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

Background: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]