Expression level of Rubisco activase negatively correlates with Rubisco content in transgenic rice

Abstract

The relationship between ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (Rca) levels was studied using transgenic rice overexpressing maize Rca (OX-mRca) and knockdown transgenic rice expressing antisense Rca (KD-Rca). The ratio of Rubisco to total soluble protein was lower in OX-mRca, whereas it was higher in KD-Rca than in WT, indicating that Rca expression was negatively correlated with Rubisco content. The expressions of other Calvin-Benson-Bassham cycle enzymes such as sedoheptulose-1,7-bisphosphatase and phosphoribulokinase analyzed by immunoblotting did not show such a negative correlation with Rca, suggesting that the effect of Rca on protein expression may be specific for Rubisco. Although Rubisco content was decreased in OX-mRca, the transcript levels of the Rubisco large subunit (OsRbcL) and the Rubisco small subunit mostly increased in OX-mRca as well as in KD-Rca. Additionally, polysome loading of OsRbcL was slightly higher in OX-mRca than it was in WT, suggesting that the OsRbcL translation activity was likely stimulated by overexpression of Rca. S-methionine labeling experiments demonstrated that there was no significant difference in the stability of newly synthesized Rubisco among genotypes. However,S-methionine-labeled Rubisco was marginally decreased in OX-mRca and increased in KD-Rca compared to the WT. These results suggest that Rca negatively affects the Rubisco content, possibly in the synthesis step.

Effects of Overproduction of Rubisco Activase on Rubisco Content in Transgenic Rice Grown at Different N Levels

“…Our results showed that although the levels of the Rubisco protein were decreased in RCA-HI plants, the mRNA levels of RBCS and of the Rubisco large subunit (RBCL) gene remained unchanged ( Figure S1). Similar results were obtained for the RCA-overexpressing rice plants reported by Fukayama et al [31,32]. These results suggest that Rubisco content is regulated post-transcriptionally.…”

“…In contrast, in the present study of RCA-transgenic plants, the amounts of transketolase and transketolase-N were not affected (Figures 2 and 3). Similarly, Fukayama et al [32] reported no changes in several Calvin-Benson cycle enzymes in RCA-transgenic rice plants. These results suggest that the changes in RCA content affected Rubisco levels selectively.…”

“…Many studies have shown that RCA and Rubisco are attractive targets for improving photosynthesis (for a review, see [4]). However, Fukayama et al [31,32] reported that the overproduction of RCA negatively affected Rubisco content. To assess whether it is possible to overproduce RCA without affecting Rubisco content, we generated and selected several transgenic rice plants exhibiting varying RCA levels under the control of two different promoters ( Figure 1) and examined the effects of changes in RCA levels on Rubisco content in plants grown with different N concentrations.…”

“…However, in such RCA-overproduced rice, Rubisco content was decreased and, consequently, photosynthesis rates were also decreased [31]. Moreover, some studies have shown that Rubisco content was increased in plants with RCA suppression [20,24,32], while other studies reported that Rubisco content did not change in these plants [21,22,25,33]. Thus, the effects of RCA levels on Rubisco content in transgenic plants with RCA overproduction or suppression remain uncertain.…”

“…Considering these studies, it is possible that the overlap in the expression of the transgenic RCA gene with that of RBCS affects Rubisco biosynthesis. In addition, Fukayama et al [32] investigated the manner in which the overexpression of RCA affects Rubisco content negatively and reported the possibility that the reduction in Rubisco content occurred at the Rubisco synthesis step. Therefore, in the present study, RCA was overexpressed under the control of the promoters of RBCS or RCA (Pro RBCS or Pro RCA ).…”