GhCAX3 Gene, a Novel Ca2+/H+ Exchanger from Cotton, Confers Regulation of Cold Response and ABA Induced Signal Transduction

As a second messenger, Ca2+ plays a major role in cold induced transduction via stimulus-specific increases in [Ca2+]cyt, which is called calcium signature. During this process, CAXs (Ca2+/H+ exchangers) play critical role. For the first time, a putative Ca2+/H+ exchanger GhCAX3 gene from upland cotton (Gossypium hirsutum cv. ‘YZ-1′) was isolated and characterized. It was highly expressed in all tissues of cotton except roots and fibers. This gene may act as a regulator in cotton’s response to abiotic stresses as it could be up-regulated by Ca2+, NaCl, ABA and cold stress. Similar to other CAXs, it was proved that GhCAX3 also had Ca2+ transport activity and the N-terminal regulatory region (NRR) through yeast complementation assay. Over-expression of GhCAX3 in tobacco showed less sensitivity to ABA during seed germination and seedling stages, and the phenotypic difference between wild type (WT) and transgenic plants was more significant when the NRR was truncated. Furthermore, GhCAX3 conferred cold tolerance in yeast as well as in tobacco seedlings based on physiological and molecular studies. However, transgenic plant seeds showed more sensitivity to cold stress compared to WT during seed germination, especially when expressed in N-terminal truncated version. Finally, the extent of sensitivity in transgenic lines was more severe than that in WT line under sodium tungstate treatment (an ABA repressor), indicating that ABA could alleviate cold sensitivity of GhCAX3 seeds, especially in short of its NRR. Meanwhile, we also found that overexpression of GhCAX3 could enhance some cold and ABA responsive marker genes. Taken together, these results suggested that GhCAX3 plays important roles in the cross-talk of ABA and cold signal transduction, and compared to full-length of GhCAX3, the absence of NRR could enhance the tolerance or sensitivity to cold stress, depending on seedling’s developmental stages.作为第二信使，钙离子（Ca2+）通过刺激特异性的细胞质游离钙离子浓度（[Ca2+]cyt）升高参与低温诱导的信号转导过程，该过程被称为钙信号特征（calcium signature）。在此过程中，CAXs（Ca2+/H+交换蛋白）发挥关键调控作用。本研究首次从陆地棉（Gossypium hirsutum cv. ‘YZ-1’）中分离并鉴定得到推定的Ca2+/H+交换蛋白编码基因GhCAX3。该基因在棉花除根系与纤维外的所有组织中均呈高表达，且可被钙离子、氯化钠（NaCl）、脱落酸（ABA）及低温胁迫上调表达，因此可能参与棉花对非生物胁迫的应答调控。与其他CAXs类似，本研究通过酵母互补实验证实，GhCAX3具备钙离子转运活性，且其N端调控区域（NRR）参与该功能的调控。在烟草中过表达GhCAX3后，种子萌发期与幼苗期均表现出对ABA不敏感的表型；当NRR被截短后，野生型与转基因植株间的表型差异更为显著。进一步的生理与分子实验证实，GhCAX3可赋予酵母与烟草幼苗低温耐受性。然而，在种子萌发阶段，转基因植株种子对低温胁迫的敏感性较野生型更高，尤其是当NRR截短型GhCAX3过表达时。此外，经ABA抑制剂钨酸钠处理后，转基因株系的敏感程度较野生型更为严重，这表明ABA可缓解GhCAX3转基因种子的低温敏感表型，在其NRR缺失时该效应尤为显著。与此同时，本研究还发现过表达GhCAX3可上调部分低温与ABA应答标记基因的表达。综上，上述结果表明GhCAX3在ABA与低温信号转导的交叉串扰中发挥重要作用；且相较于完整长度的GhCAX3，NRR的缺失可根据幼苗发育阶段的不同，增强植株对低温胁迫的耐受性或敏感性。