CDPK1 from Ginger Promotes Salinity and Drought Stress Tolerance without Yield Penalty by Improving Growth and Photosynthesis in Nicotiana tabacum

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.

在植物中，高盐、干旱、极端温度及机械损伤等胁迫条件下，胞质钙离子浓度会发生瞬时变化。钙依赖蛋白激酶（CDPKs）在将这些钙信号传递至下游效应过程中发挥关键作用。本研究从生姜的胁迫相关cDNA文库中，通过cDNA末端快速扩增（RLM-RACE）-PCR技术分离得到一个胁迫响应型CDPK基因ZoCDPK1，并对其在胁迫耐受中的功能进行了鉴定。对推导所得蛋白的分析显示，其连接结构域中的核定位序列与类钙调蛋白结构域EF手结构环中的保守序列之间存在罕见的耦合现象。ZoCDPK1在生姜根状茎中呈高丰度表达，且可被高盐胁迫、干旱胁迫及茉莉酸处理快速诱导，但不受低温胁迫或脱落酸处理的诱导。本研究利用共聚焦激光扫描显微镜，在转基因烟草表皮细胞中对ZoCDPK1与绿色荧光蛋白（GFP）的融合蛋白进行了亚细胞定位分析。在烟草中过表达生姜CDPK1基因可赋予植株盐胁迫与干旱胁迫耐受性，具体表现为种子萌发率更高、相对含水量更高、胁迫响应基因表达量上调、叶片叶绿素含量更高、光合效率及其他光合参数提升。此外，经受盐/干旱胁迫的转基因烟草在胁迫条件下的生物量较正常生长的野生型植株高出50%。在持续盐胁迫或干旱胁迫条件下，T3代转基因烟草可正常生长至成熟，提早开花并结出可育种子，且无产量损耗。ZoCDPK1可上调烟草中胁迫相关基因RD21A与ERD1的表达水平。上述结果表明，ZoCDPK1通过正向调控生姜应答盐胁迫与干旱胁迫的信号通路发挥功能，且其调控路径大概率不依赖于脱水应答元件（DRE）/C-重复序列（CRT）通路。