Post-translational regulation of plasma membrane H+-ATPase is involved in the release of biological nitrification inhibitors from sorghum roots

Abstract

It is an integral property of sorghum (Sorghum bicolor L.) to extensively release biological nitrification inhibitors (BNIs) under NH4+ nutrition, in comparison to NO3− nutrition. Our previous research indicated that plasma membrane (PM) H+-ATPase activity was stimulated by NH4+ and low rhizosphere pH, which in turn provided the driving force for BNIs release from sorghum roots. However, the regulatory mechanism of PM H+-ATPase itself in this regard is not fully elucidated. The present study thus aims at post-translational regulation of PM H+-ATPase via phosphorylation in response to NH4+ nutrition and its functional link to the release of BNIs from sorghum roots. A hydroponic system is used to grow sorghum with 1 mM NH4+ or NO3− as N source at pH 3.0 or pH 7.0 in root medium for the analysis of PM H+-ATPase and BNIs release. The effect of NH4+ on the regulation of PM H+-ATPase was further evaluated by the treatment of NO3−cultivated sorghum roots with different NH4+ concentrations (0.1~1 mM). In addition, fusicoccin (a stimulator of PM H+-ATPase) and vanadate (an inhibitor of PM H+-ATPase) were added to check the effect of PM H+-ATPase phosphorylation on BNIs release. Further, methionine sulphoximine (MSX), which inhibits glutamine synthetase, is used to analyze the effect of ammonium transport/assimilation process on the PM H+-ATPase and BNIs release. Microsomal membrane protein isolated from these roots was used for the test of PM H+-ATPase phosphorylation level by western blot technique. Meanwhile, the root exudates were collected for the analysis of BNIs. Higher amount of PM H+-ATPase protein with higher phosphorylation level were detected in sorghum roots in response to NH4+ and low rhizosphere pH, as compared to NO3− and high pH. Further, PM H+-ATPase protein amount and phosporylation level were dependent on the local supplement of NH4+ (from 0.1 ~ 1 mM) to roots. Nevertheless, the enhanced posphorylation level under all of these treatments was significantly higher than the enhanced protein level of PM H+ ATPase. Unlike protein level, phosphorylation level is closely correlated to the release of BNIs from sorghum roots. In addition, phosphorylation level of PM H+-ATPase adjusted by fusicoccin or vanadate directly affected the release of BNIs, irrespective of the protein level. In addition, ammonium assimilation inhibitor MSX caused decreased phosphorylation level of PM H+-ATPase without affecting the protein level, meanwhile inhibited the release of BNIs from sorghum roots. Our research suggests that phosphorylation of PM H+-ATPase is one of the important regulation mechanisms involved in the release of BNIs from sorghum roots. NH4+ stimulated PM H+-ATPase phosphorylation via excessive H+ generated by NH4+ assimilation in cytoplasm. The up regulation of PM H+-ATPase at post-translational level thus activated the H+ pumping activity to provide the driving force for BNIs release. A new hypothesis is proposed to elucidate the interplay of these functionally inter-linked processes involving ammonium-uptake, −assimilation, and H+-pumps activation in PM on the release of BNIs from sorghum roots.