Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide.

Raphidocelis subcapitata is one of the most frequently used species for algal growth inhibition tests. Accordingly, many microalgal culture collections worldwide maintain R. subcapitata for distribution to users. All R. subcapitata strains maintained in these collections are derived from the same cultured strain, NIVA-CHL1. However, considering that 61 years have passed since this strain was isolated, we suspected that NIVA-CHL1 in culture collections might have acquired various mutations. In this study, we compared the genome sequences among NIVA-CHL1 from 8 microalgal culture collections and one laboratory in Japan to evaluate the presence of mutations. We found single-nucleotide polymorphisms or indels at 19,576 to 28,212 sites per strain in comparison with the genome sequence of R. subcapitata NIES-35, maintained at the National Institute for Environmental Studies, Tsukuba, Japan. These mutations were detected not only in non-coding but also in coding regions; some of the latter mutations may affect protein function. In growth inhibition test with 3,5-dichlorophenol, EC50 values varied 2.6-fold among the 9 strains. In the ATCC 22662-2 and CCAP 278/4 strains, we also detected a mutation in the gene encoding small-conductance mechanosensitive ion channel, which may lead to protein truncation and loss of function. Growth inhibition test with sodium chloride suggested that osmotic regulation has changed in ATCC 22662-2 and CCAP 278/4 in comparison with NIES-35.

Effects of treated wastewater on the ecotoxicity of small streams - Unravelling the contribution of chemicals causing effects.

Wastewater treatment plant effluents are important point sources of micropollutants. To assess how the discharge of treated wastewater affects the ecotoxicity of small to medium-sized streams we collected water samples up- and downstream of 24 wastewater treatment plants across the Swiss Plateau and the Jura regions of Switzerland. We investigated estrogenicity, inhibition of algal photosynthetic activity (photosystem II, PSII) and growth, and acetylcholinesterase (AChE) inhibition. At four sites, we measured feeding activity of amphipods (Gammarus fossarum) in situ as well as water flea (Ceriodaphnia dubia) reproduction in water samples. Ecotoxicological endpoints were compared with results from analyses of general water quality parameters as well as a target screening of a wide range of organic micropollutants with a focus on pesticides and pharmaceuticals using liquid chromatography high-resolution tandem mass spectrometry. Measured ecotoxicological effects in stream water varied substantially among sites: 17ß-estradiol equivalent concentrations (EEQbio, indicating the degree of estrogenicity) were relatively low and ranged from 0.04 to 0.85 ng/L, never exceeding a proposed effect-based trigger (EBT) value of 0.88 ng/L. Diuron equivalent (DEQbio) concentrations (indicating the degree of photosystem II inhibition in algae) ranged from 2.4 to 1576 ng/L and exceeded the EBT value (70 ng/L) in one third of the rivers studied, sometimes even upstream of the WWTP. Parathion equivalent (PtEQbio) concentrations (indicating the degree of AChE inhibition) reached relatively high values (37 to 1278 ng/L) mostly exceeding the corresponding EBT (196 ng/L PtEQbio). Decreased feeding activity by amphipods or decreased water flea reproduction downstream compared to the upstream site was observed at one of four investigated sites only. Results of the combined algae assay (PSII inhibition) correlated best with results of chemical analysis for PSII inhibiting herbicides. Estrogenicity was partly and AChE inhibition strongly underestimated based on measured steroidal estrogens respectively organophosphate and carbamate insecticides. An impact of dissolved organic carbon on results of the AChE inhibition assay was obvious. For this assay more work is required to further explore the missing correlation of bioassay data with chemical analytical data. Overall, the discharge of WWTP effluent led to increased estrogenicity, PSII and AChE inhibition downstream, irrespective of upstream land use.

Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P < 0.05) and followed the same pattern in the three species. The parameter values were promoted in lower NaHCO3 concentrations (up to 252 or 336 mg L(-1)) and inhibited in higher NaHCO3 concentrations (>336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae.

Genetic elicitation by inducible expression of ß-cryptogein stimulates secretion of phenolics from Coleus blumei hairy roots.

