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Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco.

Meena MK, Ghawana S, Dwivedi V, Roy A, Chattopadhyay D - Front Plant Sci (2015)

Bottom Line: Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions.Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants.Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL) proteins sense specific temporal changes in cytosolic Ca(2+) concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs). Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologs has been reported so far. In the present study, an ortholog of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum). CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS) of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

No MeSH data available.


Related in: MedlinePlus

Kinase assay of CaCIPK25 protein. Autophosphorylation and substrate phosphorylation assay of bacterially expressed GST-CaCIPK25 and CaCIPK25T/D proteins. GST protein was used as control. Myelin basic protein (MBP) was used as substrate. A Coomassie blue-stained gel shows the amount of CaCIPK25 protein loaded.
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Figure 5: Kinase assay of CaCIPK25 protein. Autophosphorylation and substrate phosphorylation assay of bacterially expressed GST-CaCIPK25 and CaCIPK25T/D proteins. GST protein was used as control. Myelin basic protein (MBP) was used as substrate. A Coomassie blue-stained gel shows the amount of CaCIPK25 protein loaded.

Mentions: In order to biochemically characterize the CaCIPK25 protein, the CDS was expressed in E. coli as a glutathione-S-transferase (GST)-fused protein. The conserved threonine (T171) residue located at the activation domain was substituted with aspartic acid (D) and mutated recombinant protein was expressed similarly. The purified recombinant proteins were tested for auto- and substrate phosphorylation. GST-CaCIPK25 showed a low level of auto-kinase activity. The T171D substitution increased the autokinase activity of the protein by about 2-fold. GST-CaCIPK25 was able to use myelin basic protein as a substrate and the aspartic acid substitution increased the kinase activity protein by about 2.5-fold using this substrate (Figure 5).


Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco.

Meena MK, Ghawana S, Dwivedi V, Roy A, Chattopadhyay D - Front Plant Sci (2015)

Kinase assay of CaCIPK25 protein. Autophosphorylation and substrate phosphorylation assay of bacterially expressed GST-CaCIPK25 and CaCIPK25T/D proteins. GST protein was used as control. Myelin basic protein (MBP) was used as substrate. A Coomassie blue-stained gel shows the amount of CaCIPK25 protein loaded.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561800&req=5

Figure 5: Kinase assay of CaCIPK25 protein. Autophosphorylation and substrate phosphorylation assay of bacterially expressed GST-CaCIPK25 and CaCIPK25T/D proteins. GST protein was used as control. Myelin basic protein (MBP) was used as substrate. A Coomassie blue-stained gel shows the amount of CaCIPK25 protein loaded.
Mentions: In order to biochemically characterize the CaCIPK25 protein, the CDS was expressed in E. coli as a glutathione-S-transferase (GST)-fused protein. The conserved threonine (T171) residue located at the activation domain was substituted with aspartic acid (D) and mutated recombinant protein was expressed similarly. The purified recombinant proteins were tested for auto- and substrate phosphorylation. GST-CaCIPK25 showed a low level of auto-kinase activity. The T171D substitution increased the autokinase activity of the protein by about 2-fold. GST-CaCIPK25 was able to use myelin basic protein as a substrate and the aspartic acid substitution increased the kinase activity protein by about 2.5-fold using this substrate (Figure 5).

Bottom Line: Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions.Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants.Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL) proteins sense specific temporal changes in cytosolic Ca(2+) concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs). Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologs has been reported so far. In the present study, an ortholog of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum). CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS) of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

No MeSH data available.


Related in: MedlinePlus