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

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. ³⁵S-methionine labeling experiments demonstrated that there was no significant difference in the stability of newly synthesized Rubisco among genotypes. However, ³⁵S-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.