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Cloning of Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 gene (GhSnRK2) and its overexpression in transgenic Arabidopsis escalates drought and low temperature tolerance.

Bello B, Zhang X, Liu C, Yang Z, Yang Z, Wang Q, Zhao G, Li F - PLoS ONE (2014)

Bottom Line: Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants.GhSnRK2 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 GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cotton Biology, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
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 Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) 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 GhSnRK2 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 GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 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 GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.

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Physiological assay of GhSnRK2 gene silenced plants.(A) Phenotype of drought stressed plants. Water was withheld from GhSnRK2 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 GhSnRK2 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 GhSnRK2 gene silenced and non-silenced plants under salt stress. (F) The phenotype of GhSnRK2 gene silenced and non-silenced plants under salt stress.
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pone-0112269-g009: Physiological assay of GhSnRK2 gene silenced plants.(A) Phenotype of drought stressed plants. Water was withheld from GhSnRK2 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 GhSnRK2 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 GhSnRK2 gene silenced and non-silenced plants under salt stress. (F) The phenotype of GhSnRK2 gene silenced and non-silenced plants under salt stress.

Mentions: To further elucidate the function of the GhSnRK2 gene in stress tolerance, we investigated the response of GhSnRK2gene silenced plants to various stresses. Our findings validated the importance of this technique for stress tolerance studies. Under water-deficient conditions, the silenced plants wilted and drooped, regardless of the cultivar (Figure 9A). The phenotype of the non-silenced plants inoculated with empty vector (TRV) was indistinguishable from that of the WT plants. The two cotton cultivars (CRI99668 and CRI409) inoculated with the target gene displayed similar phenotypes and symptoms. The WT and control vector-treated plant survival rates were 80% and 77.3%, respectively, whereas the survival rate of the two gene silenced cotton cultivars (CRI99668 and CRI409) were 34.66% and 38.67%, respectively (Figure 9B). Student's T-test revealed a significant difference (p<0.05) between the survival rate of the gene silenced and non-silenced plants. The rate of water loss from the detached leaves was determined in both the gene silenced and non-silenced plants (Figure 9C). After monitoring the water loss for 6-h, we found that the GhSnRK2 gene silenced plants inoculated lost more water than the non-silenced plants inoculated with empty vector and the WT plants. The rate of water loss was slightly higher in the CRI99668 cultivars than in the CRI409 cultivars. Under similar conditions, the gene silenced plants, regardless of the cultivar, exhibited a substantially reduced water content compared with the non-silenced plants inoculated with empty vector and the WT plants (Figure 9D). Oxidative stress, including that mediated by salinity, alters the physiological and morphological responses of plants. To investigate the effect of salt stress on GhSnRK2 gene silenced plants, the gene silenced and non-silenced plants were treated with 150 mM NaCl for 7 days, and the effect of NaCl was measured following this stress. Our findings revealed that the chlorophyll content of the non-silenced plants was higher than that of the gene silenced plants, indicating that the GhSnRK2 gene may be involved in the oxidative stress response (Figure 9E). Growth retardation was detected in the GhSnRK2 silenced plants, and the effect of salt accumulation in the plant cells was detected as elevated blisters, which were visibly detectable on the leaf surface of GhSnRK2 gene silenced plants (Figure 9F).


Cloning of Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 gene (GhSnRK2) and its overexpression in transgenic Arabidopsis escalates drought and low temperature tolerance.

Bello B, Zhang X, Liu C, Yang Z, Yang Z, Wang Q, Zhao G, Li F - PLoS ONE (2014)

Physiological assay of GhSnRK2 gene silenced plants.(A) Phenotype of drought stressed plants. Water was withheld from GhSnRK2 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 GhSnRK2 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 GhSnRK2 gene silenced and non-silenced plants under salt stress. (F) The phenotype of GhSnRK2 gene silenced and non-silenced plants under salt stress.
© Copyright Policy
Related In: Results  -  Collection

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pone-0112269-g009: Physiological assay of GhSnRK2 gene silenced plants.(A) Phenotype of drought stressed plants. Water was withheld from GhSnRK2 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 GhSnRK2 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 GhSnRK2 gene silenced and non-silenced plants under salt stress. (F) The phenotype of GhSnRK2 gene silenced and non-silenced plants under salt stress.
Mentions: To further elucidate the function of the GhSnRK2 gene in stress tolerance, we investigated the response of GhSnRK2gene silenced plants to various stresses. Our findings validated the importance of this technique for stress tolerance studies. Under water-deficient conditions, the silenced plants wilted and drooped, regardless of the cultivar (Figure 9A). The phenotype of the non-silenced plants inoculated with empty vector (TRV) was indistinguishable from that of the WT plants. The two cotton cultivars (CRI99668 and CRI409) inoculated with the target gene displayed similar phenotypes and symptoms. The WT and control vector-treated plant survival rates were 80% and 77.3%, respectively, whereas the survival rate of the two gene silenced cotton cultivars (CRI99668 and CRI409) were 34.66% and 38.67%, respectively (Figure 9B). Student's T-test revealed a significant difference (p<0.05) between the survival rate of the gene silenced and non-silenced plants. The rate of water loss from the detached leaves was determined in both the gene silenced and non-silenced plants (Figure 9C). After monitoring the water loss for 6-h, we found that the GhSnRK2 gene silenced plants inoculated lost more water than the non-silenced plants inoculated with empty vector and the WT plants. The rate of water loss was slightly higher in the CRI99668 cultivars than in the CRI409 cultivars. Under similar conditions, the gene silenced plants, regardless of the cultivar, exhibited a substantially reduced water content compared with the non-silenced plants inoculated with empty vector and the WT plants (Figure 9D). Oxidative stress, including that mediated by salinity, alters the physiological and morphological responses of plants. To investigate the effect of salt stress on GhSnRK2 gene silenced plants, the gene silenced and non-silenced plants were treated with 150 mM NaCl for 7 days, and the effect of NaCl was measured following this stress. Our findings revealed that the chlorophyll content of the non-silenced plants was higher than that of the gene silenced plants, indicating that the GhSnRK2 gene may be involved in the oxidative stress response (Figure 9E). Growth retardation was detected in the GhSnRK2 silenced plants, and the effect of salt accumulation in the plant cells was detected as elevated blisters, which were visibly detectable on the leaf surface of GhSnRK2 gene silenced plants (Figure 9F).

Bottom Line: Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants.GhSnRK2 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 GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cotton Biology, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
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 Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) 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 GhSnRK2 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 GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 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 GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.

Show MeSH
Related in: MedlinePlus