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The red algae Kappaphycus alvarezii is the most important aquaculture species in Kappaphycus, widely distributed in tropical waters, and it has become the main crop of carrageenan production at present. The mechanisms of adaptation for high temperature, high salinity environments and carbohydrate metabolism may provide an important inspiration for marine algae study. Scientific background knowledge such as genomic data will be also essential to improve disease resistance and production traits of K. alvarezii. 43.28 Gb short paired-end reads and 18.52 Gb single-molecule long reads of K. alvarezii were generated by Illumina HiSeq platform and Pacbio RSII platform respectively. The de novo genome assembly was performed using Falcon_unzip and Canu software, and then improved with Pilon. The final assembled genome (336 Mb) consists of 888 scaffolds with a contig N50 of 849 Kb. Further annotation analyses predicted 21,422 protein-coding genes, with 61.28% functionally annotated. Here we report the draft genome and annotations of K. alvarezii, which are valuable resources for future genomic and genetic studies in Kappaphycus and other algae.

To improve H2 production, the green algae Chlamydomonas reinhardtii cc849 was co-cultured with Azotobacter chroococcum. The maximum H2 production of the co-culture was 350% greater than that of the pure algal cultures under optimal H2 production conditions. The maximum growth and the respiratory rate of the co-cultures were about 320 and 300% of the controls, and the dissolved O2 of co-cultures was decreased 74%. Furthermore, the in vitro maximum hydrogenase activity of the co-culture was 250% greater than that of the control, and the in vivo maximum hydrogenase activity of the co-culture was 1.4-fold greater than that of the control. In addition, the maximum starch content of co-culture was 1400% that of the control. Azotobacter chroococcum improved the H2 production of the co-cultures by decreasing the O2 content and increasing the growth and starch content of the algae and the hydrogenase activity of the co-cultures relative to those of pure algal cultures.

Three algae strains—Chlamydomonas reinhardtii hemHc-lbac (transgenic), cc124 and cc503—were co-cultured with Bradyrhizobium japonicum to improve H2 production. The maximum H2 productions of the three different co-cultures were 3.5-fold, 17-fold and 4.4-fold of the pure algal cultures. Meanwhile, the biomass of each of the algae strains was increased by B. japonicum. Furthermore, the respiratory rates of the co-cultures were 1.4-fold, 1.2-fold and 2.6-fold higher than the controls, respectively; the in vitro maximum hydrogenase activities of the co-cultures were 1.1-fold higher, equal to 2.4-fold higher than those of the controls, respectively, and the in vivo maximum hydrogenase activities of the co-cultures were 1.5-fold, 3.8-fold and 2.1-fold higher than the controls, respectively. The maximum starch content of the co-cultures were 8.3-fold, 8.4-fold and 4.4-fold higher than the content of the controls, respectively. Therefore, B. japonicum improved the H2 production of the co-cultures by increasing the biomass, respiratory rates, hydrogen activity and starch content of the co-cultures relative to those of pure algae cultures.

The transgenic alga lba of Chlamydomonas reinhardtii yielded H2 with 50%–180% higher than the control strain. Further experiments showed that photosynthetic rates and photosynthetic reaction center II's photochemical capacities of the transgenic algae obviously decreased 33.4%–85.9% and 30.0%–51.7%, respectively, compared with those of the control. On the contrary, respiration rates of the transgenic algae significantly increased, with 40.0%–200.0% higher than those of the control. Furthermore, starch contents of the transgenic algae were also improved significantly by 79.1%–592.8% compared with the control. Therefore, the reason of H2 yield improvement of the transgenic alga lba is not only due to its decrease of photosynthetic capacity and increase of the respiration rate, but also due to the metabolic changes related to starch metabolism, photosynthesis and respiration which is possibly caused by hetero-expression of lba gene in chloroplasts of C. reinhardtii, indicating the potential of utilization of lba gene to improve hydrogen yield of micro-green algae.

Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In prokaryotes, tryptophan synthase is a multi-enzyme complex, and it consists of α and β subunit which forms an α-ββ-α complex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophyceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rhodophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect with α subunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.

