Study of 5-aminolevulinate dehydratase in radish seedlings: are there housekeeping and light-induced enzymes?

Abstract

Aminolevulinate dehydratase (E.C. 4.2.1.24) catalyses the third step of tetrapyrrole synthesis leading to the formation of heme and chlorophylls in plant tissues. We have investigated the regulation of the enzyme by light in radish cotyledons, at the levels of protein activity, mRNA amount, using specific immunoserum and alad cDNA probe respectively. It was observed that, in the light, both 5-aminolevulinate dehydratase activity, and protein level increased 3–4 times compared to the dark-control level. However, no change in the amount of related mRNA was observed. Nevertheless, the application of an inhibitor of transcription (α-amanitin) to excised cotyledons inhibited the light-induced increases of both activity and protein level, but once again the amount of specific mRNA remained constant apparently. After the PCR amplification of a part of alad gene and the cloning of corresponding cDNA, two sequences were observed, differing in introns only or by some base changes, which did not affect the final protein sequence. However, one alteration generated a Xba I restriction site which was used to demonstrate the presence of 2 genes by Southern analysis. Thus, the apparent stability of alad mRNA could be due to the abundant expression of a housekeeping gene, which shadowed a related gene expressed in the light.