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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]

Barley (Hordeum vulgare L.) is a typical ammonium (NH4+)‐sensitive crop species and exhibits a futile‐NH4+ efflux from root cells. Plasma membrane (PM) H+‐ATPase is responsible for pumping H+ out of plant cells and providing the driving force for the transport of various substances. We hypothesized that PM H+‐ATPase is involved in this NH4+ efflux process of barley roots. Barley plants were cultivated in hydroponic solution with 1.0 mM NH4+ or 1.0 mM nitrate (NO3-) as the sole nitrogen source. Plasma membrane vesicles were isolated from the root tips for analyses of the activity. Plasma membrane H+‐ATPase was tested by Western Blot. The transcription of PM H+‐ATPase genes was analyzed using qRT‐PCR. Proton and NH4+ efflux from barley roots was analyzed by scanning ion‐selective electrode technique. The results show that root and shoot growth was repressed by NH4+ nutrition when compared to NO3- nutrition. Plasma membrane H+‐ATPase activity of barley roots under NH4+ nutrition was significantly higher than that under NO3- nutrition. Methyl‐ammonium (MeA), a non‐metabolizable analog of NH4+, had no significant effect on PM H+‐ATPase activity. The enhanced PM H+‐ATPase activity by NH4+ nutrition was consistent with a higher abundance of PM H+‐ATPase protein and higher transcription levels of six PM H+‐ATPase genes. By using the pharmacological agents vanadate and fusicoccin, a close linkage was observed between H+ efflux and NH4+ efflux from barley roots. Taken together, NH4+ nutrition stimulates PM H+‐ATPase activity, and this stimulation is possibly triggered by NH4+ assimilation in barley roots. NH4+ efflux from barley roots is associated with PM H+‐ATPase activity which could provide the driving force for NH4+ efflux. Ammonium efflux might be considered as a disposal strategy in barley roots which prevents NH4+ overloading the root cytoplasm. [ABSTRACT FROM AUTHOR]

Aims: Exudation of organic acid anions is one of the mechanisms responsible for aluminum (Al) tolerance. The plasma membrane (PM) H-ATPase is involved in the exudation of organic acid anions. However, the relationship between organic acid exudation and PM H-ATPase under Al toxicity remains unclear. This study aims to investigate the correlation among Al-induced citrate exudation, PM H-ATPase activity and counterions for citrate release from cluster roots of phosphorus-deficient (−P) white lupin. Methods: Al and various pharmacological agents were applied to incubate the cluster roots of P-deficient white lupin; the citrate exudation rate, PM H-ATPase activity and ion exudation of cluster roots were examined. Results: Citrate exudation from cluster roots of P-deficient white lupin was induced by 50 μM Al treatment within 1.5 h, but no extra increase was found when the duration of Al treatment increased. The PM H-ATPase activity of cluster roots was insensitive to Al treatment, irrespective of Al concentration and duration of Al treatment. Al treatment increased K efflux but not H efflux from cluster roots. After application of pharmacological agents to P-deficient cluster roots under Al treatment or not, vanadate decreased H efflux and increased K efflux, but had no inhibitory effect on citrate exudation; fusicoccin increased H efflux and citrate exudation, but decreased K efflux; tetraethylammonium (TEA) chloride, a K-channel inhibitor, inhibited K efflux and increased H efflux. Conclusions: These results indicate that citrate exudation induced by combined treatment with P-deficiency and Al is independent of PM H-ATPase activity, and is coupled with K efflux, which may compensate H efflux for keeping the charge balance for Al-induced citrate exudation from cluster roots of P-deficient white lupin. [ABSTRACT FROM AUTHOR]

The plasma membrane (PM) H ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice ( Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value ( K) of PM H ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H ATPase in P deficient roots was related to a slightly increase of PM H ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H-ATPase and H pump, which contributed to the rhizosphere acidification in rice roots. [ABSTRACT FROM AUTHOR]

P-type ATPases are transmembrane pumps of cations and phospholipids. They are energized by hydrolysis of ATP and play important roles in a wide range of fundamental cellular and physiological processes during plant growth and development. However, the P-type ATPase superfamily genes have not been characterized in soybean. Here, we performed genome-wide bioinformatic and expression analyses of the P-type ATPase superfamily genes in order to explore the potential functions of P-type ATPases in soybean. A total of 105 putative P-type ATPase genes were identified in the soybean genome. Phylogenetic relationship analysis of the P-type ATPase genes indicated that they can be divided into five subfamilies including P1B, P2A/B, P3A, P4 and P5. Proteins belonging to the same subfamily shared conserved domains. Forty-seven gene pairs were related to segmental duplication, which contributed to the expansion of the P-type ATPase genes during the evolution of soybean. Most of the P-type ATPase genes contained hormonal- and/or stress-related cis-elements in their promoter regions. Expression analysis by retrieving RNA-sequencing datasets suggested that almost all of the P-type ATPase genes could be detected in soybean tissues, and some genes showed tissue-specific expression patterns. Nearly half of the P-type ATPase genes were found to be significantly induced or repressed under stresses like salt, drought, cold, flooding, and/or phosphate starvation. Four genes were significantly affected by rhizobia inoculation in root hairs. The induction of two P2B-ATPase genes, GmACA1 and GmACA2, by phosphate starvation was confirmed by quantitative RT-PCR. This study provides information for understanding the evolution and biological functions of the P-type ATPase superfamily genes in soybean. [ABSTRACT FROM AUTHOR]