Expression Of A Cation/H⁺Exchanger Of Puccinellia Tenuiflora, PutCAX2, Confers Ca²⁺ And Ba²⁺Tolerance In Yeast

Plants require metal ions as mineral nutrients for growth and maintenance of metabolic activity. The distributions of Ca2+and other ions among organelles and the regulation of cytoplasmic ion concentrations are important factors in plant development, growth and adaptation to stress. Cation/H+exchangers (CAXs) are a group of proteins that efflux Ca2+and other cations out the cytosol to maintain optimal ionic concentrations in cells, which were energized by the proton gradient established by H+-ATPase or H+-pyrophosphatase. Puccinellia tenuiflora is a kide of plant which can grow up in saline soil. It is very important to research Cation/H+exchangers of Puccinellia tenuiflora.In the present study, another Puccinellia tenuiflora gene PutCAX2encoding a putative cation/proton antiporter was cloned from Puccinellia tenuiflora cDNA and characterized. The closeness of PutCAX2to OsCAX1a in the tree thus suggests that PutCAX2is a type I-A CAX the same as OsCAX1c、OsCAX1b、AtCAX1、AtCAX3、AtCAX4. PutCAX2is characterised by10transmembrane (TM) helices predicted by computer algorithms such as TMHMM.Five transformed K667yeast lines were constructed. One was transformed with empty vector pYES2as a control, while four were transformed with a vector containing PutCAX2, sNPutCAX2, sCPutCAX2or sNCPutCAX2respectively. In the presence of Zn2+or Na+, the growth of the PutCAX2transformants were weaker than the growth of the control yeast. The sNPutCAX2and sNCPutCAX2transformant grew well in the presence of50mM Ca2+or in the presence of8mM Ba2+, while the other transformants did not grow well in these media. Even in the presence of200mM Ca2+or20mM Ba2+the sNPutCAX2and sNCPutCAX2transformants still grew well. On the other hand, the other transformants didn’t grow at all. So the N-terminal region of PutCAX2has the autoinhibition function.GFP fusion proteins of PutCAX2and mutant PutCAX2genes lacking the N-terminal, C-terminal, and both N-terminal and C-terminal regions (ΔNsPutCAX2, ΔCsPutCAX2, and ΔNCsPutCAX2) localized in the plasma membrane of yeast. As we know Tonoplast-localised proton-coupled Ca2+transporters encoded by CAX genes play a critical role in sequestering Ca2+into the vacuole. CAXs may have functions beyond the vacuole. PutCAX2, a plasma membrane CAX appears to be involved in removing toxic metals from the cell. In order to determine whether ΔNPutCAX2mediates Ba2+transport, the Ba2+concentrations in the yeast transformants were determined with an atomic absorption spectrophotometer.The yeast expressing sNPutCAX2and sNCPutCAX2showed lower total Ba2+than other CAXs transformants Expression of sCPutCAX2and sNCPutCAX1.It is not clear that the function of Ba2+to plants. There is almost no reports about Ba2+ transporter. We demonstrate that expression of PutCAX2in yeast increases Ca2+and Ba2+tolerance, and may be has a role in mediating Ba2+transport out of yeast, and that it might be useful in transforming other plants to promote their Ca2+and Ba2+tolerance.