DEVELOPMENTAL STUDIES IN PORPHYRA. I. BLADE DIFFERENTIATION IN PORPHYRA PERFORATA AS EXPRESSED BY MORPHOLOGY,ENZYMATIC DIGESTION,AND PROTOPLAST REGENERATION1

Four areas containing different cell morphologies were mapped on Porphyra blades and five different cell types (i.e. tapered with long extensions, large and vacuolated, vegetative and dividing, and reproductive: males and females) were identified in them. Tissues from these areas were dissociated, and protoplasts and single cells were isolated from the dissociated tissue of each distinct region. Regeneration rates of the isolated cells and protoplasts (isolates) varied depending on their morphological type. Regeneration rates were lowest in cultured isolates from the area just above the holdfast (ca. 1 %) and increased gradually to over 80% in isolates from areas of vegetative and reproductive regions away from the holdfast. Four distinct morphological patterns were observed among the regenerating plants. Cells isolated from vegetative areas developed into leafy plants while in liquid culture, and into calli when grown on solid medium. Isolates from reproductive areas developed into either a long thin or short thick filamentous plant. Those from ripe patches of carposporangia developed into thin conchocelis filaments, while isolates from non-differentiated cells bordering the ripe reproductive patches developed into thick filaments resembling the morphology of conchosporangial branches. The blade of Porphyra appears simple as it consists of a single cell layer; however, it is complex both morphologically and physiologically.     

LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.     

DEVELOPMENTAL STUDIES IN PORPHYRA (RHODOPHYCEAE). III. EFFECT OF CULTURE CONDITIONS ON WALL REGENERATION AND DIFFERENTIATION OF PROTOPLASTS1

Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species.     

IDENTIFICATION OF CYTOKININS IN SARGASSUM MUTICUM (PHAEOPHYTA) AND PORPHYRA PERFORATA (RHODOPHYTA)1

Combined gas chromatography-mass spectrometry (GCMS) was used to identify and quantify specific cytokinins from Porphyra perforate J. Ag. and Sargassum muticum (Yendo) Fensh. The level of isopentenyladenosine was estimated to be 0.6 μ·kg^?1 fresh weight in Porphyra and 0.9 μ·kg^?1 fresh weight in Sargassum. The level of cis-zeatin riboside was estimated to be 0.2 μ·kg^?1 fresh weight in Sargassum. This is the first definitive identification of a cytokinin from a red alga, and the second report from a brown alga.     

VARIATIONS IN FLORIDOSIDE CONTENT AND FLORIDOSIDE PHOSPHATE SYNTHASE ACTIVITY IN PORPHYRA PERFORATA (RHODOPHYTA)1

The diurnal and seasonal variations in floridoside content and floridoside phosphate synthase (FPS) activity were measured in samples of Porphyra perforata J. Ag. growing in the field. Floridoside content generally increased about fourfold from early morning to the middle of the day and then dropped gradually in the afternoon and the evening. On a monthly basis, there was a steady increase in floridoside content from February to May. A similar trend in monthly increase in FPS activity was also observed from February to April. The level of FPS activity varied with the time of day. The highest activity was observed early in the morning shortly after dawn; subsequently, it decreased, reaching about 50% of the peak value late in the afternoon/evening. On a daily as well as seasonal basis, a change in FPS activity correlated with a change in floridoside content. Among environmental factors, floridoside content and FPS activity showed a positive correlation with daylength and temperature on a seasonal basis. However, on a daily basis, salinity and water temperature did not seem to affect the floridoside content or the FPS activity.     

