| dc.description.abstract | Crop plants are constantly exposed to pathogens and various environmental stresses, such as cold, salinity, and drought that cause major losses in productivity. Plants normally respond rapidly to these biotic and abiotic stresses. Efficient perception of biotic and abiotic stresses and cell-programmed signaling mechanisms for appropriate responses are important for growth and survival of plants. Calcium is an important secondary messenger in signaling pathways that respond to environmental stresses, pathogen attacks, as well as hormonal stimuli (for review, see Reddy and Reddy, 2004; Sanders et al., 2002). The transient increase of cytosolic free calcium concentration has been shown in a variety of external signals (Reddy, 2001), which in turn triggers many signal transduction pathways involved in a variety of cellular responses (Bush, 1995). The activity of many protein kinases can respond to [Ca²]cyt signals directly (Poovaiah and Reddy, 1993). These can be categorized in four classes, viz. Ca²-dependent protein kinases (CPKs), CPK-related kinases (CRKs), calmodulin (CaM)-dependent protein kinases (CaMKs), and chimeric Ca²- and CaM-dependent protein kinases (CCaMKs) (White and Broadley, 2003).
In plants, calcium-dependent protein kinases (CPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca² levels to a specific response. These enzymes are activated through calcium binding at the calmodulin-like domain and require only micromolar concentrations of free calcium for their activity (Harper et al., 1991; Roberts and Harmon, 1992). Activated CPKs alter protein phosphorylation or relative gene expression through transduction of calcium signals (Cheng et al., 2002). The biological properties and locations of different CPKs are proposed to determine their precise cellular activities, including defense/stress responses. Moreover, it is not known how specific calcium activation of a particular CPK is regulated upon stress and other signals and whether it is regulated in a tissue-specific manner.
In this study, we isolated and characterized two chickpea cDNAs encoding calcium-dependent protein kinases, designated CaCPK1 and CaCPK2. The deduced amino acid sequences of CaCPK1 and CaCPK2 contain the structures typical of the CPK family, including an N-terminal variable domain, a protein kinase domain, an autoinhibitory domain, and a calmodulin-like domain. The CaCPK1 possesses four EF hands, as predicted by a motif search, whereas CaCPK2 has three EF hands. All known plant CPK sequences contain a unique N-terminal region preceding the protein kinase catalytic domain. In the predicted CaCPK1 and CaCPK2 protein sequences, the N-terminal regions are 79 and 63 amino acids, respectively. Amino acid sequence alignment of CaCPK1 and CaCPK2 with other plant CPKs shows no homology in the N-terminal region. Genomic DNA blot analysis with the N-terminal variable regions of CaCPK1 and CaCPK2 as probes suggests that CaCPK1 and CaCPK2 exist as single-copy genes in the chickpea genome. Sequence and phylogenetic tree analyses show that CaCPK1 is similar to DcCPK, whereas CaCPK2 is similar to AtCPK7.
Chickpea CaCPK1 and CaCPK2 cDNAs encode functional CPKs when heterologously expressed in E. coli. The pH optima of CaCPK1 and CaCPK2 were found to be in a range between pH 6.8 to 8.6 and pH 7.2 to 9.3, respectively. The optimum temperature required for the activity of CaCPK1 and CaCPK2 was found in the range of 35–42°C. The Km values of CaCPK1 and CaCPK2 for histone III-S were determined to be 47 µM and 79 µM, respectively. Comparison of the ratio of Vmax/Km, which is indicative of catalytic efficiency, shows that CaCPK1 has a stronger preference for histone III-S as a substrate than CaCPK2.
The influence of Na and Mg² on the recombinant CaCPK1 and CaCPK2 substrate phosphorylation activity was tested in this work: in both cases, addition of NaCl strongly inhibited CaCPK1 and CaCPK2 activities. The inhibition of substrate phosphorylation activities of recombinant CaCPK1 and CaCPK2 by salt implies ionic interactions between the substrate and the active sites of these enzymes. In our assay, the optimum concentration of Mg² for CaCPK1 and CaCPK2 activities was found to be 8–10 mM. Inhibition of these activities was observed above 10 mM Mg², which suggests the disruption of ionic interactions between the enzymes and the substrate.
Recombinant CaCPK1 and CaCPK2 exhibit Ca² requirement for substrate phosphorylation activity. The submicromolar levels of Ca² required for maximal phosphorylation of recombinant CaCPK1 and CaCPK2 in the in vitro assays indicate the activator role of Ca² for this phosphorylation. The [Ca²] for half-maximal activity (K0.5) was found to be 0.04 µM for CaCPK1 and 0.08 µM for CaCPK2 with histone III-S as substrate. It is also interesting to note that there are differences in the number of predicted EF hands present (four in CaCPK1 and three in CaCPK2) in the CaM-like domain of these kinases. This suggests that the difference in half-maximal activity for [Ca²] observed for CaCPK1 and CaCPK2 could be due to the difference in the number of EF hands present in the CaM-like domain of each isoform. Furthermore, the failure of CaM to enhance substrate phosphorylation and autophosphorylation activities of recombinant CaCPK1 and CaCPK2 and their inhibition by W7 and calmidazolium indicated that these activities were supported by a calmodulin-like domain, which is typical of the CPK family.
To characterize the function of these CPK isoforms, their expression in various organs and in response to various phytohormones, dehydration, high salt stress, and fungal spore stimuli, as well as localization in leaf and stem tissues, were analyzed in this study. The expression of CaCPK1, accumulation of CaCPK1 protein, and its activity were ubiquitous in all tissues examined. In contrast, CaCPK2 transcript, protein, and activity were almost undetectable in reproductive tissues (flowers and fruits). Both CaCPK1 and CaCPK2 transcripts, proteins, and activities were detected in abundance in roots and in minor quantities in leaves and stems. Of the three phytohormones, viz., IAA, GA, and BA tested, only BA specifically increased both CaCPK1 and CaCPK2 transcripts, proteins, and their activities. The two genes differed in their response to GA. Accumulation of CaCPK2 transcript, protein, and activity was induced by GA but remained unaffected for CaCPK1 by exogenously supplied GA. Accumulation of transcripts, protein, and activity did not change in response to IAA for either isoform.
In leaves, the expression of CaCPK1 and CaCPK2 enhanced in response to high salt stress. Although high salt increased the expression of both genes, the induction kinetics were different. The expression of CaCPK1, its protein accumulation, and activity were increased in chickpea leaf tissue in response to treatment with fungal spores. On the other hand, treatment with fungal spores had no effect on the abundance of transcripts, proteins, and activity of CaCPK2. Furthermore, excised leaves subjected to dehydration stress showed increases in CaCPK2 transcript, protein, and activity, and no effects were noted for CaCPK1. Results of immunofluorescence and immunogold studies indicated that both isoforms were located in the plasma membrane and chloroplast membrane of leaf palisade mesophyll cells, as well as in the plasma membrane of stem xylem parenchyma cells. Together, these results suggest specific roles for CaCPK1 and CaCPK2 isoforms in phytohormone (GA/BA), defense, and stress signaling pathways. | |
