Identification of cDNA clones corresponding to two inducible nitrate reductase genes in soybean: analysis in wild-type and nr1 mutant

Abstract

Among higher plants, soybean is unique in that biochemically it has been characterized as having two constitutive nitrate reductase (cNR) isoforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while the nr1 mutant expresses only the iNR isoform. The genetic and molecular mechanisms for regulation of these isoforms have not been elucidated. We describe here the isolation, by reverse transcription-polymerase chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. They were designated as iNR1 and iNR2, respectively, since both were inducible 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 similarity 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 found to be different from those of maize and tobacco in response to tungsten inhibitor treatment, since the inhibitor decreased the steady-state levels of mRNA for soybean iNR and for NiR. Using the same 5' regions of both cDNAs as the probes, Southern blot analysis of genomic DNA revealed differences in organization between iNR1 and iNR2. The genomic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr1 mutant lacked an 8 kb fragment when probed with iNR1 cDNA. Likewise, the nr1 mutant lacked three Hae III restriction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr1 mutant showed one identical Eco RI band and two identical Hae III bands. In northern blots, the steady-state level of iNR1 mRNA was similar for the nr1 mutant and the wild-type parent after 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III restriction enzyme digestion patterns observed in Southern blot analysis of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.

Inhibitory effects of elevated endogenous cytokinins on nitrate reductase in ipt‐expressing tobacco are eliminated by short‐term exposure to benzyladenine

“…In the absence of nitrate, NR expression is at a low constitutive level. Specific isoforms of the enzyme may be responsible for the constitutive activity (Wu et al. 1995).…”

“…In the absence of nitrate, NR expression is at a low constitutive level. Specific isoforms of the enzyme may be responsible for the constitutive activity (Wu et al 1995). When nitrate starved plants are exposed to external nitrate, NR mRNA and NR activity (NRA) increase several-fold within 2-6 h of the treatment (Li and Oaks 1993).…”

Nitrate assimilation in the forage legume Lotus japonicus L.

“…The structure of the L. japonicus NR gene resembles that of other NR genes, the closest similarity being with those of the NADH-NRs of soybean ($81%; Wu et al 1995) and Phaseolus vulgaris ($79%; Hoff et al 1991). The biochemical and molecular features of nitrate reduction in L. japonicus appear to be relatively simple in comparison with some other extensively studied Fig.…”

The role of non-symbiotic haemoglobin and nitric oxide homeostasis in waterlogging tolerance in Vigna species

“…), guanine-cytosine (GC) % and melting temperature (Tm) were analyzed by using Oligoanalyzer 3.0 tool (http://www.idtdna.com/ analyzer/applications/oligoanalyzer/, Integrated DNA Technologies, Coralville, USA). Primers for various genes were designed using the data base; non-symbioticHb -Arabidopsis thaliana (Trevaskis et al 1997), Medicago sativa (Seregelyes et al 2000) and Glycine max (Anderson et al 1996), cNR -Glycine max (Wu et al 1995) and tubulin -Vigna radiata (Raha et al 1987). Sequences for various primers are listed in Table 1. RT-PCR amplified cDNAs were fractionated on agarose gel and purified.…”