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The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SPn EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots.

Betaphycus gelatinus, Kappaphycus alvarezii and Eucheuma denticulatum of Family Solieriaceae, Order Gigartinales, Class Rhodophyceae are three important carrageenan-producing red algal species, which produce different types of carrageenans, beta (β)-carrageenan, kappa (κ)-carrageenan and iota (ι)-carrageenan. So far the carrageenan biosynthesis pathway is not fully understood and few information is about the Solieriaceae genome and transcriptome sequence. Here, we performed the de novo transcriptome sequencing, assembly, functional annotation and comparative analysis of these three commercial-valuable species using an Illumina short-sequencing platform Hiseq 2000 and bioinformatic software. Furthermore, we compared the different expression of some unigenes involved in some pathways relevant to carrageenan biosynthesis. We finally found 861 different expressed KEGG orthologs which contained a glycolysis/gluconeogenesis pathway (21 orthologs), carbon fixation in photosynthetic organisms (16 orthologs), galactose metabolism (5 orthologs), and fructose and mannose metabolism (9 orthologs) which are parts of the carbohydrate metabolism. We also found 8 different expressed KEGG orthologs for sulfur metabolism which might be importantly related to biosynthesis of different types of carrageenans. The results presented in this study provided valuable resources for functional genomics annotation and investigation of mechanisms underlying the biosynthesis of carrageenan in Family Solieriaceae.

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.

Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous species which are important resources for the production of special proteins, phycobilisomes, special carbohydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lemaneiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

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.

Ishige okamurae, with leathery branched narrow fronds consisting of cylindrical hairs, is the typical species of the genus Ishige, which is considered as one of the most basal genera in the phylogeny of the Phaeophyceae. Apart from great public interest from the evolutionary respect, more attention has been brought on the abundant bioactive compounds in I. okamurae for therapeutic or economic considerations, such as diphlorethohydroxycarmalol and ishigoside. Yet little is known about related key genes or metabolic pathways involved in I. okamurae, which calls upon us to carry out global analyses of transcriptome by next generation sequencing. Altogether, we obtained 78 583 assembled scaffolds with N50 of 1 709 nucleotides, and 25 357 unigenes with significant BLAST matches (E-value cutoff of 10−5). In terms of characterization of the transcriptome of I. okamurae, we focused on anti-stress metabolic pathways and synthetic routes of bioactive compounds in an attempt to obtain a better understanding of the interactive organism-environment regulatory networks. Pathway-based analysis helped us to deepen our comprehension of the interaction between I. okamurae and its surroundings, with MAPK signal pathway as an example. Furthermore, we discovered a wide range of novel putative functional proteins that could be of wide application, such as Rab family, using sequence-based transcriptome. In conclusion, transcriptome characterization of I. okamurae (Phaeophyceae) shows strong environmental acclimation.

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.

The chloroplast and mitochondrion of brown algae (Class Phaeophyceae of Phylum Ochrophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others (Phylum Cryptophyta and Haptophyta) in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassifolia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.

The coding region of both the ferrochelatase gene, hemH, from Bradyrhizobium japonicum, and the leghemoglobin gene, lba, from Glycine max, were transferred into chloroplast of Chlamydomonas reinhardtii. As a result, transgenic C. reinhardtii cultures more rapidly consumed O(2) and increased H(2) output compared with controls in both sulfur-free and sulfur-containing medium. H(2) production of the transgenic algal cultures in sulfur-free medium was 4-fold greater than that of control cultures, approximately 3.3mlbottle(-1). Maximum expression of the hemH-lba fusion protein on day 5 coincided with the lowest levels of O(2) content and the highest H(2) evolution rate detected over 7 days of anaerobic induction in sulfur-free medium. When the concentration of sulfate in the growth medium was restored to 12.5 or 50microM, O(2) consumption and H(2) yield decreased more slowly in the transgenic algal cultures than in the control cultures. These results demonstrate that expression of the hemH-lba fusion protein in chloroplast of C. reinhardtii improved their H(2) yield by decreasing O(2) content in the medium, thereby representing the potential for H(2) production in green algae to be improved.

