Overexpression of a SBP-Box Gene (VpSBP16) from Chinese Wild Vitis Species in Arabidopsis Improves Salinity and Drought Stress Tolerance

Abstract

Salinity and drought are two major abiotic stresses that limit grape productivity. Responses to stress in grape are known to be regulated by several families of transcription factors. However, little is known about the role of grape Squamosa promoter binding protein (SBP)-box transcription factor genes in response to abiotic stress. To better understand the functions of the grape SBP-box genes in abiotic stress tolerance, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP16 was amplified from Chinese wild grapevine Vitis pseudoreticulata clone "Baihe-35-1". We observed that the VpSBP16 protein fused to the green fluorescent protein (GFP) reporter accumulated in the nucleus when transiently expressed in onion epidermal cells. Moreover, VpSBP16 was shown to have transcriptional activation activity using a yeast trans-activation assay. We performed a VpSBP16 functional analysis through the characterization of transgenic Arabidopsis thaliana plants constitutively over-expressing VpSBP16. The transgenic lines had longer roots and the seeds had a higher germination rate than the wild type (WT) under osmotic stress. In addition, the accumulation of reactive oxygen species (ROS) of transgenic seedlings was significantly lower than WT in the transgenic lines, as was electrolyte leakage. VpSBP16 overexpression also elevated expression levels of stress-response genes involved in the salt overly sensitive (SOS) pathway. These results indicate that overexpression VpSBP16 in A. thaliana enhances tolerance of salt and drought stress during seed germination, as well in seedlings and mature plants, by regulating SOS and ROS signaling cascades.

Keywords: ROS; SOS; Vitis pseudoreticulata; VpSBP16; drought; salt.

Figure 1

Cloning of VpSBP16 from V. pesudoreticulata. (A) PCR amplification of the full-length VpSBP16 cDNA and (B) DNA from Vitis pseudoreticulata. M1: DNA marker DL5000; M2: DNA marker λ-Hind III; 1: VpSBP16 PCR product from cDNA; 2 and 3: VpSBP16 PCR product.

Figure 2

The DNA, cDNA nucleotide sequence and deduced amino acid sequence of VpSBP16 from V. pesudoreticulata (A) and Exon-intron structures of VpSBP16 (B). The SBP domain is shown in red and the two zinc-binding sites of the C2HCH type (zinc finger 1 and zinc finger 2) are indicated with green and yellow boxes. The conserved basic amino acids of the nuclear location signal are shaded in dark grey.

Figure 3

Subcellular localization and transcriptional activation function of VpSBP16. (A) Subcellular localization of the VpSBP16-GFP (bottom row) fusion protein (top row) in onion epidermal cells. White arrowheads indicate the location of the nucleus in onion epidermal cell, scale bars: 50 μm. (B) Transcriptional activation function of VpSBP16 in yeast. Yeast cells containing the different plasmids grown on SD/-Trp select medium (left). Yeast cells containing the different plasmids grown on SD/-Trp-His-Ade+X-α-gal selection medium (right). 1: Positive control. (pGBKT7-Gal4); 2: pGBKT7-VpSBP16; 3: Negative control (pGBKT7). The experiments were repeated three times with consistent results.

Figure 4

Phenotypes of wild type (WT) and VpSBP16 transgenic Arabidopsis thaliana lines at the seed germination stage placed under osmotic stress (A–D) Photographs of seed germination in WT and transgenic lines 14 days after seeds were cultivated on Murashige-Skoog (MS) basal medium, MS basal medium supplemented with 150 mM NaCl or 400 mM mannitol. The experiments were repeated three times with consistent results, scale bars: 1 cm. (E) Seed germination rates of WT and transgenic lines cultivated on MS basal medium, MS basal medium supplemented with 150 mM NaCl or 400 mM mannitol for 14 days, respectively. Each data point is the mean of three replicates of 100–150 seeds. The error bars indicate the SD.

Figure 5

Analysis of the osmotic tolerance of WT and VpSBP16 transgenic A. thaliana seedlings. (A–C) Photographs of seedlings sown on MS basal medium, MS basal medium supplemented with 150 mM NaCl or 400 mM mannitol for 14, 35 and 20 days, respectively. The experiments were repeated three times with consistent results, scale bars: 1 cm. (D–G) Root growth (D–F) and root length (G) of WT and transgenic lines grown on MS basal medium, MS basal medium supplemented with 150 mM NaCl or 400 mM mannitol for 14, 35 and 20days, respectively. The experiments were repeated three times with consistent results, scale bars: 1 cm. (H) Water loss rate from 2-week-old detached transgenic and WT plants grown on the same MS basal medium, measured over a 50 min experimentation period. (I) Electrolyte leakage of seedlings of WT and the VpSBP16-1, VpSBP16-14 and VpSBP16-47 transgenic plants sampled at the last time point of dehydration. Asterisks indicated values that are significantly different from WT (n = 50 for each genotype, * 0.01< p < 0.05, one-way ANOVA). Each data point is the mean of three replicates of ten detached plants.

Figure 6

Growth of WT and VpSBP16 transgenic A. thaliana plants in pots under non-stress or osmotic stress conditions. (A) Representative photographs of 3-week-old WT and transgenic lines before salt treatment and after salt treatment for 1 week. The experiments were repeated three times with consistent results, scale bars: 1 cm.(B) Survival rates of WT and transgenic lines 3 days after re-watering. Each data point is the mean of three replicates. The error bars indicate the SD. (n = 50 for each genotype, * 0.01< p < 0.05, one-way ANOVA). (C) Representative photographs showing the phenotype of plants before drought, 18 days after drought treatment and 3 days after re-watering. The experiments were repeated three times with consistent results, scale bars: 1 cm.

Figure 7

Histochemical staining assay of ROS accumulation with nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) in 2-week-old wild type (WT), or VpSBP16-1, VpSBP16-14 and VpSBP16-47 transgenic lines after salt treatment for 1 week or after the transpirational water loss from 50 min. The experiment was repeated three times with 5–10 leaves or plants.

Figure 8

Expression levels of abiotic stress responsive genes in 3-week-old WT and VpSBP16 transgenic A. thaliana plants. AtActin1 was used as internal control for qRT-PCR. Mean values and SDs were obtained from three technical and three biological.