Medicago sativa cDNAs encoding chalcone reductase.

CHR is an enzyme that co-acts with CHS to produce a branch in the first step of the flavonoid pathway. CHS acting alone catalyzes the sequential coupling of three malonyl-COA molecules with coumaroyl-COA to produce 4,2’,4’,6’-tetrahydroxychalcone (Heller and Hahlbrock, 1980). However, in the presence of CHR and NADPH the oxygen function of the polyketide intermediate, which would produce the 6’-hydroxyl of the chalcone, is reduced and then eliminated as water prior to cyclization, resulting in the formation of 4,2’,4’-trihydroxychalcone (6’-deoxychalcone). This chalcone is the precursor of the 5-deoxy series of flavonoids and isoflavonoids (Harborne, 1988), which includes nodulation induction factors (Maxwell et al., 1989) as well as pterocarpan phytoalexins of the Leguminosae (Dixon et al., 1983). In alfalfa (Medicago sativa) increased phytoalexin production in response to elicitation or infection is a consequence of the induction of genes encoding enzymes for this pathway (Dixon et al., 1992). Although CHR is an essential first step in the synthesis of 5-deoxy isoflavonoids, CHR transcript induction has been demonstrated only in soybean (Welle and Grisebach, 1989), this being the source of the first and only other CHR gene isolated to date. An alfalfa cDNA library (Gowri et al., 1991) representing mRNA from elicited suspension-cultured cells was screened for CHR. A 650-nucleotide probe was generated by PCR from soybean genomic DNA and 20-mer primers based on the soybean CHR sequence (Welle et al., 1991). The frequency of related sequences within the alfalfa cDNA library was very high (3% of total clones) and comparable to the frequency of CHS clones within this library. Four full-length clones were examined by sequencing (Table I). Three clones (CHR4, CHRlO, and CHR12) proved to be from the same gene based on matching sequence in the coding and 3‘ untranslated region. Clone CHR12 was selected as the representative clone from this group. Although only a single putative polyadenylation signal was identified, at 107 bases downstream of the stop codon (a11 clones), polyadenylation occurred at 22 (CHR71, 23 (CHRlO), 111 (CHR4), and 133 (CHR12) bases downstream of this signal. Clone CHR7 represents a second gene based on sequence dissimilarity (66% identity) in the 3’ untrans-