biosynthesis in alfalfa

Melina López-Meyer and Nancy L. Paiva.

The Samuel Roberts Noble Foundation. P.O.Box 2180,Ardmore, OK, 73402

Alfalfa plants respond to pathogen attack by producing phytoalexins. In this plant, the major phytoalexin is medicarpin. This compound is induced in leaves that have been challenged with fungal pathogens, but not in healthy leaves. Also, medicarpin can be conjugated to malonyl and glucosyl residues to form MGM, which accumulates in roots and nodules. These observations indicate that medicarpin might be playing an important role in the defense system of the plant against fungal attack. Medicarpin is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. Isoflavone reductase (IFR) and vestitone reductase (VR) are two enzymes involved in the last part of the pathway (Guo et al.,1994; Oommen et al.,1994. Very little is known about the subcellular location of isoflavonoid-specific enzymes. Since modification of phytoalexin composition in alfalfa represents a potential method for increasing pathogen resistance, knowledge of the compartmentation of the enzymes in the pathway may have a major impact on such modification strategies. Thus, the aim of this work was to determine the subcellular location of IFR and VR in Phoma medicaginis-infected alfalfa plants. For this purpose, polyclonal antibodies raised against IFR and VR proteins were used in immunocytochemical studies. As anti-VR serum showed a single band in western analysis of infected leaf protein extracts, this serum was used to immunolocalize VR protein by transmission electron microscopy. It was determined that VR is a cytoplasmic enzyme. Western blot analysis of infected alfalfa leaf extracts challenged with anti-IFR serum showed the presence of two major and two minor bands. As this antiserum was not suitable for use in immunolocalization studies in alfalfa, transgenic tobacco plants transformed with the alfalfa IFR cDNA under the regulation of the CaMV35S promoter were used for these studies. Anti-IFR serum showed a single band in western analysis of 35S::IFR tobacco leaf extracts. Electron microscopy analysis of IFR-expressing tobacco leaves showed that IFR is also a cytoplasmic enzyme. Additional analysis of infected alfalfa leaf sections challenged with anti-IFR serum revealed that all the signal detected was localized in the cytoplasm. This confirmed the cytoplasmic localization of IFR in alfalfa, and showed that the other proteins that were recognized by anti-IFR serum were also cytoplasmic. The data presented in this work indicate that, at least up to the penultimate biosynthetic step catalyzed by VR, the biosynthesis takes place in the cytoplasm. The location of the latest step in the medicarpin pathway and the conjugation of medicarpin to MGM remain to be elucidated.

Guo L, Dixon RA, Paiva NL (1994) Conversion of vestitone to medicarpin in alfalfa (Medicago sativa L.) is catalyzed by two independent enzymes. J Biol Chem. 269: 22372-22378

Oommen A, Dixon RA, Paiva NL (1994) The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants. The Plant Cell 6:1789-1803