Photosynthetic Characteristic And Physiological Mechanism Of C₄ Photosynthetic Gene Transgenic Rice

The photosynthesis of higher plants can be divided into three types -C₃,C₄,CAM, of which the latter two, evolved from the former one, were domesticated in adverse conditions for a long term. Compared to C₃ plants, C₄ plants exhibit higher photosynthetic efficiency and better utilization of nutrition and water than C₃ plants due to the C₄ pathway serving as a CO₂ pump to concentrate CO₂ in the leaves and suppress photorespiration, especially in the conditions of high light intensity, high temperature and drought.At the end of the 20th century, with the rapid development of transgenic technology, three maize C₄ photosynthesis enzymes, such as phosphoenolpyruvate carboxylase (PEPC), pyruvate-phosphate dikinase (PPDK) and NADP-dependent malic enzyme (NADP-ME), were successfully introduced into rice through agrobacteria-mediated transformation. The transgenic rice plants express these C₄ genes at high levels and the enzymes remain functional. To study the physiological characteristic of Ca photosynthetic enzyme transgenic rice, the photosynthetic response of transgenic rice plant to light and the characteristics of chlorophyll fluorescence under photo-inhibition/photo-oxidation conditions were compared. The light-saturated photosynthetic rate of PEPC and PPDK transgenic rice plants was higher than that of untransformed rice cultivar (Oryza sativa L. subsp, japonica cv. Kitaake), for the light-saturated point of PEPC transgenic rice plants was 200μmolm^-2 s^-1more than that of untransformed rice and the light-saturated photosynthetic rate was 53.1ï¼…higher. However, the light-saturated photosynthetic rates of ME transgenic rice plants were slightly lower than those of untransformed rice. In addition, after the treatment with highlight intensity (3h) and methyl viologen (MV), a photo-oxidative reagent, the PSII photochemical efficiency (F_v/F_m), actual photochemical efficiency of PSII under light (F PSII) and photochemical quenching (q^P) of PEPC transgenic rice plants decreased less than that of untransformed one, revealing that PEPC transgenic rice plants were more tolerant to photo-inhibition or photo-oxidation. But meanwhile, F_v/F_m, F PSII and q^P of ME transgenic rice plants declined more than that of untransformed rice, which suggested that it is incidental to photodamage under photo-inhibitive or photo-oxidative conditions.F₁ hybrids of PEPC+PPDK+ME transgenic rice were obtained by further crossing PEPC+PPDK transgenic rice as the male parent with ME transgenic rice as the female parent. Subsequent to the selection of C₄ enzyme activities and photosynthetic measurement, F₂ hybrids of PEPC+PPDK+ME transgenic rice over-expressed PEPC, PPDK and ME enzyme, and exhibited high photosynthetic rate. Under strong illumination, the photosynthetic rate of the PPDK transgenic rice was not increased compared with that of the untransformed rice. The rate was decreased by 7.6ï¼…for the ME transgenic rice, increased by 52.1ï¼…for the PEPC transgenic rice, and was similar with that of the PEPC transgenic rice for the PEPC+PPDK transgenic rice. After the treatment of ATP, the photosynthetic and the rate of O₂ evolution were increased significantly for the PEPC+PPDK and PEPC+PPDK+ME transgenic rice. The latter reached to 82ï¼…of that of corn and presented some of the photosynthetic features similar to C₄ plants, indicating that ATP is the key factor for the redesigning of C₄ rice. Under the photo-oxidative conditions, the 'resistance of PEPC+PPDK+ME transgenic rice against photo-oxidation was further enhanced.Based on the studies of the physiological characteristic of different C₄ photosynthetic enzyme above mentioned, PEPC transgenic rice was observed to remarkably enhance photosynthetic capacity. This article would further investigate the photoprotective capability. The results showed that the photosynthetic rate under high light intensity had increased by 50ï¼…and photo-inhibition of photosynthesis in PEPC transgenic was alleviated after the introduction of PEPC gene from maize into rice. It was demonstrated that the increment of photosynthesis in PEPC transgenic was related to the introduction of PEPC gene using specific inhibitor of PEPC. Photo-inhibition of photosynthesis in different genotypes appears at noon under natural condition. PEPC transgenic rice