Leafy Thalli Research Articles

Role of reactive oxygen species (ROS) signaling pathway in the wound-induced spore formation of Pyropia yezoensis

Asexual spore formation of Pyropia yezoensis significantly impacts yield. After mechanical sectioning of leafy thalli into fragments, somatic cells rapidly develop into a large number of wound-induced spores (WIS, a form of asexual spores). Recent studies on P. yezoensis have demonstrated a notable increase in reactive oxygen species (ROS) during this process; however, the regulatory mechanism underlying this ROS signaling remains largely unclear. This paper shows that in P. yezoensis, ROS signals play a crucial role in WIS formation. When treated with the ROS-producing enzyme NADPH oxidase inhibitor diphenyleneiodonium chloride, P. yezoensis thalli fragments strongly repressed WIS formation, accompanied by a significant decrease of ROS levels, malondialdehyde contents, and activities of NADPH oxidase and antioxidant enzymes. The expression levels of respiratory burst oxidase homolog genes, aquaporin protein genes, superoxide dismutase genes, glutathione S-transferase genes, and glutathioneperoxidase genes were also decreased, thus inhibiting WIS formation. As an upstream signal, ROS promote ion exchange and metabolism both inside and outside of cells within the first 12 h of wound stress. Following 12 h of wound stress, transcriptional activation of specific genes associated with cell cycle progression—which promotes cell division and dedifferentiation under normal conditions—was also blocked by diphenyleneiodonium chloride. In addition, the generated ROS signals also regulated the expression of mitogen-activated protein kinase family genes under wound stress. Overall, this study provides novel insights into the molecular mechanism underlying the ROS signaling regulation of WIS formation in P. yezoensis.

Genomes Vary in Size and Spatial Patterns Within Chimeric Blades of Porphyra spp.

Genome size variation is of crucial biological importance, however variation in genome sizes within a single individual/organism is rarely reported except for some species groups such as algae where polygenomy, endopolyploidy and mixopolyploidy have previously been reported. The red algal genus Porphyra forms part of very profitable marine food products commonly known as ‘Nori’. Farming of these valuable marine crops was revolutionized by the discovery of their life cycle in the 40’s. One of the most remarkable characteristics of these taxa is the formation of chimeric gametophytic thalli. After meiosis, the four meiotic products are not released as individuals spores, but instead develop together into a single leafy thallus through successive mitotic divisions. In this study, we used flow cytometry to estimate genome sizes in 670 vegetative thallus sections from 195 blades from three Porphyra species, to determine if this chimerism could be related to the presence of multiple genome sizes and mixoploidy within thalli. Our results show a wide variety of genome sizes both within and between thalli. We interpreted these results as the presence of two different genome types of different sizes (a and b) with separate rounds of genome duplications within the vegetative thalli. By analyzing several sections per thallus, we were able to show that the different genome types and ploidy levels are not distributed haphazardly through the thallus, but are distributed along the thallus in a sectorial way in mosaics. In some individuals, the 2C genome size can either be interpreted as diploids or alternatively as haploid cells that are arrested at the G2-stage of the mitotic cycle, acting as diploid with two copies of their genome during most of their life-time. We conclude that Porphyra species belong to an aneuploid/euploid system, where genome duplications, mixoploidy, chromosomal dynamics and the presence of different genome types in the chimeric thalli play a role in shaping the genetic diversity of these taxa. Our results may have important implications to understand red algae biology and evolution and raise further questions on concepts of what constitutes an individual.

Physiological differences in photosynthetic inorganic carbon utilization between gametophytes and sporophytes of the economically important red algae Pyropia haitanensis

