The metabolism of isoflavonoid phytoalexins in alfalfa (Medicago sativa L.)

Abstract

The synthesis of isoflavonoid phytoalexins in legumes is relatively well understood, but far less is known about how these phytotoxic compounds are metabolised by the plant when no longer required. In this project medicarpin, the major isoflavonoid phytoalexin in alfalfa, was prepared in radiolabelled form and fed to cell cultures and seedlings of alfalfa. The metabolism of the radioactive phytoalexin was then studied by characterising the radiolabelled metabolites formed. Uptake of radiolabelled phytoalexin by cells was faster in elicitor-treated cultures than in untreated cultures. However, there was little difference in pattern or speed of metabolism in treated or untreated cultures. Labelled medicarpin was rapidly metabolised to a complex range of extractable medicarpin products (MPs). A very small proportion of the dose was broken down to (^14)C02. A total of 8 MPs could be resolved as distinct metabolites by HPLC and TLC. However, as incubation time increased the radioactivity became associated with multiple minor components which could not be identified. The 8 MPs were characterised by UV and mass-spectrometry and where possible by co- chromatography with authentic standards by TLC and HPLC. Four MPs were unambiguously identified as medicarpin-3-0-glucoside-6"-O-malonate (MGM), the isoflavans vestitol and sativan and the pterocarpan 6a-hydroxy-3,4'- dimethoxypterocarpan (variabilin). In addition a hydroxylated derivative of medicarpin, termed pseudomedicarpin was also tentatively identified. Of the four remaining metabolites MPl had a relative molecular mass of 166 but remained unidentified. MP2 was formed from pseudomedicarpin, but could not be characterised due to its labile nature. Similarly MP3 and MP6 remained unidentified, though the evidence suggested that MPS was a demethylated product of medicarpin. The metabolism of medicarpin in seedlings resembled that of cell cultures with the exception that rather more of the medicarpin was conjugated to form MGM.