Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco
Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30 μM aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The 13C-NMR analysis with NaH13CO3 showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500 μM Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.
KeywordsCitrate synthase Phosphoenolpyruvate carboxylase Al toxicity Al tolerance Transgenic tobacco
This work was supported in part by grants from the National Basic Research Program of China (No. 2007CB108901) and the Foundation (2004PY01-5) of Yunnan Province and Kunming University of Science and Technology for Training Adult and Young Leaders of Science and Technology.
- Coelho GTCP, Carneiro NP, Karthikeyan AS, Raghothama KG, Schaffert RE, Brandão RL, Paiva LV, Souza IRP, Alves VM, Imolesi A (2010) A phosphate transporter promoter from Arabidopsis thaliana AtPHT1;4 Gene drives preferential gene expression in transgenic maize roots under phosphorus starvation. Plant Mol Biol Rep 28:717–723CrossRefGoogle Scholar
- Hoekenga OA, Maron LG, Pineros MA, Cancxado GM, ShaV J, Kobayashi Y, Ryan PR, Dong B, Delhaize E, Sasaki T, Matsumoto H, Yamamoto Y, Koyama H, Kochian LV (2006) AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci USA 103:9738–9743PubMedCrossRefGoogle Scholar
- Ljudmila SS, Klimentina DK, Zlatimira S (2000) Total and Rubisco specific proteolytic activity during dark induced senescence of barley seedlings. Bulg J Plant Physiol 26:15–26Google Scholar
- Wu HF, Liu XL, You LP, Zhang LB, Yu JB, Zhou D, Zhao JM, Feng JH (2011) Salinity- induced effects in the halophyte Suaeda salsa using NMR-based metabolomics. Plant Mol Biol Rep. doi: 10.1007/s11105-011-0368-4