Alkamides: a new class of plant growth regulators linked to humic acid bioactivity

Background The use of humic substances as plant biostimulants has been increasingly attracting farmers and stunning researchers. The ability of these substances to enhance root growth by changing root architecture is often linked to their hormonal activities, such as auxin effects and nitric oxide production. Humeomics accesses the molecular constituents of humic substances, revealing the importance of alkyl components because of their conformations and chemical activities. Here, we describe the alkamides present in humic acids and compare their bioactivities using plasma membrane H+-ATPase activity level as a biochemical marker. Methods Humic acids isolated from vermicompost were analyzed using 13C and 15N nuclear magnetic resonance spectroscopy. The unbound fraction was extracted with ethyl acetate and submitted to gas chromatography coupled to mass spectrometry to detect the presence of N-isopropyldecanamide. We synthesized N-isopropyldecanamide and treated maize seedlings for 7 and 15 days with different concentrations. The root growth and plasma membrane H+-ATPase activity were monitored. Nitric oxide accumulation in the lateral roots was imaged using 4,5-diaminofluorescein diacetate. The results were compared with those obtained for seedlings treated with humic acids isolated from vermicompost. Results The amide functional group produced the only nitrogen signal in the 15N humic acid resonance spectrum and similar alkamide moieties were found in the unbound humic extract through comparisons using gas chromatography coupled to mass spectrometry. The synthesis of N-isopropyldecanamide had few steps and produced a high yield (86%). The effects of N-isopropyldecanamide on root growth were concentration dependent. High concentrations (10−4 M) enhanced root growth after 15 day of diminishing shoot biomass. However, low concentrations (10−8 M and 10−6 M) promoted root growth at 7 and 15 days, similar to the humic acid-induced plasma membrane H+-ATPase activity. Both N-isopropyldecanamide and humic acids enhanced nitric oxide accumulation during lateral root emergence. Conclusion We described for the first time the effects of N-isopropyldecanamide on the plasma membrane H+-ATPase activity in maize seedling roots and compared its effects with those caused by humic acids. N-Isopropyldecanamide was detected in the unbound fraction of the humic supramolecular assembly, indicating that the putative hormone-like effects of these substances result also from the presence of this new class of plant regulators, in addition to other molecules.