The accumulation of phenolic compounds in plants is often part of the defense response against stress and pathogen attack, which can be triggered and activated by elicitors. Oomycetal proteinaceous elicitor, ß-cryptogein, induces hypersensitive response and systemic acquired resistance against some pathogens. In order to test the effect of endogenously synthesized cryptogein protein on phenolic compounds accumulation in tissue, and secretion into the culture medium, Coleus blumei hairy roots were generated. Agrobacterium rhizogenes was employed to insert synthetic crypt gene, encoding ß-cryptogein, under the control of alcohol-inducible promoter. The expression of ß-cryptogein, in C. blumei hairy roots, was controlled by application of 1% and 2% ethanol, during 21 days induction period. Ethanol-induced expression of ß-cryptogein caused significant decrease of soluble phenolics and rosmarinic acid (RA) in hairy root lines and increase of phenolics, RA and caffeic acid in culture medium. These data suggest that ß-cryptogein might be a potential regulatory factor for phenolics secretion from the roots.

Transgenic sequences are frequently lost in Phytophthora parasitica transformants without reversion of the transgene-induced silenced state.

Little data exist on the mechanism and stability of transformation in Phytophthora parasitica, a major oomycete parasite of plants. Here, we studied the stability of drug-resistant protoplast transformants by analyzing single-zoospore derivatives. We show that the transgenic sequences are not stably integrated into the chromosomes, resulting in the loss of drug resistance in single-zoospore derivatives. However, in strains where the P. parasitica gene encoding the CBEL elicitor was silenced by transformation with sense or antisense constructs, silencing is not reversed when the transgenic sequences are lost. This suggests that instability of P. parasitica transformants is not an obstacle for loss-of-function studies in this organism.

Effects of chloramphenicol on photosynthesis, protein profiles and transketolase activity under extremely high CO2 concentration in an extremely-high-CO2-tolerant green microalga, Chlorococcum littorale.

An extremely-high-CO2-tolerant alga, Chlorococcum littorale, showed high quantum efficiency of PSII (PhiII) in the light at 40% CO2, as well as at 5% CO2. However, PhiII decreased greatly when chloramphenicol (CAP) was added at 40% CO2, while no such decrease was observed at 5% CO2. Cycloheximide showed no effect on PhiII at either 5% or 40% CO2. The amount of a 76 kDa polypeptide (p76) on SDS-PAGE decreased markedly in the presence of CAP at 40% CO2 but not at 5% CO2. A partial amino acid sequence of p76 was 71-100% identical (10-14 identical residues out of 14 amino acids determined) to those of transketolases (TKLs) reported in higher plants and a cyanobacterium. In agreement with these observations, the TKL activity in C. littorale was decreased by CAP at 40% CO2, but not at 5% CO2. The transient decrease in TKL activity caused by CAP under 40% CO2 was well correlated with that in PhiII. These results indicate that the addition of CAP directly or indirectly influences the stability of TKL in C. littorale at 40% CO2, but not at 5% CO2, and that photosynthetic activity was reduced by a decrease in TKL activity.

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris.

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.

The Antarctic psychrophile, Chlamydomonas subcaudata, is deficient in state I-state II transitions.

State I-State II transitions were monitored in vivo and in vitro in the Antarctic, psychrophillic, green alga, Chlamydomonas subcaudata, as changes in the low-temperature (77 K) chlorophyll fluorescence emission maxima at 722 nm (F722) relative to 699 nm (F699). As expected, the control mesophillic species, Chlamydomonas reinhardtii, was able to modulate the light energy distribution between photosystem II and photosystem I in response to exposure to four different conditions: (i) dark/anaerobic conditions, (ii) a change in Mg2+ concentration, (iii) red light, and (iv) increased incubation temperature. This was correlated with the ability to phosphorylate both of its major light-harvesting polypeptides. In contrast, exposure of C. subcaudata to the same four conditions induced minimum alterations in the 77 K fluorescence emission spectra, which was correlated with the ability to phosphorylate only one of its major light-harvesting polypeptides. Thus, C. subcaudata appears to be deficient in the ability to undergo a State I-State II transition. Functionally, this is associated with alterations in the apparent redox status of the intersystem electron transport chain and with higher rates of photosystem I cyclic electron transport in the psychrophile than in the mesophile, based on in vivo P700 measurements. Structurally, this deficiency is associated with reduced levels of Psa A/B relative to D1, the absence of specific photosystem I light-harvesting polypeptides [R.M. Morgan et al. (1998) Photosynth Res 56:303-314] and a cytochrome b6/f complex that exhibits a form of cytochrome f that is approximately 7 kDa smaller than that observed in C. reinhardtii. We conclude that the Antarctic psychrophile, C. subcaudata, is an example of a natural variant deficient in State I-State II transitions.

