Isolation and Initial Characterization of Constitutive Nitrate Reductase-Deficient Mutants NR328 and NR345 of Soybean (Glycine max)

Botany Department, Australian National University, Canberra ACT 2600, Australia.
Plant physiology (Impact Factor: 6.84). 07/1986; 81(2):572-6. DOI: 10.1104/pp.81.2.572
Source: PubMed


Two nitrate reductase deficient mutants of soybean (Glycine max [L.] Merr. cv Bragg) were isolated from approximately 10,000 M(2) seedlings, using a direct enzymic assay in microtiter plates. Stable inheritance of NR345 and NR328 phenotypes has been demonstrated through to the M(5) generation. Both mutants were affected in constitutive nitrate reductase activity. Assayable activities of cNR in nitrate-free grown seedlings was about 3 to 4% of the control for NR345 and 14 to 16% of the control for NR328. Both mutants expressed inducible NR during early plant development and were sensitive to nitrate and urea inhibition of nodulation. These new mutants will allow an extension of the characterization of nitrate reductases and their function in soybean. Preliminary evidence indicates that NR345 is similar to the previously isolated mutant nr(1), while NR328 is different.

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    • "There are some parallels between NO 3 − stimulation of lateral root growth and NO 3 − inhibition of nodulation. As discussed above, NO 3 − inhibition of nodulation is also (at least partly) a localized effect, and evidence from the use of NR-deficient mutants of pea and soybean indicates that here too the NO 3 − ion itself is able elicits the response (Carroll and Gresshoff, 1986; Jacobsen, 1984). Mutants of both pea and soybean have been isolated which are NO 3 − -tolerant for nodulation (Carroll et al., 1985; Postma et al., 1988) and these have helped to shed some light on the mechanism of NO 3 − inhibition . "
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    ABSTRACT: A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b(556) type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported.
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