Salidroside is the active ingredient in the roots of a Chinese medicine herb, Rhodiola sachalinensis A. Bor. Salidroside content in the Rh. sachalinensis differs greatly in different natural habitats. The effects of soil nutrient factors on the salidroside production in the root of Rh. sachalinensis were for the first time investigated in Chang Bai Mountain, PR China. The results showed that rich organic matter, low pH and high levels of exchangeable nitrogen and total nitrogen in soil were most essential to guarantee the high productivity of salidroside. The salidroside yield reached higher than 0.7% of root dry weight when the soil pH value was about 4.5, the soil organic matter content was higher than 17% or soil exchangeable nitrogen and total nitrogen contents were higher than 34 mg/100 mg and 0.6% of soil dry weight, respectively. However, soil bioavailable phosphorus content higher than 5 ppm and exchangeable potassium content higher than 180 ppm resulted in a low salidroside yield, only about 0.2% dry weight. This investigation will guide the soil management on how to select soil and how to fertilize the soil for the cultivation of Rh. sachalinensis to improve the salidroside productivity.

Salidroside has been identified as the most potent ingredient of the Chinese medicine herb, Rhodiola sachalinensis. Since the natural supply of this herb is rapidly decreasing, we established a compact callus aggregate (CCA) strain and culturing system for high yield salidroside production. Several callus strains induced from the explants originated from root, stem, leaf and cotyledon of R. sachalinensis were established and screened for rapid growth rate, high salidroside content and easy propagation in suspension culture condition. The CCA strain was established from a callus strain initiated from the cotyledon. The kinetics of dry weight accumulation and cellular salidroside content in various culture conditions for the strain was determined. For high salidroside production, the optimal inoculum amount was 10% and the optimal concentration for 6-benzylaminopurine and indole-3-butyric acid added in the liquid medium was 5 and 2.5 mg l-1, respectively. The acidic culture medium and a faster shaking speed favored the salidroside accumulation. The addition of 2,4-D, in the liquid MS medium and the utilization of L-tyrosol for chemical feeding enhanced salidroside production. Using a proper combination of culture condition and treatment, salidroside accumulation could reach 57.72 mg g-1 dry weight, that was 5-10-fold higher than that detected in field-grown plants. The corresponding salidroside yield was 555.13 mg l-1, a level suitable for cost effective commercial production to compensate the natural resource shortage of R. sachalinensis.

Co-cultivation of Bradyrhizobium japonicum with Chlamydomonas reinhardtii strain cc849 or the transgenic strain lba, which was hetero-expressed the gene of the soybean leghemoglobin apoprotein Lba in chloroplasts of the strain cc849, in Tris-acetate-phosphate (TAP) or TAP-sulfur free media, improved H(2) yield. H(2) production was 14 times and growth was 26% higher when strain lba and B. japonicum were co-cultured, as compared with cultivation of the algal strain alone under the same conditions. The increase in respiration rate or fast O(2) consumption by about 8 times in the co-cultures was the major reason for the improvement.

According to the codon bias in the chloroplast genome of Chlamydomonas reinhardtii, the codon-optimized coding regions of both the ferrochelatase gene, hemH, from Bradyrhizobium japonicum and the leghemoglobin gene, lba, from Glycine max were synthesized de novo and transferred into the chloroplast of C. reinhardtii. The expression level of hemH-lba protein was improved by 6.8 folds in the codon-optimized transgenic alga compared with the non-optimized one under both normal and anaerobic conditions. H(2) yield was 22% and the respiration rate was 44% higher in the codon-optimized transgenic algal cultures than those of the non-optimized ones, and was 450% and 134% higher than those of the control cultures, respectively. The transcript levels of hydA1 and hydA2 in the hemH-lba transgenic alga were also more stable and higher than those of the control alga. These results demonstrate that codon optimization increased the expression level of hemH-lba protein in the chloroplast of C. reinhardtii and improved algal H(2) yield by enhancing the respiration rate resulting in low O(2) content in the medium and up regulation of the expression of hydA1 and hydA2 in cells, thereby confirming the potential of the utilization of leghemoglobins for H(2) production in green algae.