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 H1-dependent transport system (Km 5 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

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-[(14)C]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 (K(m) = 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

Transport of sucrose into the vacuole of Vitis vinifera cell suspensions

Poster apresentado no Congresso Nacional Micro-Biotec, em Póvoa de Varzim, 2005.The accumulation of sugar in plant cells seems to be mediated by the combined action of plasma membrane and tonoplast sugar transporters, but much work remains to be done to clarify the route(s) of sugar import and how this transport and storage is regulated. Tonoplast vesicles prepared from suspension cultured cells of Vitis vinifera were used as a model system to study the transport of sucrose into the vacuole. The purity of the tonoplast preparations was checked by the marker enzymes vanadate-sensitive H+-ATPase and H+-PPase. ATP- and PPi-dependent fluorescence quenching of ACMA indicated that these vesicles were capable of building up a pH gradient (∆pH, inside acid). Ca2+-induced dissipation of ∆pH was observed on energized vesicles, as a result of Ca2+/H+ antiport activity. However, H+-dependent sucrose transport did not seem to be involved on sucrose uptake since the disaccharide did not induce ∆pH dissipation. Initial velocities of [U-14C]sucrose measured in the absence of a transmembrane H+ gradient showed a simple Michaelis-Menten kinetics, suggesting the involvement of mediated transport with the following kinetic parameters: Km, 57 mM sucrose; Vmax, 49 nmol sucrose s-1 mg-1 protein. Influx and efflux experiments suggested that the affinity of the transport system for sucrose is similar on both sides of the membrane, and the pair sucrose/[U-14C]sucrose exhibited countertransport. It is proposed that sucrose concentrated in the cytoplasm by the activity of energy-dependent plasma membrane sugar carriers is imported into the vacuole by a facilitated diffusion mechanism.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/23169/2005, SFRH/BD/10689/2002, SFRH/BD/13460/2003, SFRH/PBD/17166/2004

Mannitol accumulation as a mechanism for salt stress tolerance in Olea europaea

Poster apresentado no "Congresso Nacional Micro/Biotec 2005", na Póvoa de Varzim, Portugal, em 2005.Resource allocation is an important and highly integrated process in higher plants involving the transport of photoassimilates from source to sink organs. Mannitol and sucrose are the primary photosynthetic products and the major phloem-translocated carbohydrates in Olea europaea L., an important and widespread crop in the Mediterranean basin. In suspension cultured cells of O. europaea, we have previously identified and characterized a H+-dependent polyol transport system whose activity was correlated with OeMaT1 (Olea europaea Mannitol Transporter) expression. The addition of 100-500 mM NaCl to cultured cells led to an increase of polyol carrier capacity and OeMaT1 transcription, as well as a strong repression of mannitol dehydrogenase activity. To further understand the function of mannitol as a compatible solute, cells of O. europaea were grown in sucrose and mannitol as the sole carbon and energy source in the absence and presence of NaCl. Results showed that mannitol was preferred over sucrose, the following values for specific growth rates obtained in the absence of salt: 0.180 d-1 and 0.076 d-1, respectively. Cells grown in the presence of up to 200 mM NaCl exhibited a marked decrease in specific growth rate along with a drastic increase of intracellular Na+ concentration. Cell viability, as assessed by fluorescein diacetate and propidium iodide, decreased along with the increase of NaCl concentration in the culture medium. Furthermore, the addition of 500 mM NaCl showed that mannitol-grown cells remained viable 24h after the salt pulse; sucrose-grown cells were not able to display green fluorescence after the same treatment, and exhibited extensive loss of cell viability as evaluated by the red fluorescence. These results suggest that mannitol behaves as an important osmolyte in O. europaea, showing an enhanced intracellular accumulation and thus compensating for the decrease of external water activity.Fundação para a Ciência e a Tecnologia (FCT)

Sugar-regulated expression of a putative hexose transport gene in grape (Vitis vinifera)

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 -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 -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

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 β-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 β-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

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

AbstractASR 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.Structured summaryMINT-6743067: VvMSA (uniprotkb:Q94G23) and VvDREB (uniprotkb:A6XA90) physically interact (MI:0218) by bimolecular fluorescence complementation (MI:0809)MINT-6743043: VvMSA (uniprotkb:Q94G23) physically interacts (MI:0218) with VvDREB (uniprotkb:A6XA90) by two hybrid (MI:0018