Subfractionation of eyespot apparatuses from the green alga Spermatozopsis similis: isolation and characterization of eyespot globules.

Despite the well-characterized function of the green-algal eyespot apparatus as a combined absorption/reflection screen for the photoreceptor for phototaxis, little is known about the proteins involved in the formation of this complex organelle. We therefore purified the carotenoid-rich lipid globules, which are the most conspicuous component of the eyespot sensu strictu from Spermatozopsis similis Preisig et Melkonian. Electron microscopy and an average carotenoid:chlorophyll ratio of 51, confirmed the high purity of the fraction. The diameter of isolated globules (approx. 112 nm) fell within their in vivo range (90-120 nm). Absorption spectra in aqueous media peaked at 535 nm. The predominant carotenoids were beta/psi-, beta, beta- and delta-carotene. Freeze-fracture studies with cells and whole-mount electron microscopy of isolated globules demonstrated regularly arranged particles at the globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophresis revealed specific enrichment of 10 tightly bound major proteins and several minor proteins with the globules. Proteases were used to analyze their topology and function. Upon treatment with thermolysin, globules were released from a fraction enriched in isolated eyespot apparatuses. Major proteins of these globules, and those treated with thermolysin after isolation, were identical. However, the purified proteins were sensitive to thermolysin, indicating that domains of them are normally hidden in the globule matrix. In contrast, pronase degraded all globule-associated proteins in situ. These globules were not stable and easily fused, whereas thermolysin-treated globules were relatively stable. Lipase did not affect globule stability. These results indicate that the five thermolysin-resistant proteins (apparent Mr values: 56, 52, 32, 29, 27 kDa) are close to the surface and might be crucial for globule stabilization, whereas the thermolysin-accessible proteins are probably involved in globule/globule interactions and/or globule/eyespot-membrane interactions.

Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.

The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl(-) and Ca(2+) ions but significantly in the presence of Cl(-) ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl(-) and Ca(2+) ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.

Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes.

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM-N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM-N of cycloheximide or a chelator, 2,2'-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM-N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM-N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM-N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca2+-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.

Degradation of the Photosystem II D1 and D2 proteins in different strains of the cyanobacterium Synechocytis PCC 6803 varying with respect to the type and level of psbA transcript.

The turnover of the D1 and D2 proteins of Photosystem II (PSII) has been investigated by pulse-chase radiolabeling in several strains of the cyanobacterium Synechocystis PCC 6803 containing different types and levels of the psbA transcript. Strains lacking psbA1 and psbA3 gene and containing high levels of the psbA2 transcript showed the selective synthesis of D1 whose degradation could be slowed down by the protein synthesis inhibitor lincomycin. In contrast, in strains containing just the psbA3 gene, the intensity of the D1 protein labeling was lower and labeling of the D2 and CP43 proteins was stimulated in comparison to the psbA2-containing strains. In addition, the rate and selectivity of the D1 degradation and its dependence on the presence of lincomycin was proportional to the level of the psbA3 transcript in the particular strain. Consequently, there was parallel, lincomycin-independent and slowed-down breakdown of the D1 and D2 proteins in strains with the lowest level of psbA3 transcript. These results are discussed in terms of a model in which the rate of D1 and D2 degradation in cyanobacteria is affected not only by the rate of PSII photodamage, but also by the availability of newly synthesized D1 protein. Moreover, the comparison of the non-oxygen-evolving D1 mutants D170A** and Y161F*** differing by the presence of tyrosine Z has indicated a minor role of the oxidized form of this secondary PSII electron donor in the donor side mechanism of D1 and D2 protein breakdown.