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Ulva proliferais the dominant species of “green tide”, and has higher tolerance to environmental stresses such as temperature. However, the molecular mechanisms are still unclear. Here, transcriptome analysis, Western blot and RT-qPCR analysis ofU. proliferasuggested that, under temperature stresses (4°C, 36°C), the expression ofPCNAandCyclinAwas promoted, and the MAPK signaling was activated. Besides, the results showed that PCNA interacted with CyclinA. Interestingly, the expression ofmiR-2916, which was predicted to bind PCNA at -552∼-772, was negatively correlated with the expression ofPCNAunder temperature stresses (4°C, 36°C). In addition, the results showed that low temperature (4°C) had no obvious effect on the survival, the formation of cell walls, and the division of protoplasts. However, high temperature (36°C) had obvious effect on them. PCNA inhibitors increased the sensitivity of the protoplasts under temperature stresses. Together, our results suggested PCNA regulating the proliferation in response to the temperature stress ofU. proliferawas associated with miR-2916/PCNA/CyclinA/MAPK pathway. In conclusion, the study preliminarily illuminates the molecular mechanism in response to temperature stress ofU. prolifera, and may provide a new insight for prevention of green tide.

The genome of green microalgae has rarely been reported. Ulva prolifera is a green microalga that has received much attention. Despite research articles about U. prolifera in recent years, we know very little about its genome. Therefore, the 87.9-Mb haploid genome (containing 10 311 protein-coding genes) of U. prolifera was studied, and the genome was compared with that of U. mutabilis, which is the only published Ulva species. Results showed that the two species are closely related. A phylogenetic tree was constructed among U. prolifera and other green algae available in GenBank, revealing the evolutionary status of U. prolifera in Chlorophyta. To understand why U. prolifera could grow rapidly, we identified some genes related to growth, such as those involved in cell division, phosphorylation, and cell proliferation. In addition, genes related to stress resistance were found, which supports the notion that U. prolifera grows vigorously in nature. These results help to characterize green tides from a new perspective and reveal some important insight into the biology of U. prolifera.

Ulva prolifera is a green alga that plays an important role in green tides. Carotenoid biosynthesis is a basic terpenoid metabolism that is very important for maintaining normal life activities in algae. In this study, we first reported the complete sequences of all genes in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which is the only carotenoid synthesis pathway in U. prolifera. Then, we compared these genes with those of other species. Additionally, by detecting the carotenoid contents and expression levels of key genes participating in carotenoid biosynthesis in U. prolifera under three different light (1 000 lx, 5 000 lx and 12 000 lx) and salinity (12, 24 and 40) regimes, we found that carotenoid synthesis could be influenced by light and salinity, such that low light and high salinity could promote the synthesis of carotenoids. The results showed that the expression levels of genes involved in the MEP and the downstream pathway could affect the biosynthesis of carotenoids at the molecular level. This study contributes to a better understanding of the roles of genes participating in carotenoid biosynthesis in U. prolifera and the environmental regulation of these genes.

microRNAs (miRNA) families play a critical role in plant growth, development, and responses to abiotic stress. In this study, we characterized Up-miR-843 and its targets genes in Ulva prolifera responses to nitrogen depravation and heat stress. The data demonstrated that 184 target genes of Up-miR-843 could be successfully validated. N deficiency not heat stress stimulus induced increase in abundance of the Up-miR-843 while exhibited reverse expression of target genes, including cyclin A3 and cyclin L, which were strictly required for cell cycle progression. In addition, U. prolifera with highly expression of Up-miR-843 showed improved biomass, and photosynthesis compared with that under normal growth conditions. Thus, the N deprivation and heat responsive miRNAs might be a possible member mediating the expression of these target genes, which further regulated the growth of U. prolifera.

The influence of the social environment on healthy investment behavior is a vital research topic. This paper focuses on foreign direct investment (FDI) as an important part of its broad impact in improving the level of capital circulation and diversifying the non-systemic risk of a single country portfolio. Using data from 35 countries on direct investment in China, we find that the impact of the social environment on healthy investment behavior is mainly reflected in investors’ resistance to cultural distance and their benefit compensation across institutional distance. In addition, their joint influence is still negative, dominated by cultural distance, which can still verify that institutional distance mitigates the negative effect of cultural distance on FDI. Therefore, in order to promote international healthy investment behavior, it is feasible to improve both the mitigation effect of the institution in the short term and promote the level of cultural exchange in the long term, according to the research results of this paper.

