Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

Abstract

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.

Fig. 1

Classification of GreenCut proteins. The bar graph presents the functional classification of GreenCut proteins (on y axis) and the number of proteins placed into each class (x axis). The solid bars represent proteins with known functions while the hatched bars represent proteins of unknown functions. The proteins of unknown function have been placed in generalized functional categories based on domains or motifs within the proteins

Fig. 2

Co-expression of genes of the ATP synthase operon with CGLD22 (sll1321) in Synechocystis sp. PCC 6803. a The microarray data used to generate the expression curves were obtained from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). The atp1 gene is the putative ortholog of CGLD22; the curve showing the expression profile of atp1 is in red. The curves showing the expression profiles of slr1413 and sll0216, genes that are not part of the ATP synthase operon and were used as a control for these analyses, are a dotted and broken line, respectively. The curves showing expression profiles of all other genes of the ATP synthase operon are in gray. Microarray values were background-corrected, normalized against the median of the ratio of each sample against the reference, and log-transformed. The plotted data include microarray replicates of 38 biological experiments. b The arrangement of genes of the ATP synthase operon. The genes are depicted as arrows, with the orientation indicated by the direction of the arrow. The location of the genes on the chromosome relative to the origin is indicated. This information was obtained from CyanoBase (http://genome.kazusa.or.jp/cyanobase/) (Nakao et al. 2010). The genes of the operon are atp1 (sll1321), atpI (sll1322), atpH (ssl2615), atpG (sll1323), atpF (sll1324), atpD (sll1325), atpA (sll1326), and atpC (sll1327). slr1413 is upstream, and slr1411 and sll0216 are downstream of the ATP synthase operon, respectively, and neither is co-expressed with atp1. All of the genes of the ATP synthase operon are depicted as light gray-filled arrows, except for atp1; this arrow is red-filled. Arrows representing genes outside the operon, slr1411, slr1413, and sll0216, are unfilled and dark gray-filled

Fig. 3

Analyses of mutants defective for genes encoding GreenCut proteins. Step 1: Mutants are grown at varying light intensities on medium containing acetate or in minimal medium supplemented with CO₂. In this example, a strain with a mutation in the CGL28 gene (red box, step 1) grew slower than wild-type cells (blue box) on acetate-containing medium, and did not grow at all on minimal medium supplemented with CO₂. Step 2: F_v/F_m values, shown as a false color image, are determined for colonies grown on solid medium containing acetate. In this case, the cgl28 mutant (red box) was determined to have a markedly reduced F_v/F_m relative to wild-type cells (blue box). Step 3: The mutants are further analyzed after growth in the dark in liquid medium containing acetate for photochemical and non-photochemical quenching using fluorescence assays. This strain (blue curve) has no variable fluorescence (which can be observed in the pink curve of wild-type [WT] cells). When the horizontal bar at the top of the image is unfilled (white, outlined in black), the sample is being exposed to actinic light, while the black-filled region of the bar indicates that the sample is in the dark. All downward arrows are the times at which the sample is exposed to a pulse of saturating light, which allows for the determination of maximal fluorescence yield. Step 4: Samples are further analyzed for the contribution of each of the reaction centers to the generation of the electrochemical gradient across the thylakoid membranes by measuring the electrochromic band shift (carotenoid band shift at 520 nm) induced by illumination in the presence and the absence of the PSII inhibitors DCMU and hydroxylamine (HA). The upward arrow indicates light on, while the downward arrow indicates light off. PSII inhibitors have no effect on the electrochemical gradient generated in the cgl28 mutant in the presence of illumination, indicating that PSII cannot perform a charge separation. Step 5: In order to verify that the mutation is linked to the observed phenotype, the mutant is backcrossed with wild-type cells to determine whether the mutant phenotype is linked to the insertion (drug-resistant marker gene). A wild-type copy of the gene altered in the mutant strain is introduced into that strain (using the pSL18 plasmid and under the control of the PSAD promotor) to determine whether it rescues the mutant phenotype. If the mutation is indeed linked to the phenotype, then the mutant is further studied by additional transcriptomic, proteomic, physiological, biochemical, and biophysical analyses. Preliminary studies in this case suggest that the cgl28 mutation is not linked to the photosynthetic phenotype