Most phaeophytes (brown algae) and rhodophytes (red algae) dwell exclusively in marine habitats and play important roles in marine ecology and biodiversity. Many of these brown and red algae are also important resources for industries such as food, medicine and materials due to their unique metabolisms and metabolites. However, many fundamental questions surrounding their origins, early diversification, taxonomy, and special metabolisms remain unsolved because of poor molecular bases in brown and red algal study. As part of the 1 000 Plant Project, the marine macroalgal transcriptomes of 19 Phaeophyceae species and 21 Rhodophyta species from China’s coast were sequenced, covering a total of 2 phyla, 3 classes, 11 orders, and 19 families. An average of 2 Gb per sample and a total 87.3 Gb of RNA-seq raw data were generated. Approximately 15 000 to 25 000 unigenes for each brown algal sample and 5 000 to 10 000 unigenes for each red algal sample were annotated and analyzed. The annotation results showed obvious differences in gene expression and genome characteristics between red algae and brown algae; these differences could even be seen between multicellular and unicellular red algae. The results elucidate some fundamental questions about the phylogenetic taxonomy within phaeophytes and rhodophytes, and also reveal many novel metabolic pathways. These pathways include algal CO2 fixation and particular carbohydrate metabolisms, and related gene/gene family characteristics and evolution in brown and red algae. These findings build on known algal genetic information and significantly improve our understanding of algal biology, biodiversity, evolution, and potential utilization of these marine algae.

Saccharina is one of the most important cold-water living marine brown algal genera. In this study we analyzed the transcriptome of S. japonica, which belongs to the 1 000 Plants (OneKP) Project, by using a next-generation high-throughput DNA sequencing technique. About 5.16 GB of raw data were generated, and 65 536 scaffolds with an average length of 454 bp were assembled with SOAP de novo assembly method. In total, 19 040 unigenes were identified by BLAST; 25 734 scaffolds were clustered into 37 Gene ontology functional groups; 6 760 scaffolds were classified into 25 COG categories, as well as 2 665 scaffolds that were assigned to 306 KEGG pathways. Majority of the unigenes exhibited more similarities to algae including brown algae and diatom than other cyanobacteria, marine diatom, and plant. Saccharina japonica has the outstanding capability to accumulate halogen such as Br and I via halogenation processes from seawater. We acquired 42 different vanadium-dependent haloperoxidases (vHPO) in S. japonica transcriptome data, including 5 segments of vanadium-dependent iodoperoxidase (vIPO) and 37 segments of vanadium-dependent bromoperoxidase (vBPO). Complicated analyses of identified fulllength S. japonica vBPO1 and S. japonica vBPO2 revealed the importance of vBPO among species of brown algae and the strong relationship between marine algal vBPOs and vIPOs. This study will enhance our understanding of the biological characteristics and economic values of S. japonica species.

Brown algae (Chromista, Ochrophyta, Phaeophyceae) are a large group of multicellular algae that play important roles in the ocean’s ecosystem and biodiversity. However, poor molecular bases for studying their phylogenetic evolutions and novel metabolic characteristics have hampered progress in the field. In this study, we sequenced the de novo transcriptome of 18 major species of brown algae in China, covering six orders and seven families, using the high-throughput sequencing platform Illumina HiSeq 2000. From the transcriptome data of these 18 species and publicly available genome data of Ectocarpus siliculosus and Phaeodactylum tricornutum, we identified 108 nuclear-generated orthologous genes and clarified the phylogenetic relationships among these brown algae based on a multigene method. These brown algae could be separated into two clades: Clade Ishigeales-Dictyotales and Clade Ectocarpales-Laminariales-Desmarestiale-Fucales. The former was at the base of the phylogenetic tree, indicating its early divergence, while the latter was divided into two branches, with Order Fucales diverging from Orders Ectocarpales, Laminariales, and Desmarestiale. In our analysis of taxonomy-contentious species, Sargassum fusiforme and Saccharina sculpera were found to be closely related to genera Sargassum and Saccharina, respectively, while Petalonia fascia showed possible relation to genus Scytosiphon. The study provided molecular evidence for the phylogenetic taxonomy of brown algae.

