Nitric oxide mediates humic acids-induced root development and plasma membrane H+-ATPase activation

Abstract

It is widely reported that some humic substances behave as exogenous auxins influencing root growth by mechanisms that are not yet completely understood. This study explores the hypothesis that the humic acids' effects on root development involve a nitric oxide signaling. Maize seedlings were treated with HA 20 mg C L(-1), IAA 0.1 nM, and NO donors (SNP or GSNO), in combination with either the auxin-signaling inhibitor PCIB, the auxin efflux inhibitor TIBA, or the NO scavenger PTIO. H(+)-transport-competent plasma membrane vesicles were isolated from roots to investigate a possible link between NO-induced H(+)-pump and HA bioactivity. Plants treated with either HA or SNP stimulated similarly the lateral roots emergence even in the presence of the auxin inhibitors, whereas NO scavenger diminished this effect. These treatments induced H(+)-ATPase stimulation by threefold, which was abolished by PTIO and decreased by auxin inhibitors. HA-induced NO synthesis was also detected in the sites of lateral roots emergence. These data depict a new scenario where the root development stimulation and the H(+)-ATPase activation elicited by either HA or exogenous IAA depend essentially on mechanisms that use NO as a messenger induced site-specifically in the early stages of lateral root development.

Plant proton pumps as markers of biostimulant action

“…Biostimulants with auxin-like activity enhances in vivo root acidification in parallel with proton pump activation (Zandonadi et al, 2010). It is tempting to speculate that the H + -PPase activation could be also related to the plant cell proton extrusion shown in the present study, mainly taking into account our previous data, where humic biostimulants were able to induce a concerted activation of the plasmalemma and tonoplast proton pumps (Zandonadi et al, 2007).…”

“…There is a body of evidence of positive effects of these products that are reproduced in laboratory as well as under field conditions (Arancon et al, 2012;Zandonadi et al, 2013;Calvo et al, 2014). Humic substances from vermicompost enhance ATP hydrolysis activity, protein abundance, proton extrusion Zandonadi et al, 2010), and messenger ribonucleic acid (mRNA) levels (Quaggiotti et al, 2004) of the PM H + -ATPases in a way similar to that previously described for the plant hormone, auxin (Frías et al, 1996). These and other studies (reviewed by Zandonadi et al, 2013) have provided compelling evidence to support the original proposition by Façanha et al, (2002) on the potential of the PM H + -ATPase to be used as a biochemical marker of humus and derived substances endowed with auxin-like activity.…”

“…Protons could act as a coupling factor between the auxin action and the cell wall loosening, resulting in auxin-induced acidification and cell elongation (the acid-growth theory, Rayle and Cleland, 1992;Hager, 2003). In a previous study, we have shown the relationship between root growth and H + -ATPase activation in maize roots treated with humic acids as measured by both classical time-consuming cell fractionation and kinetic enzyme protocols, and a much easier acidification assay (Zandonadi et al, 2010). The protocol for isolation of H + -transport-competent vesicles involves many steps from buffer solution preparation, membrane preparation (including differential and density gradient centrifugations), adenosine triphosphate (ATP) or pyrophosphate (PPi) hydrolysis assays and H + transport assays.…”

“…The classic biochemical procedure for membrane isolation and H + -ATPase hydrolytic activity was carried out as described in Zandonadi et al, (2010). A simple assay to measure in vivo proton extrusion was performed using the bromocresol purple pH indicator and potentiometric pH measurements of a medium containing plants pretreated with IAA as described in Zandonadi et al, (2010), with some modifications due to the Micro-Tom plant model and…”

“…A simple assay to measure in vivo proton extrusion was performed using the bromocresol purple pH indicator and potentiometric pH measurements of a medium containing plants pretreated with IAA as described in Zandonadi et al, (2010), with some modifications due to the Micro-Tom plant model and…”