The grape aquaporin VvSIP1 transports water across the ER membrane.

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

Mannitol transport and mannitol dehydrogenase activities are coordinated in Olea europaea under salt and osmotic stresses.

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 µmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

Interaction of grape ASR proteins with a DREB transcription factor in the nucleus.

ASR proteins (abscissic acid, stress, ripening induced) are involved in plant responses to developmental and environmental signals but their biological functions remain to be elucidated. Grape ASR gene (VvMSA) encodes a new transcription factor regulating the expression of a glucose transporter. Here, we provide evidence for some polymorphism of grape ASRs and their identification as chromosomal non-histone proteins. By the yeast two-hybrid approach, a protein partner of VvMSA is isolated and characterized as an APETALA2 domain transcription factor. Interaction of the two proteins is further demonstrated by the BiFC approach and the exclusive nuclear localization of the heterodimer is visualized.

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells.

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.

OeMST2 encodes a monosaccharide transporter expressed throughout olive fruit maturation.

In olive fruits, sugars are the main soluble components providing energy and acting as precursors for olive oil biosynthesis. Large quantities of glucose, fructose and galactose are often found in olive pulp. To analyze sugar transport processes in Olea europaea, a cDNA encoding a monosaccharide transporter, designated OeMST2 (Olea europaea monosaccharide transporter 2) was cloned. An open reading frame of 1,569 bp codes for a protein of 523 amino acids and a calculated molecular weight of 57.6 kDa. The protein is homologous to other sugar transporters identified so far in higher plants. Expression of this cDNA in an hxt-null Saccharomyces cerevisiae strain deficient in glucose transport restored its capacity to grow on and to transport glucose. The encoded protein showed high affinity for D-glucose (K(m), 25 microM) and was also able to recognize D-galactose and the analogs 3-O-methyl-D-glucose and 2-deoxy-D-glucose, but not D-fructose, D-arabinose, sucrose or D-mannitol. Maximal transport activity was high at acidic pH (5.0), and the initial D-[(14)C]glucose uptake rates were strongly inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, confirming that OeMST2 is a H(+)/monosaccharide transporter. The expression of OeMST2 was studied during the ripening process. Transcript levels increased during fruit maturation, suggesting that OeMST2 takes part in the massive accumulation of monosaccharides in olive fruits. Monosaccharide:H(+) transport system activity and OeMST2 expression were negatively regulated by glucose in suspension-cultured cells. Glucose-mediated OeMST2 repression was impaired by mannoheptulose, suggesting the involvement of a hexokinase-dependent signaling pathway.

Utilization and transport of mannitol in Olea europaea and implications for salt stress tolerance.

Mannitol is one of the primary photosynthetic products and the major phloem-translocated carbohydrate in Olea europaea L., an important crop in the Mediterranean basin. Uptake of mannitol in heterotrophic cell suspensions of O. europaea was shown to be mediated by a 1 : 1 polyol : H+ symport system with a Km of 1.3 mM mannitol and a Vmax of 1.3 nmol min(-1) mg(-1) DW. Dulcitol, sorbitol and xylitol competed for mannitol uptake, whereas glucose and sucrose did not. Reverse transcription-PCR (RT-PCR) performed on mRNA extracted from cultured cells exhibiting high mannitol transport activity allowed the cloning of a partial O. europaea mannitol carrier OeMaT1. The Vmax of mannitol uptake and the amount of OeMaT1 transcripts increased along with polyol depletion from the medium, suggesting that the mannitol transport system may be regulated by its own substrate. Addition of 100-500 mM NaCl to cultured cells enhanced the capacity of the polyol : H+ symport system and the amount of OeMaT1 transcripts, whereas it strongly repressed mannitol dehydrogenase activity. Measurements of cell viability showed that mannitol-grown cells remained viable 24 h after a 250 and 500 mM NaCl pulse, whereas extensive loss of cell viability was observed in sucrose-grown cells. OeMaT1 transcripts increased throughout maturation of olive fruits, suggesting that an OeMaT is involved in the accumulation of mannitol during ripening of olive. Thus, mannitol transport and compartmentation by OeMaT are important to allocate this source of carbon and energy, as well as for salt tolerance and olive ripening.

Pathways of glucose regulation of monosaccharide transport in grape cells.

