The modification of metabolic profile in plant cell cultures as a strategy to investigate the biological role of secondary metabolites

Secondary metabolites are considered necessary for plant adaptation to the variable environment; however, only for few secondary metabolites the specific functions are known in detail. One possible approach to elucidate their roles in plant is to modify the secondary metabolite profile of in vitro cell cultures and investigate the impact of such modifications on the phenotype. The general experimental approach followed in this investigation can be summarized by these four different steps: 1) detailed analysis of the secondary metabolite profile of the chosen cell lines; 2) the metabolite profile of the cells is modified by precursor and inhibitor administration, i.e. the main object of this research project, and the effects of any treatment on the metabolome are monitored though HPLC-DAD and HPLC-ESI-MS; 3) short stresses are applied to cells and the effects of stress are characterized from a cytological point of view in order to identify specific phenotypic traits caused by stress application; 4) the effects of the metabolome modification on cellular response against the stress are evaluated. Three in vitro cell lines derived from different plant species, T2b (Ocimum basilicum), Sw4i (Petunia hybrida), R3M (Daucus carota), have been chosen for their ability to accumulate different set of secondary metabolites. In the basil cell line, T2b, a strategy using the inhibitor of the biosynthesis of coumaroyl CoA (3,4- (methylenedioxy) cinnamic acid, MDCA) coupled with the supplementation of an AC precursor (dihydroquercetin, DHQ) was performed, in order to change the ratio between rosmarinic acid (RA) and anthocyanins (ACs). In the petunia cell line, Sw4i, two different strategies were designed in order to modulate the metabolic profile. A modification of the ratio between methylated ACs (petunidin, malvidin, peonidin) and non-methylated ACs (delphinidin, cyanidin) was obtained feeding cells with DHQ, a precursor of cyanidin and delphinidin based-ACs in petunia species. In the second strategy, the use of piperonylic acid (PIP), an inhibitor of the biosynthesis of p-coumaric acid, allowed to modify the accumulation of acylated ACs versus non-acylated ACs, with a significant decrease of acylated ACs. In the carrot cell line, R3M, cells were treated with PIP in order to decrease the accumulation of acylated ACs, as obtained in petunia. Surprisingly, after the treatment, non-acylated ACs underwent a strong reduction. This unexpected result might be due to the non-specificity of acyltransferase enzymes in carrot cell line. In fact, PIP supplied to cells and cinnamic acid accumulated as substrate of the inhibited reaction, might be used to generate acylated ACs at the expense of non-acylated ACs. In R3M cell line, the protective role of specific classes of secondary metabolites after stress application could be investigated both through precursor and inhibitor administration. The heat treatment at 44°C for 1 h induced the appearance of cytoplasmic patches surrounded by endoplasmic reticulum; it was also demonstrated that the carrot cells showing this morphology were committed to a slow cell death fate. Previous experiments of feeding with hydroxycinnamic acids (HCAs) before the heat treatment caused an increase in acylated ACs and HCAs derivatives and the reduction of the number of cells with patches. By supplying PIP to R3M cells we obtained the decrease of the level of non-acylated ACs and HCA derivatives and an increase of the number of cells with stressed phenotype. Comparing the results obtained by the two complementary approaches, it was not possible to assess the role of ACs. However both strategies suggested a role of HCAs in the prevention of cellular damages induced by heat stress in R3M carrot cells.