Rice (Oryza sativa L.) plants with substantially increased Rubisco content were obtained by Agrobacteriummediated transformation with the rice rbcS sense gene under the control of the rice rbcS promoter. The primary transformants were screened for the ratio of Rubisco to leaf-N content, and the transformants with 4120% wild-type levels of Rubisco were selected. In the progeny of the selected lines of the transformants, the mRNA levels of one member of the rbcS gene family were increased from 3.9-to 6.2-fold, whereas those of other members of the rbcS gene family were unchanged. The total levels of rbcS mRNA were increased from 2.1-to 2.8-fold. The levels of rbcL mRNA were increased from 1.2-to 1.9-fold. Rubisco protein content was significantly increased by 30% on a leaf area basis. The ratio of Rubisco-N to leaf-N was also increased by 10-20%, irrespective of N treatment. The specific activity of Rubisco per unit of enzyme protein was not different. However, light-saturated photosynthesis was not enhanced even when the rate was measured at low [CO 2 ] where Rubisco becomes limiting for photosynthesis. Some lines showed lower photosynthesis at high [CO 2 ] (460 Pa). We conclude that introduction of additional sense rbcS leads to overexpression of rbcS and that this overexpression slightly up-regulates the gene expression of rbcL at the transcript level and enhances the amount of Rubisco holoenzyme. However, overproduction of Rubisco protein does not improve photosynthesis.

As ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) activity limits light-saturated photosynthesis under present atmospheric condition, the effects of an overexpression of RBCS on Rubisco content and photosynthesis were examined in the leaves at different positions in rice (Oryza sativa L.). Rubisco content in the transformant was significantly greater in the uppermost, fully expanded leaves but decreased to levels similar to those in wild-type plants in the lower leaves. The mRNA levels of total RBCS and rbcL in these leaves were much less than those in the expanding leaves, where Rubisco synthesis is active, suggesting commensurately low level of synthesis. Although the activation state of Rubisco was lower in the uppermost, fully expanded leaves of the transformant, it recovered to its full level in the lower leaves. As a result, the photosynthetic rate did not differ in leaves at the same position between the transformant and the wild type. Similarly, whole plant biomass did not differ between these genotypes. Thus, we conclude that although the overexpression of RBCS led to an enhancement of Rubisco protein content in the uppermost, fully expanded leaves, it does not result in increased photosynthetic rates or plant biomass, because of an apparent down-regulation in its activation state.

Four out of five members of the RBCS multigene family (OsRBCS2-OsRBCS5) were highly expressed in leaf blades of rice (Oryza sativa L.) irrespective of plant growth stage, whereas accumulation of all RBCS mRNAs in leaf sheaths, roots and developing spikelets was quite low. A highly positive correlation was observed between total RBCS and RBCL mRNA levels and Rubisco content at their maxima, irrespective of tissue and growth stage. The results indicate that the total RBCS mRNA level may be a primary determinant for maximal Rubisco protein content and that Rubisco gene expression is well coordinated through the whole life of rice.

No abstract

In higher plants, a small subunit of Rubisco is encoded for by an RBCS multigene family in the nuclear genome. However, it is unknown how each multigene member contributes to the accumulation of Rubisco holoenzyme. Here, four RBCS genes that are highly expressed in leaf blaedes of rice (Oryza sativa L.) were individually suppressed by RNAi, and the effects on leaf Rubisco content were examined at seedling, vegetative and reproductive stages. Rubisco contents in each transgenic line declined irrespective of growth stage, and the ratios of Rubisco-N to total N were 66-96% of wild-type levels. The mRNA levels of the suppressed RBCS genes declined significantly, whereas those of the unsuppressed ones did not change drastically. These results indicate that four RBCS genes all contribute to accumulation of Rubisco holoenzyme irrespective of growth stage and that suppression of one RBCS gene is not fully compensated by other RBCS genes. Additionally, the mRNA levels of the large subunit of Rubisco showed a change similar to that of total RBCS mRNA level irrespective of genotype and growth stage. These results suggest that gene expression of RBCS and RBCL is regulated in a coordinated manner at the transcript level in rice.