Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

Light is necessary for photosynthesis, but its absorption by pigment molecules such as chlorophyll can cause severe oxidative damage and result in cell death. The excess absorption of light energy by photosynthetic pigments has led to the evolution of protective mechanisms that operate on the timescale of seconds to minutes and involve feedback-regulated de-excitation of chlorophyll molecules in photosystem II (qE). Despite the significant contribution of eukaryotic algae to global primary production, little is known about their qE mechanism, in contrast to that in flowering plants. Here we show that a qE-deficient mutant of the unicellular green alga Chlamydomonas reinhardtii, npq4, lacks two of the three genes encoding LHCSR (formerly called LI818). This protein is an ancient member of the light-harvesting complex superfamily, and orthologues are found throughout photosynthetic eukaryote taxa, except in red algae and vascular plants. The qE capacity of Chlamydomonas is dependent on environmental conditions and is inducible by growth under high light conditions. We show that the fitness of the npq4 mutant in a shifting light environment is reduced compared to wild-type cells, demonstrating that LHCSR is required for survival in a dynamic light environment. Thus, these data indicate that plants and algae use different proteins to dissipate harmful excess light energy and protect the photosynthetic apparatus from damage.

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1 , the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy. INTRODUCTIONPlants in nature experience variations in incident light quantity over several orders of magnitude on a daily basis. Although the reactions that convert solar energy into chemical energy are remarkably efficient, the capacity of these reactions is limited; therefore, plants often absorb more light energy than they are able to use for photosynthesis. In addition, many environmental stresses, including drought, extremes of temperature, or nutrient deprivation (DemmigAdams and Adams, 1992), can further limit the ability of a plant to utilize light energy. Absorption of excessive light energy can lead to sustained depressions in photosynthetic efficiency (photoinhibition), often due to oxidative damage to the photosynthetic apparatus.Photosynthetic organisms have evolved multiple mechanisms to cope with the absorption of excessive light and its consequences. Interception of incident light can be decreased by reorientation and/or movement of chloroplasts within cells (Brugnoli and Björkman, 1992) and, in some plants, by leaf movements (Björkman and Demmig-Adams, 1994). Carotenoids, including the xanthophylls, have essential photoprotective roles as quenchers of triplet chlorophyll ( 3 Chl) and singlet oxygen ( 1 O 2 ) and as inhibitors of lipid peroxidation (Cogdell and Frank, 1987; Frank and Cogdell, 1993;Demmig-Adams et al., 1996). Another important lipidsoluble antioxidant is ␣ -tocopherol (vita...

Xanthophyll pigments have critical structural and functional roles in the photosynthetic lightharvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll f luorescence. Mutants with a defect in either the ␣-or ␤-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (␣-carotene branch), zeaxanthin, and antheraxanthin (␤-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), ␣-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

Background: Nitrogen-starvation and other stresses induce triacylglycerol (TAG) accumulation in algae, but the relevant enzymes and corresponding signal transduction pathways are unknown. Results: RNA-Seq and genetic analysis revealed three acyltransferases that contribute to TAG accumulation. Conclusion: TAG synthesis results from recycling of membrane lipids and also by acylation of DAG. Significance: The genes are potential targets for manipulating TAG hyperaccumulation.

The light-saturated rate of photosynthetic O 2 evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas lightsaturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O 2 evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.