MAJOR DEVELOPMENTAL REGULATORS AND THEIR EXPRESSION IN TWO CLOSELY RELATED SPECIES OF PORPHYRA (RHODOPHYTA)1

Little is known about the genetic and biochemical mechanisms that underlie red algal development, for example, why the group failed to evolve complex parenchyma and tissue differentiation. Here we examined expressed sequence tag (EST) data from two closely related species, Porphyra umbilicalis (L.) J. Agardh and P. purpurea (Roth) C. Agardh, for conserved developmental regulators known from model eukaryotes, and their expression levels in several developmental stages. Genes for most major developmental families were present, including MADS‐box and homeodomain (HD) proteins, SNF2 chromatin‐remodelers, and proteins involved in sRNA biogenesis. Some of these genes displayed altered expression correlating with different life history stages or cell types. Notably, two ESTs encoding HD proteins showed eightfold higher expression in the P. purpurea sporophyte (conchocelis) than in the gametophyte (blade), whereas two MADS domain‐containing paralogs showed significantly different patterns of expression in the conchocelis and blade respectively. These developmental gene families do not appear to have undergone the kinds of dramatic expansions in copy number found in multicellular land plants and animals, which are important for regulating developmental processes in those groups. Analyses of small RNAs did not validate the presence of miRNAs, but homologs of Argonaute were present. In general, it appears that red algae began with a similar molecular toolkit for directing development as did other multicellular eukaryotes, but probably evolved altered roles for many key proteins, as well as novel mechanisms yet to be discovered.     

PURIFICATION OF A SEX‐SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.     

EVOLUTION OF DEVELOPMENTAL PROGRAMS IN VOLVOX (CHLOROPHYTA)1

The volvocine green algal genus Volvox includes ～20 species with diverse sizes (in terms of both diameter and cell number), morphologies, and developmental programs. Two suites of characters are shared among distantly related lineages within Volvox. The traits characteristic of all species of Volvox—large (>500) numbers of small somatic cells, much smaller numbers of reproductive cells, and oogamy in sexual reproduction—have three or possibly four separate origins. In addition, some species have evolved a suite of developmental characters that differs from the ancestral developmental program. Most multicellular volvocine algae, including some species of Volvox, share an unusual pattern of cell division known as palintomy or multiple fission. Asexual reproductive cells (gonidia) grow up to many times their initial size and then divide several times in rapid succession, with little or no growth between divisions. Three separate Volvox lineages have evolved a reduced form of palintomy in which reproductive cells are small and grow between cell divisions. In each case, these changes are accompanied by a reduction in the rate of cell division and by a requirement of light for cell division to occur. Thus, two suites of characters—those characteristic of all Volvox species and those related to reduced palintomy—have each evolved convergently or in parallel in lineages that diverged at least 175 million years ago (mya).     

SELECTION OF INTERNAL CONTROL GENE FOR EXPRESSION STUDIES IN PORPHYRA HAITANENSIS (RHODOPHYTA) AT DIFFERENT LIFE‐HISTORY STAGES1

Accurate gene quantification depends on the use of an appropriate internal control gene, which should be verified before its use for normalizing data. Housekeeping genes, which are expressed at relatively constant levels, are generally regarded as candidate internal control genes. To determine the ideal internal control for gene expression profiles for Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) at different life‐history stages, we used absolute quantification to assess the expression levels of six housekeeping genes (18S ribosomal RNA, 30S ribosomal protein, glyceraldehyde‐3‐phosphate dehydrogenase, elongation factor 3, alpha‐tubulin, and beta‐tubulin) at the sporophyte and gametophyte stages. Housekeeping genes were selected by comparing the differences of observed copy numbers in sporophytes and in gametophytes. TubB (beta‐tubulin) was found to be the optimal internal control gene, because it showed the smallest difference of gene expression. Compared with TubB, other housekeeping genes had greater variation of expression to different degrees.     

MOLECULAR CHARACTERIZATION OF THE LECTIN,BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)1

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5′ untranslated region (UTR) and 201 bp of 3′ UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C‐terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.     

