Pterocarpan phytoalexin biosynthesis in elicitor-challenged chickpea (Cicer arietinum L.) cell cultures. Purification, characterization and cDNA cloning of NADPH:isoflavone oxidoreductase

Abstract

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 SDS/PAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa. Km values for the substrates 2'-hydroxyformononetin, 2'-hydroxypseudobaptigenin and NADPH were 6, 6 and 20 microM, 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 lambda gt10 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 10(5) 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.