Studies On The Relationship Of PEPC And Malate And Development Of C₄Photosynthetic Pathway In Maize Leaves

In C4plant maize, there are packed thylakoid lamellaes and active PS II in the bundle sheath chlorophasts of the lower position (1-3). Herein the CO2is fixed by Rubisco in C3photosynthetic pathway. But in the upper position leaves (＞4), the CO2is initially fixed by PEPC to synthesize malate in mesophyll cells and then transported to chlorophasts in the bundle sheath. Therein, malate is decarboxylated to relaese CO2and fixed by Rubisco in C3photosynthesis. In these BSC chlorophasts, the grana disappears and the activity of PS II is deficient,and NADPH is seldomly produced. Therefore, the supply of the reducing power from MC is necessary for C3cycle. As a linker and regulator in many cell metalsolisms, in the C4photosynthetic cycle of maize, malate is not only the carrier of CO2from MC to BSC but also the transporter of the reducing power to BSC from MC. As decarboxylated, malate produces NADPH in the BSC chloroplasts. As a result, the redox potential is affected by the decarboxylation of malate in these chlorophasts. It may be related to the differentiation of grana and the development of PS II activities in BSC chlorophasts and involves the regulation of C4photosynthetic pathway during the C4plant evolution and individual development. To investigate the function and mechanism of PEPC action via malate synthesis on the development of BSC chlorophasts. In this research, we measured the PEPC activities, malate contents and anatomy of leaves with different carbon assimilation pathway, observed the effect of exogenous malate on light reaction of detached leaves of maize and tobacco and on the photosynthesi of attatched leaves of transgenic PEPC rice, and constructed a plant transformation vector of RNAi for maize C4-PEPC gene. We expected that this study would provide a new thinking and theoretical basis for the exploration on the formation mechanism of C4photosynthetic pathway and the differentiation mechanism of the bundle sheath chlorophasts and also provide reference for the modification of the photosynthetic pathway in C3plants by transgenic technology. The main results are as follows:1. Close relationship between PEPC activities, malate metabolism and BSC differentiation. In this study, during the development of maize leaves, the activities of PEPC, NADP-ME, and other photosynthetic enzymes increased, C4anatomy traits were more distinctive, and malate metabolism was enhanced. The light reaction was inhibited in detached leaves of maize in exogenous malate solution and the photosynthesis was affected in attatced leaves in rice leaves fed with exogenous malate via roots. The results showed that there was a close relationship between PEPC activities, malate metabolism and development of C4anatomy.2. Mechanism of PEPC and malate action in the development of photosynthetic pathway in plants.(1) during malate treatment, in the chloroplasts, the amount of NADP decreased and the ratio of NADPH/NADP increased, resulting in the inhibition of electron transport and the production of reactive oxygen species;(2) the decarboxylation and dehydrogenation reaction of malate changed BSC redox state and then imposed effects on the differentiation of the structure and function of PS Ⅱ;(3) pyruvic acid produced by malate decarboxylation may affect cell redox state and the structure of PS Ⅱ.3. Mechanism of C4-PEPC action in transgenic rice. In the leaves of transgenic rice, the expression of PEPC gene enhanced the malate metabolism. OAA generated by PEPC was unevenly transported into different chloroplasts and impoesd a different effect on the redox state of different chloroplast.Malate produced in high reducing power chloroplasts and decarboxylated in low reducing power chloroplasts. A part of chloroplasts played the role of mesophyll chloroplasts in C4plants and the other part took a role of BSC chloroplasts.The micro cycle of C4photosynthesis run between these two types of chloroplasts.4. Cloning, plant expression vector construction of C4-PEPC gene from maize and the transformation into northern super japonica rice. In this study, the promotor and full-length sequence of C4-PEPC gene were cloned by two-step PCR from the maize inbred line Zheng58. The whole fragment length was7530bp. The pCAMBIA-1391Z-PEPC vector was attained by ligation to form linear vector using In-Fusion technology. The transformation of the maize C4-PEPC gene into northern super japonica rice was mediating by agrobacterium. The dramatic increase of PEPC activities was detected in the transgenic rice leaves.5. Construction of plant transformation vector of RNAi for maize C4-PEPC gene. A intron segments of C4-PEPC gene was selected to be RNAi sequence. At first, the forward gene segments and intron were cloned to475bp. Then it was ligated reverse gene segments (387bp). Finally, a plant transformation vector of RNAi (p7002-Z-F) was constructed succesfully. Because this interval sequence of RNAi vector was different from others, the effect of RNA interference effect needed to further explore.According to the results, we could make a preliminary conclusion. The PEPC played a role in the differentiation and development of BSC chlorophasts in maize via malate metabolism. In BSC, malate was decarboxylated to produce pyruvic acid and NADPH which consumed the NADP in cloroplasts. The deficiency of NADP might inhibit the linear electron transport and led to the generation of reactive oxygen species, which resulted in the change in the structure of PSII reaction centers. Thus we proposed that PEPC activities increased during plant evolution and synthesized more malate. Which was transported into BSC and participated in the regulation on the disappearance of grana stack and the chlorophast differentiation in BSC differring from MC chloroplasts.