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Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. Plants’ responses/adaptations to stress often involve changes in the distribution and sorting of specific proteins and molecules. Still, little attention has been given to the molecular mechanisms controlling these rearrangements. We tested the hypothesis that plants respond to stress by remodelling their endomembranes and adapting their trafficking pathways. We focused on the molecular machinery behind organelle biogenesis and protein trafficking under abiotic stress conditions, evaluating their effects at the subcellular level, by looking at ultrastructural changes and measuring the expression levels of genes involved in well-known intracellular routes. The results point to a differential response of the endomembrane system, showing that the genes involved in the pathway to the Protein Storage Vacuole and the exocyst-mediated routes are upregulated. In contrast, the ones involved in the route to the Lytic Vacuole are downregulated. These changes are accompanied by morphological alterations of endomembrane compartments. The data obtained demonstrate that plants’ response to abiotic stress involves the differential expression of genes related to protein trafficking machinery, which can be connected to the activation/deactivation of specific intracellular sorting pathways and lead to alterations in the cell ultrastructure.

International audience; Throughout their life cycle, plants face a tremendous number of environmental and developmental stresses. To respond to these different constraints, they have developed a set of refined intracellular systems including autophagy. This pathway, highly conserved among eukaryotes, is induced by a wide range of biotic and abiotic stresses upon which it mediates the degradation and recycling of cytoplasmic material. Central to autophagy is the formation of highly specialized double membrane vesicles called autophagosomes which select, engulf, and traffic cargo to the lytic vacuole for degradation. The biogenesis of these structures requires a series of membrane remodeling events during which both the quantity and quality of lipids are critical to sustain autophagy activity. This review highlights our knowledge, and raises current questions, regarding the mechanism of autophagy, and its induction and regulation upon environmental stresses with a particular focus on the fundamental contribution of lipids. How autophagy regulates metabolism and the recycling of resources, including lipids, to promote plant acclimation and resistance to stresses is further discussed.

Plant cells contain two types of vacuoles, the lytic vacuole (LV) and protein storage vacuole (PSV). LVs are present in vegetative cells, whereas PSVs are found in seed cells. The physiological functions of the two types of vacuole differ. Newly synthesized proteins must be transported to these vacuoles via protein trafficking through the endomembrane system for them to function. Recently, significant advances have been made in elucidating the molecular mechanisms of protein trafficking to these organelles. Despite these advances, the relationship between the trafficking mechanisms to the LV and PSV remains unclear. Some aspects of the trafficking mechanisms are common to both types of vacuole, but certain aspects are specific to trafficking to either the LV or PSV. In this review, we summarize recent findings on the components involved in protein trafficking to both the LV and PSV and compare them to examine the extent of overlap in the trafficking mechanisms. In addition, we discuss the interconnection between the LV and PSV provided by the protein trafficking machinery and the implications for the identity of these organelles.

Autophagy is the main catabolic process in eukaryotes and plays a key role in cell homeostasis. In vivo measurement of autophagic activity (flux) is a powerful tool for investigating the role of the pathway in organism development and stress responses. Here we describe a significant optimization of the tandem tag assay for detection of autophagic flux in planta in epidermal root cells of Arabidopsis thaliana seedlings. The tandem tag consists of TagRFP and mWasabi fluorescent proteins fused to ATG8a, and is expressed in wildtype or autophagy-deficient backgrounds to obtain reporter and control lines, respectively. Upon autophagy activation, the TagRFP-mWasabi-ATG8a fusion protein is incorporated into autophagosomes and delivered to the lytic vacuole. Ratiometric quantification of the low pH-tolerant TagRFP and low pH-sensitive mWasabi fluorescence in the vacuoles of control and reporter lines allows for a reliable estimation of autophagic activity. We provide a step by step protocol for plant growth, imaging and semi-automated data analysis. The protocol presents a rapid and robust method that can be applied for any studies requiring in planta quantification of autophagic flux.

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins of these transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV) during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana) embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EV in embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmental stages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate that the preexisting EV is reprogrammed to become a PSV in Arabidopsis.

Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophagy machinery composed of multiple Atg proteins, as well as the divergent sequence family of selective autophagy receptors. Single-particle electron microscopy (EM) is a molecular imaging approach that has become an increasingly important tool in the structural characterization of proteins and macromolecular complexes. This article summarizes the contributions single-particle EM have made in advancing our understanding of the core autophagy machinery and selective autophagy receptors. We also discuss current technical challenges and roadblocks, as well as look into the future of single-particle EM in autophagy research.

Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the prevacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.

Highlight PIN2 shows distinct trafficking and vacuolar turnover in neighbouring tricho- and atrichoblast cell files. Differential abundance of PIN2 in the root epidermis could have developmental importance for root gravitropism., The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses.

BEACH (beige and Chediak Higashi) domain-containing proteins (BDCPs) are facilitators of membrane-dependent cellular processes in eukaryotes. Mutations in BDCPs cause malfunctions of endosomal compartments in various cell types. Recently, the molecular analysis of the BDCP homolog gene SPIRRIG (SPI) has revealed a molecular function in P-bodies and the regulation of RNA stability. We therefore aimed to analyze, whether SPI has also a role in membrane-dependent processes. In this study, we show that SPI physically interacts with ESCRT (Endosomal Sorting Complex Required for Transport) associated ATPase SKD1 (Suppressor of K+-transport growth defect1) and its positive regulator, LIP5 (LYST Interacting Protein 5) and report genetic interactions between SPI and SKD1 and LIP5. We further show that the endosomal transport route of soluble proteins to the lytic vacuole is disturbed in spi lip5 double mutants but not in the single mutants. These vacuolar transport defects were suppressed by additional expression of SKD1. Our results indicate that the BEACH domain protein SPI has in addition to a role in P-bodies a function in endosomal transport routes.

Vacuolar proteins are synthesized and translocated into the endoplasmic reticulum and transported to the vacuoles through the secretory pathway. Three different types of vacuolar sorting signals have been identified, carried by N- or C-terminal propeptides or internal sequences. These signals are needed to target proteins to the different types of vacuoles that can coexist in a single plant cell. A conserved motif (NPIXL or NPIR) was identified within N-terminal propeptides, but can also function in a C-terminal propeptide and targets proteins in a receptor-mediated manner to a lytic vacuole. Binding to a family of putative sorting receptors for sequence-specific vacuolar sorting signals has been used as an assay to identify further peptides with other binding motifs. No motif was found in C-terminal sorting sequences, which need an accessible terminus, suggesting that they are recognized from the end by a still unknown receptor. The phosphatidylinositol kinase inhibitor wortmannin differentially affects sorting mediated by these two sorting sequences, suggesting different sorting mechanisms. Less is known about sorting mediated by internal protein sequences, which do not contain the conserved motif identified in N-terminal propeptides and may function by aggregation, leading to transport by coat-less dense vesicles to protein storage vacuoles. Even less is known about the sorting of tonoplast proteins, for which several sorting systems will also be needed.

Trafficking of soluble cargo to the vacuole is far from being a closed issue as it can occur by different routes and involve different intermediates. The textbook view of proteins being sorted at the post-Golgi level to the lytic vacuole via the pre-vacuole or to the protein storage vacuole mediated by dense vesicles is now challenged as novel routes are being disclosed and vacuoles with intermediate characteristics described. The identification of Vacuolar Sorting Determinants is a key signature to understand protein trafficking to the vacuole. Despite the long established vacuolar signals, some others have been described in the last few years, with different properties that can be specific for some cells or some types of vacuoles. There are also reports of proteins having two different vacuolar signals and their significance is questionable: a way to increase the efficiency of the sorting or different sorting depending on the protein roles in a specific context? Along came the idea of differential vacuolar sorting, suggesting a possible specialization of the trafficking pathways according to the type of cell and specific needs. In this review, we show the recent advances in the field and focus on different aspects of protein trafficking to the vacuoles.

