Overexpression of a Grapevine Sucrose Transporter (VvSUC27) in Tobacco Improves Plant Growth Rate in the Presence of Sucrose In vitro

Abstract

The import of sugar from source leaves and it further accumulation in grape berries are considerably high during ripening, and this process is mediated via sucrose transporters. In this study, a grape sucrose transporter (SUT) gene, VvSUC27, located at the plasma membrane, was transferred to tobacco (Nicotiana tabacum). The transformants were more sensitive to sucrose and showed more rapid development, especially roots, when cultured on MS agar medium containing sucrose, considering that the shoot/root dry weight ratio was only half that of the control. Moreover, all transformed plants exhibited light-colored leaves throughout their development, which indicated chlorosis and an associated reduction in photosynthesis. The total sugar content in the roots and stems of transformants was higher than that in control plants. No significant difference was observed in the leaves between the transformants and control plants. The levels of growth-promoting hormones were increased, and those of stress-mediating hormones were reduced in transgenic tobacco plants. The qRT-PCR analysis revealed that the expression of VvSUC27 was 1,000 times higher than that of the autologous tobacco sucrose transporter, which suggested that the markedly increased growth rate of transformants was because of the heterogeneously expressed gene. The transgenic tobacco plants showed resistance to abiotic stresses. Strikingly, the overexpression of VvSUC27 leaded to the up regulation of most reactive oxygen species scavengers and abscisic acid-related genes that might enable transgenic plants to overcome abiotic stress. Taken together, these results revealed an important role of VvSUC27 in plant growth and response to abiotic stresses, especially in the presence of sucrose in vitro.

Keywords: VvSUC27; abiotic stresses; grapevine; growth; sucrose.

Figure 1

Relationship between VvSUC27 gene expression and sugar content in grapevine berry of V. riparia DVIT1848 (A), Frontenac (V. riparia × Landot 4511) (B), V. amurensis Ruper. Zuoshan-1 (C), V. amurensis Ruper. Zuoshan-2 (D) during development after flowering. Data are expressed as the mean ± SD. from six independent experiments. Pearson correlation coefficient was calculated to determine the negative correlation between VvSUC27 gene expression and sugar content (r² = 0.43, Pearson's r = −0.67, P < 0.001) (E).

Figure 2

Uptake rate of sucrose-[¹⁴C] (1 mM) by leaf discs of VvSUC27-transformed tobacco plants. Data are expressed as the mean ± SD of determinations from three independent samples. Analysis for each sample was repeated three times, and similar results were obtained. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 3

Subcellular localization of VvSUC27 by transient expression of GFP fusion proteins in Arabidopsis protoplasts and Nicotiana benthamiana. Transient expression of GFP (as control) and VvSUC27-GFP under the control of the 35S promoter Arabidopsis protoplasts (A,B) and N. benthamiana (C,D). Localization of GFP to the plasma membrane, cytoplasm, and nucleus in Arabidopsis protoplasts (A) and N. benthamiana (C). Plasma membrane localization of VvSUC27 in Arabidopsis protoplasts (B) and N. benthamiana (D). Bars = 10 μm.

Figure 4

Effects of sucrose concentration on the seedling growth of transgenic tobacco. Seedling growth rate of transgenic tobacco in Lines 9, 15, and 16 under different sucrose concentrations. (A) Rate of Seedling growth inhibition. Transgenic and control seeds were compared on MS medium containing 60 g·L⁻¹ sucrose (B). Data are expressed as the mean ± SD from six independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 5

Phenotypes of VvSUC27-transformed tobacco plants. The plants were grown in vitro on MS medium containing 30 g·L⁻¹ sucrose or without sucrose. Thirty-day-old seedlings were used to investigate stem diameter, leaf number, leaf area, and root number (A). Time-course of stem growth in transgenic tobacco plants and CK grown in vitro. Stem length (between the root/shoot junction stem to the base of the growing apex) was measured over 7 weeks (B). Data are expressed as the mean ± SD from six independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software. Phenotypic development in 7-week-old seedlings of transformants compared with that in 7-week-old seedlings of CK (C). Leaves were cut from 7-week-old transformed plants and CK (D).

Figure 6

Dry weight and shoot/root ratio of VvSUC27-transformed tobacco plants. The plants were grown in vitro for 7 weeks after being plated on modified MS medium containing 30 g·L⁻¹ sucrose or without sucrose. The dry weight of the total plant and different plant parts (leaves, stems, roots) (A), the dry weight/seedling ratio (B), and the shoot/root ratio (C) of transgenic tobacco plants (Lines 9, 15, and 16) and CK were measured. Data are expressed as the mean ± SD from six independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 7

Cross-section of the root and leaf and expression of chlorophyll-related genes in VvSUC27 transformants and CK. Organic slice analysis of root (A) and leaf (B). Organic slices were stained by Safranin O/Fast Green staining (SO/FG). Karyons, sieve element, and chloroplasts are indicated by red, blue, and white arrowheads, respectively. Transcript ratios of light harvesting chlorophyll a/b-binding protein (lhcb) in transformants (Lines 9, 15, and 16) compared with that in CK (C). The plants were grown in vitro for 7 weeks after being plated on MS medium containing 30 g·L⁻¹ sucrose. Data are expressed as the mean ± SD from six independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 8

Sugar content of VvSUC27-transformed tobacco plants compared with that of CK. The plants were grown in vitro for 7 weeks after being plated on modified MS medium containing 30 g·L⁻¹ sucrose or without sucrose. Fructose, sucrose, and total sugar content in the root (A), stem (B), and leaf (C) of transformants (Lines 9, 15, and 16) and CK were measured separately. Data are expressed as the mean ± SD from six independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 9

Phytohormone content of VvSUC27-transformed tobacco plants compared with that of CK. The plants were grown in vitro for 7 weeks after being plated on modified MS medium containing 30 g·L⁻¹ sucrose or without sucrose. Data are expressed as the mean ± SD from three independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 10

Determination of the relative transcript levels of VvSUC27 and NtSUT1. The plants were grown in vitro for 7 weeks after being plated on modified MS medium containing 30 g·L⁻¹ sucrose or without sucrose. Transcript ratios of VvSUC27 (A) and NtSUT1 (B) in transformants (Lines 9, 15, and 16) and CK were determined. The roots, stems, and leaves of transformants and CK were obtained. Total RNAs from different tissues were isolated, and transcript levels of the internal control Nt-EF1α were used as a standard. Data are expressed as the mean ± SD from six independent experiments. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 11

VvSUC27 overexpression lines under abiotic stress. Phenotypic differences in terms of the leaf area, leaf number, elongation, and root number in MS media containing 30 g·L⁻¹ sucrose or without sucrose under normal conditions, NaCl, or mannitol from the start of the experiment for 30 d. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.

Figure 12

Expression levels of ROS scavengers and ABA-related genes in untransformed (CK) or transgenic tobaccos. Seedling growth in MS media containing 30 g·L⁻¹ sucrose or without sucrose under normal conditions (A), NaCl (B), or mannitol (C) from the start of the experiment for 30 d. Thirty-day-old seedlings were used to determine the differences in expression between transformants (Lines 9, 15, and 16) and CK. Total RNA from different tissues was isolated and tested for the presence of Nt-EF1α transcripts, which served as an internal control. Different letters indicate significant differences (P < 0.05) differences between transformants (Lines 9, 15, and 16) and CK, as determined by one-way analysis of variance followed by Tukey's test using SPSS statistical software.