Abstract
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.
Highlights
Long before the discovery of plant hormone auxin, exogenous organic substances were already described as complementary to mineral nutrients (Bottomley, 1915)
A direct correlation was found between plasmalemma proton-adenosine triphosphatase (H+-ATPase) activation and the amount of rhizosphere acidification observed in the bromocresol gel
The plasma membrane (PM) vesicles isolated from wild type tomato roots treated with indole-3-acetic acid (IAA) 1 μM, exhibited a stimulation of vanadate-sensitive H+ATPase activity (Figure 2) as well as rhizospheric acidification (Figure 3)
Summary
Long before the discovery of plant hormone auxin, exogenous organic substances were already described as complementary to mineral nutrients (Bottomley, 1915). A hundred years later, several products have been identified as biostimulants, still lacking proper scientific evaluation, and only proved to work under controlled conditions (for a review see Calvo et al, 2014). A legal definition for fertilizers and plant protection products exists, an equivalent legislation for plant biostimulants remains to be defined. There are guidelines on efficacy evaluation for the use and registration of these products, but little has been done towards developing a qualitative method for discriminating among the myriad of plant biostimulants increasingly released into the market. How can we certify whether a product is a biostimulant or not? To answer this question and establish a guideline to access biostimulant activities with simple protocols, it is worthwhile using both reproducible plant models and suitable biological markers How can we certify whether a product is a biostimulant or not? To answer this question and establish a guideline to access biostimulant activities with simple protocols, it is worthwhile using both reproducible plant models and suitable biological markers
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