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

Abstract

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 400mM NaCl treatment, the application of 100µM 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 (100µM) 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 (100µM) and transient SNP (30µM) 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 100µM 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 100µM 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.