Asymmetric localization of PIN proteins controls directionality of auxin transport and many aspects of plant development. Directionality of PIN1 within the marginal epidermis and the presumptive veins of developing leaf primordia is crucial for establishing leaf vein pattern. One mechanism that controls PIN protein distribution within the cell membranes is endocytosis and subsequent transport to the vacuole for degradation. The Arabidopsis mutant unhinged-1 (unh-1) has simpler leaf venation with distal non-meeting of the secondary veins and fewer higher order veins, a narrower leaf with prominent serrations, and reduced root and shoot growth. We identify UNH as the Arabidopsis vacuolar protein sorting 51 (VPS51) homolog, a member of the Arabidopsis Golgi-associated retrograde protein (GARP) complex, and show that UNH interacts with VPS52, another member of the complex and colocalizes with trans Golgi network and pre-vacuolar complex markers. The GARP complex in yeast and metazoans retrieves vacuolar sorting receptors to the trans-Golgi network and is important in sorting proteins for lysosomal degradation. We show that vacuolar targeting is reduced in unh-1. In the epidermal cells of unh-1 leaf margins, PIN1 expression is expanded. The unh-1 leaf phenotype is partially suppressed by pin1 and cuc2-3 mutations, supporting the idea that the phenotype results from expanded PIN1 expression in the marginal epidermis. Our results suggest that UNH is important for reducing expression of PIN1 within margin cells, possibly by targeting PIN1 to the lytic vacuole.

Vacuoles, cellular membrane-bound organelles, are the largest compartments of cells, occupying up to 90% of the volume of plant cells. Vacuoles are formed by the biosynthetic and endocytotic pathways. In plants, the vacuole is crucial for growth and development and has a variety of functions, including storage and transport, intracellular environmental stability, and response to injury. Depending on the cell type and growth conditions, the size of vacuoles is highly dynamic. Different types of cell vacuoles store different substances, such as alkaloids, protein enzymes, inorganic salts, sugars, etc., and play important roles in multiple signaling pathways. Here, we summarize vacuole formation, types, vacuole-located proteins, and functions.

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 (pat3) mutant that we identified by an epifluorescence-based forward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFP. While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compartments (PVC) with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A (VPS35A), a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lytic vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H+-pyrophosphatase and the vacuolar H+-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)–Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.

Vacuolar invertases (VIs) are highly expressed in young tissues and organs. They may have a substantial regulatory influence on whole plant metabolism as well as on photosynthetic efficiency. Therefore, they are emerging as potentially interesting biotechnological targets to increase plant biomass production, especially under stress. On the one hand, VIs are well-known as soluble and extractable proteins. On the other hand, they contain complex N-terminal propeptide (NTPP) regions with a basic region (BR) and a transmembrane domain (TMD). Here we analysed in depth the Arabidopsis thaliana VI2 (AtVI2) NTPP by mutagenesis. It was found that correct sorting to the lytic vacuole (LV) depends on the presence of intact dileucine (SSDALLPIS), BR (RRRR) and TMD motifs. AtVI2 remains inserted into membranes on its way to the LV, and the classical sorting pathway (Endoplasmic Reticulum Golgi LV) is followed. However, our data suggest that VIs might follow an alternative, adaptor protein 3 (AP3)-dependent route as well. Membrane-anchored transport and a direct recognition of the dileucine motif in the NTPP of VIs might have evolved as a simple and more efficient sorting mechanism as compared to the vacuolar sorting receptor 1/binding protein of 80 kDa (VSR1/BP80) dependent sorting mechanism followed by those proteins that travel to the vacuole as soluble proteins. ispartof: Plant & Cell Physiology vol:54 issue:8 pages:1263-1277 ispartof: location:Japan status: published

Plant vacuoles are unique, multifunctional organelles among eukaryotes. Considerable new insights in plant vacuolar protein sorting have been obtained recently. The basic machinery of protein export from the endoplasmic reticulum to the Golgi and the classical route to the lytic vacuole and the protein storage vacuole shows many similarities to vacuolar/lysosomal sorting in other eukaryotes. However, as a result of its unique functions in plant defence and as a storage compartment, some plant-specific entities and sorting determinants appear to exist. The alternative post-Golgi route, as found in animals and yeast, probably exists in plants as well. Likely, adaptor protein complex 3 fulfils a central role in this route. A Golgi-independent route involving plant-specific endoplasmic reticulum bodies appears to provide sedentary organisms such as plants with extra flexibility to cope with changing environmental conditions.

