Identification and expression of three new Nicotiana plumbaginifolia geneswhich encode isoforms of a plasma-membrane H+-ATPase, and one of which is induced by mechanical stress

To analyze in detail the multigene family encoding the plasma-membrane H+-A TPase (pma) in Nicotiana plumbaginifolia Viv., five new pma genes (pma 5-9) were isolated. Three of these (pma 6, 8, 9) were fully characterized and c lassified into new and independent subfamilies. Their cell-type expression was followed by the beta-glucuronidase (gusA) reporter-gene method. While t he pma8-gusA transgene was not expressed in transgenic tobacco, expression of the two other transgenes (pma6- and pma9-gusA) was found to be restricte d to particular cell types. In the vegetative tissues, pma6-gusA expression was limited to the head cells of the leaf short trichomes, involved in sec retion, and to the cortical parenchyma of the young nodes where the develop ing leaves and axillary flowering stalks join the stem. In the latter tissu es, gene expression was enhanced by mechanical stress, suggesting that H+-A TPase might be involved in the strength of the tissues and their resistance to mechanical trauma. The pma9-gusA transgene was mainly expressed ill the epical meristem of adventitious roots and axillary buds as well as in the phloem tissues of the stem, in which expression depended on the development al stage. In flowers, pma9-gusA expression was limited to the mature pollen grains and the young fertilized ovules, while that of pma6-gusA was identi fied in most of the organs. Reverse transcription-polymerase chain reaction of leaf and stem RNA confirmed the expression of pma 6 and 9, while pma8 w as found to be expressed in both organs at a lower level. In conclusion, al though pma 6 and 9 had a more restricted expression pattern than the previo usly characterized pma genes, they were nevertheless expressed in cell type s in which H+-ATPase had not been previously detected.