Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.     

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

Fatty acid photodecarboxylase is an ancient photoenzyme that forms hydrocarbons in the thylakoids of algae

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose–methanol–choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.     

Storage glucan and glucosyltransferase isozymes of cyanidioschyzon merolae: A primitive eukaryote

Cyanidioschyzon merolae, a primitive eukaryotic alga isolated from supposedly pure cultures of the thermoacidophilic alga, Cyanidium caldarium, has many of the characteristics of such prokaryotes as bacteria and the cyanobacteria. Cyanidioschyzon appears to have even more of these prokaryotic features than does Cyanidium. Cyanidioschyzon divides by binary fission as do most bacteria. Its thylakoids are arranged along the periphery of the cell, like the cyanobacteria. Its formation of storage glucan, as well as the type of sugar formed is more like that of the blue-green algae rather than that of the red algae. Cyanidioschyzon merolae may be much more primitive than Cyanidium caldarium, and could be the most primitive eukaryotic cell.     

Lipoxygenase pathway in brown algae: The biosynthesis of novel oxylipins ‘ectocarpins’ by hydroperoxide bicyclase CYP5164A3 of Ectocarpus siliculosus

The sequence encoding the CYP5164A3 of the brown alga Ectocarpus siliculosus (Stramenopiles, SAR) was heterologously expressed in E. coli cells. The resulting recombinant CYP74 clan-related protein CYP5164A3 possessed a selective activity towards the α-linolenic acid 13(S)-hydroperoxide (13-HPOTE) and eicosapentaenoic acid 15(S)-hydroperoxide (15-HPEPE). The major products were the heterobicyclic oxylipins. For instance, the 13-HPOTE was converted into plasmodiophorols A, B, and C formed at about 14:3:2 ratio. Plasmodiophorols A-C have been recently described as the products of enzyme hydroperoxide bicyclase CYP50918A1 of cercozoan Plasmodiophora brassicae (Rhizaria, SAR). Furthermore, an unknown compound 1 was detected. Purified product 1 (Me) was identified as a novel substituted 3-propenyl-6-oxabicyclo[3.1.0]hexane based on its MS and NMR spectral data. Conversion of 15-HPEPE by CYP5164A3 resulted in products 7 and 8, analogous to plasmodiophorols A and B. This work uncovered the CYP5164A3 as the first hydroperoxide bicyclase in brown algae. Apparently, this enzyme plays a crucial role in the biosynthesis of heterobicyclic oxylipins like hybridalactone, ecklonilactones, and related natural products, widespread in brown algae.     

A selenite-induced decrease in the lipid content of a red alga

Two unicellular marine algae (Dunaliella primolecta and Porphyridium cruentum) have been found to contain a selenium-inducible, non-enzymatic glutathione peroxidase activity when cultured in the presence of selenite. To test the possibility that selenium functions in vivo as an antioxidant in these algae, a detailed examination of the lipid content of algae cultured in the presence or absence of selenite was conducted. If selenium augments the antioxidant defenses of algal cells, an increase in the content of oxidation-sensitive lipids would be expected. The fatty acid, chlorophyll, phospholipid and glycolipid content of the green alga D. primolecta was not affected by growth in selenite. At low light intensity there was a moderate decrease in the chlorophyll and polyunsaturated fatty acid content of the red alga P. cruentum when cultured in selenite. At higher light intensity the content of all fatty acids, phospholipid, glycolipid, chlorophyll, carotenoid and phycoerythrin decreased in P. cruentum grown in selenite. Since growth in selenite did not increase the quantity of oxidation-sensitive lipids in either alga, there is no evidence for an in vivo functioning of selenium as an antioxidant. Instead, the observed decrease in lipids of the red alga P. cruentum can best be explained as a selenite-induced oxidative effect.     

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [¹⁸O₂]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.     