In Arabidopsis thaliana, different types of vacuolar receptors were discovered. The AtVSR (Vacuolar Sorting Receptor) receptors are well known to be involved in the traffic to lytic vacuole (LV), while few evidences demonstrate the involvement of the receptors from AtRMR family (Receptor Membrane RING-H2) in the traffic to the protein storage vacuole (PSV). In this study we focused on the localization of two members of AtRMR family, AtRMR1 and -2, and on the possible interaction between these two receptors in the plant secretory pathway. Our experiments with agroinfiltrated Nicotiana benthamiana leaves demonstrated that AtRMR1 was localized in the endoplasmic reticulum (ER), while AtRMR2 was targeted to the trans-Golgi network (TGN) due to the presence of a cytosolic 23-amino acid sequence linker. The fusion of this linker to an equivalent position in AtRMR1 targeted this receptor to the TGN, instead of the ER. By using a Bimolecular Fluorescent Complementation (BiFC) technique and experiments of co-localization, we demonstrated that AtRMR2 can make homodimers, and can also interact with AtRMR1 forming heterodimers that locate to the TGN. Such interaction studies strongly suggest that the transmembrane domain and the few amino acids surrounding it, including the sequence linker, are essential for dimerization. These results suggest a new model of AtRMR trafficking and dimerization in the plant secretory pathway.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H+-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H+-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

Two-pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca²⁺ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C-terminus was not essential for targeting but for channel function, while further C-terminal truncations of two or more transmembrane domains impaired protein trafficking. An N-terminal dileucine motif (EDPLI) proved to be critical for vacuolar targeting of TPC1, which was independent of the adaptor protein AP-3. Deletion or mutation of this sorting motif, which is conserved among TPCs caused redirection of the protein transport to the plasma membrane. An N-terminal region with a predicted α-helical structure was shown to support efficient vacuolar trafficking and was essential for TPC1 function. Similar to their localization in mammalian endosomes and lysosomes, MmTPC1 and MmTPC2 were targeted to small organelles and the membrane of the lytic vacuole, respectively, when expressed in plant cells. These results shed new light on the largely uncharacterized sorting signals of plant tonoplast proteins and reveal similarities between the targeting machinery of plants and mammals.

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination-independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine-rich repeat receptor-like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans-Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY-2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination-independent manner. Such MVB/PVC-mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination-independent and PVC-mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.

Potassium (K+) is a major nutrient for plant growth and development. Vacuolar K+ ion channels of the two-pore K+ (TPK) family play an important role in maintaining K+ homeostasis. Several TPK channels were previously shown to be expressed in the lytic vacuole (LV) tonoplast. Plants also contain smaller protein storage vacuoles (PSVs) that contain membrane transporters. However, the mechanisms that define how membrane proteins reach different vacuolar destinations are largely unknown. The Oryza sativa genome encodes two TPK isoforms (TPKa and TPKb) that have very similar sequences and are ubiquitously expressed. The electrophysiological properties of both TPKs were comparable, showing inward rectification and voltage independence. In spite of high levels of similarity in sequence and transport properties, the cellular localization of TPKa and TPKb channels was different, with TPKa localization predominantly at the large LV and TPKb primarily in smaller PSV-type compartments. Trafficking of TPKa was sensitive to brefeldin A, while that of TPKb was not. The use of TPKa:TPKb chimeras showed that C-terminal domains are crucial for the differential targeting of TPKa and TPKb. Site-directed mutagenesis of C-terminal residues that were different between TPKa and TPKb identified three amino acids that are important in determining ultimate vacuolar destination.

Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker–based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative β-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 β adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 β in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route.

