Molecular characterization of plasma membrane H+‐ATPase in a carrot mutant cell line with enhanced citrate excretion

The citrate efflux of a carrot mutant cell line (IPG; insoluble phosphate grower) with enhanced citrate excretion is concomitant with an electrically equivalent H+ release. Furthermore, plasma membrane H+‐ATPase activity (EC 3.6.1.35) in the IPG cell line is known to be greater than in the wild‐type cell line. In the present study, molecular characterization of plasma membrane H+‐ATPase in the IPG cell line was conducted to examine the involvement of this enzyme in enhanced citrate excretion. Four type‐II (DcPA 1, 2, 4 and 5) and two type‐I (DcPA 3 and 6) full‐sized cDNAs encoding Daucus carota plasma membrane H+‐ATPase were cloned. The transcript level of the most abundant isoform, DcPA 1, was three times higher in the IPG cell line than in the wild‐type cells. The transcript levels of the other isoforms showed no differences between cell types. The plasma membrane H+‐ATPase protein level in the IPG cell line was 1.6‐fold that of the wild‐type cell line when analysed by western blot analysis. These results indicate that the higher activity of plasma membrane H+‐ATPase in the IPG cell line is due to up‐regulation of DcPA 1. Antisense inhibition of the DcPA 1 gene also led to decreased citrate excretion from the transgenic IPG cell line, suggesting that the up‐regulation of plasma membrane H+‐ATPase is involved in enhanced citrate excretion.