Recent studies have shown that microRNA (miRNA) functions are associated with plant responses to water deficiency. Mature miRNAs are loaded onto a complex that includes the ARGONAUTE 1 (AGO1) protein; this complex then cleaves mRNAs or inhibits their translation. Here, we demonstrate that the prion-like domain of AGO1 is responsible for topological changes in AGO1 under dehydration in Arabidopsis thaliana. AGO1 undergoes liquid-liquid phase separation (LLPS), which is driven by intrinsically disordered protein domains and plays diverse roles in cellular processes. LLPS of AGO in the cytoplasm influences miRNA regulatory activity, a process related to cytoplasmic calcium levels. We found that dehydration-induced AGO1 condensation influences AGO1 activity, while contributing to dehydration tolerance in plants. Upon rehydration, the condensation-driven accumulation of AGO1 is resolved, restoring its protein levels to normal. Overall, we propose that AGO1 phase separation acts as an emergent property in response to dehydration, attenuating the energy-consuming miRNA regulatory pathway in young seedlings.

In eukaryotic cells, excess or damaged cytoplasmic constituents are targeted into lytic compartments via autophagosomal membrane trafficking. Biogenesis of autophagosomes in fungi, metazoans, and plants relies on the conserved ATG (autophagy related) proteins. The machinery responsible for autophagosome turnover has been elucidated in yeast and metazoans, but not in plants. Here we examined 14 soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) in Arabidopsis thaliana by autophagy marker and genetic analyses. We identified SYP22 (Syntaxin of Plants 22) as a SNARE that is necessary for the efficient fusion of autophagosomes with the vacuole. Genetic disruption of SYP22 led to a reduction in autophagic flux and the accumulation of autophagosomes. The vacuolar Qa-SNARE SYP22 interacted with autophagosomal proteins, such as ATG8 and the R-SNARE VAMP724. Overall, our molecular and genetic analyses of Arabidopsis SNAREs underscore the importance of autophagosome-vacuole fusion in autophagic flux, and provide an insight into how plant vacuolar SNARE proteins recognize the autophagosome and mediate its fusion. As a unique mutant defective in the turnover of autophagosomes, syp22 will be useful for overcoming bottlenecks in plant autophagy research.Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy related; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; ConA: concanamycin A; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; HOPS: homotypic fusion and protein sorting; LE: late endosome; PM: plasma membrane; PVC: prevacuolar compartment; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; SYP: Syntaxin of Plants; TEM: transmission electron microscopy; TGN: trans-Golgi network; WT: wild type.

As secretory vesicle-residing soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 and 722 (VAMP721/722) are required for stress responses as well as growth and development in plants. We previously reported the post-translational downregulation of VAMP721/722 abundance by the plant abiotic stress hormone abscisic acid (ABA) in Arabidopsis. However, how ABA induces VAMP721/722 degradation in plants is yet unclear. Here, we show that the SLOW MOTION (SLOMO) F-box protein mediates the ABA-induced degradation of VAMP721/722 in Arabidopsis. We detected higher VAMP721/722 levels in slomo-1 missense and slomo-2 null mutants than wild-type (WT) plants. We also found that VAMP722 interacts with the Leu-rich repeat (LRR) domain of SLOMO, but not with the variant LRR domain in slomo-1 mutant, in both in vitro and in planta assays. The significantly elevated growth in slomo plants compared to WT under ABA and abiotic stresses implies that plants may manage abiotic stress responses by regulating VAMP721/722 levels at least in part via SLOMO.

Autophagy is a membrane trafficking pathway through which eukaryotic cells target their own cytoplasmic constituents for degradation in the lytic compartment. Proper biogenesis of autophagic organelles requires a conserved set of autophagy-related (ATG) proteins and their interacting factors, such as signalling phospholipid phosphatidylinositol 3-phosphate (PI3P) and coat complex II (COPII). The COPII machinery, which was originally identified as a membrane coat involved in the formation of vesicles budding from the endoplasmic reticulum, contributes to the initiation of autophagic membrane formation in yeast, metazoan, and plant cells; however, the exact mechanisms remain elusive. Recent studies using the plant model species Arabidopsis thaliana have revealed that plant-specific PI3P effectors are involved in autophagy. The PI3P effector FYVE2 interacts with the conserved PI3P effector ATG18 and with COPII components, indicating an additional role for the COPII machinery in the later stages of autophagosome biogenesis. In this Update, we examined recent research on plant autophagosome biogenesis and proposed working models on the functions of the COPII machinery in autophagy, including its potential roles in stabilizing membrane curvature and sealing the phagophore.