Occurrence of steryl glycosides and acylated steryl glycosides in some marine algae

There is some controversy concerning the presence of steryl glycosides and acylated steryl glycosides in eucaryotic algae. These two classes of sterol compounds were investigated in species belonging to the three major groups of eucaryotic algae: green algae (Ulva gigantea, Cladophora rupestris), brown algae (Fucus vesiculosus, Ascophyllum nodosum), and red algae (Rhodymenia palmata, Porphyridium sp.). All these algae contain both steryl glycosides and acylated steryl glycosides. The sterol components of these compounds vary according to the alga but they are always the same as the free sterols of the alga in question. The most common sugar moiety is glucose. In the acylated steryl glycosides, the fatty acid is mainly palmitic acid. The percentage of these compounds (as a percentage of the total sterol content) is often low.     

A Lipoxygenase from Red Alga Pyropia haitanensis,a Unique Enzyme Catalyzing the Free Radical Reactions of Polyunsaturated Fatty Acids with Triple Ethylenic Bonds

Lipoxygenases (LOXs) are key enzymes to regulate the production of hormones and defensive metabolites in plants, animals and algae. In this research, a full length LOX gene has been cloned and expressed from the red alga Pyropia haitanensis (Bangiales, Rhodophyta) gametophyte (PhLOX2). Subsequent phylogenetic analysis showed that such LOX enzymes are separated at the early stage of evolution, establishing an independent branch. The LOX activity was investigated at the optimal pH of 8.0. It appears that PhLOX2 is a multifunctional enzyme featuring both lipoxygenase and hydroperoxidase activities. Additionally, PhLOX2 exhibits remarkable substrate and position flexibility, and it can catalyze an array of chemical reactions involving various polyunsaturated fatty acids, ranging from C18 to C22. As a matter of fact, mono-hydroperoxy, di-hydroperoxy and hydroxyl products have been obtained from such transformations, and eicosapentaenoic acid seem to be the most preferred substrate. It was found that at least triple ethylenic bonds are required for PhLOX2 to function as a LOX, and the resulting hydroxy products should be originated from the PhLOX2 mediated reduction of mono-hydroperoxides, in which the hydrogen abstraction occurs on the carbon atom between the second and third double bond. Most of the di-hydroperoxides observed seem to be missing their mono-position precursors. The substrate and position flexibility, as well as the function versatility of PhLOXs represent the ancient enzymatic pathway for organisms to control intracellular oxylipins.     

Low molecular weight carbohydrates in Cyanidium caldarium and some related algae

Floridoside (2-O-glycerol-α-d-galactopyranoside) and a small amount of iso-floridoside (1-O-glycerol-α-d- galactopyranoside) were found in Cyanidium caldarium. Floridoside was also found in the red algae Porphyridium cruentum and Porphyra yezoensis, although in the latter iso-floridoside was the main component. Sucrose and glucose were found in the green algae Chlorella pyrenoidosa and Scenedesmus obliquus, and also in a blue-green alga, Anacystis nidulans. Another blue-green alga, Phormidium foveolarum, contains mostly trehalose. From these results and from morphological considerations, it is suggested that Cyanidium caldarium belongs to the primitive Rhodophyta.     

Preparation of Monoclonal Antibodies against NADH: Nitrate Reductase from the Red Alga Porphyra yezoensis

Twenty-two monoclonal antibodies were raised against the nativeform of nitrate reductase (NR) from Porphyra yezoensis, a marinered alga. All the antibodies were able to bind to NR from P.yezoensis, with resultant inhibition of full (NADH:NR) and/orpartial (NADH:FR, NADH:CR, FMNH₂:NR, and MV:NR) enzymatic activity.Fifteen of the antibodies recognized the denatured form of theenzyme. Size-exclusion gel nitration and immunoblotting of theproducts of the limited proteolysis of NR from P. yezoensisby trypsin or Staphylococcus aureus V8 protease revealed that2 out of the 15 subunit-recognizing antibodies bound to theFAD domain, 6 bound to the heme domain, and 7 bound to the Mo-pterindomain. The products of limited proteolysis of NR from P. yezoensisalso revealed that the sites of proteolytic cleavage that encompassedthe heme domain were inverted as compared to the analogous sitesin NRs of higher plants. Some of the monoclonal antibodies cross-reactedwith NRs from plants belonged to different phyla. From threeto five of the antibodies bound subunits of NR from multicellularred algae, but failed to bind NRs from unicellular red algae.Three or four of the antibodies crossreacted with NRs from higherplants, such as tobacco and spinach. One of the antibodies boundNRs from several types of plant, namely, members of Cryptophyta,Chromophyta, and Chlorophyta. All of the monoclonal antibodiesthat cross-reacted with NRs from plants other than the red algaewere specific for the Mo-pterin domain of NR from P. yezoensis. (Received May 10, 1994; Accepted September 7, 1994)     

