Functional Analysis Of Cotton Ca²⁺/H⁺Exchanger Genes, GHCAX1and GHCAX3,Against Abiotic Stresses

As a second messenger, Ca2+plays 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 roles. For the first time, putative Ca2+/H+exchanger GhCAX3and GhCAXl genes from upland cotton (Gossypium hirsutum cv.’YZ-1’) were isolated and characterized. GhCAX3and GhCAX1genes may act as a regulator in cotton responsive to abiotic stresses as it could be up-regulated by Ca2+, NaCl, ABA and cold stress. Similar to other CAXs, it was proved that GhCAX3and GhCAXl also had Ca2+transport activity and the N-terminal regulatory region (NRR) through yeast complementation assay. Over-expression of GhCAX3in tobacco showed less sensitivity to ABA during seed germination and seedling stages, but the phenotypic difference between wild type (WT) and transgenic plants was more significant when the NRR was truncated. Furthermore, GhCAX3conferred cold tolerance in yeast as well as tobacco seedlings based on the investigation at physiological and molecular level. 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 ABA could alleviate cold sensitivity of GhCAX3seeds, especially in short of its NRR. Meanwhile, we also found that overexpression of GhCAX3could enhance some cold and ABA responsive marker genes. Taken together, these results suggested that GhCAX3played important roles in the cross-talk of ABA and cold signal transduction, and compared with full-length of GhCAX3, the absence of NRR could enhance the tolerance or sensitivity extent against stresses. Overexpression of GhCAXl in tobacco also showed less sensitivity to ABA, Li+and Mn2+during seed germination and seedling development. Under ABA treatment, transgenic plant seeds showed more tolerance compared to WT during seed germination, especially when expressed in N-terminal truncated version. GhCAX1conferred enhanced tolerance of transgenic tobacco plants to cold under chilling or freezing temperatures. GhCAXl also showed tolerance to cold during seedling development, extent of tolerance was same both in N-terminal truncated form and full-length. Of special note, treatment of the transgenic tobacco plants with POD inhibitors elevated the H2O2levels and greatly compromised their cold tolerance. In addition, transgenic tobacco showed more tolerant to oxidative stresses during seed germination, seedling development and maturation. Taken together, these findings exhibit that GhCAX1plays a pivotal role in cold tolerance, at least in part, by positively regulating POD-mediated reactive oxygen species elimination.