AVP_（4－8）结合点在大鼠海马内的分布和AVP_（4－8）对其受体发育的影响：放射自显影研究

本文利用放射自显影方法结合神经毒对海马神经元的选择性损毁观察ＡＶＰ（４－８）结合点在大鼠海马内的分布和定位;利用外源性ＡＶＰ（４－８）对新生大鼠的处理,观察海马ＡＶＰ（４－８）结合点的发育调节。在成年大鼠海马内,ＡＶＰ（４－８）结合点集中分布在整个海马的锥体细胞层和齿回的颗粒细胞层。秋水仙碱处理后,齿回颗粒细胞层消失,齿回区的ＡＶＰ（４－８）结合点也消失。红藻氨酸（Ｋａｉｎｉｃａｃｉｄ）处理后海马ＣＡ３－ＣＡ４的锥体细胞层消失,该区的ＡＶＰ（４－８）结合点也消失。新生大鼠海马锥体细胞层的ＡＶＰ（４－８）结合点在出生后第６天开始出现,齿回颗粒细胞层的ＡＶＰ（４－８）结合点在出生后第７天开始出现。然而,新生大鼠每天经外源性ＡＶＰ（４－８）处理,海马锥体细胞层和齿回颗粒细胞层的结合点均在出生后第５天已变得十分稠密。本文就大鼠海马ＡＶＰ（４－８）结合点的特异性分布和ＡＶＰ（４－８）处理促进海马ＡＶＰ（４－８）结合点的发育与成年后大鼠学习能力的提高的相互关系作了讨论。     

INTERSPECIFIC HYBRIDIZATION IN THE HAPLOID BLADE‐FORMING MARINE CROP PORPHYRA (BANGIALES,RHODOPHYTA): OCCURRENCE OF ALLODIPLOIDY IN SURVIVING F1 GAMETOPHYTIC BLADES1

We performed interspecific hybridization in the haploid blade‐forming marine species (nori) of the genus Porphyra, which have a heteromorphic life cycle with a haploid gametophytic blade and a diploid microscopic sporophyte called the “conchocelis phase.” The green mutant HGT‐6 of P. tenera var. tamatsuensis A. Miura was crossed with the wildtype HG‐1 of P. yezoensis f. narawaensis A. Miura; the F₁ heterozygous conchocelis developed normally and released numerous conchospores. However, almost all the conchospore germlings did not survive past the four‐cell stage or thereabouts, and only a few germlings developed into gametophytic blades. These results indicate that hybrid breakdown occurred during the meiosis, while the surviving F₁ gametophytic blades were considered a breakthrough in the interspecific hybridization of Porphyra. Organelle genomes (cpDNA and mtDNA) were found to be maternally inherited in the interspecific hybridization by molecular analyses of the organelle DNA. In particular, molecular analyses of nuclear DNA revealed that the surviving F₁ blades were allodiploids in the haploid gametophytic phase; however, there is a possibility of the occurrence of rapid chromosomal locus elimination and rearrangement in the F₁ conchocelis phase. Our findings are noteworthy to the breeding of cultivated Porphyra and will provide important information for understanding of the speciation of marine plants with high species diversity.     

PHOTOSYNTHESIS AND RESPIRATION OF THE CONCHOCELIS STAGE OF ALASKAN PORPHYRA (BANGIALES,RHODOPHYTA) SPECIES IN RESPONSE TO ENVIRONMENTAL VARIABLES1

