The Relationship Between Rubisco Activase Isoforms And Photosynthesis In Rice

Heat stress is one of the major factors that inhibit plant photosynthesis and limit crop production.Although photosynthesis of plant has the capacity to acclimate to a some range of heat environment,its biochemical principle remains unclear.Previous studies have shown that photosynthetic heat acclimation under moderate heat stress was associated with the Rubisco activation catalysed by Rubisco activase(RCA).In this study,we developed a new method for accurately quantitive analysis of RCA protein in isoform level,and investigated the role of RCA in regulating Rubisco activation and photosynthesis during photosynthetic heat acclimation in rice plant.We also studied the thermotolerance of photosynthesis in different RCA isoforms over expression transgenic rice plants.The main results obtained are shown as follows:The common protein fragment of large RCA isoform(RCA_L) and small RCA isoform(RCA_S) and the specific segment located at the C-terminus of the RCA_L were expressed respectively in E.coli.The resulting fusion proteins were used as antigen to produce the specific antibodies only recognising RCA_L and antibodied recognising both RCA_L and RCA_S.Based on these antibodies,a set of enzyme-linked immunosorbent assay(ELISA) for accurately quantitive analisis of RCA isoform protein were developed.This method has the advantages of high sensitivity and high throughput.In addition,we also developed a new method for detection of Rubisco-RCA complex which was based on sandwich ELISA.This method provided a new tool to investigate the interactions between Rubisco and RCA.Quantities for RCA isoforms showed that the RCA_S content was 6 folds more than the RCA_L content,and the RCA_L/RCA_S ratio declined during leaf aging,mRNA accumulation analysis revealed that the decline of RCA_L proportion was not caused by transcriptional regulation,but resulted from a mechanism of post-transcriptional regulation.Regression analysis showed that Pn was highly correlated with Rubisco initial activity,and that Rubisco initial activity was highly correlated with RCA isoform contents.These results implied that in rice Rubisco was mainly activated by RCA_S,while RCA_L might have the capacity of regulating RCA activity.The photosynthetic heat acclimation in rice was shown to be associated with the recovery of intial Rubisco activity and Rubisco activation state,and the RCA_S played an important role in regulating them.Quantities for RCA isoforms in mRNA level and protein level showed that high temperature might have altered the product proportion of alternative splicing,which resulted in the up-regulation of RCA_L content.In the same time,we also found that the amount of Rubisco-RCA complex increased remarkably,and that the proportion of RCA_L in Rubisco-RCA complex increased during heat acclimation.Therefore,these results suggested that the up-regulation of RCA_L played an important part in photosynthetic heat acclimation by stabilizing or restoring the activity of RCAs.RCA_L and RCA_S over expression transgenic rice plants(O-RCA_L or O-RCA_S) were obtained using Agrobacterium tumefaciens system,and their photosynthetic thermotolerance were studied.Compared with wild type rice plant(WT) and O-RCA_S, O-RCA_L exhibited better photosynthetic thermotolerance.Our results suggested that Rubisco activity in rice was mainly activated by RCA_S,but RCA_L was associated with photosynthetic thermotolerance.In addition,the results of immunoblots further suggested that the enhanced interaction between Rubisco and RCA in O-RCA_L was associated with the increased RCA_L fraction.Thus,these results further validated our previous hypothesis that RCA_L might have the capacity of regulating photosynthetic thermotolerance.In a word,our results suggested that the two RCA isoforms had different physiological functions:RCA_S was mainly responsible for activating Rubisco and regulating Pn,while RCA_L could function as a heat shock protein regulating or protecting the Rubisco activating activity of RCA_S.