ABSTRACT: In rice (Oryza sativa), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H2O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H2O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIPs, four genes (OsPIP1;3, OsPIP2;1, OsPIP2;2, and OsPIP2;4) were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H2O. In comparison, OsPIP2;2 was most supportive to H2O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H2O transport across the plasma membranes. OsPIP2;2-mediated H2O transport was accompanied by drought-tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H2O transport and drought-tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H2O transport in relevance to drought tolerance in the plant.