From 2007 to 2017, large-scale green tides occurred every year in the Yellow Sea of China, and Ulva prolifera was the main species leading to the green tide. In this study, we used the Polymerase chain reaction and 3′ Rapid-amplification of cDNA ends technique to amplify the nrDNA-LSU and IGS sequences in U. prolifera and one species of Blidingia. These techniques showed 3259 bp of nrDNA-LSU and 3388 bp of IGS in U. prolifera and 3282 bp nrDNA-LSU and 3059 bp IGS in Blidingia sp. At the same time, tandem repeats, short dyads, palindromic and multiple simple repeat sequences in the IGS sequence were found by analyzing the structure of the IGS sequence in U. prolifera and Blidingia sp. G + C contents of the IGS sequence in U. prolifera and Blidingia sp. were 52.42% and 53.09%, respectively. We divided the U. prolifera into two types according to the morphological characteristics. Although the specimens of U. prolifera from the Qingdao coastal area, Jiangsu coastal area and the Yellow Sea have different morphologies, their ITS and IGS sequences are almost identical. Therefore, the main species of the green tides in the Qingdao coastal area, Jiangsu coastal area and the Yellow Sea are the same and have the same origin.

Green tide algal blooms occur worldwide, especially in China’s Yellow Sea, and have caused serious damage to local ecological environments and economies. As a dominant agent of green tides, the green macroalga Ulva has caused widespread concern. In this study, phylogenetic clades were constructed among related Ulva species isolated from Pyropia rafts at six sites over 1.5 years based on internal transcribed spacer (ITS) and tufA sequences. In addition, traditional observation ploidy methods and flow cytometry methods were used to analyse continuous change in the biphase and sex ratios of Ulva species and to assess the changes in phase advantages over time. The results showed that the perennial Ulva populations on rafts mainly consisted of Ulva flexuosa and Ulva prolifera, and the biphasic dominance of the attached Ulva populations changed with the seasons: sporophytes were predominant mainly in winter and spring, and gametophytes were predominant mainly in summer and autumn. At the same time, there were some differences in gametophyte and sporophyte frequencies (mainly sporophyte biased) compared to the null model prediction of a √2:1 ratio, while the sex ratio of male and female gametophytes remained 1:1 throughout the year. Our results indicate the presence of both phases of bloom-forming species of Ulva in green tides year round, and that multiple generations coexist and grow continuously.

Large-scale green tides caused by Ulva prolifera, occurred for 12 consecutive years in the Yellow Sea of China. To resolve the abrupt shift in species composition between attached and floating macroalgal assemblages, field experiments were conducted from May to July 2017 to quantify the net buoyancy force and compare the floating potential of the common green macroalgae from the red algal seaweed Pyropia yezoensis rafts. At the same time, U. prolifera from different sampling locations were tested to study variable buoyancy of this species and the associated influencing factors. Our results illustrated a stronger positive buoyant force and a proportionally greater buoyancy capacity of U. prolifera, compared to the other co-occurring species. Buoyancy is a dynamic trait and is closely correlated with light intensity, morphology and physiological status. The positive buoyancy of U. prolifera is an important factor that helps explain its predominance in the Yellow Sea's large-scale green tides.

Cytological observations and sequence analysis of the ribosomal intergenic spacer (IGS) were conducted on conchospores and thalli of Pyropia yezoensis from China. The complete progress of conchospo...

Four new purified polysaccharides (PAP) were isolated and purified from the Enteromorpha prolifera by alkali extraction, and further characterization was investigated. Their average molecular weights of PAP-1, PAP-2, PAP-3, and PAP-4 were estimated as 3.44 &times, 104, 6.42 &times, 104, 1.20 &times, 105, and 4.82 &times, 104 Da, respectively. The results from monosaccharide analysis indicated that PAP-1, PAP-2, PAP-3 were acidic polysaccharides and PAP-4 was a neutral polysaccharide. PAP-1 and PAP-2 mainly consist of galacturonic acid, while PAP-3 and PAP-4 mainly contained rhamnose. Congo red test showed that no triple helical structure was detected in the four polysaccharides. The antioxidant activities were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), Superoxide, and 2, 2&prime, azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical assay. In vitro antitumor activities were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PAP-1 exhibited relatively stronger antioxidant activities among the four polysaccharides in a dose-dependent manner. At a concentration of 1.00 mg/mL, the antioxidant activities of PAP-1 on the DPPH radical scavenging rate, superoxide anion radical scavenging rate, and ABTS radical rate at 1.00 mg/mL were 56.40%, 54.27%, and 42.07%, respectively. They also showed no significant inhibitory activity against MGC-803, HepG2, T24, and Bel-7402 cells. These investigations of polysaccharides provide a scientific basis for the use of E. prolifera as an ingredient in functional foods and medicines.

