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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.

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.

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.

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.

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.

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.

Porphyra yezoensisUeda is an intertidal marine red algae that has received increasing attention as a model organism owing to its important role in biological research and the agronomic industry. The two generations ofPorphyra yezoensis, the sporophyte and the gametophyte, have the same genome but show great differences in many aspects, including structural features, habitat, and gene expression. To identify miRNAs and their probable roles inP. yezoensisdevelopment, we constructed and sequenced libraries of small RNA fromP. yezoensissporophytes and gametophytes. The sequencing data were analyzed, and 14 miRNAs were identified, with only one common to these two samples. Our results show thatP. yezoensishas a complex small RNA processing system containing novel miRNAs that have no identifiable homolog in other organisms. These miRNAs might have important regulatory roles in development of the different generations ofP. yezoensis.

Ulva, as the main genera involved in green tides in the Yellow Sea, has attracted serious concern in China. Especially,Ulva proliferais one of the causative species of the occurring. This paper focused on the complete sequences analyses of ITS, 18S, and the combined data to determine phylogenetic relationships among taxa currently attributed toUlva,Monostroma, and some other green algal. The samples are all concluded in the area of Yellow Sea, China. The results showed the content of G+C in 18S was approximately concentrated upon 49% in average of 19 subjects while the ITS region content of base G and C is obviously higher than A and T. Comparing the ITS and 18S rDNA sequences obtained in this paper to other species retrieved from GenBank, the genetic distance and the ratio of sequence divergence reflect thatU. pertusaandU. proliferahad closer genetic relationship with an 18S rDNA, which had genetic distance of 0.007 while ITS had further genetic distance. According to further comparison,Ulva proliferahas closest genetic distance withChloropelta caespitosa(0.057) andUlva californica(0.057), which is a reverification coincidedChloropelta,Enteromorpha, andUlvaare not distinct genera.

In mid-May 2008 a serious green tide caused mainly by floating Ulva prolifera (Muller) J. Agardh (Chlorophyta, Ulvales) thalli struck the coastal area of Qingdao, China. To understand the present physiological conditions of the floating alga, in this work both laboratory and field investigations were conducted on the floating U. prolifera thalli in comparison with the attached U. prolifera thalli collected from the area. The floating thalli of three distinctively different colors and attached thalli at three different stages of sporangium formation process were characterized under a microscope, while their photosynthetic parameters were determined with chlorophyll fluorescence technology. On the other hand, the sporangium formation status of the floating U. prolifera thalli was surveyed both in the laboratory and in the field. Comparisons showed that both of the paired morphological characteristics and the paired physiological parameters of the floating and attached U. prolifera thalli were consistent. Furthermore, some spores were confirmed in the field and some motile particles were found within the floating thalli. These results suggest that the floating U. prolifera thalli with different colors could be at different stages of sporangium formation. However, our results also showed that the floating alga thalli have only a limited reproductive potential. This might limit the duration and the further geographic expansion of the green algal bloom.

On the basis of previous achievements in Porphyra seedling production using enzymatically isolated thallus cells, we have investigated the influences of water content of thallus before freezing, storing temperature and enzymes on the viability of isolated cells. We have also tried outdoor cultivation of seedlings produced enzymatically in different seasons. It has been found that survival rate of isolated cells of thallus stored for two months can reach 90% if water content is 30% before freezing and storing temperature is constant at − 20 °C. The 80% cell survival rate can be reached after two years of storage under this condition. The fluctuation of storing temperature causes drastic decrease of cell survival rate. 84% of the cells have survived digestion of sea snail enzyme I, which is the highest among five enzymes used. Outdoor cultivation of seedlings produced on the first ten days of January can reach lengths of more than 20 cm upon harvesting in the middle of May. In addition, seedlings produced in spring can be stored frozen and cultivated in autumn. The harvesting date will advance 15 days in comparison with the conventional seedling producing approach. This promises multiple rounds of cultivation of Porphyra within a year.