Despite the lifecycle of the red seaweed Pyropia/Porphyra being extensively studied, photosynthesis and the CO2 concentrating mechanisms (CCMs) have comparatively not been well studied between the gametophyte phase (leafy thallus) and the sporophyte phase (filamentous conchocelis). This study found that the net photosynthetic rates of the thalli were about twice that of the conchocelis of Py. haitanensis, and the maximal quantum yield of photosystem II (Fv/Fm) and thallus respiration were significantly higher and lower than the conchocelis, respectively. Additionally, two phases did not show significant differences in phycocyanin (PC) content, but significantly higher contents of phycoerythrin (PE), allophycocyanin (APC), and chlorophyll α (Chl α) were observed in the thalli compared with the conchocelis. The external carbonic anhydrase (eCA) activity of the thalli was also higher than that of the conchocelis, but no differences in internal CA (iCA) activity were detected between the two phases. As for carbon acquisition for photosynthesis, the major carbon source of the thalli was seawater HCO3−, which was absorbed via eCA-catalyzed conversion to CO2. Conversely, the conchocelis used HCO3− transporters to directly absorb seawater HCO3−, which contributed equally to photosynthesis compared with the eCA- or iCA-mediated pathway. The discovery of different CCMs between the thallus and conchocelis phases could further our understanding of lifecycle regulation in Pyropia. In addition, a decrease in pH from 9 to 6 significantly increased the photosynthetic rates of both the thalli and conchocelis by about 100% and 600%, respectively. Thus, both phases have potential for carbon capture from flue gases and carbon sequestration.

Preliminary comparison of atmospheric CO2 enhancement to photosynthesis of Pyropia yezoensis (Bangiales, Rhodophyta) leafy thalli and filamentous thalli

SUMMARYIntertidal macroalgae are submerged in seawater at high tide and exposed to air at low tide. When they are exposed to the air, CO2 is the main inorganic carbon source. In this study, the photosynthetic performances of PSI and PSII were measured in different generations of Pyropia yezoensis (leafy thalli and filamentous thalli) that had been exposed to air containing different CO2 concentrations. Changes in photosynthesis during dehydration and salt treatment under the different CO2 concentrations were also analyzed. The results showed that in leafy thalli, the effective photochemical quantum yield of PSII (YII) was enhanced as CO2 increased, which suggested that CO2 assimilation was enhanced and that they can utilize CO2 in the air directly, even when they are subjected to moderate stress. These findings could explain why, in P. yezoensis aquaculture, moderate exposure to air does not lead to a decrease in crop yield. However, in filamentous thalli, there were no significant differences in YII at the CO2 concentrations tested. The expression of genes involved in the Calvin cycle in leafy thalli was higher than that in filamentous thalli. CO2 uptake and biomass of P. yezoensis leafy thalli is larger than filamentous thalli, which may be due to its different carbon utilization mechanism and the adaptation of intertidal environment in the evolutionary process.

Molecular cloning and expression analysis of two key genes, HDS and HDR, in the MEP pathway in Pyropia haitanensis

The 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene and the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene are two important genes in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In this study, we reported the isolation and characterization of full-length HDS (MF101802) and HDR (MF159558) from Pyropia haitanensis. Characteristics of 3-D structures of the PhHDS and PhHDR proteins were analysed respectively. The results showed that the full-length cDNA of PhHDS, which is 1801 bp long, contained a 1455 bp open reading frame (ORF) encoding a putative 484 amino acid residue protein with a predicted molecular mass of 51.60 kDa. Meanwhile, the full-length cDNA of PhHDR was 1668 bp and contained a 1434 bp ORF encoding a putative 477 amino acid 2 residue protein with a predicted molecular mass of 51.49 kDa. The expression levels of the two genes were higher in conchocelis than that in leafy thallus. Additionally, the expression levels could be influenced by light, temperature and salinity and induced by methyl jasmonate (MJ) and salicylic acid (SA). This study contributed to our in-depth understanding of the roles of PhHDS and PhHDR in terpenoid biosynthesis in Pyropia haitanensis and the regulation of the two genes by external environments.

Cloning and characterization of the geranylgeranyl diphosphate synthase (GGPS) responsible for carotenoid biosynthesis in Pyropia umbilicalis

Carotenoid metabolism in red algae is not well understood. Geranylgeranyl diphosphate (GGPP), synthesized by GGPP synthase (GGPS), is a precursor for the biosynthesis of many biologically important metabolites, including carotenoids and chlorophylls. GGPSs have been functionally characterized in many organisms, but not in species of the primitive red algal order Bangiales. Here, we cloned and characterized the gene encoding GGPS (PuGGPS) in Pyropia umbilicalis (Bangiales). PuGGPS encodes a protein of 345 amino acids with an N-terminal transit peptide. The catalytic activity of PuGGPS for the production of GGPP was verified by a color complementation assay in Escherichia coli and subsequent high-performance liquid chromatography analysis. Homology modeling of PuGGPS showed that its tertiary structure resembles that of other known GGPSs and that this structure allows for the precise docking of the enzymatic product of PuGGPS, GGPP. When leafy thalli of P. umbilicalis were treated with norflurazon, an inhibitor of the key carotenoid metabolism enzyme phytoene desaturase, the expression of PuGGPS increased by twofold compared with that of the control in the first 2 h, suggesting a prompt response to metabolic perturbation. Prolonged norflurazon treatment failed to increase PuGGPS expression. Sequence analysis showed that PuGGPS shares seven conserved motifs with other previously identified GGPSs from different organisms, including two aspartate-rich GGPS signature motifs. Phylogenetic analysis also indicated that PuGGPS is a member of the type II GGPSs found in eubacteria and plants.

