Abstract
In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) – PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.
Highlights
Environmental stresses such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural status of the environment [1]
A cDNA fragment of approximately 600 bp was amplified by RTPCR using degenerate oligos based on conserved regions of other known Calcium-dependent protein kinases (CDPKs) in the database
As many drought-inducible genes were shown to be responsive to exogenous abscissic acid (ABA), we examined the effect ABA on the expression of ZoCDPK1
Summary
Environmental stresses such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural status of the environment [1]. Calcium-dependent (calmodulinindepedent) protein kinases (CDPKs) are unique sensorresponder proteins in higher plants that decodes and translates the elevation of Ca2+ concentration into enhanced protein kinase activity and subsequent downstream signaling events [4]. The basic structural features of CDPKs are conserved. They have a five domain structure with an aminoterminal variable domain, a kinase domain, a junction domain (JD), a regulatory domain (CaM-LD, calmodulin-like domain) and a carboxy terminal variable domain [6]. The JD between the kinase and CaM-LD functions as a pseudo-substrate autoinhibitor that inhibits phosphorylation in the absence of Ca2+ and keeps the CDPK in a state of low activity [7]. The CaM-LD of CDPKs consist of two structural domains (termed the N and C “lobes”), each containing two EF hand helix-loop-helix Ca2+-binding motifs. Activation of CDPK occurs when Ca2+ levels rise to fill the two weaker affinity-binding sites in the N-lobe, thereby triggering a conformational change that leads to release of autoinhibitory region [8]
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