Differential induction of antioxidant stilbenoids in hairy roots of Vitis rotundifolia treated with methyl jasmonate and hydrogen peroxide

Abstract

Stilbenoids are polyphenolic phytoalexins that exhibit potential health applications in humans. Hairy root cultures of muscadine grape (Vitis rotundifolia Michx.) were used to study the biochemical and molecular regulation of stilbenoid biosynthesis upon treatment with 100 μM methyl jasmonate (MeJA) or 10 mM hydrogen peroxide (H2O2) over a 96-h period. Resveratrol, piceid, and ε-viniferin were identified in higher concentrations in the tissue whereas resveratrol was the most abundant stilbenoid in the medium under either treatment. An earlier increase in resveratrol accumulation was observed for the MeJA-treated group showing a maximum at 12 h in the tissue and 18 h in the medium. Furthermore, the antioxidant capacity of extracts from the tissue and medium was determined by the 2,2'-azinobis[3-ethylbenzthiazoline sulfonic acid] (ABTS) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays showing correlation with the stilbenoid content. Fourteen candidate reference genes for qPCR were tested under the described experimental conditions and resulted in the selection of 5 reference genes. Quantitative analyses of transcripts for phenylalanine ammonia-lyase (PAL), resveratrol synthase (RS), and two stilbene synthases (STS and STS2) showed the highest RNA level induction at 3 h for both treatments with a higher induction for the MeJA treatment. In contrast, the flavonoid-related chalcone synthase (CHS) transcripts showed induction and a decrease in expression for MeJA and H2O2 treatments, respectively. The observed responses could be related to an oxidative burst triggered by the exposure to abiotic stressor compounds with signaling function such as MeJA and H2O2 which have been previously related to the synthesis of secondary metabolites.

Keywords: 2,2-diphenyl-1-picrylhydrazyl; 2,2′-azinobis[3-ethylbenzthiazoline sulfonic acid]; 2,6 dimethyl-β-cyclodextrin; ABTS; Antioxidant; BDS; CHS; CTAB; DIMEB; DPPH; DW; Dunstan and Short medium; Elicitation; EtOAc; FW; H(2)O(2); HPLC; Hairy root; JA; MIQE; MS; MSV; MeJA; Minimum Information for Publication of Quantitative Real-Time PCR Experiments; Murashige and Skoog medium; Muscadine grape; NF; PAL; RNA quality indicator; ROS; RQI; RS; Rt; SD; Stilbene synthase; Stilbenoid; TAC; TEAC; Trolox equivalent antioxidant capacity; V; Vitis rotundifolia; cetyltrimethylammonium bromide; chalcone synthase; dry weight; ethyl acetate; fresh weight; high performance liquid chromatography; hydrogen peroxide; jasmonic acid; methyl jasmonate; modified Murashige and Skoog medium; normalization factor; pairwise variation; phenylalanine ammonia-lyase; qPCR; quantitative polymerase chain reaction; reactive oxygen species; resveratrol synthase; retention time; specific growth rate; standard deviation; total antioxidant capacity; μ.