THE ANTARCTIC PSYCHROPHILE,CHLAMYDOMONAS RAUDENSIS ETTL (UWO241) (CHLOROPHYCEAE,CHLOROPHYTA), EXHIBITS A LIMITED CAPACITY TO PHOTOACCLIMATE TO RED LIGHT1

The psychrophilic Antarctic alga, Chlamydomonas raudensis Ettl (UWO241), grows under an extreme environment of low temperature and low irradiance of a limited spectral quality (blue‐green). We investigated the ability of C. raudensis to acclimate to long‐term imbalances in excitation caused by light quality through adjustments in photosystem stoichiometry. Log‐phase cultures of C. raudensis and C. reinhardtii grown under white light were shifted to either blue or red light for 12 h. Previously, we reported that C. raudensis lacks the ability to redistribute light energy via the short‐term mechanism of state transitions. However, similar to the model of mesophilic alga, C. reinhardtii, the psychrophile retained the capacity for long‐term adjustment in energy distribution between PSI and PSII by modulating the levels of PSI reaction center polypeptides, PsaA/PsaB, with minimal changes in the content of the PSII polypeptide, D1, in response to changes in light quality. The functional consequences of the modulation in PSI/PSII stoichiometry in the psychrophile were distinct from those observed in C. reinhardtii. Exposure of C. raudensis to red light caused 1) an inhibition of growth and photosynthetic rates, 2) an increased reduction state of the intersystem plastoquinone pool with concomitant increases in nonphotochemical quenching, 3) an uncoupling of the major light‐harvesting complex from the PSII core, and 4) differential thylakoid protein phosphorylation profiles compared with C. reinhardtii. We conclude that the characteristic low levels of PSI relative to PSII set the limit in the capacity of C. raudensis to photoacclimate to an environment enriched in red light.     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

Resolving the phylogenetic relationship between Chlamydomonas sp. UWO 241 and Chlamydomonas raudensis sag 49.72 (Chlorophyceae) with nuclear and plastid DNA sequences

The Antarctic psychrophilic green alga Chlamy‐domonas sp. UWO 241 is an emerging model for studying microbial adaptation to polar environments. However, little is known about its evolutionary history and its phylogenetic relationship with other chlamydomonadalean algae is equivocal. Here, we attempt to clarify the phylogenetic position of UWO 241, specifically with respect to Chlamydomonas rau‐densis SAG 49.72. Contrary to a previous report, we show that UWO 241 is a distinct species from SAG 49.72. Our phylogenetic analyses of nuclear and plastid DNA sequences reveal that UWO 241 represents a unique lineage within the Moewusinia clade (sensu Nakada) of the Chlamydomonadales (Chlorophyceae, Chlorophyta), closely affiliated to the marine species Chlamydomonas parkeae SAG 24.89.     

GROWTH AND POLYSACCHARIDE PRODUCTION BY THE GREEN ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE) ON SOIL1

The polysaccharide producing soil alga Chlamydomonas mexicana Lewin was grown on soil surfaces in a growth chamber, with cell number and total polysaccharide measured weekly. Cell growth was pronounced, reaching an excess of 10⁷ cells · cm^?2 within one week. Polysaccharide production was also pronounced, with similar amounts of polysaccharide synthesized whether or not the cells were continuously given nutrients. Polysaccharide synthesis increased once the cells slowed in their growth, as in previous work with the cells in liquid medium.     

IDENTIFICATION AND DNA SEQUENCE OF A NEW H+-ATPase IN THE UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

Insertional mutagenesis was used to identify genes involved in mating and/or zygote formation in the unicellular green alga Chlamydomonas reinhardtii Dangeard. Approximately 800 insertionally mutagenized transformants were examined, and a single nonagglutinating mutant was identified. Plasmid rescue was used to clone a genomic fragment containing transforming DNA. This fragment was then used to identify the wild-type copy of the gene disrupted during mutagenesis. The wild-type gene is transcribed during all stages of the life cycle and, based on sequence similarity, encodes a P2-type proton transporting ATPase. The gene is referred to as Pmh1 for plasma membrane H ⁺ -ATPase. PMH1 displays the greatest sequence similarity to ATPases from two parasitic flagellates and a raphidophytic alga but not to the ATPase from a closely related green alga. We propose that PMH1 represents a distinct H ⁺ -ATPase isoform expressed in flagellates.     

