Overexpression of Grapevine VvIAA18 Gene Enhanced Salt Tolerance in Tobacco

Abstract

In plants, auxin/indoleacetic acid (Aux/IAA) proteins are transcriptional regulators that regulate developmental process and responses to phytohormones and stress treatments. However, the regulatory functions of the Vitis vinifera L. (grapevine) Aux/IAA transcription factor gene VvIAA18 have not been reported. In this study, the VvIAA18 gene was successfully cloned from grapevine. Subcellular localization analysis in onion epidermal cells indicated that VvIAA18 was localized to the nucleus. Expression analysis in yeast showed that the full length of VvIAA18 exhibited transcriptional activation. Salt tolerance in transgenic tobacco plants and Escherichia. coli was significantly enhanced by VvIAA18 overexpression. Real-time quantitative PCR analysis showed that overexpression of VvIAA18 up-regulated the salt stress-responsive genes, including pyrroline-5-carboxylate synthase (NtP5CS), late embryogenesis abundant protein (NtLEA5), superoxide dismutase (NtSOD), and peroxidase (NtPOD) genes, under salt stress. Enzymatic analyses found that the transgenic plants had higher SOD and POD activities under salt stress. Meanwhile, component analysis showed that the content of proline in transgenic plants increased significantly, while the content of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) decreased significantly. Based on the above results, the VvIAA18 gene is related to improving the salt tolerance of transgenic tobacco plants. The VvIAA18 gene has the potential to be applied to enhance plant tolerance to abiotic stress.

Keywords: Escherichia coli; VvIAA18; grapevine; salt tolerance; tobacco.

Figure 1

A phylogenetic tree of the VvIAA18 protein with its other plant homologous proteins. The branch lengths are proportional to the distance.

Figure 2

Subcellular localization of the VvIAA18 protein in onion epidermal cells. The VvIAA18-GFP fusion protein was localized to the nucleus. Scale bar = 100 μm.

Figure 3

Transactivation assay of the VvIAA18 protein in the yeast. Fusion protein of the GAL4 DNA-binding domain and VvIAA18 were expressed in yeast. Use the empty pBD (pGBKT7) vector (negative control) and the pGAL4 vector (positive control). The culture solution of the transformed yeast was dropped onto SD plates without tryptophan or histidine. The plates were incubated for 3 days and then subjected to β-galactosidase assay. The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.01 (**).

Figure 4

Enhanced salt tolerance in Escherichia coli. (a) Growth analysis of cells spotted on LB agar plate supplemented with 0.5 M NaCl. (b) Growth analysis of cells cultured in liquid medium supplemented with 0.5 M NaCl. Cell growth densities were measured at 600 nm at the indicated time points. The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.01 (**).

Figure 5

Molecular confirmation of transgenic plants. (a) Schematic diagram of the double plasmid pCAMBIA1301-VvIAA18 T-DNA region. LB, left border; RB, right border; hptⅡ, hygromycin phosphotransferase Ⅱ gene; VvIAA18, grape Aux/IAA transcription factor gene; gusA, β-glucuronidase gene; 35S, cauliflower mosaic virus (CaMV) 35S promoter; 35S T, CaMV 35S terminator; NOS T, nopaline synthase terminator. (b–d) β-glucuronidase (GUS) expression in leaf, stem, and root of a transgenic plant and no GUS expression in the wild-type (WT) (bar = 10 mm). (e) PCR analysis of VvIAA18-overexpressing tobacco plants. Lane M, DL2000 DNA marker; Lane W, water as negative control; Lane P, plasmid pCAMBIA1301-VvIAA18 as positive control; Lane WT, wild type; Lanes L1-L6, different transgenic lines.

Figure 6

Real-time quantitative PCR expression analysis of VvIAA18 gene in transgenic tobacco plants. The expression of VvIAA18 in vitro-grown plants of 6 transgenic plants and wild type was analyzed. The tobacco Ntactin gene was used as an internal control. Data are presented as means ± SE (n = 3). The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.01 (**).

Figure 7

The growth and rooting of transgenic plants were compared and WT was cultured for 4 weeks on MS medium with no stress or 200 mM NaCl. Data are presented as means ± SE (n=3). The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.01 (**).

Figure 8

Responses of the transgenic tobacco plants and WT grown in pots under salt stress. (a), Phenotypes of the transgenic tobacco plants grown in pots under 200 mM NaCl stress. (b), Biomass of the transgenic tobacco plants grown in pots under 200 mM NaCl stress. Data are presented as means ± SE (n = 3). The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.01 (**).

Figure 9

Southern blot analysis of the transgenic plants to detect the copy number of integrated hptⅡ gene. WT, wild type; L1, L4 and L5, enhanced salt tolerance transgenic plants.

Figure 10

Relative expression level of salt stress-responsive genes in the leaves of transgenic tobacco plants and WT under salt stress. The tobacco Ntactin gene was used as an internal control. Results are expressed as relative values with respect to WT, which was set to 1.0. Data are presented as means ± SE (n = 3). The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.05 and p < 0.01 (**).

Figure 11

The content of proline, H₂O₂ and malondialdehyde(MDA), and the activities of superoxide dismutase (NtSOD) and peroxidase (NtPOD) in the leaves of transgenic tobacco plants and WT under salt stress. Data are presented as means ± SE (n = 3). The results were analyzed by Student’s t-test in a two-tailed analysis. Significance was defined as p < 0.01 (**).

Figure 12

Hypothesis of the regulatory network of the VvIAA18 gene involved in salt stress response. Constitutive expression of VvIAA18 up-regulates the genes involved in proline biosynthesis and reactive oxygen species (ROS) scavenging, which result in significant physiological changes, including increased proline level and reduced ROS accumulation, leading to the enhanced salt tolerance.