Grape (Vitis vinifera) heterotrophic suspension-cultured cells were used as a model system to study glucose (Glc) transport and its regulation. Cells transported D-[14C]Glc according to simple Michaelis-Menten kinetics superimposed on first-order kinetics. The saturating component is a high-affinity, broad-specificity H+ -dependent transport system (Km = 0.05 mm). Glc concentration in the medium tightly regulated the transcription of VvHT1 (Vitis vinifera hexose transporter 1), a monosaccharide transporter previously characterized in grape berry, as well as VvHT1 protein amount and monosaccharide transport activity. All the remaining putative monosaccharide transporters identified so far in grape were poorly expressed and responded weakly to Glc. VvHT1 transcription was strongly repressed by Glc and 2-deoxy-D-Glc, but not by 3-O-methyl-D-Glc or Glc plus mannoheptulose, indicating the involvement of a hexokinase-dependent repression. 3-O-Methyl-D-Glc, which cannot be phosphorylated, and Glc plus mannoheptulose induced a decrease of transport activity caused by the reduction of VvHT1 protein in the plasma membrane without affecting VvHT1 transcript levels. This demonstrates hexokinase-independent posttranscriptional regulation. High Glc down-regulated VvHT1 transcription and Glc uptake, whereas low Glc increased those parameters. Present data provide an example showing control of plant sugar transporters by their own substrate both at transcriptional and posttranscriptional levels. VvHT1 protein has an important role in the massive import of monosaccharides into mesocarp cells of young grape berries because it was localized in plasma membranes of the early developing fruit. Protein amount decreased abruptly throughout fruit development as sugar content increases, consistent with the regulating role of Glc on VvHT1 expression found in suspension-cultured cells.

A grape ASR protein involved in sugar and abscisic acid signaling.

The function of ASR (ABA [abscisic acid]-, stress-, and ripening-induced) proteins remains unknown. A grape ASR, VvMSA, was isolated by means of a yeast one-hybrid approach using as a target the proximal promoter of a grape putative monosaccharide transporter (VvHT1). This promoter contains two sugar boxes, and its activity is induced by sucrose and glucose. VvMSA and VvHT1 share similar patterns of expression during the ripening of grape. Both genes are inducible by sucrose in grape berry cell culture, and sugar induction of VvMSA is enhanced strongly by ABA. These data suggest that VvMSA is involved in a common transduction pathway of sugar and ABA signaling. Gel-shift assays demonstrate a specific binding of VvMSA to the 160-bp fragment of the VvHT1 promoter and more precisely to two sugar-responsive elements present in this target. The positive regulation of VvHT1 promoter activity by VvMSA also is shown in planta by coexpression experiments. The nuclear localization of the yellow fluorescent protein-VvMSA fusion protein and the functionality of the VvMSA nuclear localization signal are demonstrated. Thus, a biological function is ascribed to an ASR protein. VvMSA acts as part of a transcription-regulating complex involved in sugar and ABA signaling.

Sugar-regulated expression of a putative hexose transport gene in grape.

Different lengths of the promoter of grape (Vitis vinifera) VvHT1 (Hexose Transporter 1) gene, which encodes a putative hexose transporter expressed during the ripening of grape, have been transcriptionally fused to the beta-glucuronidase reporter gene. In transgenic tobacco (Nicotiana tabacum) transformed with these constructs, VvHT1 promoters were clearly responsible for the sink organ preferential expression. The potential sugar effectors of VvHT1 promoter were studied in tobacco cv Bright-Yellow 2 cells transformed with chimeric constructs. Glucose (56 mM), sucrose (Suc; 58 mM), and the non-transported Suc isomer palatinose doubled the beta-glucuronidase activity conferred by the VvHT1 promoter, whereas fructose did not affect it. These effects were the strongest with the 2.4-kb promoter, which contains all putative sugar-responsive elements (activating and repressing), but they were also significant with the 0.3-kb promoter, which contains only activating sugar boxes. The induction of VvHT1 expression by both Suc and palatinose was confirmed in the homologous grape berry cell culture. The data provide the first example of a putative sugar transporter, which is induced by both glucose and Suc in higher plants. Although induction of VvHT1 expression by Suc does not require transport, the presence of glucosyl moiety is necessary for Suc sensing. These results provide new insights into sugar sensing and signaling in plants.