Water-deficit affects nearly every biological event in plants, and recent studies have shown that microRNA-functionality is associated with plant responses to water-deficiency. Liquid-liquid phase separation facilitates the condensation of biomolecules, which is driven by intrinsically disordered proteins and plays diverse roles in cellular processes. Here, we show that the prion-like domain (PrLD) of ARGONAUTE 1 is responsible for topological changes from liquid droplets to solid-condensations of AGO1 under dehydration. Unlike SERRATE, which forms functional RNP granules for miRNA biogenesis, AGO1 RNP granules are non-functional condensates, which is particularly facilitated by cytoplasmic calcium ions. We found that dehydration-induced AGO1 condensation inhibits RNA-induced Silencing Complex (RISC) activity. Following rewatering, the condensed AGO1 is degraded through three consecutive proteolytic processes, indicating that the liquid-to-solid phase transition of AGO1 is a reversible process. Overall, we propose that AGO1 phase transition may serve as a sensor for intense dehydration and attenuates the energy-consuming miRNA-regulatory pathway.

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.

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.

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.

Autophagy is a degradation pathway for cytoplasmic constituents, targeting various types of cargo to the vacuoles for recycling. Biogenesis and turnover of autophagic vesicles require a set of Autophagy-related (Atg) proteins, which are present in yeast, metazoans, and plants. Recent advances in autophagy research using yeast and mammalian cells have yielded better models describing how autophagic vesicles acquire membrane lipids and which molecules are involved in final steps in autophagy. These findings will further the understanding of how plant Atg homologs cooperate with other proteins to mediate autophagosome biogenesis and turnover. This mini-review provides an updated view of the molecular mechanisms underlying autophagosome dynamics in plant cells. Evidence supporting roles of actin filaments and microtubules in plant autophagosome biogenesis is also provided.

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 PI3Pbinding 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 x FYVE and the PI 3-kinase inhibitors.

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.

Eukaryotic cells use conserved quality control mechanisms to repair or degrade defective proteins, which are synthesized at a high rate during proteotoxic stress. Quality control mechanisms include molecular chaperones, the ubiquitin-proteasome system, and autophagic machinery. Recent research reveals that during autophagy, membrane-bound organelles are selectively sequestered and degraded. Selective autophagy is also critical for the clearance of excess or damaged protein complexes (e.g., proteasomes and ribosomes) and membrane-less compartments (e.g., protein aggregates and ribonucleoprotein granules). As sessile organisms, plants rely on quality control mechanisms for their adaptation to fluctuating environments. In this mini-review, we highlight recent work elucidating the roles of selective autophagy in the quality control of proteins and RNA in plant cells. Emphasis will be placed on selective degradation of membrane-less compartments and protein complexes in the cytoplasm. We also propose possible mechanisms by which defective proteins are selectively recognized by autophagic machinery.

Arabidopsis cargo receptor NBR1 contributes to protein quality control by promoting the formation of protein aggregates and mediating their clearance via selective autophagy.

Phosphatidylinositol 3-P (PI3P) is a signaling molecule that controls a variety of processes in endosomal, autophagic

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.

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.

Arabidopsis VPS38 is required for the intracellular localization of PI3P, an important lipid regulator of endosomal and vacuolar trafficking. 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.

In plant cells, autophagy is required for efficient recycling of cytoplasmic macromolecules in vacuoles. It was previously shown that autophagy-deficient mutants also exhibited hypersensitivity to various abiotic stresses, such as salt, osmotic changes, heat, drought, and oxidative damage. However, it has not been clearly determined whether autophagy is induced or inhibited by these environmental stressors. Using the GFP-ATG8 (green fluorescent protein fused to AUTOPHAGY-RELATED PROTEIN 8) processing assay and confocal microscopy, we assessed autophagic flux of Arabidopsis seedlings exposed to salt stress. Treatment with 150 mM NaCl resulted in an increase in the processing of GFP-ATG8. Notably, the effects of concanamycin A, an inhibitor of vacuolar proton pumps, on GFP-ATG8 processing indicated that the apparent increase in GFP-ATG8 processing by salt-induced stress was due to inefficient vacuolar degradation of the GFP moiety processed from GFP-ATG8. Salt and osmotic stresses did not increase the abundance of autophagic vesicles in the root cells. Although NaCl, KCl, and mannitol did not greatly inhibit the vacuolar trafficking of GFP-ATG8, LiCl partially inhibited autophagy. These data indicated that NaCl stress neither increases nor substantially inhibits autophagic flux. Our work illustrates the importance of autophagic flux analysis to assess the effect of abiotic stresses on plant 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.