Journal of Experimental Botany, Vol. 51, No. 347, pp. 1099-1105,
June 2000
© 2000 Oxford University Press
Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties
Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
The activity of nitrate reductase (+Mg2+, NRact) in illuminated leaves from spinach, barley and pea was 50–80% of the maximum activity (+EDTA, NRmax). However, NR from leaves of Ricinus communis L. had a 10-fold lower NRact, while NRmax was similar to that in spinach leaves. The low NRact of Ricinus was independent of day-time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinusextracts inhibiting NR were not found. NRact from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5. NRmax had a broad pH response profile that was siilar for the spinach and the Ricinus enzyme. Accordingly, the Mg2+-sensitivity of NR from Ricinus was strongly pH-dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of NR from a Ricinus extract varied dramatically with pH and Mg2+concentration. Following a light–dark transition, NRact from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NRmax. In contrast to the spinach enzyme, Ricinus-NR was hardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies against the potential 14-3-3 binding site around ser 543 of the spinach enzyme revealed that Ricinus-NR containes the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchange chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg2+-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation.
Key words: Activation state, nitrate reductase, Ricinus communis L., protein phosphorylation, 14-3-3 proteins.
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