The tetrapyrroles Mg-protoporphyrin IX (MgProto) and heme, in Chlamydomonas reinhardtii only synthesized within the chloroplast, have been implicated in retrograde control of nuclear gene expression in this unicellular green alga. However, feeding of the two tetrapyrroles to Chlamydomonas cultures growing in the dark has previously been shown to transiently induce 5 nuclear genes, among them three genes coding for the chloroplast heat shock proteins HSP70A, B and E. Here, we measured the impact of MgProto and hemin feeding in the dark on changes in gene expression at the genomic level. About 10% of the 10000 genes represented on the microarray showed a transient up or down regulation with a fold change of 4 or more (p ≤0.05). The two most prominent groups of regulated genes were those where both MgProto and heme caused either an up or down regulation. Minor regulatory groups consisted of genes which were either down- or up-regulated by one of the tetrapyrroles but not by the other. In contrast, feeding of protoporphyrin IX had no regulatory effect on a number of selected genes. Interestingly, 499 of the 982 responding genes were also regulated by heat shock; 85% of those showed the same response (up or down) as seen after MgProto/heme feeding, indicating a previously not anticipated role of MgProto and heme in stress response. Indeed, most prominent among the functional groups of annotated genes up or down regulated by the tetrapyrroles were those whose gene products are involved in protein folding and/or protein degradation. Striking is the virtual absence of regulated genes that encode constituents of the photosynthetic apparatus. This and the transient nature of changes in gene expression observed upon feeding of the tetrapyrroles suggest a signaling role of these plastid compounds in the adaptation of the alga to alterations in the environment.
Overall design
In total the analysis comprises 32 samples. A starter culture grown in the light with ~2 × 10e6 cells per ml was subdivided into 25 ml aliquots in 100 ml Erlenmeyer flasks and incubation continued in the dark for 16 h. At time 0, MgProto or hemin (final concentration 9 μM each) was added to the cultures in the dark and incubation in the dark was continued until harvest. Control cultures were treated the same way but did not receive the tetrapyrroles. Cells were harvested at time points 0 min, 60 min, 120 min and 180 min. This procedure was repeated one time, resulting in 1 biological replicate.
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