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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

Morphology of CaCIPK25 overexpressing transgenic tobacco lines. (A) Morphological comparison of vertically grown tobacco seedlings transformed with empty vector (Con), CaCIPK25 and CaCIPK25T171D genes. Transgenic seeds from two lines (L1 and L2) are represented for all the constructs, upper and lower panels are showing 2− and 15−days old seedlings after germination, respectively. (B) Comparison of root lengths of 15-days old seedlings. Averages of three measurements of 15 seedlings each are shown. * indicates statistically significant difference (p < 0.05) from the control sample. (C) Root system of soil-grown 50 days-old transgenic tobacco plants. Roots exposed to the soil surface are shown.
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Figure 6: Morphology of CaCIPK25 overexpressing transgenic tobacco lines. (A) Morphological comparison of vertically grown tobacco seedlings transformed with empty vector (Con), CaCIPK25 and CaCIPK25T171D genes. Transgenic seeds from two lines (L1 and L2) are represented for all the constructs, upper and lower panels are showing 2− and 15−days old seedlings after germination, respectively. (B) Comparison of root lengths of 15-days old seedlings. Averages of three measurements of 15 seedlings each are shown. * indicates statistically significant difference (p < 0.05) from the control sample. (C) Root system of soil-grown 50 days-old transgenic tobacco plants. Roots exposed to the soil surface are shown.

Mentions: To investigate in planta function, CaCIPK25 and CaCIPK25T171D (henceforth referred to as CaCIPK25T/D) were expressed in tobacco plants under the control of 35S promoter. More than 10 transgenic lines were raised for each construct along with the lines harboring only pBI121 (vector-control) using the Agrobacterium-mediated transformation method. T3 homozygous plants were selected for experiments on the basis of expression analysis by RT-PCR (Supplementary Figure 1). Seeds of two individual lines for each construct along with the vector-control seeds were sown on ½MS-agar plates for germination. No difference was observed among the lines with respect to the percentage of and time taken for germination, however, all the lines expressing CaCIPK25 or CaCIPK25T/D displayed longer root length within 2 days of germination. After 15 days, the primary roots of the CaCIPK25− and CaCIPK25T/D-overexpressing lines were 52 and 60% longer than the roots of the control line (Figures 6A,B). The difference in root morphology was more evident in the matured plants. The CaCIPK25− and CaCIPK25T/D− overexpressing plants showed an enlarged root system as compared to the control plants when grown in soil for 50 days (Figure 6C). Although, the leaves of the seedlings expressing both the CaCIPK25 constructs were larger than those of the control seedlings at the early stages after germination probably due to longer root, there was no significant difference in leaf sizes observed between the control and CaCIPK25-expressing tobacco plants in the later stages of growth (Supplementary Figure 2).


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)

Morphology of CaCIPK25 overexpressing transgenic tobacco lines. (A) Morphological comparison of vertically grown tobacco seedlings transformed with empty vector (Con), CaCIPK25 and CaCIPK25T171D genes. Transgenic seeds from two lines (L1 and L2) are represented for all the constructs, upper and lower panels are showing 2− and 15−days old seedlings after germination, respectively. (B) Comparison of root lengths of 15-days old seedlings. Averages of three measurements of 15 seedlings each are shown. * indicates statistically significant difference (p < 0.05) from the control sample. (C) Root system of soil-grown 50 days-old transgenic tobacco plants. Roots exposed to the soil surface are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Morphology of CaCIPK25 overexpressing transgenic tobacco lines. (A) Morphological comparison of vertically grown tobacco seedlings transformed with empty vector (Con), CaCIPK25 and CaCIPK25T171D genes. Transgenic seeds from two lines (L1 and L2) are represented for all the constructs, upper and lower panels are showing 2− and 15−days old seedlings after germination, respectively. (B) Comparison of root lengths of 15-days old seedlings. Averages of three measurements of 15 seedlings each are shown. * indicates statistically significant difference (p < 0.05) from the control sample. (C) Root system of soil-grown 50 days-old transgenic tobacco plants. Roots exposed to the soil surface are shown.
Mentions: To investigate in planta function, CaCIPK25 and CaCIPK25T171D (henceforth referred to as CaCIPK25T/D) were expressed in tobacco plants under the control of 35S promoter. More than 10 transgenic lines were raised for each construct along with the lines harboring only pBI121 (vector-control) using the Agrobacterium-mediated transformation method. T3 homozygous plants were selected for experiments on the basis of expression analysis by RT-PCR (Supplementary Figure 1). Seeds of two individual lines for each construct along with the vector-control seeds were sown on ½MS-agar plates for germination. No difference was observed among the lines with respect to the percentage of and time taken for germination, however, all the lines expressing CaCIPK25 or CaCIPK25T/D displayed longer root length within 2 days of germination. After 15 days, the primary roots of the CaCIPK25− and CaCIPK25T/D-overexpressing lines were 52 and 60% longer than the roots of the control line (Figures 6A,B). The difference in root morphology was more evident in the matured plants. The CaCIPK25− and CaCIPK25T/D− overexpressing plants showed an enlarged root system as compared to the control plants when grown in soil for 50 days (Figure 6C). Although, the leaves of the seedlings expressing both the CaCIPK25 constructs were larger than those of the control seedlings at the early stages after germination probably due to longer root, there was no significant difference in leaf sizes observed between the control and CaCIPK25-expressing tobacco plants in the later stages of growth (Supplementary Figure 2).

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