Photosynthesis and respiration of three Alaskan Porphyra species, P. abbottiae V. Krishnam., P. pseudolinearis Ueda species complex (identified as P. “pseudolinearis” below), and P. torta V. Krishnam., were investigated under a range of environmental parameters. Photosynthesis versus irradiance (P–I) curves revealed that maximal photosynthesis (P_max), irradiance at maximal photosynthesis (I_max), and compensation irradiance (I_c) varied with salinity, temperature, and species. The P_max of Porphyra abbottiae conchocelis varied between 83 and 240 μmol O₂ · g dwt^?1 · h^?1 (where dwt indicates dry weight) at 30–140 μmol photons · m^?2 · s^?1 (I_max) depending on temperature. Higher irradiances resulted in photoinhibition. Maximal photosynthesis of the conchocelis of P. abbottiae occurred at 11°C, 60 μmol photons · m^?2·s^?1, and 30 psu (practical salinity units). The conchocelis of P. “pseudolinearis” and P. torta had similar P_max values but higher I_max values than those of P. abbottiae. The P_max of P. “pseudolinearis” conchocelis was 200–240 μmol O₂ · g dwt^?1 · h^?1 and for P. torta was 90–240 μmol O₂ · g dwt^?1 · h^?1. Maximal photosynthesis for P. “pseudolinearis” occurred at 7°C and 250 μmol photons · m^?2 · s^?1 at 30 psu, but P_max did not change much with temperature. Maximal photosynthesis for P. torta occurred at 15°C, 200 μmol photons · m^?2 · s^?1, and 30 psu. Photosynthesis rates for all species declined at salinities <25 or >35 psu. Estimated compensation irradiances (I_c) were relatively low (3–5 μmol · photons · m^?2 · s^?1) for intertidal macrophytes. Porphyra conchocelis had lower respiration rates at 7°C than at 11°C or 15°C. All three species exhibited minimal respiration rates at salinities between 25 and 35 psu.     

ANALYSIS OF SPATIAL AND TEMPORAL DIVERSITY AND DISTRIBUTION OF PORPHYRA (RHODOPHYTA) IN SOUTHEASTERN NEW ZEALAND SUPPORTED BY THE USE OF MOLECULAR TOOLS1

Molecular studies have shown that New Zealand’s rocky shores are a habitat for >30 species of Porphyra, but little is known of their seasonal and zonal distribution. The spatial and temporal distribution of bladed Porphyra gametophytes at Brighton Beach, southeast New Zealand, were monitored for 32 months. Molecular markers were used for species identification, and a total of nine species was observed as being present during this time. Two species, P. cinnamomea and Porphyra sp. “ROS 54,” were the most common, and both were present for most months, while the remaining seven species were present sporadically, for only a few weeks at a time. P. cinnamomea W. A. Nelson and Porphyra sp. “ROS 54” were most common in the midintertidal, and both showed a similar seasonality with the highest presence during spring. They also showed a similar trend of seasonal dieback resulting in at least 1 month (May) in two consecutive years when they were both absent. This is one of the few studies investigating spatial and temporal distribution within a genus and over a 3‐year period. Our results show no distinct intertidal zonation patterns within the genus, and we conclude that morphologically similar species in a similar habitat rely on physiological mechanisms for survival.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

AMMONIUM AND UV RADIATION STIMULATE THE ACCUMULATION OF MYCOSPORINE‐LIKE AMINO ACIDS IN PORPHYRA COLUMBINA (RHODOPHYTA) FROM PATAGONIA,ARGENTINA1

The combined effects of ammonium concentration and UV radiation on the red alga Porphyra columbina (Montagne) from the Patagonian coast (Chubut, Argentina) was determined using short‐term (less than a week) experimentation. Discs of P. columbina were incubated with three ammonium concentrations (0, 50, and 300 μM NH₄Cl) in anilluminated chamber (PAR=300 μmol photons·m^?2·s^?1, UVA=15 W·m^?2, UVB=0.7 W·m^?2) at 15°C. Algae incubated at 300 μM ammonium showed a significant increase (P<0.05) in the concentration of mycosporine‐like amino acids (MAAs) compared with the initial value or with the other ammonium treatments. The increase of MAAs was, however, a function of the quality of irradiance received, with a higher increase in samples exposed to UVA compared with UVB (29% and 5% increase, respectively). However, UVB radiation was more effective in inducing MAA synthesis per unit energy received by the algae. Samples exposed to PAR only had an intermediate increase in MAA concentration of 16%. Chl a concentration decreased through the incubation with the greatest decrease at high ammonium concentration. Phycobiliprotein (BP) decreased through time with the smallest decrease occurring at high ammonium concentration. Photoinhibition (as a decrease of optimal quantum yield) was significantly greater under nitrogen‐deprived conditions than that under replete ammonium levels. Maximal gross photosynthesis (GP_max), as oxygen evolution, and maximal electron transport rate (ETR_max), as chl fluorescence, increased with the ammonium concentration. Positive relationships between maximal GP or ETR and pigment ratios (BP/chl a and MAAs/chl a) and negative relationships with chl a concentration were found.     