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor-ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer-mediated recycling of the plant VSR BP80 starts at the trans-Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co-expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII-mediated transport route. Retention of soluble cargo despite ongoing COPII-mediated bulk flow can only be explained by an interaction with membrane-bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER-anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR-ligand interaction. It also implies that the retromer-mediated recycling route for the VSRs leads from the TGN back to the ER.

Protein disulfide isomerase (PDI) oxidizes, reduces, and isomerizes disulfide bonds, modulates redox responses, and chaperones proteins. The Arabidopsis thaliana genome contains 12 PDI genes, but little is known about their subcellular locations and functions. We demonstrate that PDI5 is expressed in endothelial cells about to undergo programmed cell death (PCD) in developing seeds. PDI5 interacts with three different Cys proteases in yeast two-hybrid screens. One of these traffics together with PDI5 from the endoplasmic reticulum through the Golgi to vacuoles, and its recombinant form is functionally inhibited by recombinant PDI5 in vitro. Peak PDI5 expression in endothelial cells precedes PCD, whereas decreasing PDI5 levels coincide with the onset of PCD-related cellular changes, such as enlargement and subsequent collapse of protein storage vacuoles, lytic vacuole shrinkage and degradation, and nuclear condensation and fragmentation. Loss of PDI5 function leads to premature initiation of PCD during embryogenesis and to fewer, often nonviable, seeds. We propose that PDI5 is required for proper seed development and regulates the timing of PCD by chaperoning and inhibiting Cys proteases during their trafficking to vacuoles before PCD of the endothelial cells. During this transitional phase of endothelial cell development, the protein storage vacuoles become the de facto lytic vacuoles that mediate PCD.

The gravitropism defective 2 (grv2) mutants of Arabidopsis thaliana were previously characterized as exhibiting shoot agravitropism resulting from mutations in a homolog of the Caenorhabditis elegans RECEPTOR-MEDIATED ENDOCYTOSIS-8 (RME-8) gene, which is required in C. elegans for endocytosis. A fluorescent protein fusion to the GRV2 protein localized to endosomes in transgenic plants, and vacuolar morphology was altered in grv2 mutants. A defect in vacuolar membrane dynamics provides a mechanistic explanation for the gravitropic defect, and may also account for the presence of an enlarged vacuole in early embryos, together with a nutrient requirement during seedling establishment. The GRV2-positive endosomes were sensitive to Wortmannin but not brefeldin A (BFA), consistent with GRV2 operating late in the endocytic pathway, prior to delivery of vesicles to the central vacuole. The specific enlargement of GRV2:YFP structures by Wortmannin, together with biochemical data showing that GRV2 co-fractionates with pre-vacuolar markers such as PEP12/SYP21, leads us to conclude that in plants GRV2/RME-8 functions in vesicle trafficking from the multivesicular body/pre-vacuolar compartment to the lytic vacuole.

Summary Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged β-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.

Summary: The ultra-structural changes and behavior of vacuoles and related organelles(rER and Golgi bodies)during pollen development and maturation of Arabidopsis thaliana, and pollen tube elongation of Tradescantia reflexa will be described. In microspores just before generative cell formation, a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic typed vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances(MBFs)appeared.Because of the expression of the pB vacuolar processing enzyme gene in these mature pollen grains, the MBFs were considered as storage vacuoles. In pollen grains from flowers in bloom, MBFschanged to lysosomal structures with acid phosphatases(lytic vacuole). They gradually increased in number andvolume, and decomposed the cytoplasm. A large vacuole at the base of elongating pollen tube may develope by membrane supply controlled with the ER and lipid granules, in addition to the absorption of water.

Quality control in the endoplasmic reticulum (ER) prevents the arrival of incorrectly or incompletely folded proteins at their final destinations and targets permanently misfolded proteins for degradation. Such proteins have a high affinity for the ER chaperone BiP and are finally degraded via retrograde translocation from the ER lumen back to the cytosol. This ER-associated protein degradation (ERAD) is currently thought to constitute the main disposal route, but there is growing evidence for a vacuolar role in quality control. We show that BiP is transported to the vacuole in a wortmannin-sensitive manner in tobacco (Nicotiana tabacum) and that it could play an active role in this second disposal route. ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus, where it competes with other HDEL receptor ligands. When HDEL-mediated retrieval from the Golgi fails, BiP is transported to the lytic vacuole via multivesicular bodies, which represent the plant prevacuolar compartment. We also demonstrate that a subset of BiP-ligand complexes is destined to the vacuole and differs from those likely to be disposed of via the ERAD pathway. Vacuolar disposal could act in addition to ERAD to maximize the efficiency of quality control in the secretory pathway.