Volatile organic compounds in marine algae have been reported to comprise characteristic flavor of algae and play an important role in their growth, development and defensive response. Yet their biogeneration remain largely unknown. Here we studied the composition of volatile compouds in Pyropia yezoensis and their variations in response to methyl jasmonate (MeJA) and diethyldithiocarbamic acid (DIECA) treatment using gas chromatography-mass spectrometry (GC-MS). A total of 44 compounds belonging to the following chemical classes (n) were identified, including aldehydes (11), alcohols (8), acids and esters (6), alkanes (5), ketones (5), alkenes (3), and S- or N-containing miscellaneous compounds (6). External treatment with plant hormone MeJA increased the content of 1-dodecanol, 4-heptenal, and 2-propenoic acid-2-methyl dodecylester, but decreased the content of phytol, 3-heptadecene, 2-pentadecanone, and isophytol. When pretreated with DIECA, an inhibitor of the octadecanoid pathway leading to the biosynthesis of endogeneous jasmonates and some secondary metabolites, phytol and isophytol were increased, while 4-heptenal, 1-dodecanol, and 2-propenoic acid-2-methyl dodecylester were decreased, both of which were negatively correlated with their variations under MeJA treatment. Collectively, these results suggest that MeJA does affect the volatile composition of P. yezoensis, and the octadecanoid pathway together with endogenous jasmonate pathway may be involved in the biosynthesis of volatile compounds, thereby providing some preliminary envision on the composition and biogeneration of volatile compounds in P. yezoensis.

Cyanobacteria blooms possess serious threats to water resources. Herein, we report the synthesis of polyanionic membranes (PA-M) by in situ photo-crosslinking of a sulfate-based anionic monomer followed by cation-exchange with metal cations, Fe3+ (PA-Fe), Cu2+ (PA-Cu), or Zn2+ (PA-Zn). The effect of cations on the anti-cyanobacterial activities against both Microcystis aeruginosa (M. aeruginosa) and Anabaena flos-aquae (A. flos-aquae) was investigated. All the prepared metal-containing membranes (PA-Fe, PA-Cu, PA-Zn) exhibit high anti-cyanobacterial activities and long-term anti-cyanobacterial stability, demonstrating that the synthesized PA-M membranes can be used as an effective and safe inhibitor to control cyanobacterial blooms.

Background: Ulva prolifera is one of the main seaweeds (or macroalgae) species that causes “green tides”. This alga inhabits the estuarine areas that exhibit changes in nutrient contents, which include changes in nitrogen (N) levels, while the mechanisms through which these microalgae resist N deficiency remains unclear. Results: We amplified the full-length sequences and quantified expression of genes involved in the nitrogen metabolism process, the data indicated that nitrate reductase, nitrite reductase, and glutamine synthase increased after nitrogen deprivation in Ulva prolifera. Hence, although the ratio of cell-wall regeneration did not change, the apoptosis rates of protoplasts of Ulva prolifera increased after this deficiency. Furthermore, a decreased in N supplies triggered the activation of MAPK signaling, and SB239063, a p38 MAPKα/β inhibitor, enhanced the effects of N deficiency on the mortality of protoplasts and decreased the capacity for cell-wall regeneration. Conclusions: All the data provided evidence that MAPK signaling had functional roles in helping U. prolifera adapt to fluctuations in N availability within a short time. Hence, the application of biochemical reagents on cell-wall regeneration on the surface of protoplasts provided a new perspective in the genetic breeding of Ulva prolifera.

Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0–0.006, respectively) from each other, but high divergences (0.123–0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063–0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.

