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Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Isolation of various subcellular compartments followed by a high-coverage proteomic analysis provides an unparalleled foundation for the functional analyses of proteins. Analyses of tonoplast preparations free of major contaminants provide insights into vesicular fusion machinery, solute transport, and the vacuole association with the cytoskeleton, whereas analyses of the vacuolar lumen have yielded numerous soluble glycosidases, proteases, and proteins involved in stress responses. In addition, vacuolar lumen preparations have also allowed characterization of a luminal solute content in response to various abiotic stresses. Here, I revisit and update one of the most successful methodologies for vacuole and tonoplast isolation.

Coated vesicles provide a major mechanism for the transport of proteins through the endomembrane system of plants. Transport between the endoplasmic reticulum and the Golgi involves vesicles with COPI and COPII coats, whereas clathrin is the predominant coat in endocytosis and post-Golgi trafficking. Sorting of cargo, coat assembly, budding, and fission are all complex and tightly regulated processes that involve many proteins. The mechanisms and responsible factors are largely conserved in eukaryotes, and increasing organismal complexity tends to be associated with a greater numbers of individual family members. Among the key factors is the class of ENTH/ANTH/VHS domain-containing proteins, which link membrane subdomains, clathrin, and other adapter proteins involved in early steps of clathrin coated vesicle formation. More than 30 Arabidopsis thaliana proteins contain this domain, but their generally low sequence conservation has made functional classification difficult. Reports from the last two years have greatly expanded our knowledge of these proteins and suggest that ENTH/ANTH/VHS domain proteins are involved in various instances of clathrin-related endomembrane trafficking in plants. This review aims to summarize these new findings and discuss the broader context of clathrin-dependent plant vesicular transport., (© 2014 American Society of Plant Biologists. All rights reserved.)

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis βFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

Background: The onset of differentiation entails modifying the gene expression state of cells, to allow activation of developmental programs that are maintained repressed in the undifferentiated precursor cells [1, 2]. This requires a mechanism to change gene expression on a genome-scale. Recent evidence suggests that in mammalian stem cells, derepression of developmental regulators during differentiation involves a shift from stalled to productive elongation of their transcripts [3-5], but factors mediating this shift have not been identified and the evidence remains correlative., Results: We report the identification of the MINIYO (IYO) gene, a positive regulator of transcriptional elongation that is essential for cells to initiate differentiation in Arabidopsis. IYO interacts with RNA polymerase II and the Elongator complex and is required to sustain global levels of transcriptional elongation activity, specifically in differentiating tissues. Accordingly, IYO is expressed in embryos, meristems, and organ primordia and not in mature tissues. Moreover, differential subcellular protein distribution further refines the domain of IYO function by directing nuclear accumulation, and thus its transcriptional activity, to cells initiating differentiation. Importantly, IYO overexpression induces premature cell differentiation and leads to meristem termination phenotypes., Conclusions: These findings identify IYO as a necessary and sufficient factor for initiating differentiation in Arabidopsis and suggest that the targeted nuclear accumulation of IYO functions as a transcriptional switch for this fate transition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Sortin1 is a chemical genetic-hit molecule that causes specific mislocalization of plant and yeast-soluble and membrane vacuolar markers. To better understand its mode of action, we designed a Sortin1-hypersensitive screen and identified several Sortin1-hypersensitive and flavonoid-defective mutants. Mechanistically, Sortin1 mimics the effect of the glutathione inhibitor buthionine sulfoximine and alters the vacuolar accumulation of flavonoids, likely blocking their transport through vacuole-localized ABC transporters. Structure-activity relationship studies conducted in Arabidopsis revealed the structural requirements for Sortin1 bioactivity and demonstrated that overlapping Sortin1 substructures can be used to discriminate between vacuolar-flavonoid accumulations and vacuolar-biogenesis defects. We conclude that Sortin1 is a valuable probe for dissecting novel links among flavonoid transport, vacuolar integrity, and the trafficking of vacuolar targeted cargoes in Arabidopsis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Two different gene families have been proposed to act as sorting receptors for vacuolar storage cargo in plants: the vacuolar sorting receptors (VSRs) and the receptor homology-transmembrane-RING H2 domain proteins (RMRs). However, functional data on these genes is scarce and the identity of the sorting receptor for storage proteins remains controversial. Through a genetic screen we have identified the mtv2 mutant, which is defective in vacuolar transport of the storage cargo VAC2 in shoot apices. Map-based cloning revealed that mtv2 is a loss of function allele of the VSR4 gene. We show that VSR1, VSR3 and VSR4, but not the remaining VSRs or RMRs, participate in vacuolar sorting of VAC2 in vegetative tissues, and 12S globulins and 2S albumins in seeds, an activity that is essential for seedling germination vigor. Finally, we demonstrate that the functional diversification in the VSR family results from divergent expression patterns and also from distinct sorting activities of the family members., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