STUDIES ON THE ANOMALOUS CAMBIAL ACTIVITY IN DOXANTHA UNGUIS-CATI (BIGNONIACEAE). II. A CASE OF DIFFERENTIAL PRODUCTION OF SECONDARY TISSUES

The peculiar secondary growth in Doxantha unguis-cati provides several developmental problems concerning cambial activity. One of the most interesting of these problems is the presence of both unidirectional and bidirectional arcs of cambium within the same stem. This investigation reports the ontogenetic development of these two kinds of cambial arcs. The first cambial divisions are observed in the fascicular regions of the 11th to 16th internodes from the shoot tip. This event is initiated after internode elongation is completed. In the initial stages, secondary tissues have a cylindrical configuration, but subsequently four grooves become apparent. These grooves signify the first evidence of unidirectional cambial activity. The four unidirectional arcs occur near the four major vascular strands to which all of the leaf traces connect. As secondary growth continues, the bidirectional and unidirectional arcs of cambium become separated and radial fissues can be seen between the furrows of phloem and the lobes of secondary xylem. Additional furrows originate either as sets of four between the original set of four or as single furrows to either or both sides of an existing furrow. All furrows are bordered by multiseriate rays. The initials of the bidirectional and unidirectional cambial arcs are non-stratified and are similar in size and appearance. The phloem produced within the furrow differs in several respects from that produced by the bidirectional arcs. The two types of cambial activity and the precise locations of the unidirectional cambial arcs in the stem (i.e. near the four major strands) suggests that transported products from the leaves are involved in the control of unidirectional cambial activity.     

STRUCTURE AND PHYSIOLOGY OF THE HAPTONEMA IN CHRYSOCHROMULINA (PRYMNESIOPHYCEAE). II. MECHANISMS OF HAPTONEMATAL COILING AND THE REGENERATION PROCESS1

The axoneme in the free part of the haptonema in Chrysochromulina acantha Leadbeater & Manton and C. simplex Estep et al. consists of seven single microtubules, except in the extreme distal swelling where, in C. simplex, there are only three microtubules. In the extended haptonema, the microtubules are arranged in a ring though they are not evenly spaced, the gap between two of the microtubules being larger than that between any other neighboring pairs. In the coiled haptonema, rearrangement of the microtubules occurs so that the ring becomes distorted and the microtubules form two superposed arcs. A sliding microtubule mechanism is considered as a means by which haptonematal movement might be affected, and this is discussed in relation to the fine structure of both embedded material and negatively stained demembranated cells. We show that haptonematal coiling is dependent on the presence of calcium ions and that an external concentration of between 10^?6 and 10^?7 M Ca²⁺ is the threshold below which the frequency of coiling on cell death is reduced. The results of experiments using ethylene bis-(oxyethylenenitrilo)-tetracetic acid (EGTA) and lanthanum ions to control extracellular and intracellular Ca²⁺ concentrations are discussed in terms of both external free calcium and intracellular pools. We also show that haptonematal regeneration following excision begins with a short lag phase. This is followed by a period of rapid growth, decreasing after approximately 4 h. Full haptonematal regrowth is not achieved until after 12–15 h. The rate of haptonematal regeneration is strongly affected when the flagella are regenerating simultaneously. These observations are interpreted in terms of competition for intracellular precursors.