Chlr_gun4_retrograde_signaling
Retrograde signaling and photoprotection in a gun4 mutant of Chlamydomonas reinhardtii
GUN4 is a regulatory subunit of Mg-chelatase involved in the control of tetrapyrrole synthesis in plants and cyanobacteria. By random insertion mutagenesis and screening for a pale green/low fluorescence phenotype we have isolated a first gun4 knock-out mutant of the unicellular green alga Chlamydomonas reinhardtii. The mutant contains 50% of chlorophyll compared to wild type and accumulates the tetrapyrrole precursor ProtoIX. Chlorophyll biosynthesis in the chloroplast is coordinated with nuclear gene expression that could be altered upon perturbation of tetrapyrrole flow. Genome uncoupled (gun) mutants impaired in plastid-to-nucleus retrograde signaling were first identified in Arabidopsis thaliana because of de-repression of LHC genes upon nurflurazon treatment that disrupts chloroplast function. However, this procedure raised criticisms because of possible side effects of nurflurazon and alternative systems for the study of retrograde signalling in more physiological conditions are required. To this aim we have isolated and studied a gun4 mutant in C. reinhardtii. In C. reinhardtii the involvement of GUN4 in coupling nuclear genome expression to chloroplast metabolism is detectable under physiological conditions. In contrast to an increased level of LHC transcripts, the accumulation of most chlorophyll-binding LHC proteins is drastically diminished as compared to wild type, implying post-transcriptional down-regulation in the absence of transcriptional coordination. We have then determined the nuclear transcriptome in dark-grown cells to identify novel transcripts whose expression is regulated by the tetrapyrrole-dependent plastid signal. We chose to use dark-grown cells to avoid effects on gene expression due to other signaling pathways such as those triggered by ROS and the reduction/oxidation state of the photosynthetic electron transport chain in the light. Besides LHCs, we obtained that 803 genes change their expression level in gun4 as compared to wild type, giving an unprecedented characterization of nuclear gene transcription in a gun mutant in physiological condition.
We have carried out a transcriptional analysis in a gun4 mutant of the unicellular green alga Chlamydomonas reinhardtii as compared to the wild type, in dark-grown cells. GUN4 is a regulatory subunit of Mg-chelatase that is involved in the control of chlorophyll biosynthesis and coordinated nuclear gene expression. Previously, the involvement of GUN4 protein in coupling nuclear genome expression to chloroplast metabolism was demonstrated through norflurazon treatment that disrupts chloroplast function in Arabidopsis and LHCs for chlorophyll-binding subunits of photosystem antennae were the most responsive genes. The present transcript analysis has been instead performed under physiological condition, showing that, besides LHCs, 803 genes change their expression level in gun4 as compared to wild type. In Chlamydomonas the function of GUN4 becomes apparent under normal growth condition and implies a wide range genome-coupling effect.