We report a functional characterization of AtVPS45 (for vacuolar protein sorting 45), a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex. A null allele of AtVPS45 was male gametophytic lethal, whereas stable RNA interference lines with reduced AtVPS45 protein levels had stunted growth but were viable and fertile. In the silenced lines, we observed defects in vacuole formation that correlated with a reduction in cell expansion and with autophagy-related defects in nutrient turnover. Moreover, transport of vacuolar cargo with carboxy-terminal vacuolar sorting determinants was blocked in the silenced lines, suggesting that AtVPS45 functions in vesicle trafficking to the vacuole. These trafficking defects are similar to those observed in vti12 mutants, supporting a functional relationship between AtVPS45 and VTI12. Consistent with this, we found a decrease in SYP41 protein levels coupled to the silencing of AtVPS45, pointing to instability and malfunction of the SYP41/SYP61/VTI12 SNARE complex in the absence of its cognate Sec1/Munc18 regulator. Based on its localization on the TGN, we hypothesized that AtVPS45 could be involved in membrane fusion of retrograde vesicles recycling vacuolar trafficking machinery. Indeed, in the AtVPS45-silenced plants, we found a striking alteration in the subcellular fractionation pattern of vacuolar sorting receptors, which are required for sorting of carboxy-terminal vacuolar sorting determinant-containing cargo. We propose that AtVPS45 is essential for recycling of the vacuolar sorting receptors back to the TGN and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.

Vacuoles are very prominent compartments within plant cells, and understanding of their function relies on knowledge of their content. Here, we present a simple vacuole purification protocol that was successfully used for large-scale isolation of vacuoles, free of significant contamination from other endomembrane compartments. This method is based on osmotic and thermal disruption of mesophyl-derived Arabidopsis protoplasts, followed by a density gradient fractionation of the cellular content. The whole procedure, including protoplast isolation, takes approximately 6 h.

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.

Background: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants., Results: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens., Conclusions: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

Chemical genomics is an interdisciplinary approach that unites the power of chemical screens and genomics strategies to dissect biological processes such as endomembrane trafficking. We have taken advantage of the evolutionary conservation between plants and Saccharomyces cerevisiae to identify such chemicals. Using S. cerevisiae, we screened a library of diverse chemical structures for compounds that induce the secretion of carboxypeptidase Y, which is normally targeted to the vacuole. Among 4,800 chemicals screened, 14 compounds, termed sorting inhibitors (Sortins), were identified that stimulated secretion in yeast. In Arabidopsis seedlings, application of Sortin1 and -2 led to reversible defects in vacuole biogenesis and root development. Sortin1 was found to redirect the vacuolar destination of plant carboxypeptidase Y and other proteins in Arabidopsis suspension cells and cause these proteins to be secreted. Sortin1 treatment of whole Arabidopsis seedlings also resulted in carboxypeptidase Y secretion, indicating that the drug has a similar mode of action in cells and intact plants. We have demonstrated that screening of a simple eukaryote, in which vacuolar biogenesis is not essential, can be a powerful tool to find chemicals that interfere with vacuolar delivery of proteins in plants, where vacuole biogenesis is essential. Our studies were done by using a sublethal dose of Sortin1, demonstrating the powerful ability of the chemical to control the induced phenotype in a manner that would be difficult to achieve using conventional genetics.

Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report in vivo evidence that the precursor protease vesicle-localized vacuolar processing enzyme-gamma (VPEgamma) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPEgamma is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpegamma mutants but not in WT plants, suggesting a unique role of VPEgamma in protein processing and degradation in Arabidopsis.

Plant cells contain several types of vacuoles with specialized functions. Although the biogenesis of these organelles is well understood at the morphological level, the machinery involved in plant vacuole formation is largely unknown. We have recently identified an Arabidopsis mutant, vcl1, that is deficient in vacuolar formation. VCL1 is homologous to a protein that regulates membrane fusion at the tonoplast in yeast. On the basis of these observations, VCL1 is predicted to play a direct role in vacuolar biogenesis and vesicular trafficking to the vacuole in plants. In this work, we show that VCL1 forms a complex with AtVPS11 and AtVPS33 in vivo. These two proteins are homologues of proteins that have a well-characterized role in membrane fusion at the tonoplast in yeast. VCL1, AtVPS11, and AtVPS33 are membrane-associated and cofractionate with tonoplast and denser endomembrane markers in subcellular fractionation experiments. Consistent with this, VCL1, AtVPS11, and AtVPS33 are found on the tonoplast and the prevacuolar compartment (PVC) by immunoelectron microscopy. We also show that a VCL1-containing complex includes SYP2-type syntaxins and is most likely involved in membrane fusion on both the PVC and tonoplast in vivo. VCL1, AtVPS11, and AtVPS33 are the first components of the vacuolar biogenesis machinery to be identified in plants.