Structure and expression of the Chlamydomonas reinhardtii alad gene encoding the chlorophyll biosynthetic enzyme, ?-aminolevulinic acid dehydratase (porphobilinogen synthase)

Abstract

cDNA clones for the alad gene encoding the chlorophyll biosynthetic enzyme ALA dehydratase (ALAD) from Chlamydomonas reinhardtii were isolated by complementation of an Escherichia coli ALAD mutant (hemB). The C. reinhardtii alad gene encodes a protein that has 50 to 60% sequence identity with higher plant ALADs, and includes a putative Mg(2+)-binding domain characteristic of plant ALADs. Multiple classes of ALAD cDNAs were identified which varied in the length of their 3'-untranslated region. Genomic Southern analysis, using an ALAD cDNA as a probe, indicates that it is a single-copy gene. This suggests that the differently sized ALAD cDNAS are not the products of separate genes, but that a primary ALAD transcript is polyadenylated at multiple sites. A time course determination of ALAD mRNA levels in 12-h light:12-h dark synchronized cultures shows a 7-fold increase in ALAD mRNA at 2 h into the light phase. The ALAD mRNA level gradually declines but continues to be detectable up to the beginning of the dark phase. ALAD enzyme activity increases 3-fold by 6 h into the light phase and remains high through 10 h. Thus, there is an increase in both ALAD mRNA level and ALAD enzyme activity during the light phase, corresponding to the previously observed increase in the rate of chlorophyll accumulation.