Abstract: Using wheat as a case study, this work presents a comprehensive in planta assessment of the relationship between cytosolic exclusion of Na+ in roots, and salinity tolerance. It offers convenient methods for the functional assessment of activity of plasma membrane and tonoplast Na+/H+ exchangers, two major transport systems contributing salinity trait in plants. The protocols developed here could be used for targeted screening of plants for salinity tolerance.
Abstract: In 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.
Abstract: This work investigates early events in root ion and water transport under acute salt stress. Using a range of microelectrode and imaging techniques, kinetics of the radial Na+ and K+ transport and loading into the xylem was resolved. We show that sequential depolarization of root cortical and stelar cells is the main driving force behind the above processes. The reported results are important for understanding of the complex physiology of salt stress as well as for practical purposes of using low quality irrigation water for saline agriculture.
Abstract: The secretory pathway encloses functionally interlinked organelles for the synthesis and deposition of most of the building blocks of eukaryotic cells, such as lipids, proteins and sugars. The coat protein complex II (COPII) is a specialized protein complex for the transport between secretory organelles, specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. This review focuses on the developments on COPII research in the plant system. Here, we address the most recent advances in the distribution and regulation of ER-to-Golgi protein transport intermediates and functional analyses of COPII isoforms. New studies support that such isoforms might not be functionally redundant and that they might have unanticipated roles in maintaining the integrity of the ER.
Abstract: ARF-GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which facilitate the activation or inactivation of ARF-GTPases, respectively. There are 15 predicted proteins that contain an ARF-GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF-GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF-GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans-Golgi network (TGN), where it co-localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post-Golgi structures of unknown nature. Taking advantage of the in vivo AGD5-ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF-GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post-Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN-localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane-localised ADP ribosylation factor B (ARFB), confirming that ARF-GAP specificity for ARF-GTPases within the cell environment may be spatially regulated.
Abstract: A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST-GFP, to other organelles. GOLD36 showed partial distribution of ST-GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein-like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk-flow marker to the cell surface was also compromised. Using a combination of classical mapping and next-generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus (At1g54030). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock-out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post-ER compartments and suggest that its export from the ER is a requirement to ensure steady-state maintenance of the organelle's organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.
Abstract: Proteins and traces of polysaccharide are the only polymeric colloids consistently transported in the xylem sap of plants. The hypothesis that such proteins could have physical inhibitory effects on xylem water transport was investigated. Ovalbumin, with a molecular weight of 45 kDa and a molecular diameter of 5.4 nm, is an inert, water-soluble protein that is midway along the size range of endogenous xylem sap proteins. Solutions of ovalbumin conjugated to a fluorescent marker and supplied to transpiring shoot explants of tobacco (Nicotiana tabacum L.) and olive (Olea europaea L.) were shown by confocal laser scanning microscopy to accumulate specifically at wall-based pit membranes that connect neighbouring xylem conduits. In addition, pressure-induced perfusion of micro-filtered ovalbumin solutions, at concentrations similar to those of endogenous xylem sap proteins, through the xylem of tobacco stem or olive twig segments resulted in the retention of c. 40% of the ovalbumin and reductions in the axial hydraulic conductance of the xylem. Smaller molecules such as Texas Red 3000 (MW 3 kDa) and Alexafluor 488-cadaverin conjugates (MW 0.64 kDa) did not show similar characteristics. The partial reduction in xylem hydraulic conductance appeared to be related to the accumulation of ovalbumin at xylem pit membranes and the consequent fouling of trans-membrane water-conducting pores with smaller diameters than those of the ovalbumin molecules. Potential implications of these novel findings for whole-plant water relations are considered.
Abstract: The study of electrical network systems, integrated with chemical signaling networks, is becoming a common trend in contemporary biology. Classical techniques are limited to the assessment of signals from doublets or triplets of cells at a fixed temporal bin width. At present, full characteristics of the electrical network distribution and dynamics in plant cells and tissues has not been established. Here, a 60-channels multielectrode array (MEA) is applied to study spatiotemporal characteristics of the electrical network activity of the root apex. Both intense spontaneous electrical activities and stimulation-elicited bursts of locally propagating electrical signals have been observed. Propagation of the spikes indicates the existence of excitable traveling waves in plants, similar to those observed in non-nerve electrogenic tissues of animals. Obtained data reveal synchronous electric activities of root cells emerging in a specific root apex region. The dynamic electrochemical activity of root apex cells is proposed to continuously integrate internal and external signaling for developmental adaptations in a changing environment.
Abstract: BACKGROUND AND AIMS: Flavonoids have the potential to serve as antioxidants in addition to their function of UV screening in photoprotective mechanisms. However, flavonoids have long been reported to accumulate mostly in epidermal cells and surface organs in response to high sunlight. Therefore, how leaf flavonoids actually carry out their antioxidant functions is still a matter of debate. Here, the distribution of flavonoids with effective antioxidant properties, i.e. the orthodihydroxy B-ring-substituted quercetin and luteolin glycosides, was investigated in the mesophyll of Ligustrum vulgare leaves acclimated to contrasting sunlight irradiance. METHODS: In the first experiment, plants were grown at 20 % (shade) or 100% (sun) natural sunlight. Plants were exposed to 100 % sunlight irradiance in the presence or absence of UV wavelengths, in a second experiment. Fluorescence microspectroscopy and multispectral fluorescence microimaging were used in both cross sections and intact leaf pieces to visualize orthodihydroxy B-ring-substituted flavonoids at inter- and intracellular levels. Identification and quantification of individual hydroxycinnamates and flavonoid glycosides were performed via HPLC-DAD. KEY RESULTS: Quercetin and luteolin derivatives accumulated to a great extent in both the epidermal and mesophyll cells in response to high sunlight. Tissue fluorescence signatures and leaf flavonoid concentrations were strongly related. Monohydroxyflavone glycosides, namely luteolin 4'-O-glucoside and two apigenin 7-O-glycosides were unresponsive to changes in sunlight irradiance. Quercetin and luteolin derivatives accumulated in the vacuoles of mesophyll cells in leaves growing under 100 % natural sunlight in the absence of UV wavelengths. CONCLUSIONS: 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.
Abstract: Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes. In addition, although ARF1 appears to be central to the recruitment of both COPI components and the golgin, we have established a different subcellular distribution of these ARF1 effectors. Live cell imaging demonstrates that GDAP1 and COPI are distributed on Golgi membranes. However, GDAP1 is also found on ARF1-labeled structures that lack coatomer, suggesting that the membrane environment, rather than ARF1 alone, influences the differential recruitment of ARF1 effectors. In support of this hypothesis, fluorescence recovery after photobleaching analyses demonstrated that GDAP1 and COPI have different kinetics on membranes during the cycle of activation and inactivation of ARF1. Therefore, our data support a model where modulation of the cellular functions of ARF1 in plant cells encompasses not only the intrinsic activities of the effectors, but also differential recruitment onto membranes that is spatially regulated.
Abstract: Trafficking 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.
Abstract: ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1-GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus.
Abstract: 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.
Abstract: The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.
Abstract: Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.
