Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering

Abstract

Sugar compartmentation into vacuoles of higher plants is a very important physiological process, providing extra space for transient and long-term sugar storage and contributing to the osmoregulation of cell turgor and shape. Despite the long-standing knowledge of this subcellular sugar partitioning, the proteins responsible for these transport steps have remained unknown. We have identified a gene family in Arabidopsis consisting of three members homologous to known sugar transporters. One member of this family, Arabidopsis thaliana vacuolar glucose transporter 1 (AtVGT1), was localized to the vacuolar membrane. Moreover, we provide evidence for transport activity of a tonoplast sugar transporter based on its functional expression in bakers' yeast and uptake studies in isolated yeast vacuoles. Analyses of Atvgt1 mutant lines indicate an important function of this vacuolar glucose transporter during developmental processes like seed germination and flowering.

Fig. 1.

Tonoplast localization of the AtVGT1–GFP fusion protein in yeast and Arabidopsis. (A and B) In yeast, the AtVGT1–GFP fusion protein is localized in internal membranes (A) and after separation of subcellular compartments can only be seen in vacuolar membranes (B). (Scale bars, A, 5 μm; B, 1 μm.) (C and D) When expressed transiently in Arabidopsis protoplasts, the AtVGT1–GFP fusion protein is clearly inserted into the tonoplast, which separates the vacuolar volume from the cytoplasm containing chloroplasts (white arrows). (E and F) After protoplast lysis, GFP fluorescence can only be seen in the remaining membrane of intact vacuoles. (Scale bar, 10 μm.)

Fig. 2.

Transport properties of AtVGT1 in yeast. (A) The transport capacity for glucose of yeast cells expressing the AtVGT1 cDNA in sense orientation (strain SAY114s; black circles) or antisense orientation (strain SAY114as; gray circles) in the presence of ATP and in sense orientation in the absence of ATP (white circles) was analyzed. Values represent the mean of seven independent transport analyses for sense and antisense and three for sense without added ATP (±SE). (B) Substrate specificity for AtVGT1 is shown as relative uptake rates of different sugars in AtVGT1-expressing yeast cells (sense: black bars; antisense: gray bar), determined at an initial outside concentration of 1 mM and in the presence of 4 mM ATP if not stated otherwise. (Inset) Lineweaver–Burk plot of concentration-dependent glucose uptake. v, nmol·sec⁻¹; S, mM. All data represent average values of at least two independent transport tests.

Fig. 3.

AtVGT1 expression analyzed by GUS reporter plants and RT-PCR. (A) Influorescence with GUS histochemical staining showing reporter expression in anthers of GUS reporter plants starting from flower stage 10, according to Bowman (53). (Inset) Anthers at higher magnification showing GUS staining in pollen grains. (Magnification: ×10; Inset, ×66.) (B) RT-PCR analysis using equal amounts of total RNA from wild-type leaves, flowers, roots, and stems. PCRs with 30, 35, and 40 cycles were done with AtVGT1- or Actin2-specific primers.

Fig. 4.

Identification of T-DNA insertions in the AtVGT1 gene. (A) The position and orientation of the T-DNA insertion in the AtVGT1 gene of the mutant lines SALK_000988 and SAIL_669_D03. The gene has 13 introns (white boxes), and the insertions are located at position −1 (SALK line) and +2760 relative to the start codon. The orientation of the left border (LB) is indicated, and the opposite end of the insertion has not been characterized. (B) Genomic PCR analysis demonstrating homozygous T-DNA insertion in the SALK_000988 line. By using a T-DNA primer and a gene-specific primer, a 428-bp PCR product was amplified from mutant DNA (lane 3) but not from wild-type (wt) DNA (lane 1); whereas, by using gene-specific primers spanning the insertion site, a 1,788-bp PCR product was amplified from wild-type DNA (lane 2) but not from mutant DNA (lane 4). (C) RT-PCR analysis demonstrating the lack of AtVGT1 transcript in the SAIL_669_D03 line. By using gene-specific primers spanning the insertion site, a 407-bp RT-PCR product was amplified from wild-type flower cDNA but not from mutant flower cDNA.

Fig. 5.

Delay in flowering of Atvgt1 mutant lines. (A) Initiation of flowering diagrammed as the percentage of plants showing growth of primary shoots was determined for Col-0 wild-type (wt) plants (black circles), SALK_000988 plants (gray squares), and SAIL_669_D03 plants (white triangles). (B) Atvgt1 mutant lines in comparison with wild-type plants (Col-0) 1 week after the induction of flowering (36 days after germination).