Summary The plasma membrane H+‐ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+‐ATPase) genes. Four of these are classified into two gene subfamilies, pma1‐2‐3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5′ end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma–gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell‐type specific manner. Expression in these organs was confirmed at the protein level, using subfamily‐specific antibodies. (ii) pma4–gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+‐ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+‐ATPases with different enzymatic properties.

A cDNA clone was isolated for a fourth pma gene encoding a putative plasma membrane H(+)-ATPase of Nicotiana plumbaginifolia. The sequence of the predicted 952 residue PMA4 polypeptide was compared with those of other known plant PMAs, revealing a higher identity with the Arabidopsis thaliana proteins (86-89%) than with the other three N. plumbaginifolia PMA proteins (80-82%). This supports the view that there are two pma subfamilies which probably arose from a gene duplication predating the separation of the Dilleniidae and Asteridae plant subclasses. Measured pma4 transcript levels indicate that pma4 is similarly expressed in root, stem, leaf, and flower tissues, contrary to the pmal-3 subfamily whose members displayed differential expression according to the organ.

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