Nitric oxide enhances salt secretion and Na+ sequestration in a mangrove plant, Avicennia marina, through increasing the expression of H+-ATPase and Na+/H+ antiporter under high salinity

Modulation of nitric oxide (NO) on ion homeostasis, by enhancing salt secretion in the salt glands and Na+ sequestration into the vacuoles, was investigated in a salt-secreting mangrove tree, Avicennia marina (Forsk.) Vierh. The major results are as follows: (i) under 400 mM NaCl treatment, the application of 100 mu M sodium nitroprusside (SNP), an NO donor, significantly increased the density of salt crystals and salt secretion rate of the leaves, along with maintaining a low Na+ to K+ ratio in the leaves. (ii) The measurement of element contents by X-ray microanalysis in the epidermis and transversal sections of A. marina leaves revealed that SNP (100 mu M) significantly increased the accumulation of Na+ in the epidermis and hypodermal cells, particularly the Na+ to K+ ratio in the salt glands, but no such effects were observed in the mesophyll cells. (iii) Using non-invasive micro-test technology (NMT), both long-term SNP (100 mu M) and transient SNP (30 mu M) treatments significantly increased net Na+ efflux in the salt glands. On the contrary, NO synthesis inhibitors and scavenger reversed the effects of NO on Na+ flux. These results indicate that NO enhanced salt secretion by increasing net Na+ efflux in the salt glands. (iv) Western blot analysis demonstrated that 100 mu M SNP stimulated protein expressions of plasma membrane (PM) H+-ATPase and vacuolar membrane Na+/H+ antiporter. (v) To further clarify the molecular mechanism of the effects of NO on enhancing salt secretion and Na+ sequestration, partial cDNA fragments of PM H+-ATPase (HA1), PM Na+/H+ antiporter (SOS1) and vacuolar Na+/H+ antiporter (NHX1) were isolated and transcriptional expression of HA1, SOS1, NHX1 and vacuolar H+-ATPase subunit c (VHA-c1) genes were analyzed using real-time quantitative polymerase chain reaction. The relative transcript abundance of the four genes were markedly increased in 100 mu M SNP-treated A. marina. Moreover, the increase was reversed by NO synthesis inhibitors and scavenger. Taken together, our results strongly suggest that NO functions as a signal in salt resistance of A. marina by enhancing salt secretion and Na+ sequestration, which depend on the increased expression of the H+-ATPase and Na+/H+ antiporter.