Seed germination of the WT and <i>GhSnRK2</i> plants subjected to NaCl and exogenous ABA treatment and biomass accumulation of these plants.

<p>(A) Seed germination frequency of the WT and <i>GhSnRK2</i> transgenic plants cultured on MS medium supplemented with different concentrations of NaCl (50 mM or 100 mM) or ABA (0 µM, 0.3 µM, or 0.5 µM). (B) The survival rate of the WT and <i>GhSnRK2</i> transgenic plants cultured in MS medium containing 50 mM or100 mMNaCl. The mean values were compared using Student's T-test (p<0.05). (C) The germination rate in MS medium supplemented with 0.3 µM or 0.5 µM ABA. The values are presented as the mean germination rates (%) of approximately 200 seeds. Asterisk denotes a significant difference (P<0.05). (D) Biomass accumulation of the <i>GhSnRK2</i> transgenic and WT plants. For dry weight biomass, the dry weight in the roots and shoots was recorded after drying in an oven to a constant weight at 70°C for 48 h. (E) Fresh weight biomass of <i>GhSnRK2</i> transgenic plants and corresponding WT plants. The fresh weight of the roots and shoots was measured immediately after harvesting. Each of the three biological replicates consisted of 12 plants. Student's T-test was performed. Asterisk denotes a significant difference (P<0.05).</p

Plant transformation vector and the expression pattern of the <i>GhSnRK2</i> gene in the transgenic lines.

<p>(A) Schematic representation of the T-DNA region of the binary vector <i>pCAMBIA2301-GhSnRK2</i>. (B) Expression pattern of the <i>GhSnRK2</i> gene in the transgenic plants. Various upregulated expression patterns of the <i>GhSnRK2</i> gene in transgenic lines were detected, as indicated by the vertical axis. The values are presented as the means of three experimental replicates; the error bars indicate the standard deviations. The <i>AtACT2</i> gene was used as an internal control for normalization of gene expression.</p

Cloning of <i>Gossypium hirsutum</i> Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (<i>GhSnRK2</i>) and Its Overexpression in Transgenic <i>Arabidopsis</i> Escalates Drought and Low Temperature Tolerance

<div><p>The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the <i>Gossypium hirsutum</i> sucrose non-fermenting 1-related protein kinase 2 (<i>GhSnRK2</i>) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that <i>GhSnRK2</i> participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the <i>GhSnRK2</i>-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing <i>GhSnRK2</i> exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that <i>GhSnRK2</i> acts as a positive regulator in response to cold and drought stresses. Plants overexpressing <i>GhSnRK2</i> displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of <i>GhSnRK2</i> expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as <i>AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1</i>, and <i>AtABI5</i>, were increased in the <i>GhSnRK2</i>-overexpressing plants compared to the wild-type plants. <i>GhSnRK2</i> gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the <i>GhSnRK2</i> gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.</p></div

Phylogenetic analysis, subcellular localization, and expression pattern of <i>GhSnRK2</i> in cotton plant.

<p>(A) A phylogenetic tree of <i>GhSnRK2</i> and other <i>SnRK2</i> proteins from different plants was constructed using the neighbor-joining method with MEGA 5. The sequences used for analysis are listed by accession number: <i>Litchi chinensis</i> (<i>LcSnRK2</i>), AFX72761.1; <i>Arabidopsis thaliana</i> (<i>AtSNRK2.2</i>), CP002686.1; <i>Arabidopsis thaliana</i> (<i>AtSnRK2.3</i>), AED98274.1; <i>AtSnRK2.1</i>, AED91326.1; <i>AtSnRK2.9</i>, AEC07398.1; <i>AtSnRK2.10</i>, AEE33751.1; <i>AtSnRK2.4</i>, AEE28666.1; <i>AtSnRK2.5</i>, AED97781.1; <i>AtSnRK2.7</i>, AEE87152.1; <i>Populus tremula</i> (<i>PtreSnRK2.6a</i>), AGW51610.1; <i>Zea mays</i> (<i>ZmSnRK2.2</i>), NM_001137717.1; <i>Solanum tuberosum</i> (<i>StSnRK2.8</i>), AFR68945.1; <i>Oryza sativa</i> (<i>RK1</i>), ABB89146.1; <i>Oryza sativa</i> (<i>SAPK4)</i>, BAD18000.1; <i>Sorghum bicolor</i> (<i>SAPK4</i>), AGM39623.1; and <i>Zea mays</i> (<i>SAPK5</i>), ACG42286.1. The bootstrap values are shown on the tree branches. (B) Subcellular localization of the <i>GhSnRK2-GFP</i> protein. (2 and 5) GFP alone; (4 and 6) <i>GhSnRK2-GFP</i> in onion epidermal cells; (1 and 3) corresponding bright-field images. (C) The expression pattern of the <i>GhSnRK2</i> gene in cotton plants subjected to 10% PEG stress. The gene expression data were normalized to that of the cotton histone 3 gene. The values are presented as the means of three experimental replicates. The vertical axis represents the relative expression level. The values from1 to 6 indicate the time (h) of PEG treatment. Asterisk denotes a significant difference (P<0.05) compared with the control (0 h). (D) Relative expression levels of the <i>GhSnRK2</i> gene in various cotton plant tissues. Samples from root (RT) stem (ST), cauline leaves (CL), rosettes leaves (RL) and flowers (FL) were analyzed. The vertical axis represents the relative expression level. The letters denote significant differences (P<0.05) based on Duncan's multiple range tests. The cotton histone 3 gene was used as an internal control for normalization of the gene expression data.</p

Survival rates of <i>GhSnRK2</i> transgenic plants under drought and low temperature stresses.