NOVEL ICE‐BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE,CHLOROPHYCEAE)1

Many cold‐adapted unicellular plants express ice‐active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice‐binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze.     

THE ROLE OF ZINC FINGER PROTEIN IN RNAi INTERFERENCE IN A UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

In our previous study, we generated a strain of 19‐P (1030) in which artificial RNA interference (RNAi) was induced by transcribing a hairpin RNA of ~780‐bp stem. We utilized this RNAi‐induced strain to uncover RNAi‐related genes. Random insertional mutagenesis was performed to generate tag‐mutants that show a RNAi deficient phenotype. The 92‐12C is one such tag‐mutant, which bears a 14‐kb deletion in chromosome 1. Complementation of 92‐12C revealed that a protein gene, including a Cys‐Cys‐Cys‐His‐type zinc finger motif and an ankyrin repeat motif, is essential for effective RNAi in Chlamydomonas reinhardtii (Dangeard). BLAST analysis revealed that the zinc finger protein is homologous to an mRNA splicing‐related protein of other species. Therefore, one of the probable scenarios is that mRNA coding for RNAi‐related proteins cannot be properly spliced, which causes RNAi deficiency in the 92‐12C tag‐mutant.     

PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced-min^-1.mg^-1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N-ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo-tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg²⁺. The monovalent cation K⁺ acts as a strong activator. The K_m values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with V_max for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5-bisphosphate, adenosine monophosphate (AMP), ribose-5-phosphate, and glyceraldehyde-3-phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3-diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).     

A NEW HOMOTHALLIC VARIETY OF CHLAMYDOMONAS MOEWUSH (CHLOROPHYCEAE)1

A new homothallic variety of Chlamydomonas moewusii Gerloff var. monoica is described. When first isolated, the alga exhibited a very strong mating reaction (80–90% zygotes on 2 wk BBM agar slants), but after 2 mo in axenic culture, the reaction was significantly reduced in intensity. Attempts were made to restore the initial mating intensity by varying environmental conditions, but met with limited success. The alga did not grow heterotrophically in carbon-supplemented BBM medium.     

PHYLOGENY OF CARTERIA (CHLOROPHYCEAE) INFERRED FROM MOLECULAR AND ORGANISMAL DATA1

Comparative ultrastructural data have shown that at least two distinct groups exist within Carteria. Similarly, interpretations of variation in gross morphological features have led to the discovery of morphologically distinct groups within the genus. Partial sequences from the nuclear-encoded small- and large-subunit ribosomal RNA molecules of selected Carteria taxa were studied as a means of 1) testing hypotheses that distinct groups of species exist within the genus and 2) assessing monophyly of the genus. Parsimony analysis of the sequence data suggests that three Carteria species, C. lunzensis, C. crucifera, and C. olivieri, form a monophyletic group that is the basal sister group to all other ingroup flagellate taxa (including species of Chlamydomonas, Haematococcus, Stephanosphaera, Volvox, and Eudorina). Two other Carteria taxa, C. radiosa and Carteria sp. (UTEX isolate LB 762), form a clade that is the sister group to a clade that includes Haematococcus spp., Chlamydomonas spp., and Stephanosphaera. Thus, the sequence data support the interpretations of ultrastructural evidence that described two distinct Carteria lineages. Moreover, the sequence data suggest that these two Carteria groups do not form a monophyletic assemblage. Parsimony analysis of a suite of organismal (morphological, ultra-structural, life history, and biochemical) character data also suggest two distinct lineages among the five Carteria taxa; however, the organismal data are ambiguous regarding monophyly of these Carteria taxa. When the two independent data sets are pooled, monophyly of Carteria is not supported; therefore, the weight of available evidence, both molecular and organismal, fails to support the concept of Carteria as a natural genus.     

