Robert K. Togasaki

Oxygen sensitivity of algal H2- production.

Photoproduction of H2 by green algae utilizes electrons originating from the photosynthetic oxidation of water and does not require metabolic intermediates. However, algal hydrogenases are extremely sensitive to O(2), which limits their usefulness in future commercial H2-production systems. We designed an experimental technique for the selection of O2-tolerant, H2-producing variants of Chlamydomonas reinhardtii based on the ability of wild-type cells to survive a short (20 min) exposure to metronidazole in the presence of controlled concentrations of O2. The number of survivors depends on the metronidazole concentration, light intensity, preinduction of the hydrogenase, and the presence or absence of O2. Finally, we demonstrate that some of the selected survivors in fact exhibit H2-production capacity that is less sensitive to O2 than the original wild-type population.

Purification and cDNA Isolation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

Chloroplastic phosphoglycerate kinase (PGK) was purified to homogeneity from a soluble fraction of chloroplasts of a cell-wall-deficient mutant strain of Chlamydomonas reinhardtii (cw-15) using ammonium sulfate fractionation, Reactive Blue-72 column chromatography, and native polyacrylamide gel electrophoresis. PGK activity was attributed to a single polypeptide with a molecular mass of 42 kD. Relative purity and identity of the isolated enzyme was confirmed by N-terminal amino acid sequence determination. Antiserum against this enzyme was raised and a western blot analysis of whole-cell lysate from cw-15 cells using this anti-chloroplastic PGK serum detected a single polypeptide with a molecular mass of 42 kD. The cDNA clone corresponding to the Chlamydomonas chloroplastic PGK was isolated from a Chlamydomonas cDNA expression library using the anti-PGK serum. The cDNA sequence was determined and apparently codes for the entire precursor peptide, which consists of 461 codons. The results from Southern and northern blot analyses suggest that the chloroplastic PGK gene exists as a single copy in the nuclear genome of C. reinhardtii and is expressed as a 1.8-kb transcript. The C. reinhardtii chloroplastic PGK cDNA has 71 and 66% homology with wheat chloroplastic PGK and spinach chloroplastic PGK, respectively. Based on the deduced amino acid sequence, the chloroplastic PGK of C. reinhardtii has more similarity to plant PGKs than to other PGKs, having both prokaryotic and eukaryotic features.

A cDNA clone encoding a ferredoxin-NADP+ reductase from Chlamydomonas reinhardtii.

FNR (EC 1.18.1.2) is a flavoenzyme that plays an important role in the metabolism of photosynthetic organisms. FNR catalyzes the final step of the linear photosynthetic electron transfer chain by mediating the passage of electrons from reduced Fd to NADP+. FNR is situated at a branch point in electron flow, playing a key role in regulating the relative amounts of cyclic and noncyclic electron flow to meet the demands of the plant for ATP and reducing power. Besides these photosynthetic functions, recently FNR has been implicated in the defense response mechanisms against oxidative stress in Escherichia coli (Liochev et al., 1994). We report here the isolation of a cDNA clone encoding an FNR of the unicellular green alga Chlamydomonas reinhardtii (Table I). This clone was obtained as a false-positive signal from a cDNA library (Merchant and Bogorad, 1987) by antiphosphoglycerate kinase (EC 2.7.2.3) antibodies (Kitayama and Togasaki, 1992). The size of the cDNA insert of Chlamydomonas FNR is 1565 bp. The DNA sequence of Chlamydomonas FNR has 63, 57, and 58% homology with rice (Aoki and Ida, 1994), pea (Newman and Gray, 1988), and spinach FNR (Jansen et al., 1988), respectively. The deduced amino acid sequence of Chlamydomonas FNR has 59, 46, and 45% identity with rice, pea, and spinach FNR, respectively. Studies on the three-dimensional x-ray structure of FNR have focused on spinach FNR and the functions of amino acid residues have been assigned by Karplus et al. (1991). A11 of the amino acid residues that have been assigned specific functions, such as flavin adenine dinucleotide-binding residues and NADP-binding residues, have been well conserved in the FNR of Chlamydomonas. The homology within the putative N-terminal amino acid regions are very low when compared with N-terminal amino acid regions of a11 other species of FNRs. In addition, there is no significant identity of the putative transit peptide regions to the organellar FNR from spinach, pea, rice, and Cyanophora paradoxa (Jakowitsch et al., 1993).

