Coaction of light, nitrate and a plastidic factor in controlling nitrite-reductase gene expression in spinach

Abstract

It is well established that nitrite reductase (NIR; EC 1.7.7.1) a key enzyme of nitrate reduction - is "induced" by nitrate and light. In the present study with the spinach (Spinacia oleracea L.) seedling the dependency of NIR appearance on nitrate, light and a 'plastidic factor' was investigated to establish the nature of the coaction between these controlling factors. A cDNA clone coding for spinach NIR was available as a probe. The major results we have obtained are the following: (i) The light effect on the appearance of NIR activity occurs through phytochrome. No specific bluelight effect is involved, (ii) Immunotitration data indicate that light affects the appearance of NIR by inducing the de-novo synthesis of the NIR protein, (iii) A multiplicative relationship exists between the action of nitrate and light on NIR appearance. This indicates that the actions of light and nitrate are indeed independent of each other but that both factors operate on the same causal sequence, (iv) Anion-exchange chromatography revealed only a single form of NIR in spinach. Experiments involving plastid photooxidation indicate that this NIR is exclusively plastidic. (v) Northern blot analysis of NIR mRNA showed a strong increase of the steady-state level in the presence of nitrate whereas light had no effect; NIR mRNA was almost undetectable when the plastids were damaged by photooxidation. It is concluded that NIR gene expression in spinach requires positive control by a 'plastidic factor'. Moreover, nitrate exerts a coarse control at the mRNA level whereas fine tuning of NIR protein synthesis is post-transcriptional and is exerted by light, operating via phytochrome.