<p>(<b>A</b>) WT and <i>GhSnRK2</i> transgenic plants before and after drought stress. (<b>B</b>) Seedlings were cultured for 2 weeks with constant watering before the withholding of water. After 9 days without water, all of the plants were irrigated, and plant re-growth was scored 4 days later. The plants were scored as survivors if there were healthy green young leaves after re-watering. The survival rate was calculated as the ratio of the number of surviving plants to the total number of treated plants in the flower pot. Asterisk denotes a significant difference (P<0.05). (<b>C</b>) WT and <i>GhSnRK2</i> transgenic plants before and after low temperature stress. (<b>D</b>) Survival rates under low temperature stress conditions. The survival rate after transferring 4-week-old <i>GhSnRK2</i> transgenic and WT plants to a low temperature chamber at 4°C, −4°C or −8°C for 10 h, followed by returning the plants to normal growth conditions. Clearly green plants after returning to the normal growth condition were scored as survivors, and plants exhibiting clear signs of wilting were denoted as dead. The mean survival rates of the WT line were compared with those of the transgenic lines using Student's T-test. (E) The water use efficiency of the WT and <i>GhSnRK2</i> transgenic plants. Asterisk denotes a significant difference (P<0.05).</p

Physiological assay of <i>GhSnRK2</i> gene silenced plants.

<p>(A) Phenotype of drought stressed plants. Water was withheld from <i>GhSnRK2</i> gene silenced and non-silenced plants for 5 days approximately two weeks post-inoculation. Each of the ten treated groups consisted of five plants. A photograph of each group representative was captured. (B) The survival rate was determined by withholding water from <i>GhSnRK2</i> gene silenced and non-silenced plants for 5 days approximately two weeks post-inoculation, and the survival rate was recorded as the percentage of plants that survived after re-watering for 3 days. (C) The relative water loss was determined two weeks after inoculation. The reduction in the fresh weight from the initial weight was determined at the indicated time and represented as the percentage of water loss. The experiment was replicated three times. (D) The mean RWC was analyzed after immersing the fresh detached leaves in distilled water for 4 h and oven drying at 80°C for 48 h. The values are presented as the means of three biological replicates. Asterisk denotes a significant difference (P<0.05). (E) The change in the chlorophyll content of <i>GhSnRK2</i> gene silenced and non-silenced plants under salt stress. (F) The phenotype of <i>GhSnRK2</i> gene silenced and non-silenced plants under salt stress.</p

Flow Cytometry Analysis of Cell Death Induced by VD-toxins.

<p>The protoplasts isolated from the T₃ transgenic lines (KB1) and non-transformed negative controls were treated with 30 mg/L and 60 mg/L VD-toxin for 3 hours and analyzed by flow cytometry after PI staining. Flow cytometry analysis was conducted using 10,000 protoplasts per sample. Percentages of dead protoplasts are indicated. Vertical axis refers to PI fluorescence intensity and the abscissa refers to the number of protoplast. Data are representative of three independent experiments.</p

Western blot assays of expression of P35 and OP-IAP protein in T₃ transgenic cotton lines.

<p>The total soluble protein extracted from the young leaves of the T₃ transgenic lines and non-transformed negative controls. (A) P35 expression. (B) OP-IAP expression. Lane 1 the negative control plant; lane 2, 3, 4 the three T₃ transgenic lines (KB1, KB2, KB3); lane M molecular mass marker is PageRuler ^TM prestained protein ladder (MBI Fermentas, Maryland, USA).</p

Seedling growth of the WT and <i>GhSnRK2</i>-overexpressing lines in response to NaCl and ABA treatment.

<p>(A) WT and <i>GhSnRK2</i> transgenic plants subjected to different concentrations of NaCl. (B) Approximately one-week-old seedlings were transferred to 1/2MS medium supplemented with different concentrations of NaCl; the root length was measured after 7 days. Each of the three biological replicates consisted of 16 plants. The mean values were compared using Student's T-test (p<0.05). (C) The root growth of the WT and <i>GhSnRK2</i> transgenic plants treated with different concentrations of ABA. (D) The plants were cultured vertically on MS-containing medium treated with different concentrations of ABA (0.5 <i>µ</i>m, 1 <i>µ</i>m, or 2 <i>µ</i>m); the root length was measured after 7 days. Each of the three biological replicates consisted of 16 plants. Student's T-test was used to compare the mean values of the WT and transgenic lines. Asterisk denotes a significant difference (P<0.05).</p

Biochemical and physiological assays of plants overexpressing the <i>GhSnRK2</i> gene.

<p>(A) Rate of water loss from <i>GhSnRK2</i> transgenic plants. Six plants of each transgenic and WT line were analyzed. Three biological replicates produced similar results. Asterisk denotes a significant difference (P<0.05). (B) The RWC of <i>GhSnRK2</i> transgenic plants. (C) Stomata aperture size of WT and <i>GhSnRK2</i> transgenic plants treated with different concentrations of ABA (D) The stomata apertures were measured after 2 h of treatment with different concentrations of ABA, and the mean values of the WT and transgenic lines at each ABA concentration were compared using Student's T-test (P<0.05). Asterisk denotes a significant difference (P<0.05). (E) Proline accumulation in WT and <i>GhSnRK2</i> transgenic plants. The proline content of <i>GhSnRK2</i> transgenic lines was consistently higher than that of the WT line. Student's T-test revealed a significant difference (p<0.05) between the transgenic and WT lines (n = 3). (F) The chlorophyll content of the WT and <i>GhSnRK2</i> transgenic plants. The mean values were compared using Student's “t-test”. Asterisk denotes a significant difference (P<0.05).</p