The ribosomal intergenic spacer (IGS) variation was compared for the nine main cultivated varieties of Pyropia yezoensis in China obtained from the Chinese National Pyropia Germplasm Repository. The full length of the IGS is 5984 bp including several sequence repeats and some complex structures. The sequence analysis of four partial IGS amplicons ranged from 3628 bp to 3776 bp; and the G+C content is 52.5%. Through similarity analysis and multiple sequence alignment of these partial IGS amplicons from the nine varieties, we have found 278 variable sites which account for approximately 7.5% of the whole IGS length. Both comparison of partial IGS sequences and phylogenetic analysis indicate high levels of genetic variation among different P. yezoensis cultivars in different areas of China. In comparison to the IGS of P. haitanensis, this study highlights the important function of classification of IGS in P. yezoensis. Therefore, the IGS sequence may be used to identify intraspecific variation in P. yezoensis.

The complete nuclear ribosomal DNA (nrDNA) cistron sequence of Pyropia yezoensis has been sequenced, of which each unit is composed of the intergenic spacer (IGS), the small-subunit (SSU) ribosomal RNA (rRNA) gene, the internal transcribed spacer 1 (ITS1), the 5.8S rRNA gene, the internal transcribed spacer 2 (ITS2), and the large-subunit (LSU) rRNA gene. This cistron occurs in tandem repeats that can number in 100s to 1000s. Furthermore, in the analysis of the sequence data, we determined that introns were present in the small-subunit rRNA genes, for which the exon sequences (1834 bp) are almost identical. The intron numbers ranging from zero to two in the small-subunit rRNA genes are different between multiple individuals. However, the full length of the large-subunit rRNA genes is 4770 bp without any introns, and the 5.8S rRNA gene is 159 bp. Moreover, the 5′ and 3′ terminal sequences of the genic regions of the ribosomal cistron are extremely conservative even among various species in red alga. In addition, the range for ITS1 is 371–372 bp and for ITS2 is 532 to 535 bp. The IGS is 5984 bp in length and possesses upstream direct repeats, downstream repeats, and middle inverted repeats that make sequencing untoward. This is the first time that the complete nucleotide of the IGS region of P. yezoensis has been sequenced. The provision of the full length of nrDNA cistron will offer useful information to be widely utilized for identification, taxonomic relationships, and phylogenies in red algae.

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.

The metabolic pathway of 8-heptadecene in red algae was investigated. The results showed that the amounts of 8-heptadecene in the primitive bangiophycidean red algae Pyropia and Bangia were 30–50 % of the volatile compounds, much higher than that in the green alga Ulva pertusa, the brown alga Sargassum thunbergii and the florideophycidean red alga Gracilaria lemaneiformis. Studies on the metabolism of 8-heptadecene in Pyropia found that its enzymatic system has no significant catalytic activity on palmitic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid. However, the isolated enzymatic solution showed activity in the presence of eicosapentaenoic acid. This activity produced about four times the amount of 8-heptadecene compared with other substrates and the control, indicating the solution had a specific catalytic function for eicosapentaenoic acid. Furthermore, the enzyme solution was strongly inhibited by NaN3 but not by phenidone and phenanthroline suggesting that the enzyme is structurally related to heme protein. Thus, it is believed that a constant amount of 8-heptadecene is maintained in the primitive red alga Pyropia and the 8-heptadecene is a metabolite of eicosapentaenoic acid and may catalyzed by enzymes including a heme lipoxygenase-like enzyme.

(Bangiales, Rhodophyta)((T.J. Chang

Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer (ITS) regions and the rbc L genes in seven organisms and obtained 536–566 bp long ITS sequences, 1 377–1 407 bp long rbc L sequences and 1 718–1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbc L genes. The sequencing results showed that the three Ulva prolifera (or U. pertusa) gene sequences from Qingdao and Nan’ao Island were identical. The ITS, 18S rDNA and rbc L genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U. flexuosa had the least evolutionary distance from U. californica in both the ITS regions (0.009) and the 18S rDNA (0.002). These data verified that Ulva and Enteromorpha are not separate genera.

