SummaryTrafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

SummaryIn plant cells, the endoplasmic reticulum (ER) and Golgi apparatus form a unique system in which single Golgi stacks are motile and in close association with the underlying ER tubules. Arabidopsis has three RHD3 (ROOT HAIR DEFECTIVE 3) isoforms that are analogous to the mammalian atlastin GTPases involved in shaping ER tubules. We used live-cell imaging, genetic complementation, split ubiquitin assays and western blot analyses in Arabidopsis and tobacco to show that RHD3 mediates the generation of the tubular ER network and is required for the distribution and motility of Golgi stacks in root and leaf epidermal cells. We established that RHD3 forms homotypic interactions at ER punctae. In addition, the activity of RHD3 on the tubular ER is specifically correlated with the cellular distribution and motility of Golgi stacks because ER to Golgi as well as Golgi to plasma membrane transport was not affected by RHD3 mutations in the conserved GDP/GTP motifs. We found a possible partial redundancy within the RHD3 isoforms in Arabidopsis. However, yeast Sey1p, a functional atlastin homologue, and RHD3 are not interchangeable in complementing the respective loss-of-function mutants, suggesting that the molecular mechanisms controlling ER tubular morphology might not be entirely conserved among eukaryotic lineages.

This work investigates the role of cytosolic Na + exclusion in roots as a means of salinity tolerance in wheat, and offers in planta methods for the functional assessment of major transporters contributing to this trait. An electrophysiological protocol was developed to quantify the activity of plasma membrane Na + efflux systems in roots, using the microelectrode ion flux estimation (MIFE) technique. We show that active efflux of Na + from wheat root epidermal cells is mediated by a SOS1-like homolog, energized by the plasma membrane H + -ATPase. SOS1-like efflux activity was highest in Kharchia 65, a salt-tolerant bread wheat cultivar. Kharchia 65 also had an enhanced ability to sequester large quantities of Na + into the vacuoles of root cells, as revealed by confocal microscopy using Sodium Green. These findings were consistent with the highest level of expression of both SOS1 and NHX1 transcripts in plant roots in this variety. In the sensitive wheat varieties, a greater proportion of Na + was located in the root cell cytosol. Overall, our findings suggest a critical role of cytosolic Na + exclusion for salinity tolerance in wheat and offer convenient protocols to quantify the contribution of the major transporters conferring this trait, to screen plants for salinity tolerance.Abbreviations: NHX, tonoplast Na + /H + exchanger; SOS1, plasma membrane Na + /H + exchanger.

The above findings lead to the hypothesis that flavonoids play a key role in countering light-induced oxidative stress, and not only in avoiding the penetration of short solar wavelengths in the leaf.

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.n addition to meeting cellular energy needs through photosynthesis, chloroplasts are centers of anabolic metabolism that contain complete biosynthetic pathways (e.g., for de novo synthesis of fatty acids, amino acids, tocopherols, and carotenoids) and participate in numerous pathways that span multiple subcellular compartments (e.g., for synthesis of membrane lipids, monoterpenes, diterpenes, and photorespiration). Such metabolism requires exchange of a multitude of polar and nonpolar metabolites with the extraplastidic environment and consistent with this, proteomic and bioinformatic analysis of the chloroplast envelope identified 102 transporter candidates (Dataset S1). Sixty-six have recognized functions as ion or metabolite transporters, but only one transports nonpolar metabolites. This apparent paucity of nonpolar metabolite transporters in the envelope, despite the large numbers of nonpolar metabolites synthesized by plastids, highlights a significant gap in our understanding of plant metabolism.Tocochromanols are one well-studied group of nonpolar compounds synthesized and localized in plastids that include the biosynthetically related tocopherols, tocotrienols, and plastochromanol-8 (PC8) (Fig. 1). Tocochromanol biosynthesis has been fully elucidated, null mutants with well-defined biochemical phenotypes are available for each reaction, and with the exception of p-hydroxyphenylpyruvate dioxygenase, all biosynthetic activities localize to the plastid inner envelope where synthesis occurs (1, 2). Because tocochromanols are only present in chloroplast membranes (1, 3), it was assumed that their functions would be restricted to this organelle; however, many tocopheroldeficient mutant phenotypes are, instead, consistent with impacts on extraplastidic processes. These include alterations in membrane lipids, formation of secretory pathway-derived vesicles, cell-wall developmen...

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.

SUMMARYThe mechanisms underlying the organization and dynamics of plant endomembranes are largely unknown. Arabidopsis RHD3, a distant member of the dynamin superfamily, has recently been implicated in plant ER morphology and Golgi movement through analyses of dominant-negative mutants of the putative GTPase domain in a heterologous system. Whether RHD3 is indispensable for ER architecture and what role regions other than the putative GTPase domain play in RHD3 function are unanswered questions. Here we characterized an EMS mutant, gom8, with disrupted Golgi movement and positioning and compromised ER shape and dynamics. gom8 mapped to a missense mutation in the RHD3 hairpin loop domain, causing accumulation of the mutant protein into large structures, a markedly different distribution compared with wild-type RHD3 over the ER network. Despite the GOM8 distribution, tubules fused in the peripheral ER of the gom8 mutant. These data imply that integrity of the hairpin region is important for the subcellular distribution of RHD3, and that reduced availability of RHD3 over the ER can cause ER morphology defects, but does not prevent peripheral fusion between tubules. This was confirmed by evidence that gom8 was phenocopied in an RHD3 null background. Furthermore, we established that the region encompassing the RHD3 hairpin domain and the C-terminal cytosolic domain is necessary for RHD3 function. We conclude that RHD3 is important in ER morphology, but is dispensable for peripheral ER tubulation in an endogenous context, and that its activity relies on the C-terminal region in addition to the GTPase domain.

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