Species of Sargassum are distributed worldwide, and are of great ecological and economic importance in marine ecosystems and bioresources. In this study, transcriptome sequencings of six Sargassum species were performed for the first time using an Illumina platform. For each sample, a total of 2.1–2.5 Gb of nucleotides are collected and assembled into 69 871–116 790 scaffolds, with an average length of 410–550 bp and N50 length of 756–1 462 bp. A total of 20 512–28 684 unigenes of each sample were annotated and compared well with known gene sequences from nr database. Clusters of Orthologous Groups (COG), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also performed for further understanding of gene functions and regulation pathways. Gene expression levels were calculated based on RPKM values and compared among these species, especially for those genes related to carbohydrate metabolism. Cluster analyses indicated that the differences of global gene expression between S. fusiforme, which was nominated as Hizikia fusiformis before, and other five species were not significant. Further phylogenetic analysis of 108 orthologous genes confirmed that S. fusiforme had closer relationship with S. hemiphyllum rather than S. horneri. These transcriptome data provided valuable information for better understanding of genome and gene characteristics of Sargassum algae and benefiting comparative and phylogenetic studies of Phaeophyceae species in future studies.

The chloroplast and mitochondrion of red algae (Phylum Rhodophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using red algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Rhodophyta. We have found that red algae were divided into three clades of orders, Florideophyceae, Bangiophyceae and Cyanidiophyceae. Taxonomy resolution for Class Florideophyceae showed that Order Gigartinales was close to Order Halymeniales, while Order Gracilariales was in a clade of Order Ceramials. We confirmed Prionitis divaricata (Family Halymeniaceae) was closely related to the clade of Order Gracilariales, rather than to genus Grateloupia of Order Halymeniales as reported before. Furthermore, we found both mitochondrial and chloroplastic genes in Rhodophyta under negative selection (Ka/Ks < 1), suggesting that red algae, as one primitive group of eukaryotic algae, might share joint evolutionary history with these two organelles for a long time, although we identified some differences in their phylogenetic trees. Our analysis provided the basic phylogenetic relationships of red algae, and demonstrated their potential ability to study endosymbiotic events.

An ORF cDNA fragment of one of leghemoglobin genes, lba was cloned from Glycine max and transferred into chloroplasts of Chlamydomonas reinhardtii. More rapidly O2 consumption, lower O2 content and higher H2 output were monitored in the transgenic algal cultures than those in WT cultures either in S-free or S-containing medium. Maximum expression of lba in the transgenic algae consisted with the time when minimal O2 contents and maximal H2 evolution occurred. The highest H2 production achieved in sulfur-free medium for both algal cultures. When restoring sulfate in the medium, H2 production in the transgenic algal cultures kept steadily around 130–145 μl per bottle while that in WT cultures decreased gradually from 98 μl per bottle at 12.5 μM sulfate to 40 μl per bottle at 100 μM sulfate. The results indicated that heteroexpression of lehemoglaobin genes in chloroplasts of green algae improved H2 yield by decreasing O2 content in the medium. This protein had potential to be used in improvement of H2 production in green algae.

Both Calvin-Benson-Bassham (C3) and Hatch-Slack (C4) cycles are most important autotrophic CO2 fixation pathways on today’s Earth. C3 cycle is believed to be originated from cyanobacterial endosymbiosis. However, studies on evolution of different biochemical variants of C4 photosynthesis are limited to tracheophytes and origins of C4-cycle genes are not clear till now. Our comprehensive analyses on bioinformatics and phylogenetics of novel transcriptomic sequencing data of 21 rhodophytes and 19 Phaeophyceae marine species and public genomic data of more algae, tracheophytes, cyanobacteria, proteobacteria and archaea revealed the origin and evolution of C4 cycle-related genes. Almost all of C4-related genes were annotated in extensive algal lineages with proteobacterial or archaeal origins, except for phosphoenolpyruvate carboxykinase (PCK) and aspartate aminotransferase (AST) with both cyanobacterial and archaeal/proteobacterial origin. Notably, cyanobacteria may not possess complete C4 pathway because of the flawed annotation of pyruvate orthophosphate dikinase (PPDK) genes in public data. Most C4 cycle-related genes endured duplication and gave rise to functional differentiation and adaptation in different algal lineages. C4-related genes of NAD-ME (NAD-malic enzyme) and PCK subtypes exist in most algae and may be primitive ones, while NADP-ME (NADP-malic enzyme) subtype genes might evolve from NAD-ME subtype by gene duplication in chlorophytes and tracheophytes.