The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis

Abstract

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)+ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40,000 clones in each library revealed 44 clones which hybridize preferentially with a [32P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [32P] cDNA probe synthesized from poly(A)+ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.

Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops inChlamydomonas

“…Cells were permeabilized by a freeze-thaw cycle and labeled with 32 P-UTP for 15 min (Gagné and Guertin, 1992). RNA was extracted and hybridized to a filter containing several chloroplast gene probes.…”

“…The reaction was performed as described by Gagné and Guertin (1992) with few modifications. Briefly, 2 3 10 8 cells from a culture in exponential phase were pelleted at 4°C for 5 min at 2500g, washed with cold run-on washing buffer (10 mM HEPES, pH 7.5, 250 mM Suc, 150 mM KCl, 1 mM EDTA, and 0.4 mM PMSF freshly supplemented), frozen, and thawed to allow for cell wall permeabilization.…”

Comparative genomics ofPhyscomitrella patensgametophytic transcriptome andArabidopsis thaliana: Implication for land plant evolution

“…The cab homologue LI818 was first identified in Chlamydomonas eugametos as a light-inducible mRNA that encoded a new class of the cab gene family (56). This class of cab homologues has not been reported in other land plants, and may have been lost during the evolution of the vascular plant lineage.…”

The sulfur acclimation SAC3 kinase is required for chloroplast transcriptional repression under sulfur limitation in Chlamydomonas reinhardtii

“…For run-on transcription assays, WT and sac3 cells were grown in TAP medium, harvested, adjusted to 1 ϫ 10 6 cells per ml, and transferred to TAP-S. Permeabilized cells for the assay were prepared by the freeze͞thaw method (13), and aliquots were stored at Ϫ80°C until use. Of the pelleted permeabilized cells, 30 l (Ϸ5 ϫ 10 7 cells) were mixed with 15 l of 4ϫ run-on transcription buffer (1 M sucrose͞60 mM MgCl 2 ͞15 mM DTT͞50 mM NaF͞50 mM Hepes, pH 7.5), 2 l of rRNasin RNase inhibitor (Promega), 0.25 mM GTP, 0.5 mM ATP, 0.25 CTP, and 125 Ci (1 Ci ϭ 37 GBq) of [␣-32 P]UTP (43).…”

“…To assess the contribution of transcriptional changes, run-on assays were performed. In these experiments, chloroplast transcription activity was compared at time 0 (ϩS medium) and 8 h after starvation by using WT and sac3 cells permeabilized by freeze͞ thaw cycles, followed by a short incubation in the presence of [␣-32 P]UTP to extend previously initiated transcripts (13). RNAs were labeled and extracted identically from both strains in duplicate and then used as probes on DNA dot blots to estimate transcription rates of nine chloroplast genes encoding components of the photosynthesis and gene expression machineries.…”