Porphyra yezoensis has a macroscopic foliage gametophyte phase with only a single cell layer, and is ideally suited for the study of the sexual differentiation process, from the vegetative cell to the spermatia. Firstly, we compared variations in the responses of the vegetative and male sectors to desiccation. Later, cell tracking experiments were carried out during the formation of spermatia from vegetative cells. The two sectors showed similar tolerance to desiccation, and the formation of spermatia from vegetative cells was independent of the degree of desiccation. Both light and scanning electron microscopy (SEM) observations of the differentiation process showed that the formation of spermatia could be divided into six phases: the one-cell, two-cell, four-cell, eight-cell, pre-release and spermatia phases. Photomicrographs of Fluorescent Brightener staining showed that the released spermatia had no cell walls. Photosynthetic data showed that there was a significant rise in Y(II) in the four-cell phase, indicating an increase in photosynthetic efficiency of PSII during this phase. We propose that this photosynthetic rise may be substantial and provide the increased energy needed for the formation and release of spermatia in P. yezoensis.

Ulva sp. (Chlorophyta) is a representative species of the intertidal macro-algae responsible for the green tides that occurred along the shores of Qingdao in 2008 and had detrimental effects on the preparation for the 2008 Beijing Olympic Games sailing competition. In view of its significance, we have investigated the photosynthetic performance of the photosystems and the changes in photosynthetic electron transport that occur during desiccation and rehydration of Ulva sp. The PSII activity in Ulva sp. declined gradually during the course of desiccation, which was reflected by the decreased maximum quantum yield and effective quantum yield, whereas the PSI activity fluctuated significantly. In contrast, the electron transport rates of PSII approached zero at severe levels of desiccation, but the electron transport of PSI, which still operated, could be suppressed effectively by a specific inhibitor. Furthermore, the electron transport of PSI during rehydration of desiccated thalli was recovered faster than that of PSII. All these results implied that the linear electron flow was abolished in desiccated Ulva sp., whereas the cyclic PSI activity was significantly elevated, was still active at severe levels of desiccation and could be restored faster than PSII activity. Based on these results, we concluded the PSI-driven cyclic electron flow might provide desiccation tolerance and additional flexibility for the cell physiology of Ulva sp. under desiccation conditions, which might be one of the most important factors that make Ulva sp. well suited to experience daily cycles of desiccation at low tide and rehydration at high tide.

To establish a micropropagation system of three Laurencia complex species (Laurencia okamurai, Laurencia tristicha, and Chondrophycus undulatus) by tissue culture techniques, we studied the regeneration characteristics and optimal culture conditions of axenic algal fragments cultured on solid medium and in liquid medium. Regeneration structures were observed and counted regularly under a reverse microscope to investigate the regeneration process, polarity and optimal illumination, and temperature and salinity levels. The results show that in most cultures of the three species, we obtained bud regeneration on solidified medium with 0.5% agar and in liquid medium. Rhizoid-like regeneration was filamentous and developed from the lower cut surface of fragments in L. okamurai, but was discoid and developed from the apical back side of bud regeneration in L. tristicha and C. undulatus. Regeneration polarity was localized to the apical part of algal fronds in all three species, and on fragments cut from the basal part of algae buds could develop from both the upper and the lower cut surfaces. Buds could develop from both the medullary and the cortical portions in L. okamurai and C. undulatus, while in L. tristicha, buds only emerged from the cortex. The optimal culture conditions for L. okamurai were 4 500 lx, 20°C and 35 (salinity); for C. undulatus, 4 500 lx, 20°C and 30; and for L. tristicha, 4 500 lx, 25°C and 30.

The rbcL gene of Ulva pertusa, Enteromorpha prolifera and Monostroma grevillei was amplified, sequenced and analyzed. By comparing the rbcL sequences with seven other Ulvales species retrieved from GenBank, the sequence divergences and the phyletic evolution were analyzed and the phylogenetic tree was constructed. From the phylogenetic tree, it can be found that U. pertusa, E. prolifera and U. californica group in one branch, while E. compressa, U. rigida and U. fenestrata cluster in another clade. Obviously, unlike the Enteomorpha species, the Ulva species do not gather in one branch. Therefore Ulva and Enteomorpha might be affiliates of one genus. E. compressa and E. intestinalis gathered together, which coincided with the morphological characters. However, the thallus of U. pertusa is thick and with many holes, which is different from E. prolifera in morphology. They cluster together in the phylogenetic tree with a genetic distance of 0.005. The results indicate that Ulva and Enteromorpha are not distinguished strictly.

