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.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.light capture/conversion | carbon capture/conversion | smart canopy | enabling plant biotechnology tools | sustainable crop production Increasing demands for global food production over the next several decades portend a huge burden on the world's shrinking farmlands. Increasing global affluence, population growth, and demands for a bioeconomy (including livestock feed, bioenergy, chemical feedstocks, and biopharmaceuticals) will all require increased agricultural productivity, perhaps by as much as 60-120% over 2005 levels (e.g., refs. 1 and 2), putting increased productivity on a collision course with environmental and sustainability goals (3). The 45 y from 1960 to 2005 saw global food production grow ∼160%, mostly (135%) by improved production on

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO2 is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration ofCO2 required for the half-maximal rate of photosynthetic 02 evolution (Ko4C021) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO2 fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K165(CO2) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextranbound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO2 is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO2 from HC03-at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HC03-transport for internal accumulation might occur at the level of the chloroplast envelope.

Aquatic photosynthetic microorganisms account for almost 50% of the world's photosynthesis (19). These organisms face several challenges in acquiring CO 2 from the environment. The first challenge is presented by the properties of ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is an unusually slow enzyme with a low affinity for CO 2 . At atmospheric levels of CO 2 , Rubisco can function at only about 25% of its catalytic capacity because the concentration of dissolved CO 2 is less than the K m (CO 2 ) of Rubisco and due to the relatively high concentration of O 2 which competes with CO 2 . A second challenge these organisms face is that the diffusion of CO 2 in an aqueous solution is 10,000 times slower than the diffusion of CO 2 in air. Thus, the ability to scavenge CO 2 as quickly as it becomes available is highly advantageous to aquatic photosynthetic organisms. Third, algae often experience significant fluctuations in inorganic carbon (C i ϭ CO 2 ϩ HCO 3 Ϫ ) levels and pH, which change the availability of CO 2 and HCO 3 Ϫ for photosynthesis. At an acidic pH, the vast majority of C i is in the form of CO 2 , while at an alkaline pH,

Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO(2) (having no active CCM) versus cells growing on low levels of CO(2) (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO(2) in C. reinhardtii.

Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3− and are ubiquitous in nature. Higher plants contain three evolutionarily distinct CA families, αCAs, βCAs, and γCAs, where each family is represented by multiple isoforms in all species. Alternative splicing of CA transcripts appears common; consequently, the number of functional CA isoforms in a species may exceed the number of genes. CAs are expressed in numerous plant tissues and in different cellular locations. The most prevalent CAs are those in the chloroplast, cytosol, and mitochondria. This diversity in location is paralleled in the many physiological and biochemical roles that CAs play in plants. In this review, the number and types of CAs in C3, C4, and crassulacean acid metabolism (CAM) plants are considered, and the roles of the α and γCAs are briefly discussed. The remainder of the review focuses on plant βCAs and includes the identification of homologs between species using phylogenetic approaches, a consideration of the inter- and intracellular localization of the proteins, along with the evidence for alternative splice forms. Current understanding of βCA tissue-specific expression patterns and what controls them are reviewed, and the physiological roles for which βCAs have been implicated are presented.

Carbonic anhydrases catalyse the reversible hydration of CO 2 , increasing the interconversion between CO 2 and HCO 3 -+ H + in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated a-, b-and g-CA. Animals have only the a-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C 3 dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.

The ability of photosynthetic organisms to use CO 2 for photosynthesis depends in part on the properties of Rubisco. Rubisco has a surprisingly poor affinity for CO 2 , probably because it evolved in an atmosphere that had very high CO 2 levels compared with the present atmosphere. In C 3 plants the K m (CO 2 ) of Rubisco ranges between 15 and 25 m. In cyanobacteria Rubisco has an even lower affinity for CO 2 , and the K m (CO 2 ) can be greater than 200 m. In comparison, the concentration of CO 2 in water in equilibrium with air is approximately 10 m. From these numbers it becomes apparent that Rubisco is operating at no more than 30% of its capacity under standard atmospheric conditions. This is one of the reasons that C 3 plants contain such large amounts of Rubisco. Exacerbating this situation is the fact that O 2 is a competitive substrate with respect to CO 2 .In the atmosphere, where the O 2 level is 21% and the CO 2 level is 0.035%, the competition by O 2 accounts for as much as 30% of the reactions catalyzed by Rubisco. A number of photosynthetic organisms have developed ways to increase the level of CO 2 at the location of Rubisco in the plant. This results in an increase in CO 2 fixation and a decrease in the deleterious oxygenation reaction. An excellent example of a CO 2 -concentrating mechanism in higher plants is C 4 photosynthesis, which has arisen independently in a number of plant families. Aquatic photosynthetic organisms such as the microalgae have also adapted to low CO 2 levels by concentrating CO 2 internally. This Update will focus on CO 2 -concentrating mechanisms in the microalgae. For more detailed reviews of the CO 2 concentration by algae, the reader is referred to the special issue of the Canadian Journal of Botany (1998, Vol. 76) and the article by Raven (1997). TYPES OF CO 2 -CONCENTRATING MECHANISMS AND THE PROBLEM OF LEAKAGE OF ACCUMULATED CO 2C 4 plants are the best-studied organisms that concentrate CO 2 to enhance the carboxylation reaction of Rubisco. They have high levels of PEP carboxylase in leaf mesophyll cells, whereas Rubisco is located primarily in the bundle-sheath cells. CA within the mesophyll converts CO 2 entering the leaf into HCO 3 Ϫ , which is the substrate for PEP carboxylase. The advantages that PEP carboxylase has over Rubisco are its high affinity for HCO 3 Ϫ and its insensitivity to O 2 . At physiological CO 2 levels and pH, the HCO 3 Ϫ concentration in the cytoplasm of mesophyll cells is about 50 m, whereas the K m (HCO 3 Ϫ ) of PEP carboxylase is estimated to be about 8 m. Therefore, in contrast to Rubisco, PEP carboxylase is saturated for HCO 3 Ϫ at ambient CO 2 levels. To finish the CO 2 -concentrating effect of C 4 metabolism, the C 4 acid generated in the mesophyll cells is then transported to the bundle-sheath cells and decarboxylated, creating an elevated CO 2 level specifically within these cells.The problem faced by all photosynthetic organisms that concentrate CO 2 is that it can easily diffuse through biological membranes. How can such a slipp...