Significance of PPi Hydrolysis Function of H+-Pyrophosphatase in Vegetative Growth of Arabidopsis thaliana

H⁺-pyrophosphatase (H⁺-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the membrane. PPi is a by-product released from nearly 200 distinct biosynthetic processes, such as macromolecule biosyntheses of DNA, RNA, and proteins. H⁺-PPase has been found in the vacuolar membranes of plants but not in yeast or animal. Overexpression of H⁺-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H⁺-PPase in Arabidopsis thaliana have postgerminative developmental defects. Here, to further clarify the physiological significance of this important enzyme, we newly generated three varieties of H⁺-PPase overexpressing lines with different levels of activity that we analyzed together with the loss-of-function mutant fugu5-3. The H⁺-PPase overexpressors exhibited enhanced activity of H⁺-PPase during vegetative growth, but no change in the activity of vacuolar H⁺-ATPase. Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24% and 44%, compared to the wild type and fugu5-3, respectively. Consistently, the overexpressors had larger rosette leaves and ~30% more cells in the leaves than the wild type. When uncoupling mutated variants of H⁺-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H⁺-PPase was introduced. Taken together, our findings clearly demonstrate that additional expression of H⁺-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.