Cellular Levels of Glutamyl-tRNA Reductase and Glutamate-1-Semialdehyde Aminotransferase Do Not Control Chlorophyll Synthesis in Chlamydomonas reinhardtii

Abstract

Abstract 5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. In plants, algae, and most bacteria, ALA is generated from glutamate. First, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNAGlu. Activated glutamate is then converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR). Finally, GSA is rearranged to ALA by GSA aminotransferase (GSAT). In the unicellular green alga Chlamydomonas reinhardtii, GTR and GSAT were found in the chloroplasts and were not detected in the mitochondria by immunoblotting. The levels of both proteins (assayed by immunoblotting) and their mRNAs (assayed by RNA blotting) were approximately equally abundant in cells growing in continuous dark or continuous light (fluorescent tubes, 80 μmol photons s−1 m−2), consistent with the ability of the cells to form chlorophyll under both conditions. In cells synchronized to a 12-h-light/12-h-dark cycle, chlorophyll accumulated only during the light phase. However, GTR and GSAT were present at all phases of the cycle. The GTR mRNA level increased in the light and peaked about 2-fold at 2 h into the light phase, and GTR protein levels also increased and peaked 2-fold at 4 to 6 h into the light phase. In contrast, although the GSAT mRNA level increased severalfold at 2 h into the light phase, the level of GSAT protein remained approximately constant in the light and dark phases. Under all growth conditions, the cells contained significantly more GSAT than GTR on a molar basis. Our results indicate that the rate of chlorophyll synthesis in C. reinhardtii is not directly controlled by the expression levels of the mRNAs for GTR or GSAT, or by the cellular abundance of these enzyme proteins.