Speciation in the marine crop Pyropia yezoensis (Bangiales,Rhodophyta)

In the marine red alga Pyropia yezoensis, commonly known in Japan as nori, sympatric occurrence of two cryptic species Pyropia sp. 2 and Pyropia sp. 3 on the same rock in a natural habitat has been confirmed by molecular analysis and detailed morphological observations. To confirm whether Pyropia sp. 2 and Pyropia sp. 3 were reproductively isolated in the sympatric population, 170 blades that had previously been studied using a maternally inherited plastid marker were examined with a nuclear gene marker. The results suggested that Pyropia sp. 2 and Pyropia sp. 3 with identical morphological features were reproductively isolated in the sympatric population and that they were different species based on the biological species concept. Although gametophytic blades of Pyropia were usually assumed to be haploid, 18 of 170 blades possessed both of the two genotypes derived from Pyropia sp. 2 and from Pyropia sp. 3. These results inferred that allodiploid blades were generated from the interspecific hybridization between these two cryptic species. The present findings provide insights for future studies on the speciation mechanism in seaweeds, particularly for genera that contain numerous species.     

A selenium-induced peroxidation of glutathione in algae

Two unicellular marine algae cultured in media containing sodium selenite were examined for glutathione peroxidase activity. The 400 g supernatant from disrupted cells of both the green alga Dunaliella primolecta and the red alga Porphyridium cruentum were able to enhance both the H₂O₂ and the tert-butyl hydroperoxide dependent oxidation of glutathione. The glutathione peroxidation activity of D. primolecta was reduced only slightly by heating the 400 g supernatant, a 30% decrease in the rate with H₂O₂ and 10% decrease in the rate with t-BuOOH being observed. Heating caused the H₂O₂ dependent activity in P. cruentum to be reduced by only 30%, but the activity with t-BuOOH was reduced by 90%. Freezing decreased the t-BuOOH dependent activity of P. cruentum by 90%, but did not lower the t-BuOOH dependent activity of D. primolecta or the H₂O₂ dependent activity of either alga. It was concluded that the heat and cold stable, glutathione peroxidation was non-enzymatic in nature. A variety of small molecules (ascorbate, Cu(NO₃)₂, selenocystine, dimethyldiselenide and selenomethionine) were shown to be able to enhance the hydroperoxide dependent oxidation of glutathione in the assay system employed in this study. Such compounds could be responsible for the activity observed in algae. The heat and cold labile t-BuOOH reductase activity of P. cruentumwas possibly enzymatic, but was not attributable to the presence of glutathione-S-transferase. Both algae, when cultured in the presence of added selenite, displayed an approximate doubling of the non-enzymatic H₂O₂ and t-BuOOH dependent glutathione oxidase activities. The heat and cold labile t-BuOOH reductase activity of P. cruentum was unaltered when the alga was grown in the presence of added selenite. These observations are consistent with the hypothesis that selenium compounds present in the algae are responsible for the selenium induced glutathione peroxidation.     

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe^IIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe^IIINO dissociation rate (k_d) and NO association rate (k_on) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k_on and k_d by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O₂ catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O₂ intermediate, but instead were due to the slower NO k_on and k_d and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.     

