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Nucleotide Sequences of Three Chalcone Reductase Genes from Alfalfa

An important reaction in the biosynthesis of many phytoalexins in leguminous plants is the formation of 4,2‘,4‘trihydroxychalcone from 4-coumaroyl-COA and malonylCOA, first demonstrated by Ayabe et al. (1988). Welle and Grisebach (1988) purified a reductase from soybean cell cultures and demonstrated that the enzyme, named CHR, co-acts, with NADPH as cofactor, with CHS in the formation of the 4,2’,4’-trihydroxychalcone. They showed also that the two enzymes are induced coordinately after treatment of the cell cultures with elicitor-type molecules. Moreover, Welle et al. (1991) isolated the encoding cDNA. This reductase can play a fundamental role in planta in the balancing of the production of 5-deoxyflavonoids or 5-hydroxyflavonoids, the synthesis of which depends on the co-action of CHS and CHR or on CHS alone, respectively. Despite this pivotal role and except for the pioneering work already cited, the CHR has not been extensively studied, either at the enzymatic or gene expression levels. We have previously shown that during an incompatible interaction, Medicago sativalPseudomonas syringae pv pisi, severa1 genes involved in phytoalexin production (CHS and isoflavone reductase) were induced (Esnault et al., 1993). This led us to address the question of an involvement of CHR. Our strategy was based first on the isolation of cDNA encoding CHR in alfalfa as well as its genomic organization, and second on the analysis of its expression. In this communication, we report the isolation of three cDNAs encoding CHR. The soybean CHR cDNA clone (Welle et al., 1991) was used to screen a AgtlO cDNA library made from poly(A)+ RNA extracted from leaves harvested 6 h after infection with the incompatible bacterium P. syringae pv pisi. After three rounds of screening and subcloning in the pBS SKplasmid, seven clones were obtained and sequenced. Three of them, MsCHRla, MsCHRlc, MsCHRZa, were full length and their features are described in Table I. They contain an insert of 1202,1115, and 1134 bp, respectively, with an open reading frame of 936 nucleotides. The predicted size of the reductase is 312 amino acids with a molecular mass of around 35 kD. The deduced amino acid sequences are more than 90% homologous to the soybean CHR, and they are related to the aldo-keto reductase family. No signal or ~~