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So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a  Glycine soja  transcriptional profile, and identified three PPCK genes ( GsPPCK1 ,  GsPPCK2  and  GsPPCK3 ) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of  GsPPCK3  under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed  GsPPCK3  in  Medicago sativa  and found that  GsPPCK3  overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the  GsPPCK3  and  SCMRP  genes into alfalfa, and demonstrated the increased alkali tolerance of  GsPPCK3 - SCMRP  transgenic lines. Further investigation revealed that  GsPPCK3 - SCMRP  co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of  PEPC ,  CS  (citrate synthase),  H + -ATPase  and  NADP-ME  genes in  GsPPCK3 - SCMRP  transgenic alfalfa under alkali stress. As expected, we demonstrated that  GsPPCK3 - SCMRP  transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of  GsPPCK3  in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.

Ectopic Expression of GsPPCK3 and SCMRP in Medicago sativa Enhances Plant Alkaline Stress Tolerance and Methionine Content