exhibited a less decrease in F_v/F_m, a less photo-inhibition and a higher efficiency of light energy conversion to chemical energy and lower thermal energy dissipation.The eighth generation of PEPC transgenic rice with the good photosynthetic characteristic and photoprotection effect was achived through systematic reselection and re-identification. With photosynthetic rate and chlorophyll fluorescence parameters by applying the stable and radioactive carbon isotope studied, the results showed that the germplasm of PEPC transgenic rice was a C₃ plant with certain C₄ photosynthetic characteristics, but the photosynthetic characteristic was improved, for the original photoproduct of C₄ plant increased. Compared with the previous generations, the germplasm of PEPC transgenic rice has been steady in genetic with high photosynthetic characteristic, tolerance to photo-oxidization, which may become a new germplasm resource in the breeding for high photosynthetic efficiency.F₁ hybrids were obtained by crossing PEPC transgenic rice germplasm as male parent with japonica rice cultivar 9516. Processed with the cultivation of pollen lines, selection by photosynthetic characteristic and enzyme activity, and systematic reselection and re-identification of three generations, stable JAAS45 pollen lines of pure-bred diploid plants were achieved. By investigation of the content of photosynthetic pigment, net photosynthetic rate and utilization efficiency of water for JAAS45 and its parents, it was revealed that with the proceeding of growth, chlorophyll per area and Car content were higher than 9516, and retained a much higher level close to PC after tillering stage. The net photosynthetic rate (Pn) of JAAS45 approximated that of 9516 at the seeding stage, but it exceeded 9516 at tillering stage, which was even higher than PC at heading and filling stage. From seeding to heading period, utilization efficiency of water for JAAS45 was evidently higher than 9516, whereas close to PC, accounting for the higher Pn and superior ability resistant to photo-inhibition, as well as the full accessing of the weaker light intensity at morning and dusk for photosynthesis, which was similar with the male parent PC. Additionally, JAAS45 has higher WUE during development, favorable to economically making use of water in agriculture.On the other side, the characteristics of chlorophyll fluorescence and the performance of membrane-lipid peroxidation in JAAS45 pollen lines and its resistance to photo-oxidation were studied. After the treatment of photo-oxidation, Fv/Fm, PSIIand q^P in JAAS45 pollen lines decreased less than those in 9516—the male parent, while q^N increased more, which suggested that more light energy absorbed by JAAS45 pollen lines was converted into chemical energy and the excessive light energy was exhausted through thermal energy dissipation, resulting in the alleviation of photodamage. Meantime, the higher activities of scavenging-active-oxygen enzymes such as SOD, POD in JAAS45 pollen lines led to the less accumulation of 0₂^?. As a result, the content of MDA—the product of membrane-lipid peroxidation, was less than that in 9516, and the contents of chlorophyll and protein in JAAS45 pollen lines decreased less than those in 9516, while more close to the male parent, demonstrating the stronger tolerance of JAAS45 pollen lines to photo-oxidation.To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of JAAS45 pollen lines and its parents, physiological indices of photosynthetic CO₂ exchange and chlorophyll fluorescence parameters were measured in leaves. The results revealed that the PEPC gene could be stably inherited and transferred from the male parent to JAAS45 pollen line. Moreover, JAAS45 pollen line exhibited high levels of PEPC activity, higher saturated photosynthetic rate, photosynthetic apparent quantum yield (AQY), Fv/Fm, q^P and q^N, indicating that JAAS45 pollen line has a high tolerance to photo-inhibition/photo- oxidation under strong light and high temperature. Furthermore, JAAS45 pollen lines were confirmed to remain a C₃ plant by ¹³C carbon isotope determination, and have a limited photosynthetic C₄ microcycle feeding with exogenous C₄ primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explained the physiological inherited properties of PEPC transgenic rice and provided a basis for the integration of traditional breeding and biological technology.