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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

Analysis of CaCIPK25 promoter for tissue-specific expression. (A) Schematic diagram of CaCIPK25 promoter-GUS reporter construct (pCaCIPK25-GUS) and its two truncated versions (pCaCIPK25D1-GUS and pCaCIPK25D2-GUS). Positions (not to scale) of ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements are shown in gray and red lines. Transcription and translation start sites (+1 and ATG) are marked. (B) Expression of GUS driven by different deletion constructs of CaCIPK25 promoters in whole seedlings, root apex, cotyledon, and leaves and flowers are shown.
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Figure 4: Analysis of CaCIPK25 promoter for tissue-specific expression. (A) Schematic diagram of CaCIPK25 promoter-GUS reporter construct (pCaCIPK25-GUS) and its two truncated versions (pCaCIPK25D1-GUS and pCaCIPK25D2-GUS). Positions (not to scale) of ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements are shown in gray and red lines. Transcription and translation start sites (+1 and ATG) are marked. (B) Expression of GUS driven by different deletion constructs of CaCIPK25 promoters in whole seedlings, root apex, cotyledon, and leaves and flowers are shown.

Mentions: The 2.2 kb promoter sequence/5′UAS was analyzed for the presence of putative cis-acting elements. There were several dehydration responsive element/C-repeat (GTCGAC), ABA-responsive element (ACGTG), ARR1AT (NGATT), auxin responsive elements (TGTCTC), and W-box element (TTGAC/TGAC) are present within this region of the promoter explaining differential expression of CaCIPK25 upon treatments with salt, PEG, ABA, BAP, and IAA. In addition to those, multiple copies of ROOTMOTIFTAPOX1 (ATATT) (24 copies in the positive strand) and POLLEN1LELAT52 (AGAAA) (9 copies in the positive strand) elements, which drive root-specific and anther-specific expression, respectively, were found. Detail analysis of cis-acting elements in the 5′UAS of CaCIPK25 is presented in Supplementary Text. ROOTMOTIFTAPOX1 element was first identified in the promoter region of the rolD gene of Agrobacterium rhizogenes. The GUS gene driven by rolD promoter strongly expressed in the roots and expressed at a very low level in stem and leaves of tobacco plants. The distinctive expression pattern of the rolD promoter-GUS construct was that the strongest GUS activity was observed in the root elongation zone and vascular tissue and a very low expression in the root apex (Elmayan and Tepfer, 1994), highly consistent with the expression pattern of pCaCIPK25-GUS construct. A high GUS activity driven by CaCIPK25 promoter was observed in the root, cotyledon and reproductive organs and a very low GUS activity was observed in the true leaves. The pollen-specific cis-acting element POLLEN1LELAT52 was previously reported in the 5′UAS of tomato endo-β-mannase 5 gene (LeMAN5). Transgenic Arabidopsis plants expressing GUS driven by LeMAN5 promoter showed strong GUS activity in the anthers and pollens. In anthers, the highest LeMAN5 mRNA expression was observed in the later stages of flower development (Filichkin et al., 2004), similar to pCaCIPK25-GUS expression in flower. To delineate the CaCIPK25 promoter regions driving the root and flower-specific expression, two promoter deletion constructs were used. The first deletion construct (pCaCIPK25D1) was made by retaining −1 to −1047 bases of the promoter. The first deletion of 1150 bases from the 5′-end removed 13 ROOTMOTIFTAPOX1 elements out of twenty-four and one POLLEN1LELAT52 elements out of nine (Figure 4A). Arabidopsis plants expressing GUS driven by pCaCIPK25D1 showed a substantial reduction of GUS-staining in cotyledons, petals, anthers, and stigma. However, decrease in GUS stain in roots was not so profound, suggesting the −1046 to −2196 region of the promoter was more relevant for the expression of the gene in cotyledon, petal and anther tissues. The second deletion construct (pCaCIPK25D2) removed next 700 bases and all the ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements. This deletion totally abolished GUS expression in petals and cotyledons. There was a substantial reduction in GUS expression in roots and anthers, but the removal of all the ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements did not totally abolish the GUS expression in these two tissues, suggesting that the immediate 378 bases from the transcription start site of the promoter was also responsible, although modestly, for root- and anther-specific expression (Figure 4B).