Characterization of the protein phosphatase 2c gene from Porphyra yezoensis and functional analysis under dessicating conditionsa

Abstract To characterize the structure and function of the protein phosphatase 2C (PP2C) gene in Porphyra yezoensis, full-length cDNA of the locus (denoted pypp2c) was obtained by electronic cloning, rapid amplification of cDNA ends, and reverse-transcription PCR. The nucleotide sequence of PyPP2C was 1988 bp, including a 5′-untranslated region (UTR) of 301 bp, a 3′-UTR of 256 bp, and an open reading frame of 1431 bp that can be translated into 476 amino acids with a molecular mass of 50.96 kDa and a putative isoelectric point of 8.34. The amino acids had 30–36% identity with those encoded by PP2C genes in protozoa and higher plants. A PP2C-like catalytic domain was found in the 57–397 region, and several residues involved in the metal ion binding sites were conserved. mRNA expression levels in the three life stages of leafy thalli, filaments, and conchospores were examined by real-time fluorescent quantitative PCR. Highest expression of pypp2c occurred in conchospores. Gene expression levels were similar among the three life stages. pypp2c expression at different thallus relative water contents (RWCs) varied, with the highest expression occurring at 60% RWC.

CHARACTERIZATION OF THE ALTERNATIVE OXIDASE GENE IN PORPHYRA YEZOENSIS (RHODOPHYTA) AND CYANIDE-RESISTANT RESPIRATION ANALYSIS(1).

The full-length cDNA of the alternative oxidase (AOX) gene from Porphyra yezoensis Ueda (PyAOX) [currently assigned as Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (http://www.algaebase.org)] an ancient member of the Rhodphyta, was cloned by electronic cloning, rapid amplification of cDNA ends (RACE), and reverse transcription PCR. The nucleotide sequence of PyAOX consists of 1,650 bp, including a 5' untranslated region (UTR) of 170 bp, a 3' UTR of 148 bp, and an open reading frame (ORF) of 1,332 bp that can be translated into a 443-amino-acid residue with a molecular mass of 47.33 kDa and a putative isoelectric point (pI) of 9.71. The putative amino acids had 50%-61% identity with AOX genes in Eukaryota and higher plants and had AOX-like characteristics. The expression of PyAOX mRNA in different stages of the life cycle, conchospores, filamentous thalli (conchocelis stage), and leafy thalli, was detected by real-time quantitative PCR (qPCR). The highest level of expression, which was observed in filamentous thalli, was three times higher than that observed in leafy thalli. The next highest level, which was observed in the conchospores, was twice as high as that observed in leafy thalli. We showed that an alternative respiration pathway existed in P. yezoensis with a noninvasive microsensing system. The contribution of the alternative pathway to total respiration in filamentous thalli was greater than that in leafy thalli. This result was consistent with the level of AOX gene expression observed in different stages of the life cycle.

A SIMPLE METHOD FOR ISOLATING FILAMENTS AS "ALGAL SEED STOCK" FROM MONOSTROMA LATISSIMUM (CHLOROPHYTA) GERMLINGS, AND APPLICATION FOR MASS CULTIVATION(1).

Germlings were grown from Monostroma latissimum Wittr. reproductive cells on nylon ropes. Holdfast threads and some uniseriate filaments were observed to have penetrated the fibers of the dispersed ropes. The algal filaments were easily isolated and prepared for cultivation, in comparison to the methods of enzymatically isolated algal protoplasts. Under low light (60-100 μmol photons · m(-2) · s(-1) ), the algal filaments grew to form a filamentous mass. When cultivated under stronger light (300-600 μmol photons · m(-2) · s(-1) ), they grew to initially form tubular thalli and then, when cultivated under light intensities >700 μmol photons · m(-2) · s(-1) , formed foliaceous thalli. Consequently, the filaments were homogenized into small sections and then sewed on the nylon rope for algal mass cultivation. Under high-intensity natural light, they grew to form leafy thalli.

