Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtii.

Phototropin (PHOT) is a photoreceptor involved in a variety of blue-light-elicited physiological processes including phototropism, chloroplast movement and stomatal opening in plants. The work presented here tests whether PHOT is involved in expression of light-regulated genes in Chlamydomonas reinhardtii. When C. reinhardtii was transferred from the dark to very low-fluence rate white light, there was a substantial increase in the level of transcripts encoding glutamate-1-semialdehyde aminotransferase (GSAT), phytoene desaturase (PDS) and light-harvesting polypeptides (e.g. LHCBM6). Increased levels of these transcripts were also elicited by low-intensity blue light, and this blue-light stimulation was suppressed in three different RNAi strains that synthesize low levels of PHOT. The levels of GSAT and LHCBM6 transcripts also increased following exposure of algal cells to low-intensity red light (RL). The red-light-dependent increase in transcript abundance was not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, implying that the influence of RL on transcript accumulation was not controlled by cytoplasmic redox conditions, and that a red-light photoreceptor(s) may be involved in regulating the levels of transcripts from specific photosynthesis-related genes in C. reinhardtii. Interestingly, elevated GSAT and LHCBM6 transcript levels in RL were significantly reduced in the PHOT RNAi strains, which raises the possibility of co-action between blue and RL signaling pathways. Microarray experiments indicated that the levels of several transcripts for photosystem (PS) I and II polypeptides were also modulated by PHOT. These data suggest that, in C. reinhardtii, (i) PHOT is involved in blue-light-mediated changes in transcript accumulation, (ii) synchronization of the synthesis of chlorophylls (Chl), carotenoids, Chl-binding proteins and other components of the photosynthetic apparatus is achieved, at least in part, through PHOT-mediated signaling, and (iii) a red-light photoreceptor can also influence levels of certain transcripts associated with photosynthetic function, although its action requires normal levels of PHOT.

Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii.

The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (approximately 300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only approximately 20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with approximately 10,000 unique array elements (approximately 70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).

Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii is a particularly important model organism for the study of photosynthesis since this alga can grow heterotrophically, and mutants in photosynthesis are therefore conditional rather than lethal. The recently developed tools for genomic analyses of this organism have allowed us to identify most of the genes required for chlorophyll and carotenoid biosynthesis and to examine their phylogenetic relationships with homologous genes from vascular plants, other algae, and cyanobacteria. Comparative genome analyses revealed some intriguing features associated with pigment biosynthesis in C. reinhardtii; in some cases, there are additional conserved domains in the algal and plant but not the cyanobacterial proteins that may directly influence their activity, assembly, or regulation. For some steps in the chlorophyll biosynthetic pathway, we found multiple gene copies encoding putative isozymes. Phylogenetic studies, theoretical evaluation of gene expression through analysis of expressed sequence tag data and codon bias of each gene, enabled us to generate hypotheses concerning the function and regulation of the individual genes, and to propose targets for future research. We have also used quantitative polymerase chain reaction to examine the effect of low fluence light on the level of mRNA accumulation encoding key proteins of the biosynthetic pathways and examined differential expression of those genes encoding isozymes that function in the pathways. This work is directing us toward the exploration of the role of specific photoreceptors in the biosynthesis of pigments and the coordination of pigment biosynthesis with the synthesis of proteins of the photosynthetic apparatus.

Chlamydomonas reinhardtii in the landscape of pigments.

This review focuses on the biosynthesis of pigments in the unicellular alga Chlamydomonas reinhardtii and their physiological and regulatory functions in the context of information gathered from studies of other photosynthetic organisms. C. reinhardtii is serving as an important model organism for studies of photosynthesis and the pigments associated with the photosynthetic apparatus. Despite extensive information pertaining to the biosynthetic pathways critical for making chlorophylls and carotenoids, we are just beginning to understand the control of these pathways, the coordination between pigment and apoprotein synthesis, and the interactions between the activities of these pathways and those for other important cellular metabolites branching from these pathways. Other exciting areas relating to pigment function are also emerging: the role of intermediates of pigment biosynthesis as messengers that coordinate metabolism in the chloroplast with nuclear gene activity, and the identification of photoreceptors and their participation in critical cellular processes including phototaxis, gametogenesis, and the biogenesis of the photosynthetic machinery. These areas of research have become especially attractive for intensive development with the application of potent molecular and genomic tools currently being applied to studies of C. reinhardtii.

Analysis of light and CO(2) regulation in Chlamydomonas reinhardtii using genome-wide approaches.

Over the past decade new technologies have been developed to elucidate ways in which cells acclimate to environmental change. Many of these techniques have allowed the identification of specific transcripts that change in abundance in response to particular environmental stimuli; such transcripts represent genes that are potentially differentially regulated. Two techniques that foster identification of differentially regulated genes are differential display and expression profiling using high density DNA microarrays. The former technology amplifies cDNA fragments from mRNAs that differentially accumulate under specific environmental conditions, while the latter provides a more global view of changes in gene expression in response to environmental stimuli. Coupling these technologies with the analysis of mutants aberrant for regulatory molecules that participate in acclimation processes will allow the identification of groups of genes controlled by specific regulatory elements. In this article we describe the use of differential display and DNA microarray profiling to examine environmentally-regulated gene expression. We also show specific experiments using the unicellular green alga Chlamydomonas reinhardtii, in which mRNA abundance is evaluated in response to both changing light and CO(2) conditions.

Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii.

We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light-activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2-induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low-CO2 conditions. Genes specifically activated by high light, even under high-CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.