Abstract The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting transport of cargo. Vacuolar from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) assumed occur via clathrin-coated vesicles, although direct proof their participation missing. Here, we present evidence that post-TGN toward lytic vacuoles occurs independently clathrin MVBs/LEs are derived through maturation....

Abstract We generated fusions between three Arabidopsis (Arabidopsis thaliana) tonoplast intrinsic proteins (TIPs; α-, γ-, and δ-TIP) yellow fluorescent protein (YFP). also produced soluble reporters consisting of the monomeric red (RFP) either C-terminal vacuolar sorting signal phaseolin or sequence-specific proricin. In transgenic leaves, mature roots, root tips, all TIP localized to central vacuole both lumenal RFP were found within TIP-delimited vacuoles. embryos from developing, mature,...

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved shaping ER tubules. Here, we characterized the localization, topology, effect, interactions five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 13, ER. Our results indicate that RTNLB13 RTNLB1-4 colocate to constrict tubular membrane. All RTNs preferentially accumulate on...

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, membranous rod derived from cortical ER. Little is known about forces that model ER during formation. Here, we show members reticulon (RTNLB) family ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play role formation...

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. cortical ER plants pleomorphic structured as tubular network capable of morphing into flat cisternae, mainly at three-way junctions, back to tubules. Plant reticulon family proteins (RTNLB) tubulate the by dimerization oligomerization, creating localized membrane tensions result curvature. Some RTNLB ER-shaping are present plasmodesmata...

To acquire information on the relationships between structural maturation of proteins in endoplasmic reticulum (ER) and their transport along secretory pathway, we have analyzed destiny an assembly-defective form trimeric vacuolar storage glycoprotein phaseolin. In leaves transgenic tobacco, where assembly-competent phaseolin is correctly targeted to vacuole, defective remains located ER or a closely related compartment it represents major ligand chaperone BiP. Defective maintained...

Phaseolin, one of the major legume proteins for human nutrition, is a trimeric glycoprotein 7S class that accumulates in protein storage vacuoles common bean. Phaseolin cotranslationally introduced into lumen endoplasmic reticulum; from there, it transported through Golgi complex to vacuoles. also vacuole vegetative tissues transgenic plants. By transient and permanent expression tobacco leaf cells, we show here vacuolar sorting phaseolin saturable saturation leads Golgi-mediated secretion...

We have cloned a member of the reticulon (RTN) family Arabidopsis thaliana (RTNLB13). When fused to yellow fluorescent protein (YFP) and expressed in tobacco leaf epidermal cells, RTNLB13 is localized endoplasmic reticulum (ER). Coexpression soluble ER luminal marker reveals that YFP‐tagged, myc‐tagged or untagged induces severe morphological changes lumen ER. show, using fluorescence recovery after photobleaching (FRAP) analysis, overexpression greatly reduces diffusion proteins within...

Saffron is the dried stigmas of Crocus sativus and most expensive spice in world. Its red color due to crocins, which are apocarotenoid glycosides that accumulate vacuole a level up 10% stigma dry weight. Previously, we characterized first dedicated enzyme crocin biosynthetic pathway, carotenoid cleavage dioxygenase2 (CsCCD2), cleaves zeaxanthin yield crocetin dialdehyde. In this work, identified six putative aldehyde dehydrogenase (ALDH) genes expressed C. stigmas. Heterologous expression...

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, complex mixture apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up 10% dry weight. We developed general approach, based on coexpression analysis, heterologous expression yeast (Saccharomyces cerevisiae), vitro transportomic assays using microsomes total plant metabolite extracts, identification putative vacuolar...

Aquaporins (AQPs) are water channels allowing fast and passive diffusion of across cell membranes. It was hypothesized that AQPs contribute to elongation processes by influx the plasma membrane tonoplast maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), highly abundant AQP isoforms AtTIP1;1, AtTIP1;2, AtTIP2;1 facilitate emergence new lateral root primordia (LRPs). The number roots strongly reduced triple tip mutant, whereas single, double,...