Grateloupia huangiae (Halymeniaceae, Rhodophyta), a new species from Taiwan previously confused with Polyopes lancifolius, with emphasis on the development of the auxiliary-cell ampullae

Lin S.-M., and Liang H.-Y. 2011. Grateloupia huangiae (Halymeniaceae, Rhodophyta), a new species from Taiwan previously confused with Polyopes lancifolius, with emphasis on the development of the auxiliary-cell ampullae. Phycologia 50: 232–240. DOI: 10.2216/10-29.1Grateloupia huangiae S.-M. Lin & H.-Y. Liang sp. nov. was previously recorded as G. okamurae Yamada [currently recognized as Polyopes lancifolius (Harvey) Kawaguchi & H.W. Wang] in Taiwan. Molecular phylogenetic analysis shows that G. huangiae is unrelated to P. lancifolius but is closely related to Grateloupia taiwanensis S.-M. Lin & H.-Y. Liang. Although G. huangiae superficially resembles P. lancifolius in having a leafy thallus and short proliferations borne on the thallus surface, the structure of the auxiliary-cell ampulla is very different. The auxiliary-cell ampulla of G. huangiae is composed of three orders of unbranched filaments before diploidization, each of which is 11–13 cells long. The auxiliary cell is the first cell of the third-order ampullar filament, which is cut off from the first cell of the second-order ampullar filament. This type of auxiliary-cell ampulla is the same as that found in G. taiwanensis. On the other hand, P. lancifolius has a more complex auxiliary-cell ampulla in which the ampullar filaments may bear up to five to six orders of branches before diploidization. A detailed study of the development of auxiliary-cell ampullae before and after diploidization is needed in other species and genera within the Halymeniaceae to better delineate generic concepts in the family.

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROMPORPHYRA YEZOENSIS

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO2 and HCO3−, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full-length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5′ untranslated region (UTR) of 177 bp, a 3′ UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274-amino-acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the β-CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real-time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4-fold higher and 10-fold higher, respectively.

Nitophyllum adhaerens (Ceramiales, Delesseriaceae), uma nova ocorrência para o litoral brasileiro

Durante estudo sobre as Rodofíceas do infralitoral do Estado do Espírito Santo foram encontrados exemplares de Nitophyllum adhaerens M.J. Wynne em coletas realizadas a 6 metros de profundidade. Os espécimens foram estudados detalhadamente e comparados com espécies morfologicamente semelhantes. As principais características da espécie são talo foliáceo delicado, monostromático, sem nervura central e sem vênulas, presença de rizóides marginais e soros de tetrasporângios localizados próximo às margens. Esta é a primeira ocorrência de Nitophyllum adhaerens para o litoral brasileiro.

Effect of nitrogen and phosphorus on the development and differentiation of vegetative cells of Porphyra yezoensis on solid agar medium

To identify the optimal nitrogen and phosphorus concentrations for development and differentiation of vegetative cells of Porphyra yezoensis cultured on solidified agar medium, vegetative cells were isolated with marine gastropod enzymes from mature thalli and varied concentrations of nitrogen and phosphorus were added to the culture media. Our results showed that: (1) nitrate is a good nitrogen source in solid culture even at a concentration as high as 18.80 mM. In contrast, ammonium is not, because few vegetative cells developed into germlings over 14 days in medium containing this ion; (2) at 0.47 mM and higher nitrogen concentrations, isolated cells produced large numbers of normal leafy thalli within 14 days, while at lower concentrations of 0.23 mM–0.47 mM, regenerated cells showed obvious nitrogen deficiency after 20 days in culture; (3) optimum phosphorus concentrations were between 0.07 mM–0.14 mM and produced >52% normal vegetative cells within 14 days. Most germlings were abnormal and showed no further development when the phosphorus concentration was below 0.03 mM or over 0.30 mM, and (4) the differentiation of germlings was significantly correlated to the ratio of N and P (a ratio of 5:1 produced best results).

