wortmannin treatment, indicated that ATG11, PI3K, and ATG9 act upstream of ATG2. Our data support a model in which plant ATG1 and PI3K complexes play a role in the initiation of autophagy, whereas ATG2 is involved in a later step during the biogenesis of autophagic vesicles.

AUTOPHAGY-RELATED11 plays a critical role in general autophagy- and senescence-induced mitophagy in Arabidopsis.

Autophagy-mediated turnover removes damaged organelles and unwanted cytoplasmic constituents and thus plays critical roles in cellular housekeeping and nutrient recycling. This "self eating" is tightly regulated by the AUTOPHAGY-RELATED1/13 (ATG1/13) kinase complex, which connects metabolic and environmental cues to the vacuolar delivery of autophagic vesicles. Here, we describe the Arabidopsis thaliana accessory proteins ATG11 and ATG101, which help link the ATG1/13 complex to autophagic membranes. ATG11 promotes vesicle delivery to the vacuole but is not essential for synthesizing the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts that are central to autophagic vesicle assembly. ATG11, ATG101, ATG1, and ATG13 colocalize with each other and with ATG8, with ATG1 tethered to ATG8 via a canonical ATG8-interacting motif. Also, the presence of ATG11 encourages starvation-induced phosphorylation of ATG1 and turnover of ATG1 and ATG13. Like other atg mutants, ATG11-deficient plants senesce prematurely and are hypersensitive to nitrogen and fixed-carbon limitations. Additionally, we discovered that the senescence-induced breakdown of mitochondria-resident proteins and mitochondrial vesicles occurs via an autophagic process requiring ATG11 and other ATG components. Together, our data indicate that ATG11 (and possibly ATG101) provides important scaffolds connecting the ATG1/13 complex to both general autophagy and selective mitophagy.

Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.

Arabidopsis Qc-SNARE gene AtSFT12 is involved in salt and osmotic stress responses and Na(+) accumulation in vacuoles.

KEY MESSAGE: AtSFT12, an Arabidopsis Qc-SNARE protein, is localized to Golgi organelles and is involved in salt and osmotic stress responses via accumulation of Na (+) in vacuoles. To reduce the detrimental effects of environmental stresses, plants have evolved many defense mechanisms. Here, we identified an Arabidopsis Qc-SNARE gene, AtSFT12, involved in salt and osmotic stress responses using an activation-tagging method. Both activation-tagged plants and overexpressing transgenic plants (OXs) of the AtSFT12 gene were tolerant to high concentrations of NaCl, LiCl, and mannitol, whereas loss-of-function mutants were sensitive to NaCl, LiCl, and mannitol. AtSFT12 transcription increased under NaCl, ABA, cold, and mannitol stresses but not MV treatment. GFP-fusion AtSFT12 protein was juxtaposed with Golgi marker, implying that its function is associated with Golgi-mediated transport. Quantitative measurement of Na(+) using induced coupled plasma atomic emission spectroscopy revealed that AtSFT12 OXs accumulated significantly more Na(+) than WT plants. In addition, Na(+)-dependent fluorescence analysis of Sodium Green showed comparatively higher Na(+) accumulation in vacuoles of AtSFT12 OX cells than in those of WT plant cells after salt treatments. Taken together, our findings suggest that AtSTF12, a Golgi Qc-SNARE protein, plays an important role in salt and osmotic stress responses and functions in the salt stress response via sequestration of Na(+) in vacuoles.

The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis.

Autophagy is an intracellular recycling route in eukaryotes whereby organelles and cytoplasm are sequestered in vesicles, which are subsequently delivered to the vacuole for breakdown. The process is induced by various nutrient-responsive signaling cascades converging on the Autophagy-Related1 (ATG1)/ATG13 kinase complex. Here, we describe the ATG1/13 complex in Arabidopsis thaliana and show that it is both a regulator and a target of autophagy. Plants missing ATG13 are hypersensitive to nutrient limitations and senesce prematurely similar to mutants lacking other components of the ATG system. Synthesis of the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts, which are essential for autophagy, still occurs in ATG13-deficient plants, but the biogenesis of ATG8-decorated autophagic bodies does not, indicating that the complex regulates downstream events required for autophagosome enclosure and/or vacuolar delivery. Surprisingly, levels of the ATG1a and ATG13a phosphoproteins drop dramatically during nutrient starvation and rise again upon nutrient addition. This turnover is abrogated by inhibition of the ATG system, indicating that the ATG1/13 complex becomes a target of autophagy. Consistent with this mechanism, ATG1a is delivered to the vacuole with ATG8-decorated autophagic bodies. Given its responsiveness to nutrient demands, the turnover of the ATG1/13 kinase likely provides a dynamic mechanism to tightly connect autophagy to a plant's nutritional status.

Delivery of prolamins to the protein storage vacuole in maize aleurone cells.