DNA barcoding of a new record of epi-endophytic green algae Ulvella leptochaete (Ulvellaceae,Chlorophyta) in India

Epi-endophytic green algae comprise one of the most diverse and phylogenetically primitive groups of green algae and are considered to be ubiquitous in the world’s oceans; however, no reports of these algae exist from India. Here we report the serendipitous discovery of Ulvella growing on intertidal green algae Cladophora glomerata and benthic red algae Laurencia obtusa collected from India. DNA barcodes at nuclear ribosomal DNA Internal Transcriber Spacer (nrDNA ITS) 1 and 2 regions for Indian isolates from the west and east coasts have been generated for the first time. Based on morphology and DNA barcoding, isolates were identified as Ulvella leptochaete. Phylogenetic reconstruction of concatenated dataset using Maximum Likelihood method differentiated Indian isolates from other accessions of this alga available in Genbank, albeit with low bootstrap support. Monophyly of Ulvella leptochaete was obvious in both of our phylogenetic analyses. With this first report of epi-endophytic algae from Indian territorial waters, the dire need to catalogue its cryptic diversity is highlighted and avenues of future research are discussed.     

Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of <Emphasis Type="Italic">Gracilaria vermiculophylla </Emphasis>(Ohmi) Papenfuss (Gracilariales,Rhodophyta)

Background  

Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae.     

Gene Regulation of Iron-Deficiency Responses Is Associated with Carbon Monoxide and Heme Oxydase 1 in Chlamydomonas reinhardtii

Carbon monoxide (CO) as an endogenous gaseous molecule regulates a variety of biological processes in animals. However, CO regulating nutrient stress responses in green alga is largely unknown. On the other hand, heme oxydase (HO1 as a rate-limiting enzyme of the first step for heme degration and to catalyze heme into biliverdin (BV), which is concomitant with releasing of CO and ferrous ions, probably participates in the process of CO-regulating response to nutrient stress in green alga. In this paper, we described an observation that CO could regulate iron-homeostasis in iron-starving Chlamydomonas reinhardtii. Exogenous CO at 8 µM was able to prevent the iron deficient-inducing chlorosis and improve chlorophyll accumulation. Expression pattern of FOX1, FTR1 and ferredoxin was up-regulated by CO exposure in iron-deficient mediam. treatment with external CO increasing iron accumulation in iron-deficient C. reinhardtii. Moreover, to get insights into the regulatory role of HO1, we constructed a transgenic alga overexpressing HO1 and HO1 knock-out mutants. The results show that there was no significant influence on chlorosis with HO1 overexpression of C. reinhardtii under iron-deficiency and the chlorophyll accumulation, and gene expression associated with iron deficiency of mutant were greatly improved. Otherwise, those results from HO1 knock-out mutants were opposite to HO1 overexpression mutants. Finally, CO exposure induced NO accumulation in cells. However, such an action could be blocked by NO scavenger cPTIO. These results indicate that CO/HO1 may play an important role in improving green algae adaptation to iron deficiency or cross-talking with NO under the iron deficiency.     

Degradation of arsenobetaine by microorganisms associated with marine macro algae,Monostroma nitidium and Hizikia fusiforme

• 1.1. Arsenobetaine-containing growth media (ZoBell 2216E; solution of inorganic salts) were mixed with each of two marine macro algae, a green alga Monostroma nitidum and a brown alga Hizikia fusiforme, as a source of microorganisms.
• 2.2. The conversion of arsenobetaine to trimethylarsine oxide and/or dimethylarsinic acid by the microorganisms associated with the marine macro algae was confirmed in both the media.
• 3.3. A striking contrast, however, in the conversion pattern was observed between the two algae: arsenobetaine was converted to trimethylarsine oxide and trimethylarsine oxide to dimethylarsine acid successively with M. nitidum, while the reverse was observed with H. fusiforme.

     

Occurrence of sucrose phosphatase in vascular and non-vascular plants

Green plants including representatives of angiosperms, gymnosperms, ferns, mosses, liverworts and green algae were shown to contain a specific sucrose phosphatase, the last enzyme in the pathway of sucrose synthesis. The enzyme from all species required Mg²⁺ for activity and it was partially inhibited by sucrose. It was not detected in a red alga, brown algae, or mushroom species which contain little or no sucrose.