LOCALIZATION OF OXYTETRACYCLINE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Oxytetracycline (OTC) is an important antimicrobial used in aquaculture. However, residues of OTC have been isolated from nontarget aquatic organisms, sediments, and water located near aquaculture facilities. Identifying OTC in plant material is particularly difficult due to interference from pigments and polyphenol substances but is important especially for algae since they are a primary food source for fish in early life stages. In this study, we describe the effect of OTC (0.1, 1, 10, 25, 50, 100 μg · mL^?1) on cell growth, and the localization of OTC (0, 1, 25, 100 μg · mL^?1) in vacuoles of Chlamydomonas reinhardtii P. A. Dang. (wildtype, ATCC 18798). We also present a method for semiquantifying OTC in living cells using fluorescent microscopy and Adobe Photoshop. We exposed algal cells to OTC and sampled after 2 or 7 d exposure. On day 7, OTC significantly inhibited algal growth at 1, 10, 25, 50, and 100 μg · mL^?1. When viewed with fluorescent microscopy, cells exposed to the 25 and 100 μg · mL^?1 contained yellow fluorescent areas, ≤1 μm in diameter that were easily discernable against the red fluorescence of the intracellular chl. The fluorescent areas corresponded to small spherical vacuoles (i.e., polyphosphate bodies that contain calcium and magnesium complexed with polyphosphate) seen in the cells by LM. Since OTC has a high affinity for divalent cations, we suggest that OTC is localized in these vacuoles.     

FATTY ACID AND LIPID COMPOSITION OF THE ACIDOPHILIC GREEN ALGA CHLAMYDOMONAS SP.1

The lipid and fatty acid compositions of Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii were compared, and the effects of pH of the medium on lipid and fatty acid components of Chlamydomonas sp. were studied. The fatty acids in polar lipids from Chlamydomonas sp. were more saturated than those of C. reinhardtii. The relative percentage of triacylglycerol to the total lipid content in Chlamydomonas sp. grown in medium at pH 1 was higher than that in other cells grown at higher pH. A probable explanation might be that Chlamydomonas sp. has two low pH adaptation mechanisms. One mechanism is the saturation of fatty acids in membrane lipids to decrease membrane lipid fluidity, and the other is the accumulation of triacylglycerol, as a storage lipid, to prevent the osmotic imbalance caused by high concentrations of H₂SO₄.     

FUNCTIONAL CHARACTERIZATION AND EXPRESSION ANALYSIS OF p‐HYDROXYPHENYLPYRUVATE DIOXYGENASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The enzyme p‐hydroxyphenylpyruvate dioxygenase (HPPD) is very important in prenylquinone biosynthesis in all photosynthetic organisms. In this study, we present the functional characterization and expression analysis of HPPD from the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. Recombinant HPPD1 enzyme was purified and characterized. Kinetic analysis revealed a K_m of 49 μM for p‐hydroxyphenylpyruvate, similar to other HPPDs. The size of HPPD subunit was estimated as 47 kDa by SDS‐PAGE, in accordance with the predicted molecular size after HPPD1 cDNA sequence. However, native HPPD1 enzyme showed an apparent molecular mass of 188 kDa and a homotetrameric structure, which suggests a reconsideration of the idea that all eukaryotic HPPDs have a homodimeric structure while all prokaryotic HPPDs are homotetramers. Expression analysis by Northern blot revealed that hppd1 expression is strongly up‐regulated by low temperature and poorly regulated by high temperature, darkness, or moderate light changes, suggesting that Chlamydomonas HPPD may play an important role in the synthesis of tocopherols and/or plastoquinones under stress conditions in the physiological context of the adaptation to growth at low temperatures.     

SELF-STERILE AND MATURATION-DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA,CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

Homothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform-resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly-outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation-defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self-sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully-matured, chloroform-resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.     

ACETATE IS A CHEMOATTRACTANT FOR THE COLONIAL GREEN ALGA ASTREPHOMENE GUBERNACULIFERA (CHLOROPHYCEAE)1

Astrephomene gubernaculifera Pocock is capable of growth on sodium acetate, even in the absence of light. From an evolutionary standpoint, it would be beneficial for this motile heterotrophic organism to be able to sense and swim to such an energy source. We used three assays to test for chemoattraction of A. gubernaculifera. In each case, there was strong chemoattraction to acetate. Propionate, a molecule structurally similar to acetate, also elicited a strong chemoattraction response, even though it was incapable of supporting growth in the dark in this strain. This implies that chemoattraction in A. gubernaculifera is based on a receptor‐mediated signaling response. Video analysis of colonies exhibiting the attraction response indicated that most of them remained motile in the vicinity of the acetate. This rules out the simplest possible model for accumulation in which colonies stop when they encounter increased acetate concentrations. We also tested a variety of other organic molecules for their ability to chemoattract A. gubernaculifera. Of the 11 sugars, 18 amino acids, and three other organic molecules tested, only the amino acid phenylalanine elicited chemoattraction.     

