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

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.

Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

“…Transcription of many of the genes for pigment biosynthesis in C. reinhardtii is regulated by light (Matters and Beale, 1995;Lohr et al, 2005;Nogaj et al, 2005), even though the enzyme products of the light-regulated genes are comparably abundant in the cells irrespective of the light conditions (Nogaj et al, 2005; this work) and/or are not rate limiting for end product formation. The rate of pigment synthesis catalyzed by the expressed proteins appears to be regulated by posttranslational processes such as allosteric enzyme activity modulation, rather than by changes in enzyme abundance.…”

“…Light/dark-synchronized cells subjected to an imposed circadian cycle showed no significant variation in the PPO and FeC levels during the light phase. Synchronized cells steadily increase their chlorophyll content throughout the light phase and do not synthesize chlorophyll in the dark phase (Nogaj et al, 2005). It would therefore be expected that the PPO level would increase during the light phase, but this was not observed.…”

“…This suggests a different type of regulation of these two enzymes in the synchronized cells compared to continuous light and dark conditions. The synchronized cells were previously verified to exhibit the typical synchronization behavior of dividing once during the dark phase and not synthesizing chlorophyll in the dark (Nogaj et al, 2005).…”

Enzymes of the Heme Biosynthetic Pathway in the Nonphotosynthetic Alga Polytomella sp

“…From the present results, it is inferred that amyloplasts are the major site of heme synthesis in Polytomella sp., thus resembling the chloroplasts of C. reinhardtii and plants. At present, it cannot be concluded whether GSAT, PPO, and FeC are located exclusively in the amyloplasts of Polytomella sp., as they are in the chloroplast of C. reinhardtii (36,51). Further knowledge about the intracellular localization of Polytomella sp.…”

Growth- and nutrient-dependent gene expression in the toxigenic marine dinoflagellate Alexandrium minutum

“…An upregulation of RubBisCO on the mRNA level might partially compensate this effect. Synthesis of the chlorophyll tetrapyrrole skeleton is dependent on the availability of amino acid precursors (reviewed in Nogaj et al, 2005), and lower chlorophyll quotas in Nlimited cells are common (Sciandra et al, 2000;Verhoeven et al, 1997). This can be associated with reduced amounts of light harvesting complexes, while other components of the light harvesting machinery shift abundance (Peltier and Schmidt, 1991).…”