Effect of humic acids on phosphate level and energetic metabolism of tobacco BY-2 suspension cell cultures

To investigate the role of natural humic substances on plant cell phosphate level and metabolism, tobacco BY-2 suspension cell cultures were grown in the presence of humic samples of different chemical composition: soil humic acid and its three size-fractions (I-III) separated by High Pressure Size Exclusion Chromatography. The humic samples were characterized by CPMAS-NMR spectroscopy and on-line pyrolysis-gas chromatography-mass spectrometry. Suspension cell cultures, after 7 days of incubation, were facing Pi starvation. The fraction III, the most hydrophilic and smallest in molecular size among humic samples, induced a partial relief from Pi starvation, increasing total cell phosphate amount, ATP and glucose-6-phosphate levels, as well as the activity of secreted acid phosphatases. Furthermore, fraction III induced a decrease of KCN-insensitive respiration, evaluated in both suspension cells and isolated mitochondria. The low amount of acidic groups in fraction III excluded that its observed effect in relieving cells from Pi deficiency may be attributed to a partial replacement of the chelating ability of secreted acids (mainly citric and malic acids) in releasing Pi from metal-phosphate complexes. The molecular characteristics of fraction III are conducive to a flexible conformational structure due to hydrophilic domains, which are still contoured by hydrophobic moieties such as alkyl and aromatic compounds. Such flexible molecular associations may induce an efficient release of Pi from organic sources (e.g. nucleic acids), released in the media by damaged or dead cells, exerting a sort of positive effect on either the production or activity of extracellular Pi hydrolytic enzymes. This work shows that only by combining advanced molecular characterization of natural humic molecules with their effect on plant cells, it is possible to formulate sound hypotheses for structure-activity relationships.