The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO 2 and about 90% with ambient CO 2 . In addition, it is likely that pyrenoidal Rubisco is active in CO 2 fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO 2 -fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO 2 fixation in C. reinhardtii and other unicellular algae containing CO 2 -concentrating mechanisms.Pyrenoids are electron-dense structures that are found in the plastids of most algae. When pyrenoids are isolated, the most abundant protein present is Rubisco (Kuchitsu et al., 1991; McKay and Gibbs, 1991; Okada, 1992). However, the amount of Rubisco present in the pyrenoid is controversial. Estimates of the amount of Rubisco in the pyrenoid versus the amount in the entire chloroplast range from 60% or higher (Vladimirova et al., 1982; Lacoste-Royal and Gibbs, 1987; Morita et al., 1997) to as low as 5% (Sü ss et al., 1995).The function of Rubisco in the pyrenoid is also unclear. Lacoste- Royal and Gibbs (1987) found that a higher percentage of Rubisco was localized to the pyrenoid in stationary cells rather than in actively growing cells of Chlamydomonas reinhardtii, an observation that is consistent with the hypothesis that the pyrenoid is a storage body. Sü ss et al. (1995) interpreted their localization data to indicate that there were two forms of Rubisco in C. reinhardtii: form I, which bound to the thylakoid membranes in the stroma, and form II, which was found in the pyrenoid. They further speculated that the pyrenoid-localized form II might function as part of the CO 2 -concentrating mechanism.In photosynthetic organisms that possess a CO 2 -concentrating mechanism, Rubisco has a very specific localization. In higher plants with C4-type photosynthesis, Rubisco is specifically localized to chloroplasts of bundlesheath cells (Hatch, 1992). In unicellular cyanobacteria with CO 2 -concentrating mechanisms, Rubisco is specifically localized to structures known as carboxysomes (Allen, 1984; Codd and Marsden, 1984). Recent studies of the cyanobacteria's CO 2 -concentrating mechanism indicate that HCO 3 Ϫ accumulated by the cell is dehydrated specifically in the carboxysome, where the resulting CO 2 can be fixed before leaking out of the cell (Badger and Pric...

Abstract. The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-l,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO2 concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO2-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO2 conditions. These growth conditions also partly inhibit the induction of the CO2-concentrating mechanism. When cells are grown with acetate in the dark, the CO2-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO2-concentrating mechanism under low-CO 2 conditions. We suggest that this ultrastructural reorganization under low-CO 2 conditions plays a role in the CO2-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3–. Aquatic photosynthetic organisms have evolved different forms of CO2-concentrating mechanisms to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CO2-concentrating mechanisms is the critical roles played by various specially localized extracellular and intracellular CAs. There are three evolutionarily unrelated CA families designated α-, β-, and γ-CA. In the green alga, Chlamydomonas reinhardtii Dangeard, eight CAs have now been identified, including three α-CAs and five β-CAs. In addition, C. reinhardtii has another CA-like gene, Glp1 that is similar to known γ-CAs. To characterize these different CA isoforms, some of the CA genes have been overexpressed to determine whether the proteins have CA activity and to generate antibodies for in vivo immunolocalization. The CA proteins Cah3, Cah6, and Cah8, and the γ-CA-like protein, Glp1, have been overexpressed. Cah3, Cah6, and Cah8 have CA activity, but Glp1 does not. At least two of these proteins, Cah3 and Cah6, are localized to the chloroplast. Using immunolocalization and sequence analyses, we have determined that Cah6 is located to the chloroplast stroma and confirmed that Cah3 is localized to the chloroplast thylakoid lumen. Activity assays show that Cah3 is 100 times more sensitive to sulfonamides than Cah6. We present a model on how these two chloroplast CAs might participate in the CO2-concentrating mechanism of C. reinhardtii. Key words: carbonic anhydrase, CO2-concentrating mechanism, Chlamydomonas, immunolocalization.

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.