ALBERT

Description: Aims Sorghum ( Sorghum bicolor ) roots release biological nitrification inhibitors (BNIs) to suppress soil nitrification. Presence of NH 4 + in the rhizosphere stimulates BNIs release and it is hypothesized to be functionally associated with plasma membrane (PM) H + -ATPase activity. However, whether the H + -ATPase is regulated at the transcriptional level, and if so, which isoforms of the H + -ATPases are involved in BNIs release are not known. Also, it is not clear whether the stimulation on BNIs release from roots is due to NH 4 + uptake or its assimilation, which are addressed in this study. Methods Root exudates from intact sorghum plants were collected using aerated solutions of NH 4 + or methyl-ammonium (MeA); and the BNI-activity release was determined. PM vesicles were isolated from fresh roots using a two-phase partitioning system; and the hydrolytic H + -ATPase activity was determined. All genes encoding PM H + -ATPases were searched in sorghum genome, and their expression in response to NH 4 + or MeA were analyzed by quantitative RT-PCR in sorghum roots. Results BNIs release and PM H + -ATPase activity increased with NH 4 + concentration (≤1.0 mM) in the root-exudate collection solutions, but at higher concentrations, it did not respond further or declined in case of the PM H + -ATPase activity. Twelve PM H + -ATPase genes were identified in sorghum genome; and these isoforms were designated SbA1 to SbA12 . Five H + -ATPase genes were stimulated by NH 4 + in the rhizosphere, and have similar expression pattern, which is consistent with the variation in H + -ATPase activity. MeA, a non-metabolizable analogue of NH 4 + , had no significant effects on BNIs release, H + -ATPase activity, or expression of the H + -ATPase genes. Conclusions Our results suggest that the functional link between PM H + -ATPase activity and BNIs release is evident only at NH 4 + levels of ≤1.0 mM in the rhizosphere. The variation in PM H + -ATPase activity by NH 4 + is due to transcriptional regulation of five isoforms of the H + -ATPases. The stimulatory effect of NH 4 + on BNIs release is functionally associated with NH 4 + assimilation and not just with NH 4 + uptake alone.