Study of the (+)-Pisatin Biosynthetic Pathway by RNAi and Development of a Novel Method to Elicit the Production of Plant Secondary Metabolites

by Kaimoyo, Evans.

(+)-Pisatin, ([+]-[6aR,11aR]-6a-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan) is the major phytoalexin of the garden pea (Pisum sativum L.). Despite being the first phytoalexin to be chemically characterized, its biosynthesis remains to be fully elucidated. RNA-mediated genetic interference (RNAi) was used to gain further insights into the (+)-pisatin biosynthetic pathway. The expression of three genes, isoflavone reductase (IFR) catalyzing the reduction of 7,2?-dihydroxy-4?,5?methylenedioxyisoflavone to (-)-sophorol, sophorol reductase (SOR) involved in reducing (-)-sophorol to (-)-7,2?-dihydroxy-4?,5?-methylenedioxyisoflavanol and hydroxymaackiain-3-O methyltransferase (HMM) involved in methylation of (+)-6ahydroxymaackiain to (+)-pisatin was silenced. The genes are transcriptionally coregulated during (+)-pisatin biosynthesis, with the IFR and SOR proposed to function upstream of the HMM gene. Hairy roots expressing the HMM RNAi construct, deficient in (+)-pisatin biosynthesis were identified. However, these did not accumulate (+)-6ahydroxymaackiain, precursor to (+)-pisatin. Instead they accumulated 2,7,4?trihydroxyisoflavanone, daidzein, liquiritigenin and isoformononetin. The amino acid sequence of HMM is very similar to that of another methyltransferase, hydroxyisoflavanone-4?-O-methyltransferase (HI4?MOT), found in most legumes. HI4?MOT catalyzes the methylation of 2,7,4?-trihydroxyisoflavanone (THI) to 2,7dihydroxy-4?-methoxyisoflavanone, one of the earliest enzymatic steps in isoflavonoid biosynthesis. In pea, HI4?OMT may be the same enzyme as “HMM” catalyzing the 13 methylation of both THI and (+)-6a-hydroxymaackiain. Preventing the methylation of THI could divert pea intermediates to the production of daidzein and isoformononetin instead of (+)-pisatin. None of the transgenic hairy roots expressing the IFR RNAi construct were totally deficient in (+)-pisatin biosynthesis. However, all produced reduced amounts of (+)pisatin, with one culture accumulating 7,2?-dihydroxy-4?,5?-methylenedioxyisoflavone, the substrate for IFR. Hairy roots expressing the SOR RNAi construct deficient in (+)pisatin biosynthesis were identified. These accumulated (-)-sophorol, the substrate for SOR. These data provide evidence for the involvement of these genes and the intermediates with (-)-optical activity in (+)-pisatin biosynthesis. The elicitation of the biosynthesis of secondary metabolites in plant cell and tissue cultures by electric current was explored. Electric current was demonstrated to elicit the biosynthesis of secondary metabolites in pea hairy and intact roots, seedling, root and cell suspension cultures of various plant species. Electric current has potential for use as an elicitor of secondary metabolites in basic and commercial research ventures. 14