Developing pea seeds contain two functionally distinct vacuoles – lytic vacuoles and protein storage vacuoles (PSV). The Golgi apparatus of these cells has to discriminate between proteins destined for these vacuolar compartments. Whereas it is known that sorting into the lytic vacuole is performed via the conserved clathrin-coated vesicle pathway, sorting of proteins into the protein storage vacuole remains enigmatic. In developing pea cotyledons, the major storage proteins are sorted via ‘dense vesicles’. In this report we examined the sorting of a minor protein of the protein storage vacuole, the sucrose-binding-protein homolog (SBP), along the secretory pathway employing immunoelectron microscopy on cryosectioned pea cotyledons. SBP follows the same vesicular route into the PSV as the main storage proteins legumin and vicilin, via the dense-vesicles. Furthermore, legumin and SBP are sorted together into the same dense vesicle population at the stack. Although soluble cargo proteins of the dense vesicles, they show a stratified distribution in the lumen of the dense vesicles. Whereas the legumin label is equally distributed across the lumen, the SBP label is concentrated at the membrane of the vesicle. This observation is discussed with respect to a putative receptor-mediated sorting of the proteins into the dense vesicles.

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.

Expression of the Aspergillus nigerbeta-glucosidase gene, BGL1, in Nicotiana tabacum plants (cv. Xanthi) had a profound effect on the volatile emissions of intact and crushed leaves. BGL1 was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and targeted to the cytoplasm, cell wall, lytic vacuole (LV), chloroplast or endoplasmic reticulum (ER). Subcellular localization was confirmed by gold immunolabelling, followed by transmission electron microscopy (TEM). Significant beta-glucosidase activity was observed in transgenic plants expressing BGL1 in the cell wall, LV and ER. Compared with controls, all intact transgenic leaves were found to emit increased levels of 2-ethylhexanol, as determined by gas chromatography-mass spectrometry (GC-MS) analysis of the headspace volatiles. Plants expressing BGL1 in the cell wall (Tcw) emitted more trans-caryophyllene than did non-transgenic controls, whereas plants expressing BGL1 in the ER (Ter) and LV (Tvc) emitted more cembrene than did non-transgenic controls. Volatiles released from crushed transgenic leaves and collected with solid-phase microextraction (SPME) polydimethylsiloxane fibre were distinctly enhanced. Significant increases in linalool, nerol, furanoid cis-linalool oxide, 4-methyl-1-pentanol, 6-methyl-hept-5-en-2-ol and 2-ethylhexanol were detected in transgenic plants when compared with wild-type controls. 3-Hydroxyl-beta-ionone levels were increased in crushed Tcw and Ter leaves, but were undetectable in Tvc leaves. The addition of glucoimidazole, a beta-glucosidase inhibitor, abolished the increased emission of these volatiles. These results indicate that the expression of a fungal beta-glucosidase gene in different subcellular compartments has the potential to affect the emission of plant volatiles, and thereby to modify plant-environment communication and aroma of agricultural products.

