Abstract
Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway for the initial fixation of atmospheric CO2 into the four-carbon (C4) acid, oxaloacetate. Here, we report that a vector carrying the intact maize C4pepc gene was used to transform C3 monocot japonica rice cultivar Tainung 67 (TNG67) which is an important cultivar in Taiwan, and C3 dicot Cleome spinosa which is closely related to the C4 dicot Cleome gynandra, via an Agrobacterium-mediated method. In total, 29 transgenic rice and 4 transgenic C. spinosa plants were obtained. Genomic PCR and southern blot analyses revealed that the maize pepc and selective antibiotic resistant genes were present in almost all randomly-selected transgenic plants but not in wild-type plants. Reverse transcription-PCR showed that maize pepc mRNA was detected in transgenic plants. Western blot analysis confirmed the presence of 110 kDa maize PEPC protein in transgenic plants but not in wild-type plants. Furthermore, higher PEPC enzyme activities were detected in transgenic rice plants than in transgenic C. spinosa plants. Taken together, we clearly demonstrated monocot maize C4pepc gene is functional in both C3 monocot rice and C3 dicot C. spinosa plants. Two T5 homozygous rice lines, each harboring a single insertion of maize pepc gene, showed 7- to 9.4-fold and 40–54% PEPC enzyme activity as compared to the untransformed rice and maize, respectively. These stable transgenic rice lines will be valuable material for studying the effect of maize PEPC on rice photosynthesis under different conditions.
Highlights
Most important crops including rice, wheat, soybean and potato are C3 plants that assimilate atmospheric CO2 directly through the C3 photosynthetic pathway, in which a C3 acid phosphoglycerate is formed via ribulose 1,5-bisphosphate carboxylase (Rubisco) in mesophyll cells
phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway, catalyzing the initial fixation of atmospheric CO2; the expression of C4-type pepc gene in C3 rice plants is thought to be a primary event in the establishment of C4 photosynthetic activity in rice to improve the
The vector PEPCgenome/pBIH2 carrying intact maize pepc genomic DNA fragment and two selective antibiotic resistant genes was used to transform the monocot rice and dicot C. spinosa plants by Agrobacterium-mediated method. Both pepc-specific and hygromycin phosphotransferase II (hptII)-specific fragments were detected in 28 transgenic plants regenerated from a total of 819 seed-derived embryogenic calli (Fig. 4a, b)
Summary
Most important crops including rice, wheat, soybean and potato are C3 plants that assimilate atmospheric CO2 directly through the C3 photosynthetic pathway, in which a C3 acid phosphoglycerate is formed via ribulose 1,5-bisphosphate carboxylase (Rubisco) in mesophyll cells. It is generally agreed that overexpression of a single maize pepc gene encoding PEPC protein or even quadruple transformants overexpressing all four key enzymes in the C4 photosynthetic pathway [i.e., PEPC, pyruvate, orthophosphate dikinase (PPDK), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME)] into rice plants do not show dramatic improvements in photosynthesis under normal conditions (Taniguchi et al 2008; Kajala et al 2011; Miyao et al 2011). The enzyme activities of PPDK and PEPC in these wheat lines were only about twofold and fourfold, respectively, higher than those of the control plants These expression levels are much lower than those achieved in the transgenic rice plants (Ku et al 1999; Taniguchi et al 2008). Whether the differences in expression levels of these maize genes in the C3 rice and wheat affect their photosynthetic performance and other physiological traits under different conditions remain to be investigated
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