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Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco.

Xiang Y, Lu YH, Song M, Wang Y, Xu W, Wu L, Wang H, Ma Z - PLoS ONE (2015)

Bottom Line: Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants.Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions.Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.

View Article: PubMed Central - PubMed

Affiliation: Guizhou Rapeseed Institute, Guizhou Academy of Agricultural Sciences, Guiyang, China; Crop Genomics and Bioinformatics Center and National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China.

ABSTRACT
Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.

No MeSH data available.


Related in: MedlinePlus

Purification of the 6xHis:TaCRT1 protein (A) and Scatchard plot of calcium binding to the affinity-purified TaCRT1 protein (B).The 6xHis and 6xHis:TaCRT1 proteins isolated from non-induced cells (lanes 1 and 3) and IPTG-induced cells (lanes 2 and 4) and purified TaCRT1 proteins obtained after thrombin cleavage (lane 5) were resolved on an SDS-PAGE gel and stained with Coomassie Blue. Lanes 1–4 contain 2 μg of protein each.
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pone.0140591.g003: Purification of the 6xHis:TaCRT1 protein (A) and Scatchard plot of calcium binding to the affinity-purified TaCRT1 protein (B).The 6xHis and 6xHis:TaCRT1 proteins isolated from non-induced cells (lanes 1 and 3) and IPTG-induced cells (lanes 2 and 4) and purified TaCRT1 proteins obtained after thrombin cleavage (lane 5) were resolved on an SDS-PAGE gel and stained with Coomassie Blue. Lanes 1–4 contain 2 μg of protein each.

Mentions: The complete ORF of TaCRT1 was cloned in a pET32a vector and expressed in E. coli. The recombinant protein was produced in E. coli after induction with IPTG. The 6xHis:TaCRT1 protein migrated on a SDS-PAGE gel with a mobility corresponding to a protein of approximately 70 kD (Fig 3A, lane 4), and the purified TaCRT1 protein, after thrombin cleavage, migrated with a mobility corresponding to a protein of approximately 50 kD (Fig 3A, lane 5), a weight that is slightly larger than the predicted molecular mass (47.2 kD). This difference might be due to glycosylation of the calreticulin. For the 6xHis-only control, the expected band at a position corresponding to 20 kDa was observed (Fig 3A, lane 2). The calcium binding assay showed that the TaCRT1 protein bound approximately 33.3 mol of Ca2+ per mol of protein (Fig 3B). Taken together, these results provide direct evidence that the gene product of TaCRT1 is an authentic calcium-binding protein.


Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco.

Xiang Y, Lu YH, Song M, Wang Y, Xu W, Wu L, Wang H, Ma Z - PLoS ONE (2015)

Purification of the 6xHis:TaCRT1 protein (A) and Scatchard plot of calcium binding to the affinity-purified TaCRT1 protein (B).The 6xHis and 6xHis:TaCRT1 proteins isolated from non-induced cells (lanes 1 and 3) and IPTG-induced cells (lanes 2 and 4) and purified TaCRT1 proteins obtained after thrombin cleavage (lane 5) were resolved on an SDS-PAGE gel and stained with Coomassie Blue. Lanes 1–4 contain 2 μg of protein each.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4607401&req=5

pone.0140591.g003: Purification of the 6xHis:TaCRT1 protein (A) and Scatchard plot of calcium binding to the affinity-purified TaCRT1 protein (B).The 6xHis and 6xHis:TaCRT1 proteins isolated from non-induced cells (lanes 1 and 3) and IPTG-induced cells (lanes 2 and 4) and purified TaCRT1 proteins obtained after thrombin cleavage (lane 5) were resolved on an SDS-PAGE gel and stained with Coomassie Blue. Lanes 1–4 contain 2 μg of protein each.
Mentions: The complete ORF of TaCRT1 was cloned in a pET32a vector and expressed in E. coli. The recombinant protein was produced in E. coli after induction with IPTG. The 6xHis:TaCRT1 protein migrated on a SDS-PAGE gel with a mobility corresponding to a protein of approximately 70 kD (Fig 3A, lane 4), and the purified TaCRT1 protein, after thrombin cleavage, migrated with a mobility corresponding to a protein of approximately 50 kD (Fig 3A, lane 5), a weight that is slightly larger than the predicted molecular mass (47.2 kD). This difference might be due to glycosylation of the calreticulin. For the 6xHis-only control, the expected band at a position corresponding to 20 kDa was observed (Fig 3A, lane 2). The calcium binding assay showed that the TaCRT1 protein bound approximately 33.3 mol of Ca2+ per mol of protein (Fig 3B). Taken together, these results provide direct evidence that the gene product of TaCRT1 is an authentic calcium-binding protein.

Bottom Line: Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants.Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions.Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.

View Article: PubMed Central - PubMed

Affiliation: Guizhou Rapeseed Institute, Guizhou Academy of Agricultural Sciences, Guiyang, China; Crop Genomics and Bioinformatics Center and National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China.

ABSTRACT
Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.

No MeSH data available.


Related in: MedlinePlus