Arsenic tolerance in a Chlamydomonas photosynthetic mutant is due to reduced arsenic uptake even in light conditions

Arsenate resistance has been used for screening for photosynthetic mutants of Chlamydomonas, since photosynthetic mutants, such as CC981 defective in phosphoribulokinase, were shown to have arsenate resistance. Also, another type of arsenate-resistant mutants, including AR3 that lacks a homolog of a phosphate (Pi) transporter, PTB1, has been isolated. We investigated the uptake of Pi and arsenate, and the gene expression of Pi transporters, which are involved in both Pi and arsenate transport, in mutants CC981 and AR3. In the wild type, both Pi and arsenate uptake were initially high, but were inactivated in the presence of arsenate with time, especially in the dark. In contrast, both mutants were shown to exhibit higher Pi uptake, but lower arsenate uptake than the wild type, regardless of the presence or absence of light. Then, the gene expression of Pi transporters in the cells used for the uptake measurements was investigated and compared between the mutants and the wild type. In CC981, the mRNA levels of PTA2 and PTA4 were higher, while those of PTB3 and PTB5 were lower, as compared with in the wild type. In AR3, those of PTA2 and PTB2 were higher, but that of PTB5 was lower than in the wild type. These findings suggest that the arsenate resistance shown by the mutants in light is due to reduction of arsenate uptake probably through the down-regulation of some Pi transporter expression, while the Pi uptake maintained even in the dark is possibly related to higher expression of other Pi transporter(s) than in the wild type.

Isolation of Intact Chloroplasts from Chlamydomonas Reinhardtii with Beckman Centrifugal Elutriation System

The unicellular green alga Chlamydomonas reinhardtii has been widely used in genetic, molecular, and biochemical studies of photosynthesis (1, 2). Isolation of intact chloroplasts from this alga has been difficult, due to the large size of the single chloroplast present within each cell. Klein et al. (3) have reported a method of isolating functional chloroplasts from wild type cells of Chlamydomonas, utilizing autolysine (4) to remove the cell wall and detergents to differentially solubilize the plasma and organellar membranes. Belknap (5) used autolysine for cell wall removal and pressure shock with a Yeda press (6) for plasma membrane disruption to isolate functional intact chloroplasts from both the wild type and a mutant, F60, lacking phosphoriulokinase. L. Mendiola-Morgenthaler et al. (7) combined the Yeda press method and cell wall deficient mutant, CW-15–2, to isolate intact chloroplasts with high rates of both photosynthesis and light dependent organellar protein synthesis. In all of the above published procedures, Percoli step gradients were used to separate intact chloroplasts from unbroken cells and other subcellular components. Recently, Lin et al. (8) applied centrifugal elutriation to the isolation of intact chloroplasts from wheat. In this paper, we describe the application of the same Beckman centrifugal elutriation system to the isolation of intact chloroplasts from CW-15 cells.

Perspectives on Early Research on Photosynthesis in Chlamydomonas

Personal perspectives of the authors on research conducted in the laboratory of R. P. Levine at the Biological Laboratories, Harvard University in the latter half of the 1960s to the early 1970s is described. The chapter summarizes the state of research in photosynthesis and chloroplast biology in the early 1960s. The authors recall experiments and events that led to the establishment of Chlamydomonas reinhardtii as a model organism for the study of photosynthesis and the molecular biology of chloroplasts. These reminiscences include personal anecdotes that try to convey the excitement, and elation and disappointments, that were experienced during these pioneering times in Chlamydomonas research.

Characterization of Transformant of Chlamydomonas Reinhardtii with Antisense Directed cDNA of Phosphoglycerate Kinase

In green algae, including C. reinhardtii more than 70% of the rubisco present in the chloroplast is confined to a crystal-like body called the pyrenoid (1), unlike the situation in the chloroplasts of flowering plants wherein rubisco is freely distributed throughout. Thus, green algae apparently exhibit an unique system for maintenance and/or regulation of calvin cycle enzyme(s). Recently, Henk et al. studied rubisco deficient mutants by electron microscopy and found that the appearance of the pyrenoid body and the activity of rubisco were correlated (2).