The sequences of the ITS (internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli (NB, PT and ST) were amplified, sequenced and analyzed. In addition, the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated. The results are as follows: the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains. The sequences of ITS1 were 331 bp to 334 bp, while those of the 5.8S rDNA were 158 bp and the sequences of ITS2 ranged from 673 bp to 681 bp. The sequences of the ITS had a high level of homology (up to 99.5%) with that of P. haitanensis (DQ662228) retrieved from GenBank, but were only approximately 50% homologous with those of other species of Porphyra. The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis. These results suggest that the three cultivated strains of P. haitanensis evolved conservatively and that the ITS showed evolutionary consistency. However, the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations. Therefore, the relationship of Porphyra interspecies phyletic evolution could be judged, which provides the proof for Porphyra identification study. However, proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.

l-Rhamnose (Rha) is an important constituent of pectic polysaccharides, a major component of the cell walls of Arabidopsis, which is synthesized by three enzymes encoded by AtRHM1, AtRHM2/AtMUM4, and AtRHM3. Despite the finding that RHM1 is involved in root hair formation in Arabidopsis, experimental evidence is still lacking for the in vivo enzymatic activity and subcellular compartmentation of AtRHM1 protein. AtRHM1 displays high similarity to the other members of RHM family in Arabidopsis and in other plant species such as rice and grape. Expression studies with AtRHM1 promoter-GUS fusion gene showed that AtRHM1 was expressed almost ubiquitously, with stronger expression in roots and cotyledons of young seedlings and inflorescences. GFP::AtRHM1 fusion protein was found to be localized in the cytosol of cotyledon cells and of petiole cells of cotyledon, indicating that AtRHM1 is a cytosol-localized protein. The overexpression of AtRHM1 gene in Arabidopsis resulted in an increase of rhamnose content as much as 40% in the leaf cell wall compared to the wild type as well as an alteration in the contents of galactose and glucose. Fourier-transform infrared analyses revealed that surplus rhamnose upon AtRHM1 overexpression contributes to the construction of rhamnogalacturonan.

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR (ISSR PCR). Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity (h*) and Shannon index of diversity (I*) in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones (0.190 3 and 0.274 8), which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intraspecies genetic analysis.

Enteromorpha prolifera (Muell.) J. Agardh (Chlorophyta, Ulvophyceae), which is distributed widely in the inter-tidal zone of the ocean, is one of the most common fouling green algae. However, the present understandings of the life history of E. prolifera have been insufficient to explain their seasonal abundances. Thus it is essential to investigate how many reproductive strategies are likely to contribute to the successful colonization and flourishing of the green alga. In the present study the reproduction diversity of E. prolifera was observed and studied systematically by culturing chopped tissues. Our results showed that there are in total seven pathways of reproduction for E. prolifera including sexual, asexual and vegetative reproduction. It was indicated that the variety of the reproductive ways and the large quantity of reproductive cells produced and released during the reproductive season are the two key factors that facilitate colonization of E. prolifera. The reproduction of the alga E. prolifera mainly depends on asexual methods. The results presented here contribute to increasing our understanding about how the opportunistic macroalgae successfully maintain colonization and excessive growth.

In order to investigate the possible effects of the ecological environment on photosynthetic activity and the major light harvesting complex, the oxygen evolution rates and composition of phycobilisome from marine red alga Porphyra yezoensis Ueda and freshwater red alga Compsopogon coeruleus (Balbis) Montagne, which could grow and reproduce under salinity up to 35 ppt, were studied. The results showed that the oxygen evolution rate of P. yezoensis in seawater was significantly higher than that of C. coeruleus in freshwater, and P. yezoensis tolerated inorganic ions at a relatively higher concentration than C. coeruleus. Moreover, the phycoerythrin (PE) of P yezoensis was R-phycoerythrin containing alpha, beta, and gamma subunits comprised phycoerythrobilin and phycourobilin. In contrast, the PE from C. coeruleus consisted of alpha, beta, and gamma subunits comprised only phycoerythrobilin but not phycourobilin, suggesting that the PE from C. coeruleus was of a new type.