COPPER TOLERANCE OF CHLAMYDOMONAS ACIDOPHILA (CHLOROPHYCEAE) ISOLATED FROM ACIDIC,COPPER-CONTAMINATED SOILS1

Cells of Chlamydomonas acidophila Negoro, isolated from three soils with different available copper contents (74, 80, and 87 μg·g^?1), were assayed for their responses to copper. Soil pH ranged from 3.3–3.9. Responses were evaluated using algistatic assays involving five day exposure to copper concentrations from 0.1–100 mg·L^?1 at pH 3.8 and 6.6 in defined liquid media. Interspecies and intraspecies comparisons were made between the soil isolates and laboratory strains of C. reinhardtii and C. acidophila, respectively. Algistatic copper concentrations of soil isolates were 20–125 times greater than those of the laboratory strain of C. reinhardtii. Concentrations of 0.1 mg Cu·L^?1, or greater, killed the laboratory strain of C. acidophila. Soil isolates of C. acidophila appeared to be copper tolerant; however, there was no conclusive evidence to indicate that the level of copper tolerance in the soil isolates was positively correlated with the level of available copper in the soil.     

LOSS OF HYBRID LETHALITY DURING BACKCROSS PROGRAMS INVOLVING CHLAMYDOMONAS EUGAMETOS AND CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE)1

Wild-type strains of the interfertile species Chlamydomonas eugametos (UTEX 9 and 10) and Chlamydomonas moewusii (UTEX 96 and 97) male readily and reciprocally; however, considerable lethality occurs among F₁ hybrid meiotic products. We prepared two hybrid backcross lineages using C. eugametos and C. moewusii. One lineage began with the cross C. eugametos mating-type-plus (mt⁺) × C. moewusii mating-type-minus (mt^?). An F₁ mt⁺ hybrid from this cross was back-crossed to C. moewusii mt^?, and a B₁ mt⁺ hybrid was recovered. The B₁ hybrid was again backcrossed to C. moewusii mt^?, and this process was repeated through the fifth backcross. The other backcross lineage began with the reciprocal cross C. moewusii mt⁺× C. eugametos mt^? and employed C. eugametos as the recurring mt^? parent. This lineage also was continued through the fifth backcross. Meiotic product survival in the reciprocal interspecific crosses was less than 10%. In successive back-cross generations associated with both lineages, this value increased progressively to a maximum of 85–90%, the level observed for the intraspecific crosses. These results are consistent with the hypothesis that multiple genetic differences exist between C. eugametos and C. moewusii and that these are the major source of meiotic product lethality associated with the interspecific crosses. The inheritance of chloroplast genetic markers for resistance to streptomycin (sr-2) and for resistance to erythromycin (er-nM1) was also scored w the interspecific crosses and in the backcrosses. Most hybrid zygospores transmitted the resistance markers of the mt⁺ parent only, or of both parents, with the former zygospore type being more common. Although the intraspecific C. eugametos and C. moewusii crosses differ conspicuously with respect to the fraction of zygospores which transmit chloroplast genetic markers of both parents, the inheritance of chloroplast genetic markers in the interspecific crosses and backcrosses at' scribed here failed to clarify the genetic basis for this difference.     

INFLUENCE OF GROWTH STATUS AND NUTRIENTS ON EXTRACELLULAR POLYSACCHARIDE SYNTHESIS BY THE SOIL ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE)1

Increased levels of nitrogen in liquid growth medium bring about increased growth and a delay in extracellular polysaccharide production by Chlamydomonas mexicana Lewin on a per-cell basis. Addition of nitrogen to stationary phase cultures causes renewed growth and a temporary lag in polysaccharide synthesis until growth again ceases. Removal of nitrogen terminates growth, causing an immediate increase in polysaccharide synthesis. Phosphate-starved cells show a response similar to nitrogen-starved cells, indicating that the beginning of stationary phase and not nitrogen depletion causes the stimulation in extracellular polysaccharide synthesis. As similar results are assumed to occur on soil, the significance of this response is discussed.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.