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)

Analysis of CaCIPK25 promoter for tissue-specific expression. (A) Schematic diagram of CaCIPK25 promoter-GUS reporter construct (pCaCIPK25-GUS) and its two truncated versions (pCaCIPK25D1-GUS and pCaCIPK25D2-GUS). Positions (not to scale) of ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements are shown in gray and red lines. Transcription and translation start sites (+1 and ATG) are marked. (B) Expression of GUS driven by different deletion constructs of CaCIPK25 promoters in whole seedlings, root apex, cotyledon, and leaves and flowers are shown.
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Related In: Results  -  Collection

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Figure 4: Analysis of CaCIPK25 promoter for tissue-specific expression. (A) Schematic diagram of CaCIPK25 promoter-GUS reporter construct (pCaCIPK25-GUS) and its two truncated versions (pCaCIPK25D1-GUS and pCaCIPK25D2-GUS). Positions (not to scale) of ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements are shown in gray and red lines. Transcription and translation start sites (+1 and ATG) are marked. (B) Expression of GUS driven by different deletion constructs of CaCIPK25 promoters in whole seedlings, root apex, cotyledon, and leaves and flowers are shown.
Mentions: The 2.2 kb promoter sequence/5′UAS was analyzed for the presence of putative cis-acting elements. There were several dehydration responsive element/C-repeat (GTCGAC), ABA-responsive element (ACGTG), ARR1AT (NGATT), auxin responsive elements (TGTCTC), and W-box element (TTGAC/TGAC) are present within this region of the promoter explaining differential expression of CaCIPK25 upon treatments with salt, PEG, ABA, BAP, and IAA. In addition to those, multiple copies of ROOTMOTIFTAPOX1 (ATATT) (24 copies in the positive strand) and POLLEN1LELAT52 (AGAAA) (9 copies in the positive strand) elements, which drive root-specific and anther-specific expression, respectively, were found. Detail analysis of cis-acting elements in the 5′UAS of CaCIPK25 is presented in Supplementary Text. ROOTMOTIFTAPOX1 element was first identified in the promoter region of the rolD gene of Agrobacterium rhizogenes. The GUS gene driven by rolD promoter strongly expressed in the roots and expressed at a very low level in stem and leaves of tobacco plants. The distinctive expression pattern of the rolD promoter-GUS construct was that the strongest GUS activity was observed in the root elongation zone and vascular tissue and a very low expression in the root apex (Elmayan and Tepfer, 1994), highly consistent with the expression pattern of pCaCIPK25-GUS construct. A high GUS activity driven by CaCIPK25 promoter was observed in the root, cotyledon and reproductive organs and a very low GUS activity was observed in the true leaves. The pollen-specific cis-acting element POLLEN1LELAT52 was previously reported in the 5′UAS of tomato endo-β-mannase 5 gene (LeMAN5). Transgenic Arabidopsis plants expressing GUS driven by LeMAN5 promoter showed strong GUS activity in the anthers and pollens. In anthers, the highest LeMAN5 mRNA expression was observed in the later stages of flower development (Filichkin et al., 2004), similar to pCaCIPK25-GUS expression in flower. To delineate the CaCIPK25 promoter regions driving the root and flower-specific expression, two promoter deletion constructs were used. The first deletion construct (pCaCIPK25D1) was made by retaining −1 to −1047 bases of the promoter. The first deletion of 1150 bases from the 5′-end removed 13 ROOTMOTIFTAPOX1 elements out of twenty-four and one POLLEN1LELAT52 elements out of nine (Figure 4A). Arabidopsis plants expressing GUS driven by pCaCIPK25D1 showed a substantial reduction of GUS-staining in cotyledons, petals, anthers, and stigma. However, decrease in GUS stain in roots was not so profound, suggesting the −1046 to −2196 region of the promoter was more relevant for the expression of the gene in cotyledon, petal and anther tissues. The second deletion construct (pCaCIPK25D2) removed next 700 bases and all the ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements. This deletion totally abolished GUS expression in petals and cotyledons. There was a substantial reduction in GUS expression in roots and anthers, but the removal of all the ROOTMOTIFTAPOX1 and POLLEN1LELAT52 elements did not totally abolish the GUS expression in these two tissues, suggesting that the immediate 378 bases from the transcription start site of the promoter was also responsible, although modestly, for root- and anther-specific expression (Figure 4B).

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