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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

Activity of CaCIPK25 gene promoter in different tissues in transgenic Arabidopsis. CaCIPK25 gene promoter up to the translation start codon was fused to GUS gene and introduced in Arabidopsis to study promoter activity in different tissues. Representative samples were shown after histochemical GUS staining. Tissues shown from left were radicle, whole seedling, root apex, lateral root initial flower buds and flowers, flower at higher magnification.
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Figure 3: Activity of CaCIPK25 gene promoter in different tissues in transgenic Arabidopsis. CaCIPK25 gene promoter up to the translation start codon was fused to GUS gene and introduced in Arabidopsis to study promoter activity in different tissues. Representative samples were shown after histochemical GUS staining. Tissues shown from left were radicle, whole seedling, root apex, lateral root initial flower buds and flowers, flower at higher magnification.

Mentions: Expression profiling of CaCIPK25 by qRT-PCR in chickpea, showed its highest expression in flower was about 2.5-fold, while the expressions in stem and leaf were 0.5− and 0.3−folds, respectively, of its expression level in root. Similar tissue-specific expression profile of the corresponding transcript (TC05858) was reported using RNASeq data (Garg et al., 2011) (Figure 2A). Upon treatment with 250 mM of sodium chloride, CaCIPK25 transcript level increased at 1 h by 2.8-fold, reached at 5-fold by the 6th h and then declined to 3.9-fold at the 24th h. CaCIPK25 expression steadily increased by PEG treatment from 4-fold increase after 1 h to 48-fold increase after 24 h of treatment. Exposure to low-temperature did not affect its expression significantly. Both the ABA and auxin treatments enhanced CaCIPK25 expression, however, with different time kinetics. The expression quickly increased to 4.4-fold within 1 h of IAA treatment and then slowly declined to 1.8-fold at 24th h, whereas, transcript level increased slowly to 4.1-fold after 12 h of treatment with ABA and decreased to 3.9-fold after 24th h. Auxin and cytokinin function antagonistically and synergistically in root development. Accordingly, BAP treatment reduced CaCIPK25 expression by more than 2-fold after 6 and 24 h of treatment. Treatment with methyl jasmonate (MeJA) and salicylic acid (SA) resulted in similar expression profiles with a slow increase in transcript level to about 4-fold after 12 h and then decrease to about 2-fold after 24 h (Figure 2B). 2.2 kb upstream activation sequence (5′UAS/promoter) together with the 5′UTR (Supplementary Text 1) of CaCIPK25 gene was cloned and fused with the reporter gene β-glucuronidase (GUS) (pCaCIPK25-GUS) and introduced in Arabidopsis to monitor tissue-specific expression of the gene at different growth stages. pCaCIPK25-GUS displayed a strong expression all over the radicle just after germination. However, with growth, the primary and the lateral roots, except the root tips and lateral root initials comprising of meristem cells, showed strong expression of the gene. This differential GUS staining in root indicated that CaCIPK25 promoter activity is suppressed in tissues with high auxin concentration. The cotyledons, mostly the veins, showed moderate GUS expression. However, the true leaves showed a low GUS staining. The stem did not show any detectable GUS expression. As expected, the strongest expression was observed in flower, specifically, in the petals, anthers, and stigma. The promoter activity in flower was dependent on growth stage as GUS activity was not visible in the immature flowers (Figure 3).


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)

Activity of CaCIPK25 gene promoter in different tissues in transgenic Arabidopsis. CaCIPK25 gene promoter up to the translation start codon was fused to GUS gene and introduced in Arabidopsis to study promoter activity in different tissues. Representative samples were shown after histochemical GUS staining. Tissues shown from left were radicle, whole seedling, root apex, lateral root initial flower buds and flowers, flower at higher magnification.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: Activity of CaCIPK25 gene promoter in different tissues in transgenic Arabidopsis. CaCIPK25 gene promoter up to the translation start codon was fused to GUS gene and introduced in Arabidopsis to study promoter activity in different tissues. Representative samples were shown after histochemical GUS staining. Tissues shown from left were radicle, whole seedling, root apex, lateral root initial flower buds and flowers, flower at higher magnification.
Mentions: Expression profiling of CaCIPK25 by qRT-PCR in chickpea, showed its highest expression in flower was about 2.5-fold, while the expressions in stem and leaf were 0.5− and 0.3−folds, respectively, of its expression level in root. Similar tissue-specific expression profile of the corresponding transcript (TC05858) was reported using RNASeq data (Garg et al., 2011) (Figure 2A). Upon treatment with 250 mM of sodium chloride, CaCIPK25 transcript level increased at 1 h by 2.8-fold, reached at 5-fold by the 6th h and then declined to 3.9-fold at the 24th h. CaCIPK25 expression steadily increased by PEG treatment from 4-fold increase after 1 h to 48-fold increase after 24 h of treatment. Exposure to low-temperature did not affect its expression significantly. Both the ABA and auxin treatments enhanced CaCIPK25 expression, however, with different time kinetics. The expression quickly increased to 4.4-fold within 1 h of IAA treatment and then slowly declined to 1.8-fold at 24th h, whereas, transcript level increased slowly to 4.1-fold after 12 h of treatment with ABA and decreased to 3.9-fold after 24th h. Auxin and cytokinin function antagonistically and synergistically in root development. Accordingly, BAP treatment reduced CaCIPK25 expression by more than 2-fold after 6 and 24 h of treatment. Treatment with methyl jasmonate (MeJA) and salicylic acid (SA) resulted in similar expression profiles with a slow increase in transcript level to about 4-fold after 12 h and then decrease to about 2-fold after 24 h (Figure 2B). 2.2 kb upstream activation sequence (5′UAS/promoter) together with the 5′UTR (Supplementary Text 1) of CaCIPK25 gene was cloned and fused with the reporter gene β-glucuronidase (GUS) (pCaCIPK25-GUS) and introduced in Arabidopsis to monitor tissue-specific expression of the gene at different growth stages. pCaCIPK25-GUS displayed a strong expression all over the radicle just after germination. However, with growth, the primary and the lateral roots, except the root tips and lateral root initials comprising of meristem cells, showed strong expression of the gene. This differential GUS staining in root indicated that CaCIPK25 promoter activity is suppressed in tissues with high auxin concentration. The cotyledons, mostly the veins, showed moderate GUS expression. However, the true leaves showed a low GUS staining. The stem did not show any detectable GUS expression. As expected, the strongest expression was observed in flower, specifically, in the petals, anthers, and stigma. The promoter activity in flower was dependent on growth stage as GUS activity was not visible in the immature flowers (Figure 3).

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