Ectopic expression of GsPPCK3 and SCMRP in Medicago sativa enhances plant alkaline stress tolerance and methionine content

Abstract

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.

Figure 1. Expression patterns of the Glycine soja PPCK family genes under 50 mM NaHCO₃ (pH 8.5) treatment based on the microarray data.

a. Expression patterns of the PPCK family genes under alkali stress in Glycine soja roots. b. Expression patterns of the PPCK family genes under alkali stress in Glycine soja leaves.

Figure 2. Multiple alignment between GsPPCK3 and homologous PPCKs from Arabidopsis and soybean based on the full-length amino acid sequences.

The 11 subdomains of the catalytic domain were marked as solid lines. The protein kinase ATP-binding signature (GxGxxG), the Ser/Thr kinase active site signature (VAHRDIKPDNILF) and the conserved G-T/S-XX-Y/F-X-APE motif were marked as black solid boxes. Sequences were aligned by using ClustalW, and gaps were introduced to maximize alignment.

Figure 3. Expression patterns of GsPPCK3 in Glycine soja.

a. Expression levels of GsPPCK3 were up-regulated by alkali stress in both roots and leaves. Total RNA was extracted from leaves and roots of the 3-week-old Glycine soja seedlings treated with 50 mM NaHCO₃ (pH 8.5) for the indicated time points, respectively. Relative transcript levels were determined by quantitative real-time PCR analysis with GAPDH as an internal control. The mean values from three fully independent biological repeats and three technical repeats were shown. *P<0.05; **P<0.01 by Student’s t-test. b. Tissue expression specificity of GsPPCK3 in Glycine soja. Total RNA was extracted from different tissues of Glycine soja seedlings. Significant differences (P<0.05 by Duncan’s Multiple Range Test) were indicated by different lowercase letters.

Figure 4. GsPPCK3 overexpression in alfalfa conferred enhanced alkaline tolerance.

a. Semi-quantitative RT-PCR identification of GsPPCK3 transgenic alfalfa. b. Growth performance of WT and transgenic lines under control conditions or 100 mM NaHCO₃ treatment. Photographs were taken 12 days after initial treatment. c. The relative membrane permeability of WT and transgenic plants. d. The MDA content of WT and transgenic plants. e. The root activity of WT and transgenic plants. f. The total chlorophyll content of WT and transgenic plants. For phenotypic analysis of GsPPCK3 transgenic alfalfa, the 3-week-old WT and GsPPCK3 transgenic plants with similar sizes were treated with 1/8 Hoagland nutrient solution containing either 0, or 50, or 100 mM NaHCO₃ every 3 days for a total of 12 days.

Figure 5. Increased alkaline tolerance of GsPPCK3-SCMRP overexpression transgenic alfalfa.

a. Semi-quantitative RT-PCR analysis of GsPPCK3 and SCMRP expression levels in WT and trangenic lines. b. Western blot identification of GsPPCK3-SCMRP transgenic alfalfa. c. Growth performance of WT and transgenic lines after alkali treatment. Photographs were taken 15 days after initial treatment. d. Shoot length of the WT and transgenic lines. e. Shoot weight and root weight of the WT and transgenic lines. For phenotypic analysis of GsPPCK3-SCMRP transgenic alfalfa, the 4-week-old plants were treated with 1/8 Hoagland nutrient solution containing either 0, or 100, or 150 mM NaHCO₃ every 3 days for a total of 15 days.

Figure 6. GsPPCK3 overexpression altered a set of physiological indices and expression levels of stress responsive genes.

a. The PEPC activity of the WT and GsPPCK3-SCMRP transgenic lines under alkali treatment. b. The net photosynthetic rate of the WT and GsPPCK3-SCMRP transgenic lines. c. The citric acid content of the WT and transgenic lines. d. Increased expression levels of PEPC in GsPPCK3-SCMRP transgenic plants under alkali stress (50 mM NaHCO₃, pH 8.5). e. Increased expression levels of NADP-ME in GsPPCK3-SCMRP transgenic plants. f. Increased expression levels of H⁺-ATPase in GsPPCK3-SCMRP transgenic plants. g. Increased expression levels of CS in GsPPCK3-SCMRP transgenic plants. To explore expression patterns of stress-responsive genes, the 4-week-old WT and GsPPCK3-SCMRP transgenic seedlings after shoot cottage were treated with 50 mM NaHCO₃ (pH 8.5) for 0, 3, 6, 12 and 24 h, respectively. Relative transcript levels were determined by quantitative real-time PCR with GAPDH as internal reference, and were normalized to WT plants at 0 h. Values represented the means of three fully independent biological replicates, and three technological replicates for each.