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 pl… Show more
“…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).…”
Section: Discussionsupporting
confidence: 69%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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…”
Section: Methodsmentioning
confidence: 99%
“…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 standard protocol to evaluate the effects of biostimulants on plant physiology is still lacking. The proton pumps present in the vacuolar and plasma membranes are the primary agents responsible for the regulation of the electrochemical gradient that energizes the nutrient uptake system and acid growth mechanism of plant cells. In this study, two of these enzymes were characterized as biochemical markers of biostimulant activity. A simple and fast protocol based on the degree of root acidification using a pH sensitive dye and the Micro-Tom tomato as a plant model is proposed as an efficient methodology to prove the efficacy of biostimulants that are claimed to improve nutrient acquisition and root growth. The results agree with the data from more conventional, expensive and time-consuming proton pump assays. A direct correlation was found between plasmalemma proton-adenosine triphosphatase (H + -ATPase) activation and the amount of rhizosphere acidification observed in the bromocresol gel. Moreover, roots of the diageotropica (dgt) Micro-Tom plants, defective in auxin responses, barely acidify bromocresol purple gel even in the presence of indole-3-acetic acid (IAA, 1 μM). The biostimulant TEA (vermicompost water extract, 25 %) enhances proton extrusion by 40 % in wild type (WT) plants, but no effect was induced in dgt plants. These results reinforce the notion that the class of biostimulant known as humic substances stimulates plant proton pumps and promotes root growth by exerting an auxin-like bioactivity and establish the usefulness of an economically and technically feasible assay to certify this kind of biostimulant.
“…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).…”
Section: Discussionsupporting
confidence: 69%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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…”
Section: Methodsmentioning
confidence: 99%
“…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 standard protocol to evaluate the effects of biostimulants on plant physiology is still lacking. The proton pumps present in the vacuolar and plasma membranes are the primary agents responsible for the regulation of the electrochemical gradient that energizes the nutrient uptake system and acid growth mechanism of plant cells. In this study, two of these enzymes were characterized as biochemical markers of biostimulant activity. A simple and fast protocol based on the degree of root acidification using a pH sensitive dye and the Micro-Tom tomato as a plant model is proposed as an efficient methodology to prove the efficacy of biostimulants that are claimed to improve nutrient acquisition and root growth. The results agree with the data from more conventional, expensive and time-consuming proton pump assays. A direct correlation was found between plasmalemma proton-adenosine triphosphatase (H + -ATPase) activation and the amount of rhizosphere acidification observed in the bromocresol gel. Moreover, roots of the diageotropica (dgt) Micro-Tom plants, defective in auxin responses, barely acidify bromocresol purple gel even in the presence of indole-3-acetic acid (IAA, 1 μM). The biostimulant TEA (vermicompost water extract, 25 %) enhances proton extrusion by 40 % in wild type (WT) plants, but no effect was induced in dgt plants. These results reinforce the notion that the class of biostimulant known as humic substances stimulates plant proton pumps and promotes root growth by exerting an auxin-like bioactivity and establish the usefulness of an economically and technically feasible assay to certify this kind of biostimulant.
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