Expression of an activated plasma membrane ATPase results in plant cell expansion

The plant plasma membrane H+-ATPase couples ATP hydrolysis with proton transport outside the cell and thus creates an electrochemical gradient, which activates secondary transporters. Plant growth depends not only on cell division, but also on cell elongation. The acid growth theory proposes that the plasma membrane H+-ATPase is an important player in plant cell elongation, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. The aim of this thesis consisted of testing this hypothesis by expressing H+-ATPase isoforms in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. As the plasma membrane H+-ATPase is a highly regulated enzyme, we wondered whether expressing an isoform from a distant species, AHA2 (Arabidopsis H+-ATPase isoform 2) would result in activity deregulation. Although H+-ATPase expression was observed, the proton pumping activity in vivo and in vitro was unchanged and we did not observe any modification of the phenotype. We then expressed a homologous isoform, PMA4 (plasma membrane H+-ATPase) from Nicotiana plumbaginifolia, deleted from its C-terminal auto-inhibitory domain. The resulting pump (ΔCPMA4) is expected to be constitutively activated. Expression of activated ΔCPMA4 resulted in a lower external pH by 0.5 – 1.1 pH unit as well as in a 70 – 306% increased H+-ATPase activity (ATP hydrolysis and proton pumping) of isolated plasma membranes. In addition, after heat shock, ΔCPMA4-expressing cells were 17 - 57% larger than wild-type cells and displayed abnormal shapes. Longer induction of ΔCPMA4 expression turned out to be also lethal for N. tabacum cells. In conclusion, data obtained in this work show a role of H+-ATPase in controlling cell growth and thus support the acid growth theory. They also indicate the importance for H+-ATPases to be tightly regulated.