IDENTIFICATION OF CDNA CLONES CORRESPONDING TO 2 INDUCIBLE NITRATE REDUCTASE GENES IN SOYBEAN - ANALYSIS IN WILD-TYPE AND NR(1) MUTANT

Among higher plants, soybean is unique in that biochemically it has be en characterized as having two constitutive nitrate reductase (cNR) is oforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while t he nr(1) mutant expresses only the iNR isoform. The genetic and molecu lar mechanisms for regulation of these isoforms have not been elucidat ed. We describe here the isolation, by reverse transcription-polymeras e chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. The y were designated as iNR1 and iNR2, respectively, since both were indu cible by nitrate. The iNR1 and iNR2 cDNAs cover total encoding regions of 2661 and 2673 nucleotides, respectively. The iNR1 clone shows a 12 bp deletion at the 5' end, relative to iNR2. They show overall simila rity of 89% at the nucleotide level, and 87% at the amino acid level. Like all plant NRs cloned so far, deduced amino acid sequences between iNR1 and iNR2 show greatest variation at the N-terminal region while no difference was observed at the C-terminus. Soybean iNR mRNAs were f ound to be different from those of maize and tobacco in response to tu ngsten inhibitor treatment, since the inhibitor decreased the steady-s tate levels of mRNA for soybean INR and for NiR. Using the same 5' reg ions of both cDNAs as the probes, Southern blot analysis of genomic DN A revealed differences in organization between iNR1 and iNR2. The geno mic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr(1) mutant lacked an 8 kb fragment when prob ed with iNR1 cDNA. Likewise, the nr(1) mutant lacked three Hae III res triction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr(1) mutant showed one identical Eco RI band and tw o identical Hae III bands. In northern blots, the steady-state level o f iNR1 mRNA was similar for the nr, mutant and the wild-type parent af ter 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III r estriction enzyme digestion patterns observed in Southern blot analysi s of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.