Observation of female gamete (Carpogonia) germination ofPorphyra yezoensis andP. oligospermatangia

Several papers have reported that part or whole leafy thallus seemingly consisting of zygotospores can give rise to both blades and conchocelis in the same culture ofPorphyra. Study on samples of wild and cultivatedPorphyra yezoensis andP. oligospermatangia were conducted to clarify the origination of the young blades in the culture. It is confirmed that single cells on the blade of both species, which normally intermixed with zygotospores, germinated into young blades. TEM and SEM observation has shown that the single cells ofPorphyra yezoensis had typical features of female gamete (carpogonia) but archeospore. Therefore, the female gametes are responsible in developing leafy thalli.

Ultrastructure of single cells, callus-like and monospore-like cells inPorphyra yezoensis ueda on semisolid culture medium

It had been demonstrated that individual cells or protoplasts isolated fromPorphyra thallus by enzyme could develop into normal leafy thalli in the same way as monospores, and that isolated cells develop in different way in liquid and on semi-solid media. The authors observed the ultrastructure of isolated vegetative cells cultured on semi-solid media and compared them with those of monospores and isolated cells cultured in liquid media. The results showed that subcellular structures were quite different among cells in different conditions. In their development, isolated cells on semi-solid media did not show the characteristic subcellular feature of monospore formation, such as production of fibrous vesicles. Callus-like cells formed on semi-solid media underwent a distinctive modification in cellular organization. They developed characteristic cell inclusions and a special 2-layer cell covering. Golgi bodies, ER, starch grains, mitochondria. Vacuoles were not commonly found in them.

The ultrastructure of separated and cultured cell ofPorphyra yezoensis

There are many reports that cells (protoplasts) separated from the thallus ofPorphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells.

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal “seed” stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R‐10 and 2% Macerozyme R‐10. The highest number of protoplasts was obtained on a 50‐rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as“seed” stock for algal mass culture.

The life history of Porphyra endiviifolium from the South Shetland Islands, Antarctica

This paper describes the reproduction and life history of an intertidal species, Porphyra endiviifolium, from Antarctica. Field specimens were examined microscopically, prepared for electron microscopy and used to establish cultures. Wild populations comprised two kinds of leafy thalli, morphologically similar but distinguished by their mode of reproduction, either sexual or asexual. Carpospores from monoecious leafy gametophytes developed into conchocelis filaments in culture, and under “winter-spring” conditions these formed conchospores that germinated to produce leafy thalli. Monospores from asexual leafy thalli developed directly into two different forms of leafy thalli. Only one of the cultured morphotypes became fertile, reproducing asexually by monospores. We conclude that the phases of the life history of P. endiviifolium show different ecological strategies, the conchocelis phase reproducing in response to short days unlike the leafy thalli in which growth and reproduction respond primarily to irradiance.

Enhanced growth of the red algaPorphyra yezoensis Ueda in high CO2 concentrations

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO2. It was found that the higher the CO2 concentration, the faster the growth of the thalli. Aeration with elevated CO2 lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO3− to OH− and CO2 proceeded much faster than the dissociation of hydrated CO2 releasing H+. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO2 + HCO3− + CO3−). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO2 by marine algae is discussed.

Cell suspension culture from Porphyra linearis (Rhodophyta) a multicellular marine red alga

A cell-suspension culture suitable for continuous propagation was established from protoplasts of the red alga Porphyra linearis Grev., an edible, winter annual species of nori. Protoplast-derived cells that did not regenerate into thalli were used to establish a culture line of uniform-sized (average about 25 μm diam.) cells, which resembled the vegetative cells of this species in the leafy thallus phase. Cell division occurred about once per 24 to 30 h in uncrowded (1–2 cells per culture well) culture. This cell-suspension culture has now been maintained as continuously growing subcultures for more than four years without formation of organized thalli; however, the latter can be obtained at will by altering culture conditions (lowering temperature from 20° to 10 °C, lengthening photoperiod from 10: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaa0aaaeaaca% aIXaGaaGinaaaaaaa!3777!\[\overline {14} \] or 8 : % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaa0aaaeaaca% aIXaGaaGOnaaaaaaa!3779!\[\overline {16} \] to 14: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaa0aaaeaaca% aIXaGaaGimaaaaaaa!3773!\[\overline {10} \] or 16: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaa0aaaeaaca% aI4aaaaaaa!36C0!\[\overline 8 \] and increasing irradiance from 10 to ≥ 30 μmol m-2 s-1). This appears to be the first continuous non-clonal cell-suspension culture developed for a multicellular alga.