Down-regulation of sucrose synthase expression and activity in transgenic hairy roots of Lotus japonicus

Sucrose synthase is central to carbon metabolism in roots and nodules of legumes. In order to analyze its role we have used antisense RNA strategies to down-regulate its expression in the model legume Lotus japonicus. In contrast to other legumes the sucrose synthase gene from Lotus was expressed highly not only in nodules, but also in roots. This enabled us to use transgenic hairy roots to evaluate the potential of the antisense RNA strategy. The sucrose synthase sense message was drastically reduced in 4 out of 5 antisense sucrose synthase hairy root lines. Sucrose synthase protein levels were significantly reduced, and sucrose synthase enzyme activities declined to between 20% and 25% of the controls. By contrast, activities of UDPGlucose pyrophosphorylase, fructokinase, glucokinase and acid invertase remained unchanged, while unexpectedly, alkaline invertase activity declined in the antisense sucrose synthase lines. The significantly higher fresh weight of the control cultures compared to the antisense hairy roots observed after 14 days of growth, suggest that the changes in enzyme activities affected the growth rate. Overall, this work demonstrates that the antisense RNA strategy can be used successfully in L. japonicus to alter gene expression