Regulation Of PM H~+ ATPase Involved In The Release Of BNIs From Sorghum Roots

Ammonium(NH4+) and nitrate(NO3-)are the two primary nitrogen(N)species utilized by the crop plants.In agricultural production systems,however,N is applied mainly through NH4+ based fertilizers.Nitrification is the microbiologically mediated oxidation of relatively immobile NH4+ into highly mobile NO3-.This causes potential loss through NO3-leaching,and gaseous N emissions in the form of NO,N2O and N2 with undesirable impacts on environment and human health,ultimately leading to notoriously poor(N)-use efficiency(NUE)of agricultural plants.Inhibiting the activity of nitrifiers thus can help retain soil N in NH4+form to minimize the chances of N losses through NO3-leaching and gaseous emissions,thus improving environmental health and agronomic nitrogen use efficiency.Plant roots have been reported to release certain organic compounds having the potential to inhibit the process of nitrification and are termed as biological nitrification inhibitors(BNIs).Among field crops,sorghum roots have the great potential to extensively release BNIs.Contrary to NO3-,NH4+ in the rhizosphere or in root medium triggers the release of BNIs from sorghum roots.In addition,uptake as well assimilation of NH4+causes net H+release from plant roots.It is not clear,which factors had effect on the release of BNIs from sorghum roots.Plasma membrane proton ATPase(PM H+ATPase)is a master enzyme that pumps H+outside the plant cells at the expense of ATP.It was found through earlier research that uptake and assimilation of NH4+,rhizosphere pH and hydrophilic BNIs release are linked together through the activity of PM H+ ATPase.However,the underlying mechanism of involvement of H+ ATPase in this regard is still a question.Present study was conducted in a hydroponic system with different treatments based on N forms and pH levels.Three genetic stocks of sorghum('Hybridsorgo',PVK 801 and 296B)were used as the test crops depending upon the experiments.Root exudates were collected from intact plant roots and BNI activity was determined.Plasma membrane was isolated using two-phase partitioning system,followed by the determination of PM H+ATPase activity.The involvement of PM H+ATPase in BNIs release from sorghum root system was evaluated by modifying its activity through the application of different pharmacological agents[(fusicoccin and vanadate)and anion-channel blockers(-niflumic acid(NIF)and anthracene-9-carboxylate(A9C)],depending upon the experiment design,in the collection solutions by using split root system.Western blotting and qRT-PCR were used for the protein quantification and analysis of expression of the genes encoding PM H+ATPase.The results were found as follows:1.Results from the present study reveal strong response of PM H+ATPase activity and BNIs release in sorghum roots towards NH4+ nutrition and low pH in the rhizosphere.The presence of NH4+ in the rhizosphere either at pH 3.0 or pH 7.0 stimulated both the H+ATPase activity as well as the BNIs release from sorghum roots.Interestingly,NO3--raised sorghum plants when subjected to short-term incubation with NH4+,compared to NO3-in the collection solution,released more BNIs and showed higher H+ATPase activity.In addition,a significant increase in H+ATPase activity and hydrophilic BNIs release was observed at low pH as compared to high pH in the collection solution.Fusicoccin,a stimulator of H+ATPase activity,stimulated BNIs release;whereas,vanadate,an inhibitor of H+ATPase activity,suppressed the release of BNIs.2.An increasing trend of hydrophilic-BNIs release from roots of both sorghum genotypes(PVK 801 and 296B)was observed in response to decreasing pH in the growth medium.Fusicoccin had a stimulating effect on H+extrusion and hydrophilic-BNIs release,however,vanadate inhibited H+extrusion and the release of hydrophilic-BNIs.Anion-channel blockers did not have a blocking effect on the release of hydrophilic BNIs,instead boosted both H+-extrusion and hydrophilic-BNIs release.These results indicated the release of protonated BNIs operated by some unknown membrane transporters,which is supposed to compensate for charge balance when the transport of other anions is suppressed by anion-channel blockers.3.Hydrophilic BNIs release and H+ATPase activity exhibited a strong correlation either under NH4+ nutrition or NO3-nutrition at pH 3.0 or pH 7.0.Plasma membrane(PM)H+ ATPase activity was higher under NH4+ nutrition and low pH than under NO3-and high pH.A pH optimum achieved for the activity of PM H+ATPase under above-described treatments ranged from pH 6.0 to pH 6.2.Results from qRT-PCR showed stimulated expression of five genes in sorghum roots treated with NH4+.Western blotting results revealed more abundance of PM H+ATPase in sorghum roots in response to NH4+ as compared to NO3-under low pH.Moreover,NH4+ treatment yielded higher Vmax and lower Km as compared to NO3-at either of the pH levels.In contrast,methylammonium(MeA),an analog of NH4+,did not cause the induction of ATPase activity.Taken together,it is concluded that NH4+ nutrition and low rhizosphere pH has a strong effect on the release of hydrophilic BNIs,which is due to the stimulated activity of PM H+ATPase in sorghum roots.The regulation of PM H+ATPase at transcriptional level and post translational level is the major effect to enhance the activity of this enzyme in sorghum roots,which in turn directly control the BNI release.