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.

The phosphatidylinositol 3-phosphate effector FYVE3 regulates FYVE2-dependent autophagy in Arabidopsis thaliana.

Phosphatidylinositol 3-phosphate (PI3P) is a signaling phospholipid that play a key role in endomembrane trafficking, specifically autophagy and endosomal trafficking. However, the mechanisms underlying the contribution of PI3P downstream effectors to plant autophagy remain unknown. Known PI3P effectors for autophagy in Arabidopsis thaliana include ATG18A (Autophagy-related 18A) and FYVE2 (Fab1p, YOTB, Vac1p, and EEA1 2), which are implicated in autophagosome biogenesis. Here, we report that FYVE3, a paralog of plant-specific FYVE2, plays a role in FYVE2-dependent autophagy. Using yeast two-hybrid and bimolecular fluorescence complementation assays, we determined that the FYVE3 protein was associated with autophagic machinery containing ATG18A and FYVE2, by interacting with ATG8 isoforms. The FYVE3 protein was transported to the vacuole, and the vacuolar delivery of FYVE3 relies on PI3P biosynthesis and the canonical autophagic machinery. Whereas the fyve3 mutation alone barely affects autophagic flux, it suppresses defective autophagy in fyve2 mutants. Based on the molecular genetics and cell biological data, we propose that FYVE3 specifically regulates FYVE2-dependent autophagy.

ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci.

Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin-fold proteins Autophagy-related (ATG)-8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8-PE adduct, we also show that ATG8 lipidation requires the ATG12-ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12-ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12-ATG5 adduct is essential for ATG8-mediated autophagy in plants by promoting ATG8 lipidation.

Plant SMU-1 and SMU-2 homologues regulate pre-mRNA splicing and multiple aspects of development.

In eukaryotes, alternative splicing of pre-mRNAs contributes significantly to the proper expression of the genome. However, the functions of many auxiliary spliceosomal proteins are still unknown. Here, we functionally characterized plant homologues of nematode suppressors of mec-8 and unc-52 (smu). We compared transcript profiles of maize (Zea mays) smu2 endosperm with those of wild-type plants and identified pre-mRNA splicing events that depend on the maize SMU2 protein. Consistent with a conserved role of plant SMU-2 homologues, Arabidopsis (Arabidopsis thaliana) smu2 mutants also show altered splicing of similar target pre-mRNAs. The Atsmu2 mutants occasionally show developmental phenotypes, including abnormal cotyledon numbers and higher seed weights. We identified AtSMU1 as one of the SMU2-interacting proteins, and Atsmu1 mutations cause similar developmental phenotypes with higher penetrance than Atsmu2. The AtSMU2 and AtSMU1 proteins are localized to the nucleus and highly prevalent in actively dividing tissues. Taken together, our data indicated that the plant SMU-1 and SMU-2 homologues appear to be involved in splicing of specific pre-mRNAs that affect multiple aspects of development.

Subcellular Localization of PI3P in Arabidopsis.

Phosphatidylinositol-3-phosphate (PI3P) is a signaling phospholipid enriched in the membranes of late endosomes (LE) and vacuoles. PI3P mediates vacuolar and endosomal trafficking through recruiting PI3P-binding effector proteins to the LE. PI3P is produced from phosphatidylinositol by the PI 3-kinase complex containing VACUOLAR PROTEIN SORTING 34 (VPS34). The role of PI3P has been elucidated by using genetically encoded PI3P biosensors. We previously showed that Arabidopsis VPS38, a component of the VPS34 complex, localized at the LE and that VPS38 is essential for proper PI3P distribution in endosomal and vacuolar trafficking routes. In this chapter, we describe methods for microscopic imaging of PI3P using the PI3P biosensor citrine-2 × FYVE and the PI 3-kinase inhibitors.

How phosphoinositides shape autophagy in plant cells.

Plant cells use autophagy to degrade their own cytoplasm in vacuoles, thereby not only recycling their breakdown products, but also ensuring the homeostasis of essential cytoplasmic constituents and organelles. Plants and other eukaryotes have a conserved set of core Autophagy-related (ATG) genes involved in the biogenesis of the autophagosome, the main autophagic compartment destined for the lytic vacuole. In the past decade, the core ATG genes were isolated from several plant species. The core ATG proteins include the components of the VACUOLAR PROTEIN SORTING 34 (VPS34) complex that is responsible for the local production of phosphatidylinositol 3-phosphate (PI3P) at the site of autophagosome formation. Dissecting the roles of PI3P and its effectors in autophagy is challenging, because of the multi-faceted links between autophagosomal and endosomal systems. This review highlights recent studies on putative plant PI3P effectors involved in autophagosome dynamics. Molecular mechanisms underlying the requirement of PI3P for autophagosome biogenesis and trafficking are also discussed.