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

Abstract

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.

Figure 1

Schematic representation of VvHT1 promoter regions used for the constructs. Successive deletions of the distal part of the promoter (p2.4VvHT1, p0.8 VvHT1, and p0.3VvHT1), with specific sites for restriction enzymes and primers used for chimeric pVvHT1/GUS fusions, are shown. The positions of putative sugar-responsive elements are shown with respect to the translation initiation start. Suc box 3, black triangles; SURE1 motif, white triangles; AMYBOX1, gray triangle; AMYBOX2, hatched triangles. Position over or under the promoter line stands for plus or minus strand, respectively. Triangles corresponding to positive sugar response elements are presented upside up, and those corresponding to negative sugar response elements are presented upside down.

Figure 2

GUS activity and uidA expression in different organs of tobacco plants expressing the reporter gene under the control of different lengths of VvHT1 promoter. A, GUS activity of roots, stems, and young leaves was expressed relative to the activity measured in mature leaves for each independent transformant (13–20 per construct). The p35S/GUS activity in mature leaves was taken as 100%. Data are given ± se. B, Northern-blot analysis of reporter gene transcripts in source and sink organs under control of VvHT1 and 35S promoters hybridized with a probe corresponding to iudA coding region. Twenty micrograms (leaves) and 25 μg (roots) of total RNA were loaded on the gel.

Figure 3

Relative GUS activity under the control of different VvHT1 promoters, 35S promoter, or for the promoterless construct, in BY2 transgenic cell suspensions at the 7th d of subculture. Inset shows at a larger scale the activity conferred by different truncated VvHT1 promoters. Data are the mean values of three independent replicates ± se. GUS activity in the p35S-GUS-transformed cells was 386 pmol methyl-umbelliferone (MU) min⁻¹ mg protein⁻¹.

Figure 4

Cell growth (A), sugar content of the medium and GUS activity of BY2 transgenic cell suspensions expressing p2.4VvHT1-GUS (B), or p35S-GUS fusion (C). Suc (black squares), Glc (black circles), and Fru (white circles) content of the medium.

Figure 5

GUS relative activity in suspension cells carrying p0.3VvHT1/GUS, p0.8VvHT1/GUS, p2.4VvHT1/GUS, or p35S/GUS in response to the addition of different sugars. For each construct, the control is the same transformed cell suspension without treatment. The different compounds were added 4 d after the beginning of subculture and measurements were made 2 d later. Data are representative for three independent transformation procedures of BY2 cells for each construct and treatments produced in each of the transgenic cultures. Values differing significantly (Student's t test, P = 0.05) from the control are indicated by asterisks.

Figure 6

RNA gel-blot analysis of GUS transcripts accumulation under Glc induction of VvHT1 promoter activity in tobacco BY2 transgenic cell suspension and mRNA quantification by using a Bio-Imaging Analyzer. For each lane, 20 μg of total RNA was loaded on gel and membranes were hybridized with iudA-specific probe and rRNA probe.

Figure 7

Growth of tobacco BY2 cells and GUS activity of BY2 cells in the presence of different disaccharides. A, Effect of different disaccharides provided as the unique source of carbon on cell growth. B, Effects of different disaccharides on 2.4VvHT1 promoter-conferred GUS activity. Values differing significantly (Student's t test, P = 0.05) from the control are indicated by asterisks.

Figure 8

Effect of Suc and palatinose treatment on VvHT1 expression in grape cell suspension culture. For each lane, 20 μg of total RNA was loaded on gel. Membranes were hybridized with VvHT1 and rRNA probes. Data are representative for three independent treatments of cell culture and for the corresponding RNA gel-blot experiments.