The auxin-like activity of humic substances is related to membrane interactions in carrot cell cultures

A detailed characterization of two humic fractions was performed: One with low relative molecular mass (LMr<3,500 Da) and one with high relative molecular mass (HMr >3,500 Da). Distinct 1H NMR spectroscopic patterns were observed for the two fractions. HMr showed an aromatic proton region, an intense and broad region (3.0–5.0 ppm) attributed to sugar-like and polyether components, and an intense doublet at 1.33 ppm (identified as protons of the β-CH3 in lactate). In contrast, LMr did not show resonances due to aromatic protons and was characterized by a broad unresolved region, assigned to sugar-like components. The 13C NMR spectra showed that the LMr humic fraction was richer in carboxylic and aliphatic C groups compared to HMr fraction. These substances were fluorescein-labeled [fluorescein isothiocyanate (FITC)], and their interaction with carrot cells in culture was monitored for 10 d, and compared to FITC–indole-3-acetic acid (IAA) to clarify their mechanisms of biological activity. After different incubation times, fluorescein staining of carrot cells and decrease of fluorescein concentration in the culture medium were evaluated. Fluorescent membrane staining was only present in IAA and the LMr humic fraction treated cell cultures. A consequential decrease of fluorescein concentration in the culture media was also observed. Pretreatment of carrot cells with unconjugated IAA or LMr humic fraction markedly reduced fluorescein staining of both FITC–IAA and FITC–LMr humic fraction. Blocking tests gave indirect evidence ofpossible binding of the LMr humic fraction to IAA cell membrane receptors. These results indicate that the two humic fractions behave differently. Only LMr humic fraction, like IAA, interacts with cellular membranes in carrot cell cultures