Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content by Antisense RNA Reduces Photosynthesis in Transgenic Tobacco Plants

Abstract

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO(2) assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO(2), and 25 degrees C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO(2) partial pressures (p(i)). In control leaves, Rubisco activity only limited the rate of CO(2) assimilation below a p(i) of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p(i). The unchanged conductance and lower CO(2) assimilation resulted in a higher p(i), which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.