Biochemical studies on the iojap mutant of maize

The plastids in the white tissue of green-white striped or entirely white leaves of the nuclear gene induced iojap mutant are much smaller than the normal chloroplasts of green mesophyll or bundle sheath cells. Their matrix contains membranous structures and DNA fibrils whereas no grana thylakoids and no ribosomes could be found by electron microscopy (L. K. Shumway and T. E. Weier, 1967). This mutant may serve as an appropriate tool for the study of the function of plastid DNA and ribosomes and of the interaction between the genetic systems of the plastid and the nucleo-cytoplasm during biogenesis of the chloroplasts.

We checked green and white leaf tissue of iojap maize for the presence of fraction I protein and plastid coupling factor CF1 in order to test the function of the protein-synthesizing system of the plastids (subunits of both proteins are synthesized on chloroplast ribosomes). Fraction I protein was shown to be present in green tissue by reaction with antibodies against fraction I protein from barley by means of crossed immunoelectrophoresis. However, no precipitation peak could be observed in the case of white tissue. The plastid coupling factor CF1 was solubilized by EDTA treatment of washed plastid membranes. After polyacrylamide gel electrophoresis of the ten-fold concentrated extract the gels were stained for ATPase activity (T. Borner, R. Manteuffel, A. R. Wellburn, Protoplasma 98:153-161). In extracts of plastids from white tissue no activity could be detected whereas EDTA extracts from green tissue yielded an intense white ATPase band in the gels. The identity of this ATPase with plastid coupling factor was shown by reaction with antiserum against the plastid coupling factor CF1. These results indicating a plastid ribosome deficiency support the observations of V. Walbot and E. H. Coe Jr. (PNAS 76:2760-2764) that plastids of white iojap tissue contain no rRNA and are unable to incorporate amino acids.

In contrast the activity of PEP carboxylase could easily be found in white leaf tissue. Extracts of white leaves (ij/ij) show about 40% of the activity (fresh weight basis) observed in green leaves:
  Mutant Control
Activity in µmol/min ml 0.57 ± 0.1 1.45 ± 0.16
Activity in % 39.3 100

Since the plastid ribosomes are obviously missing in the white tissue, this enzyme should be synthesized on cytoplasmic ribosomes. The absorption spectrum of white leaves exhibits a very reduced content of carotenoids and chlorophyll as compared with green leaves. The height of the peaks at 482 nm and 670 nm corresponds to 1% to 2% of carotenoid content and to less than 0.1% of chlorophyll a content of green leaves. In agreement with the reduced content of photosynthetic pigments the white iojap tissue shows no trace of photosynthetic activity as examined by delayed light emission. In order to check the influence of the iojap mutation on mitochondria we measured the oxygen consumption (nmol per min per 50 mg) of small pieces of tissue by a Clark type electrode. Mitochondria of white tissue are much less active than those of green tissue:
  Mutant Control
Dark 3.3 ± 0.2 8.7 ± 0.4
Light 3.3 ± 0.2 5.1 ± 0.02

This might be caused by a further effect of the mutation or by a shortage of substrate, respectively. (We thank E. H. Coe for kindly supplied gifts of seeds of iojap maize.)

A. Siemenroth, Th. Borner and R. Hagemann

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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