Nuclear divisions of carpospores, conchocelis and conchospores of Porphyra yezoensis, P. haitanensis, P. katadai var. hemiphylla and P. oligospermatangia from China were investigated. The observations showed diploid chromosome numbers of 2n = 6 for P. yezoensis and P. oligospermatangia, and 2n = 10 for P. haitanensis and P. katadai var. hemiphylla. For all four species, somatic pairing of chromosome sets was observed in late prophase. Sister chromosomes separated at anaphase as mitosis took place in carpospores, conchocelis filamentous cells, conchosporangial branch cells and sporangial cells (conchospore formation). Chromosome configurations of tetrad and ring-shaped in conchospore germination were observed, demonstrating the occurrence of meiosis. The characteristics of diploid nuclear division in 2n = 6 species are the same as those of 2n = 10 species. The influence of somatic pairing on nuclear division of diploid cells in Porphyra was discussed.

Chloroplast formation is associated with embryo development and seedling growth. However, the relationship between chloroplast differentiation and embryo development remains unclear. Five FtsHi genes that encode proteins with high similarity to FtsH proteins, but lack Zn2+-binding motifs, are present in the Arabidopsis genome. In this study, we showed that T-DNA insertion mutations in the Arabidopsis FtsHi4 gene resulted in embryo arrest at the globular-to-heart-shaped transition stage. Transmission electron microscopic analyses revealed abnormal plastid differentiation with a severe defect in thylakoid formation in the mutant embryos. Immunocytological studies demonstrated that FtsHi4 localized in chloroplasts as a thylakoid membrane-associated protein, supporting its essential role in thylakoid membrane formation. We further showed that FtsHi4 forms protein complexes, and that there was a significant reduction in the accumulation of D2 and PsbO (two photosystem II proteins) in mutant ovules. The role of FtsHi4 in chloroplast development was confirmed using an RNA-interfering approach. Additionally, mutations in other FtsHi genes including FtsHi1, FtsHi2, and FtsHi5 caused phenotypic abnormalities similar to ftshi4 with respect to plastid differentiation during embryogenesis. Taken together, our data suggest that FtsHi4, together with FtsHi1, FtsHi2, and FtsHi5 are essential for chloroplast development in Arabidopsis.

The xanthophyll cycle (Xc), which involves violaxanthin de-epoxidase (VDE) and the zeaxanthin epoxidase (ZEP), is one of the most rapid and efficient responses of plant and algae to high irradiance. High light intensity can activate VDE to convert violaxanthin (Vx) to zeaxanthin (Zx) via antheraxanthin (Ax). However, it remains unclear whether VDE remains active under low light or dark conditions when there is no significant accumulation of Ax and Zx, and if so, how the ΔpH required for activation of VDE is built. In this study, we used salicylaldoxime (SA) to inhibit ZEP activity in the intertidal macro-algae Ulva sp. (Ulvales, Chlorophyta) and then characterized VDE under low light and dark conditions with various metabolic inhibitors. With inhibition of ZEP by SA, VDE remained active under low light and dark conditions, as indicated by large accumulations of Ax and Zx at the expense of Vx. When PSII-mediated linear electron transport systems were completely inhibited by SA and DCMU, alternative electron transport systems (i.e., cyclic electron transport and chlororespiration) could maintain VDE activity. Furthermore, accumulations of Ax and Zx decreased significantly when SA, DCMU, or DBMIB together with an inhibitor of chlororespiration (i.e., propyl gallate (PG)) were applied to Ulva sp. This result suggests that chlororespiration not only participates in the build-up of the necessary DpH, but that it also possibly influences VDE activity indirectly by diminishing the oxygen level in the chloroplast. © 2013 Xie et al.

The endosperm is an essential part of the seed which sustains embryo development and contains storage reserves. Endosperm development involves a series of nuclear divisions in the absence of cytokinesis. In this study, it is shown that a mutant Arabidopsis plant carrying the disrupted At4g24900 gene exhibits severe defects during seed development. At4g24900 encodes a nuclear-localized C2H2-containing protein and the transcripts of this gene are alternatively spliced, consequently producing at least nine differentially spliced isoforms. The heterozygous T-DNA insertion mutation in this gene resulted in abortion of 25% of seeds, and the homozygous mutant allele displayed embryo lethality. Differential interfernce contrast (DIC) analyses revealed that the mutant has endosperm nuclei with more than one nucleolus and an embryo arrested at the globular to heart stage transition. Because this mutant exhibits a titan-like phenotype, it was designated ttl. The TTL gene is preferentially expressed in tissues with quickly dividing cells as revealed in P(TTL)::GUS (β-glucuronidase) transgenic plants. It is proposed that TTL is likely to function as a key regulator of endosperm nuclear division.