Limits...
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

Protein sequence alignment of AtCIPK25 and its ortholog CaCIPK25 in chickpea using ClustalW. The activation loop is shown in shaded region with the conserved threonine residue marked with an arrow. The NAF/FISL module responsible for interaction with CBL is shown in box.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561800&req=5

Figure 1: Protein sequence alignment of AtCIPK25 and its ortholog CaCIPK25 in chickpea using ClustalW. The activation loop is shown in shaded region with the conserved threonine residue marked with an arrow. The NAF/FISL module responsible for interaction with CBL is shown in box.

Mentions: A previously reported screening to identify dehydration-inducible chickpea expression sequence tags (ESTs) (Boominathan et al., 2004) yielded an EST (GenBank: CD051323) with high expression under dehydration. 5′ and 3′ RACE (Rapid amplification of cDNA ends) resulted in a cDNA clone of 1727 base pair (bp) in length. Deduced protein sequence of the clone displayed highest homology (67% identity and 81% similarity) with CIPK25 of Arabidopsis and annotated as chickpea CIPK25 by NCBI (National Center for sBiotechnology Information), hence, referred to as CaCIPK25 (C. arietinum CIPK25, NCBI: XP_004498818). The protein sequence showed 88% identity with a Medicago truncatula CBL-interacting protein kinase (XP_003588823.1) and 74% identity with CIPK25 of Theobroma cacao (XP_007011728.1). In addition to the 1257 bp long protein-coding sequence (CDS), the cDNA clone also possessed a 5′-untranslated region (5′UTR) of 174 bases in length and a 3′-untranslated region (3′UTR) of 295 bases in length (Supplementary Text 1). Sequencing of the genomic DNA clone and comparison with the genome sequence showed that the gene was intronless and was located on linkage group 4. The deduced protein sequence was 418 amino acids in length with estimated molecular mass 47.55 kDa. CaCIPK25 was quite shorter in size than its Arabidopsis ortholog, which is of 488 aa, however, like other CIPKs, possessed an N-terminal SNF-1-related serine/threonine protein kinase domain (12–266 aa) and a C-terminal regulatory domain (295–409 aa) with a CBL-interacting NAF/FISL module. The activation loop (DFG…APE) and the threonine residue (Thr171), substitution of which by aspartic acid in SOS2 resulted in constitutive kinase activity, were conserved in CaCIPK25 (Figure 1).


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)

Protein sequence alignment of AtCIPK25 and its ortholog CaCIPK25 in chickpea using ClustalW. The activation loop is shown in shaded region with the conserved threonine residue marked with an arrow. The NAF/FISL module responsible for interaction with CBL is shown in box.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Protein sequence alignment of AtCIPK25 and its ortholog CaCIPK25 in chickpea using ClustalW. The activation loop is shown in shaded region with the conserved threonine residue marked with an arrow. The NAF/FISL module responsible for interaction with CBL is shown in box.
Mentions: A previously reported screening to identify dehydration-inducible chickpea expression sequence tags (ESTs) (Boominathan et al., 2004) yielded an EST (GenBank: CD051323) with high expression under dehydration. 5′ and 3′ RACE (Rapid amplification of cDNA ends) resulted in a cDNA clone of 1727 base pair (bp) in length. Deduced protein sequence of the clone displayed highest homology (67% identity and 81% similarity) with CIPK25 of Arabidopsis and annotated as chickpea CIPK25 by NCBI (National Center for sBiotechnology Information), hence, referred to as CaCIPK25 (C. arietinum CIPK25, NCBI: XP_004498818). The protein sequence showed 88% identity with a Medicago truncatula CBL-interacting protein kinase (XP_003588823.1) and 74% identity with CIPK25 of Theobroma cacao (XP_007011728.1). In addition to the 1257 bp long protein-coding sequence (CDS), the cDNA clone also possessed a 5′-untranslated region (5′UTR) of 174 bases in length and a 3′-untranslated region (3′UTR) of 295 bases in length (Supplementary Text 1). Sequencing of the genomic DNA clone and comparison with the genome sequence showed that the gene was intronless and was located on linkage group 4. The deduced protein sequence was 418 amino acids in length with estimated molecular mass 47.55 kDa. CaCIPK25 was quite shorter in size than its Arabidopsis ortholog, which is of 488 aa, however, like other CIPKs, possessed an N-terminal SNF-1-related serine/threonine protein kinase domain (12–266 aa) and a C-terminal regulatory domain (295–409 aa) with a CBL-interacting NAF/FISL module. The activation loop (DFG…APE) and the threonine residue (Thr171), substitution of which by aspartic acid in SOS2 resulted in constitutive kinase activity, were conserved in CaCIPK25 (Figure 1).

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