NADPH : isoflavone oxidoreductase (IFR) is the first soluble enzyme of the pterocarpan-specific part of phytoalexin biosynthesis in chickpea (Cicer arietinum L.). The enzyme was purified to apparent homogeneity by a five-step procedure from chickpea cell cultures treated with yeast extract as elicitor. Analysis by gel filtration and SDSjPAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa. K , values for the substrates 2'-hydroxyformononetin, 2'-hydroxypseudobaptigenin and NADPH were 6, 6 and 20 pM, respectively. The IFR showed pronounced specificity for the substitution pattern of isoflavones. We found a 2'-hydroxy group and a 4',5'-methylenedioxy or 4-methoxy function to be essential for acceptance as substrate. The isoelectric point of the protein was determined as 6.3 by IEF and there is no evidence for the existence of isoenzymes.Partial amino acid sequences of IFR were determined from internal peptides obtained by tryptic digestion of the protein and corresponding oligonucleotides were synthesized. A ilgtlO cDNA library was constructed using poly(A)-rich RNA isolated from chickpea cell cultures treated with Ascochyta rabiei elicitor. 150 positive clones were obtained by screening 2 x lo5 clones with an IFR-specific oligonucleotide. The identity of sequenced clones was confirmed by comparison of the deduced amino acid sequence with the internal peptide sequences of purified IFR. The sequence of a 1183-bp clone contained a continuous open reading frame of 954 bases encoding a polypeptide of 318 amino acids with a calculated molecular mass of 35.4 kDa, indicating that a full-length cDNA coding for IFR was isolated.

Chickpea (Cicer ariet~n~ L.) cell suspension cultures transferred into a medium containing yeast extract accumulate the phytoalexins medicarpin and maackiain. Concomitant with accumuIation of the pterocarpans a new enzyme acttvity is induced which was characterized as NADPH:isoflavone oxidoreductase. Maximum enzyme activity was reached 16 h after transfer of cells and then activity rapidly declined. The soluble enzyme was partially purified and shown to catalyze the reduction of the isoflavone 2'-hydroxyformononetin to the isoflavanone vestitone which is an intermediate in medicarpin biosynthesis. The enzyme data suggest that 2'-hydroxylation is a prerequisite for the conversion of isoflavones to pterocarpans.

Yuki Ichinose3-b, K arin T iem ann3, C laudia Schw enger-Erger3, K azuhiro T oyoda3 C, F rauke H e in 3, Thom as H an selle3, H olger C ornels3 and W olfgang B arz3 * a Institu t für B iochem ie und B iotechnologie d er Pflanzen, W estfälische W ilhelm s-U niversität M ünster, D-48143 M ünster, G erm an y b Science and Technology for E nergy C onversion, G ra d u ate School of N atural Science and Technology, O kayam a U niversity, O kayam a 700 -8 5 3 0 , Japan c L aborato ry of P lant Pathology & G en etic E ngineering, College of A griculture, O k ay am a U niversity, O kayam a 700-8530, Japan * A utor for co rresp o n d en ce and re p rin t requests. Fax: (+49) 2518328371. E-m ail: barz@ uni-m uenster.de Z. N aturforsch. 55c, 4 4 -5 4 (2000), received S ep tem b er 9/O ctober 28, 1999 P lant D efen ce G enes, G en e A ctivation, G ene Suppression, A scochyta rabiei, Cicer arietinum L.In response to the exogenous application of elicitors and attem p ted invasion by pathogens, plants exhibit a wide range of defense reactions. To un d erstan d the defense m echanism s at the level of gene activation and deactivation, differential screenings w ere p erform ed to iso late cD N A clones which are differentially expressed in p ath ogen-inoculated resistant chick pea plants and elicitor-treated cell cultures. A plenty of genes were isolated and arran g ed in 5 groups, nam ely d efen se-related pathw ays, signal transduction pathw ays, regulation of gene expression, catabolic pathw ays and prim ary m etabolism . M ost of these genes w ere activated although several genes w ere also fo u n d to be suppressed. We discuss the plausible functions of cD N A pro d u cts in plan t defense responses. The cD N A s provide a variety of tools to investigate m o lecular m echanism s o f defense responses and clearly reflect the massive g en o mic and m etabolic changes which occur during m anifestation of antim icrobial defense. 0939-5075/2000/0100-0044 $ 06.00 © 2000 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D Unauthenticated Download Date | 6/21/16 8:05 AM fence -a broad perspective. Physiol. Mol. Plant Pathol. 51, 3 4 7 -3 6 6 . Bourois M., M oore P., Ruel L., Grau Y., Heitzler P. and Simpson P. (1990), An early embryonic product of the gene shaggy encodes a serine/threonine protein ki nase related to the C D C 28/cdc2+ subfamily. E M B O J. 9, 2 8 7 7 -2 8 8 4 . Cervantes E ., Rodriguez A. and Nicolas G. (1994), E th ylene regulates the expression of a cysteine proteinase gene during germination of chickpea ( Cicer arietinum L .). Plant Mol. Biol. 25, 2 0 7 -2 1 5 . Chang M. M., Hadwiger L. A. and Horovitz D. (1992), M olecular characterizarion of a pea ß-l,3-glucanase induced by Fusarium solani and chitosan challenge. Plant Mol. Biol. 20, 6 0 9 -6 1 8 . Chomczynski P. (1992), One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 1 3 4 -1 3 9 Chomczynski P. and Sacchi N. (1987), Single-step method of RN A isolation by acid guanidinium thiocyanate-phe...

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Cell-suspension cultures of Ascochyta rabiei-resistant (ILC 3279) and -susceptible (ILC 1929) chickpea (Cicer arietinum L.) cultivars were compared with regard to their elicitor-induced accumulation of pterocarpan phytoalexins and increases in the activities of biosynthetic enzymes. The growth performances and protein patterns of the two cell-culture lines were essentially identical. Treatment of cell cultures with a polysaccharide elicitor from A. rabiei induced fivefold-higher amounts of the phytoalexins medicarpin and maackiain in the cells of the resistant than in the susceptible cultivar. Glucose 6-phosphate dehydrogenase and eight enzymes representing the general phenylpropanoid pathway, the flavonoid-forming steps and the pterocarpanspecific branch of phytoalexin biosynthesis were found to be elicitor-induced. Phenylalanine ammonia-lyase and chalcone synthase reached sharp, transient optima some 8 h after elicitor application in the cells of both cultivars. The activities of isoflavone 2'- and 3'-hydroxylases were only induced in cells of the resistant cultivar with a maximum after 8 h. Cinnamic acid 4-hydroxylase, chalcone isomerase, 2'-hydroxyisoflavone reductase and pterocarpan synthase showed a later or no sharp optimum. The isoflavone-specific 7-O-glucosyltransferase was not induced in either cell-culture line. Cells of the susceptible cultivar failed to induce significant activities of isoflavone 2'-hydroxylase and these cells produced only very low amounts of phytoalexins. Isoflavone 2'-hydroxylase is postulated to be the main limiting enzyme for pterocarpan biosynthesis in cells of the susceptible cultivar. The pterocarpan biosynthetic pathway in chickpea cells represents a suitable model for investigations of differential gene activation in connection with the expression of antimicrobial defence reactions.