Zinc’s Regulation Effect On Carbonic Anhydrase And Photosynthesis In Rice( Oryza Sativa L.)

Zinc is one of the essential biological micro-nutrients for normal growth and development processes in plant, and it is an essential component of biological metabolic enzymes and involved in the regulation of enzyme activity. The carbonic anhydrase(CA) is regarded as a zinc-containing enzyme involved in plant photosynthesis. In higher plant, CAs are divided into three classes, α-, β- and γ-CA. The β-CA can efficiently catalyze the reversible hydration reaction of CO2, plays a prominent role in transportation and fixation of CO2 in plant photosynthesis. The γ-CA gene product is subtype of mitochondrial Complex I, and involved in plant respiratory metabolism process. Multiple subtypes of CAs have been found in rice until now, but the associations of CA with zinc and photosynthesis are not clear. This study attempts to analyze the response of the CA activity with zinc ions, and investigate the relationship between different subtypes of CAs and photosynthesis, which will provide strong evidence and the basic theory for increasing crop yields by the way of improving photosynthetic performances.In the research, three rice varieties(N22、R49、9311) were chosen as the plant materials, which were treated with 0.2% Zn SO4 foliar spray during the tillering or booting stage, then net photosynthetic rate and SPAD value were measured at different grain filling stages(0, 10 and 20 d after flowering) in rice flag leaves, and β-CA/γ-CA genes and proteins expression, activity of CA were analyzed, in order to clarify the interaction between zinc, carbonic anhydrase and photosynthesis.The main results are as follows:1. With 0.2%ZnSO4 foliar spray, SPAD value, CA activity and net photosynthetic rate of flag leaves of three rice varieties in different grain filling stages were found to be generally higher than the control group. And these three physiological indexes maintain a relatively high level during 0d and 10 d in rice flag leaves, moreover, are lowest in N22. Correlation analysis of the physiological indexes showed that, SPAD value, CA activity with net photosynthetic rate is significantly positive correlation.2. By RT-qPCR Technology, the expression of β-CA/γ-CA genes showed that, the expression of chloroplast β-CA1/β-CA2 genes was far higher than others in rice flagleaves and sheaths, and was increased in the treated rice flag leaves and sheaths, this was consistent with the tendency of SPAD value and net photosynthetic rate. These data highlight that β-CA1/β-CA2 were major genes in the rice CA gene family. Used Western Blotting Technology with β-CA1 antibody, the expression of β-CA1 protein showed that, the β-CA1 protein accumulation in treated groups was slightly higher than the control group.3. The rice were treated with 0.2%Zn SO4 under the blooming stage, including the tillering or booting stage, The result showed that the effects of zinc treatment were different on rice growth in different treat time. On the flowering day, after zinc treatment in the tillering stage, CA activity and genes expression of rice tissues were higher than that in the booting stage, but On the tenth and twenty day after flowering, the results were exactly opposite. Based on the above results, we can speculate that zinc may extend the time of grain filling time and maintain the photosynthetic function of rice leaf.4. Compared CA activity and genes expression between rice flag leaves and flag sheaths showed that, CA was mainly presence in rice flag leaves, especially in grain filling early stage, CA activity and the main genes expression in rice flag leaves were higher than that in flag sheaths, but in grain filling later stage, the results were basically the same. there are several possible explanations for these results, including the flag leaf is the key photosynthetic organs, but the aging rate of rice sheaths is slower than rice leaves, and rice sheath can be used as one of the photosynthesis organs, moreover, the potential function of rice sheath should not be ignored.5. The primary and secondary structures of β-CA/γ-CA proteins were analyzed by using online software PSRPRED/Target P /Wolf Psort/Prot Param Tools and Clustal W2. The results presented demonstrate that the β-CA proteins were Alkaline-labile protein, which were located in the chloroplast,the γ-CA protein was Acidic-stabilizing protein, located in the mitochondria. Additionally, we found that the rice β-CAs shared significant identity with the conservative active sites reported in other higher plants, including three residues binging the catalytic zinc sites of Cys(114)-His(174)-Cys(177).The study demonstrated that there was significantly positive correlation between CA activity and net photosynthetic rate in rice, and β-CA1/β-CA2 genes located in chloroplast were major genes in rice CA gene family, otherwise, these data highlight that zinc can increase CA activity and genes expression of β-CA1/β-CA2 of rice, and improve the rice photosynthesis, so these results can be used as an important reference for improving the performance of rice photosynthesis to increase the rice yield,and provide valuable clues to the research on the fuction of CAs.