The Society for Experimental Biology symposium on Membrane Trafficking in Plants took place in Glasgow between 23 and 26 August 2003 and was organized by M. Blatt and G. Thiel. ![][1] In August 2003, the plant endomembrane community gathered under the auspices of the Society for Experimental Biology to consider the latest developments in the rapidly growing field of the plant secretory pathway. The meeting was preceded by the two‐day annual gathering of the European Plant Secretory Pathway Group, during which postgraduates and postdocs presented their latest data. Plants have homologues of the majority of the vertebrate and yeast genes that encode both the structural and the regulatory proteins of the secretory pathway. However, it is clear that plant cells are different in (1) the organization and operation of the components of the pathway, such as highly motile Golgi stacks, (2) the necessity to sort proteins into different vacuoles and (3) the need to organize a precisely orientated apparatus for cell division (Fig 1). Here we consider the highlights of the meeting and the controversies surrounding the mechanisms by which the plant cell exports material from the site of synthesis to the site of action and imports material from the external environment. Figure 1. A simplified map of the secretory pathway of higher plants. Cargo synthesized and processed in the endoplasmic reticulum (ER) is transferred directly to the closely apposed and mobile Golgi apparatus (G). However, direct transfer of some storage proteins to the vacuole may occur (route 1). Proteins destined for the protein storage vacuole may leave the Golgi at the cis ‐ and trans ‐compartments (route 2), whilst exocytotic cargo predominantly leaves at the trans‐ face (route 3). Product destined for the lytic vacuole is sorted at the trans ‐Golgi and routed in a clathrin‐ and receptor‐dependent fashion through a prevacuolar … [1]: /embed/graphic-1.gif

Using the cryo-fixation/freeze-substitution method, we studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during microspore and pollen development, and pollen maturation of Arabidopsis thaliana. In young microspores forming exine (pollen outer cell wall), vacuoles looked like those of somatic cells. In microspores during the formation of intine (inner cell wall), a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic type vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs were considered to be storage vacuoles. In pollen grains from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the cytoplasm. The autolysis of pollen grains is the first finding in this study, which may contribute to the loss of ability of pollen germination after anthesis.

We have studied the transport of soluble cargo molecules by inhibiting specific transport steps to and from the Golgi apparatus. Inhibition of export from the Golgi via coexpression of a dominant-negative GTP-restricted ARF1 mutant (Q71L) inhibits the secretion of alpha-amylase and simultaneously induces the secretion of the vacuolar protein phytepsin to the culture medium. By contrast, specific inhibition of endoplasmic reticulum export via overexpression of Sec12p or coexpression of a GTP-restricted form of Sar1p inhibits the anterograde transport of either cargo molecule in a similar manner. Increased secretion of the vacuolar protein was not observed after incubation with the drug brefeldin A or after coexpression of the GDP-restricted mutant of ARF1 (T31N). Therefore, the differential effect of inducing the secretion of one cargo molecule while inhibiting the secretion of another is dependent on the GTP hydrolysis by ARF1p and is not caused by a general inhibition of Golgi-derived COPI vesicle traffic. Moreover, we demonstrate that GTP-restricted ARF1-stimulated secretion is observed only for cargo molecules that are expected to be sorted in a BP80-dependent manner, exhibiting sequence-specific, context-independent, vacuolar sorting signals. Induced secretion of proteins carrying C-terminal vacuolar sorting signals was not observed. This finding suggests that ARF1p influences the BP80-mediated transport route to the vacuole in addition to transport steps of the default secretory pathway to the cell surface.

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

Dynamin, a high-molecular-weight GTPase, plays a critical role in vesicle formation at the plasma membrane during endocytosis in animal cells. Here we report the identification of a new dynamin homolog in Arabidopsis named Arabidopsis dynamin-like 6 (ADL6). ADL6 is quite similar to dynamin I in its structural organization: a conserved GTPase domain at the N terminus, a pleckstrin homology domain at the center, and a Pro-rich motif at the C terminus. In the cell, a majority of ADL6 is associated with membranes. Immunohistochemistry and in vivo targeting experiments revealed that ADL6 is localized to the Golgi apparatus. Expression of the dominant negative mutant ADL6[K51E] in Arabidopsis protoplasts inhibited trafficking of cargo proteins destined for the lytic vacuole and caused them to accumulate at the trans-Golgi network. In contrast, expression of ADL6[K51E] did not affect trafficking of a cargo protein, H(+)-ATPase:green fluorescent protein, destined for the plasma membrane. These results suggest that ADL6 is involved in vesicle formation for vacuolar trafficking at the trans-Golgi network but not for trafficking to the plasma membrane in plant cells.

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of α-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.