Fulvic acid affects proliferation and maturation phases in Abies cephalonica embryogenic cells

Abstract

Embryogenic cell masses (ECM) of Abies cephalonica were grown on proliferation media in the presence and absence of fulvic acid (FA), whose molecular composition and conformational rigidity were evaluated by CPMAS-¹³C NMR spectroscopy. To assess the physiological effects of this humic material during proliferation and maturation stages of somatic embryogenesis (SE), proliferation rate, proportion of consecutive developmental stages of pro-embryogenic masses (PEM), cellular ATP and glucose-6-phosphate were evaluated at regular intervals. FA increased the proliferation rate, especially during the early sampling days, and the percentage of PEM in their advanced developmental stage. Cellular ATP and glucose-6-phospahte were increased by FA pre-treatment during the maturation phase. Furthermore, the effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), such as a decrease of growth and the enhancement of PEM III induction, were inverted by FA. Proton pumping ATPase and PPase activities were decreased in microsomes from PCIB-treated ECM, while they increased in the presence of FA. This fulvic matter also induced a delay in somatic embryo formation during the maturation phase. Both the improvement of the PEM proliferation and the reduction of the subsequent maturation process of A. cephalonica are explained by a release from the complex humic structure of low molecular-weight molecules, which may interact with the plant hormonal signaling pathway. These effects appear to be related to the hydrophilic and conformationally labile nature of FA. The structure-activity relationship observed here suggests that the influence of FA on ECM may be attributed to specific bioactive molecules that are preferentially released from the FA loose superstructure.