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.

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The apparent high molecular mass of humic acids (HAs) hardly seems compatible with their direct effects in plant physiology. However, previous evidence has indicated that HAs are non-covalent associations of relatively small molecules, which can be broken down by the action of organic acids. The aim of this work was to evaluate the effects of organic acids on the structure of HAs by spectroscopy and on their bioactivity by following the responses of maize root growth. Changes in the exudation of organic acids from maize seedlings treated with HAs at 50 mg C L 21 were evaluated by high-performance liquid chromatography. The results are in agreement with the concept that HAs are chemical aggregates that acquire characteristics typical of low-molecular-mass humic substances when exposed to organic acids exuded by the roots. Maize seedlings grown in solutions supplemented with HAs plus citric acid at 0.0005, 0.005 and 0.05 mM exhibited significant changes in their root area, primary root length, number of lateral roots and lateral root density and increases in plasma membrane H + -ATPase activity. Furthermore, the root exudation profile of plants treated with HAs exhibited an increase in the efflux of oxalic and citric acids, with a concurrent decrease in malic and succinic acids. These data reveal a crosstalk between HAs and plants where the exudation of organic acids from the roots influences and is influenced by bioactive molecules released from HAs during root development.

Chemical reactions (hydrolysis, oxidation, reduction, methylation, alkyl compounds detachment) were applied to modify the structure of humic substances (HS) isolated from vermicompost. Structural and conformational changes of these humic derivatives were assessed by elemental analyses, size exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance ((13)C CPMAS-NMR), and diffusion ordered spectroscopy (DOSY-NMR), whereas their bioactivity was evaluated by changes in root architecture and proton pump activation of tomato and maize. All humic derivatives exhibited a large bioactivity compared to original HS, both KMnO(4)-oxidized and methylated materials being the most effective. Whereas no general relationship was found between bioactivity and humic molecular sizes, the hydrophobicity index was significantly related with proton pump stimulation. It is suggested that the hydrophobic domain can preserve bioactive molecules such as auxins in the humic matter. In contact with root-exuded organic acids the hydrophobic weak forces could be disrupted, releasing bioactive compounds from humic aggregates. These findings were further supported by the fact that HS and all derivatives used in this study activated the auxin synthetic reporter DR5::GUS.

A series of humic matter samples isolated from a soil sequence, different oxisols, size‐fractionated from a vermicompost humic acid and subjected to chemical modifications, were characterized by CPMAS 13C‐NMR spectroscopy. The relative signal areas in chemical shift regions of NMR spectra of the four sets of samples were analysed by principal component analysis (PCA). Hierarchical cluster analysis (HCA) was applied to build a classification model, which allowed the recognition of humic matter according to its origin. The relationship between carbon species and biological activity of humic acids, as promoters of lateral root emergence, was obtained by applying PLS multivariate analysis. This showed that lateral root emergence was mostly related to NMR parameters such as the hydrophobicity index (HB/HI) and the 40–110 and 160–200 ppm chemical shift regions (hydrophilic carbon HI), while the content of hydrophobic (HB) carbon in humic samples was negatively correlated with induction of lateral root hair. Our results represent a step further in the structure‐bioactivity relationship of natural humic substances and confirm their role as plant root growth promoters.