U lrike M ack en b ro ck an d W olfgang Barz L ehrstuhl für Biochem ie d er Pflanzen, W estfälische W ilhelm s-U niversität, H in d en b u rg p latz 55, D -4400 M ü n ster, B undesrepublik D eutschland Z. N atu rfo rsch . 46c, 4 3 -5 0 (1991); received O cto b er 9, 1990 Cicer arietinum , Cell C u ltu re, E licitor, P tero carp an Phytoalexins, F o rm o n o n etin 7 -0 -g lu co sid e -6 "-0 -m a lo n a te A fter inhibition o f p h en y lalan in e am m o n ia lyase by L -a-am inooxy-ß-phenylpropionic acid, the constitutively form ed fo rm o n o n etin 7 -0 -g lu co sid e-6 "-0 -m alo n ate is m etabolized w ith the isoflavone aglycone being used as an interm ediate in the elicitor-induced fo rm atio n o f p te ro carp an phytoalexins in ch ickpea cell suspension cultures. In elicited cultures n o t treated with the in h ib ito r phytoalexins are synthesized de novo from phenylalanine. T herefore, in chickpea cells the constitutive isoflavone conjugate m etabolism and the elicitor-induced p te ro carp an fo rm atio n show m etabolic linkage un d er specific physiological conditions. Brought to you by | Mount Royal University Authenticated Download Date | 6/12/15 8:55 PM

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Cell suspension cultures of chickpea (Cicer arietinum L.) were used to prepare protoplasts and vacuoles. The vacuolar preparation revealed only slight contaminations of cytoplasmic marker enzymes. H PLC analysis of the vacuolar extract showed that the malonylglucosides of isoflavones, isoflavanones and pterocarpans are exclusively located in the vacuole. Experi ments designed to determine the subcellular localization of the isoflavone malonylglucoside: malonylesterase suggest an association of this enzyme with the vacuolar membrane. Finally, a ß-l,3-glucanase and a chitinase with basic isoelectric points were also found to be localized in the chickpea vacuoles.

Constitutive phenolics of chickpea cell suspension cultures are the isoflavones formononetin and biochanin A, the isoflavanones homoferreirin and cicerin and the pterocarpans medicarpin and maackiain. They accumulate as vacuolar malonylglucosides. The biosynthetic pathways to isoflavones, pterocarpans and malonylglucoside conjugates together with their enzymes are explained. Elicitation of cell cultures leads to pronounced increases in the activities of biosynthetic enzymes with differential effects on the enzymes involved in conjugate metabolism. Low elicitor doses favour pterocarpan conjugate formation whereas high doses lead to pterocarpan aglycone accumulation accompanied by vacuolar efflux of formononetin and pterocarpan malonylglucosides. Elicitor-induced changes in enzyme activities and vacuolar efflux of conjugates are prevented by application of 10-3M concentrations of cinnamic acid. Cinnamate is alternatively metabolized to a glucose ester, a S-glutathionyl conjugate and to cell wall bounds forms; these reactions are intensified by elicitation. Isoflavone and pterocarpan biosynthesis and conjugate metabolism as regulated by elicitation and cinnamate is depicted in a metabolic grid to explain the complex regulatory pattern of phenolic accumulation in chickpea cell cultures.Abbreviations: AOPP -L-a-aminooxy-/3-phenylpropionic acid, BGM -biochanin A 7-0-glucoside-6"--0-malonate, FGM -formononetin 7-0-glucoside--6"-0-malonate, HPLC -high performance liquid chromatography, MaGM -maackiain 3-0-glucoside--6'--0-malonate, MeGM -medicarpin 3-0-glucoside--6'-0-malonate

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