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration lytic embryos [M. Feeney et al. , Plant Physiol. 177, 241–254 (2018)], PSVs undergo division the later stages maturation. Here, we study biophysical mechanism PSV morphogenesis vivo, discovering that micrometer-sized liquid droplets containing form within vacuolar lumen phase...

Abstract Background Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms encoded by the Arabidopsis genome. For several isoforms, tissue and cell specific pattern of expression not known. Results We generated fluorescent protein fusions to genomic sequences all members family whose is predicted occur root tissues (TIP1;1 1;2; TIP2;1, 2;2 2;3; TIP4;1) expressed these fusions, both individually selected pairwise...

Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape ER into tubules. It has been hypothesized their unusually long conserved hydrophobic regions cause reticulons assume a wedge-like topology induces curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana reticulon (RTNLB13) localized to, and induced constrictions in, cortical Ectopic expression RTNLB13 was sufficient induce tubulation in mutant (pah1...

Significance This study demonstrates, in vivo, that reticulon (RTN) proteins, responsible for the shaping and maintenance of endoplasmic reticulum (ER) membrane tubules, rely on a highly conserved C-terminal amphipathic helix (APH) their morphogenic function. Previously it was thought RTN could bend ER both by assuming wedge-like topology forming oligomeric arcs. We show here deleting or mutating APH region abolishes function plant but does not affect its capacity to oligomerize. These...

Natural rubber (polyisoprene) from the tree Hevea brasiliensis is synthesized by specialized cells called laticifers. It not clear how particles arise, although one hypothesis that they derive endoplasmic reticulum (ER) membrane. Here we cloned genes encoding four key proteins found in association with and studied their intracellular localization transient expression Nicotiana benthamiana leaves. We show that, while cis-prenyltransferase (CPT), responsible for synthesis of long polyisoprene...

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about origins these transient organelles. PSV hypothesized to either arise de novo or originate from preexisting embryonic vacuole (EV) during seed maturation. Here, we tested hypotheses by studying formation Arabidopsis (

Aquaporins influence water flow in plants, yet little is known of their involvement the water-driven process seed germination. We therefore investigated role seeds laboratory and under field global warming conditions. mapped expression tonoplast intrinsic proteins (TIPs) during dormancy cycling germination normal stress found that two key aquaporins, TIP3;1 TIP3;2, which have previously been implicated or solute transport, respectively, act antagonistically to modulate response abscisic...

Ricin is a heterodimeric toxin that accumulates in the storage vacuoles of castor bean (Ricinus communis) endosperm. Proricin synthesized as single polypeptide precursor comprising catalytic A chain and Gal-binding B joined by 12-amino acid linker propeptide. Upon arrival vacuole, removed. Here, we replicate these events transfected tobacco (Nicotiana tabacum) leaf protoplasts. We show internal propeptide responsible for vacuolar sorting sufficient to redirect ricin heterodimer vacuole when...

When expressed in tobacco cells, the catalytic subunit of dimeric ribosome inactivating protein, ricin, is first inserted into endoplasmic reticulum (ER) and then degraded a manner that can be partially inhibited by proteasome inhibitor clasto-lactacystin beta-lactone. Consistent with implication cytosolic proteasomes, degradation ricin A chain brefeldin A-insensitive polypeptides accumulate presence are not processed vacuole-specific fashion. Rather, these stabilized part deglycosylated...

The catalytic A subunit of ricin can inactivate eukaryotic ribosomes, including those Ricinus communis where the toxin is naturally produced. How such plant cells avoid intoxication has remained an open question. Here we report transient expression a number chain-encoding cDNA constructs in tobacco protoplasts. Ricin chain entered endoplasmic reticulum lumen, it was efficiently glycosylated, but toxic to and disappeared with time brefeldin A-insensitive manner, suggesting reverse...

In this study, we demonstrate that the folding and assembly of IgG in transgenic tobacco plants is orchestrated by BiP (binding protein), an endoplasmic reticulum resident chaperone. Expression calreticulin was examined express immunoglobulin chains, either singly or combination to form antibody. mRNA expression lowest wild-type nontransformed those expressed light chain alone. Higher levels were detected expressing fully assembled (light heavy chains), most abundant RNA transcript found...