Redox Regulation Of Rubisco Activase From Tobacco Mediated By Thioredoxin F

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzymewhich catalyzes the first step reaction of both photosynthetic CO2assimilation andphotorespiratory carbon oxidation, whereby to determine the magnitude of netphotosynthetic rate. Rubisco must be activated by Rubisco activase (RCA) to becomecatalytically competent in vivo. Recently, it was found that α isoform is regulated bythioredoxin-f in redox status in Arabidopsis which contain two isoforms of Rubiscoacivase in vivo. But, like Tobacco, which only has β isoform whether is regulated bythioredoxin-f has not be reported. Through the study of the redox regulation ofRubisco and Rubisco activase from Tobacco and Arabidopsis, we raise twoconclusions initially as follows:1. We proved that Rubisco activity is also regulated by redox status in Tobacco.Furthermore, it also is through Rubisco activase mediated by thioredixin f. Weobtained the thioredoxin-f and RCA from Arabidopsis and Tobacco purified fromleaves or prokaryotic expression, then measured the changes of the numbers ofsulfhydryl groups, Rubisco activiation activity and the ATPase activity under thedifferent redox states with or without thioredoxin f. Similar to RCA from Arabidopsis,the results showed that Tobacco RCA activity including Rubisco activation activity andATPase activity were increased only when thioredoxin f and DTT (NaHSO3) both inassay, on the contrary, the activity of Rubisco activity was decreased when GSSG andthioredoxin f was added. The similar result was observed in the number of sulfhydrylgroups of RCA from Tobacco.2. Similar to Rubisco activation activity of RCA, the reuglation of tobacco RCAactivity by the redox status also has the species specificity. The changes in redoxstatus of RCA from the species of one isoform could be mediated through Trx-f fromTobacco but not that from Arabidopsis, and the redox status of RCA from the speciesof two isoform could be changed when Trx-f from Arabidopsis presented.Furthermore, through sequence alignment of RCA, among speices of two isoformsthere are three conserved amino acids sites (335,359,378) in small isoform of RCA, which are different among species of one isoform. We constructed3RCA mutant ofArabidopsis RCA small isoform, in which three amino acids sites of335,359,378wassubstituted for the amino acids from Tobacco, respectively. After measurement ofATPase of these3RCA mutants in the presence of tobacco thioredoxin f underdifferent redox states, the results showed that only the ATPase activity of M335Lcould be regulated in the presenceTrx-f of Tobacco. When DTT was added, the ATPaseactivity of M335L increased33%, and it decreased about30%when GSSG was added.Therefore, we suggested that L335maybe an important amino acid site which isrelated to specificity of redox regulation of RCA although the detail mechanism isunclear yet.