Enhanced Cell Expansion in a KRP2 Overexpressor is Mediated by Increased V-ATPase Activity

Decreased cell numbers during leaf development often trigger increased cell size, a phenomenon called compensation. In compensation-exhibiting mutants, the unusually high cell expansion activity occurs through two different mechanisms during the post-mitotic stage of leaf development, except in the KIP-RELATED PROTEIN 2-overexpressing line (KRP2 o/e), whose cell sizes are 2-fold greater during proliferative growth. However, the molecular basis of compensated cell expansion (CCE) has not been characterized. The det3-1 mutant has a mutation in the C-subunit of the vacuolar-type H+-ATPase (V-ATPase) complex that causes a 50% decrease in its activity and cell size. To determine the contribution of V-ATPase activity to CCE, the cellular phenotypes of double mutants between det3-1 and compensation-exhibiting fugu5-1, an3-4, fas1-5 and KRP2 o/e were analyzed in detail. Interestingly, while decreased V-ATPase activity caused by det3-1 did not suppress CCE in fugu5-1, fas1-5 and an3-4, CCE in KRP2 o/e was totally suppressed. Furthermore, measurements revealed that the activity and quantity of the A-subunit of the V-ATPase complex were significantly increased in the shoots of KRP2 o/e plants. Importantly, the unusually increased size of actively dividing KRP2 o/e cells was restored to normal in the det3-1 background. Taken together, our data strongly suggest that CCE in KRP2 o/e, but not in other compensation-exhibiting mutants, occurs exclusively through the increase of V-ATPase activity.