Low content of nitrate reductase in chloroplast-ribosome deficient tissue of the 'iojap' mutant

The nuclear allele 'iojap', in homozygous condition, was previously shown to interfere with chloroplast biogenesis by causing a chloroplast ribosome deficiency (V. Walbot and E.H. Coe, Jr., PNAS 76:2760-2764, 1979; A. Siemenroth et al., Plant Physiol. 65:1108-1110, 1980, MNL 54:53-54, 1980). We analyzed the offspring of a cross, ij ij x ++, consisting of 3 types of seedlings: pure green, pure white, and pure yellowish. We measured the activities of nitrate reductase (NR) and nitrite reductase (NiR) according to published procedures (R.R. Mendel and A. Miffler, Mol. Gen. Genet. 177: 145-153, 1979) and determined the content of the NR apoprotein by immunological methods (J. Schiemann and A. Muller, Biochem. Physiol. Pflanzen 180:63-74, 1985) using antiserum against NR purified from squash cotyledons. The results clearly demonstrate that chloroplast-ribosome deficient white and yellowish leaves exhibit only low levels of NR activity, whereas NiR activities are about 70% of normal wild-type activity:

The low NR activity is caused by a comparable low quantity of the NR apoprotein as tested by a 'protection of inhibition assay' using antiserum against NR. The molybdenum cofactor necessary for NR activity is present in all leaf types in equal quantities (Mendel unpubl.). NiR is a chloroplast enzyme (cf. E. Kessler, Progr. Bot. 43:74-82, 1981). Thus, it may not be surprising that it is affected in its activity by a mutation leading to the appearance of entirely undifferentiated plastids (although NiR is encoded in nuclear DNA and synthesized on cytoplasmic ribosomes, otherwise it would not be found in a chloroplast-ribosome deficient mutant). But it is striking that the NR shows low activity and even low protein content in the chloroplast-ribosome deficient leaves, because NR is located outside the plastids in the cytosol (Kessler, 1981). We conclude that the 'iojap' mutation, by inhibiting normal chloroplast development, leads to a reduced accumulation of the NR apoprotein. The reduced rate of accumulation might be caused by a repression (or missing derepression) of the synthesis or, less probably, by a specific and fast degradation of this protein.

It is known that the inactivation of photosynthesis may lead to a decrease in NR activity (cf. S.H. Duke and S.O. Duke, Physiol. Plant. 62:485-493, 1984). Neither the white nor the yellowish leaves of 'iojap'maize show any trace of photosynthetic activity as determined by measurements of delayed light emission (A. Siemenroth et al., Plant Physiol. 65:1108-1110, 1980; Borner and Matorin unpubl.). Therefore, the 'iojap' mutation could act via its effect on photosynthesis on NR activity. A direct and specific effect of the products of photosynthesis on the synthesis (or degradation) of the NR protein, however, is highly improbable. We postulate, therefore, that there exists in maize a dependence of NR accumulation on normal chloroplast development. There are a few examples for a control on the transcription or translation of the genetic information for certain chloroplast proteins in the nucleo-cytoplasmic compartment by the developmental stage of the chloroplast (J.W. Bradbeer et al., Nature 279:816-817, 1979; M.H. Harpster et al., Plant Mol. Biol. 3:59-71, 1984; L. Herrera-Estrella et al., Nature 310:115-120, 1984; S.P. Mayfield and W.C. Taylor, Eur. J. Biochem. 144:79-84, 1984; P. Eckes et al., Mol. Gen. Genet. 199:216-224, 1985). Our results suggest that the developmental stage of the chloroplast may also be important for the synthesis of at least one non-chloroplastic protein, the nitrate reductase. (We thankfully acknowledge generous gifts of seeds of 'iojap' maize by E.H. Coe, Jr., and of antiserum against NR by W.H. Campbell.)

Th. Borner, J. Schiemann and R.R. Mendel

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