Exon shuffling potentiates a diverse repertoire of brown algal NB-ARC-TPR candidate immune receptor proteins via alternative splicing.

Like other organisms, brown algae are subject to diseases caused by bacteria, fungi, and viruses. Brown algal immunity mechanisms are not well characterized; however, there is evidence suggesting that pathogen receptors exist in brown algae. One key protein family likely associated with brown algal innate immunity possesses an NB-ARC domain analogous to innate immune proteins in plants and animals. In this study, we conducted an extensive survey of NB-ARC genes in brown algae and obtained insights into the domain organization and evolutionary history of the encoded proteins. Our data show that brown algae possess an ancient NB-ARC-tetratricopeptide repeat (NB-TPR) domain architecture. We identified an N-terminal effector domain, the four-helix bundle, which was not previously found associated with NB-ARC domains. The phylogenetic tree including NB-ARC domains from all kingdoms of life suggests the three clades of brown algal NB-TPRs are likely monophyletic, whereas their TPRs seem to have distinct origins. One group of TPRs exhibit intense exon shuffling, with various alternative splicing and diversifying selection acting on them, suggesting exon shuffling is an important mechanism for evolving ligand-binding specificities. The reconciliation of gene duplication and loss events of the NB-ARC genes reveals that more independent gene gains than losses have occurred during brown algal evolution, and that tandem duplication has played a major role in the expansion of NB-ARC genes. Our results substantially enhance our understanding of the evolutionary history and exon shuffling mechanisms of the candidate innate immune repertoire of brown algae.

Integrative analysis of chloroplast DNA methylation in a marine alga-Saccharina japonica.

KEY MESSAGE: We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, methylated DNA immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

The oxylipin messenger 1-octen-3-ol induced rapid responses in kelp Macrocystis pyrifera.

Oxylipins are important oxygenated derivatives of fatty acids that regulate a variety of plant physiological and pathological processes in response to specific external challenges. A large body of evidence has indicated that algae can also produce a surprisingly diverse array of volatile oxylipins, yet little is known about the roles of volatile oxylipins as defense signals in macroalgae. In this study, the kelp Macrocystis pyrifera was treated by the oxylipin messenger 1-octen-3-ol and then a genome-wide gene expression profile and fatty acid spectrum analysis were performed. We found that M. pyrifera responded rapidly to the exposure of the oxylipin messenger 1-octen-3-ol. It regulated the expression levels of genes mainly involved in signal transduction, lipid metabolism, oxidation prevention, cell wall synthesis, photosynthesis, and development. Moreover, 1-octen-3-ol treatments decreased several types of total fatty acid contents and increased free fatty acid contents, especially for the C18 and C20 fatty acids. In addition, it decreased the content of indole-3-acetic acid, abscisic acid, and zeatin and increased the gibberellic acid content. Our findings demonstrated that 1-octen-3-ol is an available inducer for M. pyrifera, which is capable of rapidly upregulating kelp's defense response.

Exploring Core Response Mechanisms to Multiple Environmental Stressors Via A Genome-Wide Study in the Brown Alga Saccharina japonica (Laminariales, Phaeophyceae).

Saccharina japonica is an important large brown alga and a major component of productive beds on the northwest coast of the Pacific Ocean. Abiotic stress response mechanisms are receiving considerable attention because global climate change is increasing their abiotic stress levels. However, our knowledge of how S. japonica broadly responds to stress is limited. In this study, we investigated the S. japonica responsive genes underlying acclimation to diverse stressors of acidification, high light, high temperature, hypersalinity, and hyposalinity and identified 408 core genes constantly and differentially expressed in response to all stressors. Our results confirm that stressors had strong effects on genes participating in photosynthesis, amino acid metabolism, carbohydrate metabolism, halogen metabolism, and reactive oxygen species defense. These findings will improve our understanding of brown algal response mechanisms linked to environmental stress and provide a list of candidate genes for improving algal stress tolerance in light of environmental stress in future studies.

Elevated CO2 affects kelp nutrient quality: A case study of Saccharina japonica from CO2 -enriched coastal mesocosm systems.

Kelps provide critical services for coastal food chains and ecosystem, and they are important food source for some segments of human population. Despite their ecological importance, little is known about long-term impacts of elevated CO2 (eCO2 ) on nutrient metabolites in kelps and the underlying regulation mechanisms. In this study, the kelp Saccharina japonica was cultured in CO2 -enriched coastal mesocosm systems for up to 3 months. We found that, although eCO2 significantly increased the growth rate, carbon concentrations, and C/N ratio of S. japonica, and it had no effect on total nitrogen and protein contents at the end of cultivation period. Meanwhile, it decreased the lipid, magnesium, sodium, and calcium content and changed the amino acid and fatty acid composition. Combining the genome-wide transcriptomic and metabolic evidence, we obtained a system-level understanding of metabolic response of S. japonica to eCO2 . The unique ornithine-urea cycle (OUC) and aspartate-argininosuccinate shunt (AAS), coupled with TCA cycle, balanced the carbon and nitrogen metabolism under eCO2 by providing carbon skeleton for amino acid synthesis and reduced power for nitrogen assimilation. This research provides a major advance in the understanding of kelp nutrient metabolic mechanism in the context of global climate change, and such CO2 -induced shifts in nutritional value may induce changes in the structure and stability of marine trophic webs and affect the quality of human nutrition resources.

Single-base methylome profiling of the giant kelp Saccharina japonica reveals significant differences in DNA methylation to microalgae and plants.

Brown algae have convergently evolved plant-like body plans and reproductive cycles, which in plants are controlled by differential DNA methylation. This contribution provides the first single-base methylome profiles of haploid gametophytes and diploid sporophytes of a multicellular alga. Although only c. 1.4% of cytosines in Saccharina japonica were methylated mainly at CHH sites and characterized by 5-methylcytosine (5mC), there were significant differences between life-cycle stages. DNA methyltransferase 2 (DNMT2), known to efficiently catalyze tRNA methylation, is assumed to methylate the genome of S. japonica in the structural context of tRNAs as the genome does not encode any other DNA methyltransferases. Circular and long noncoding RNA genes were the most strongly methylated regulatory elements in S. japonica. Differential expression of genes was negatively correlated with DNA methylation with the highest methylation levels measured in both haploid gametophytes. Hypomethylated and highly expressed genes in diploid sporophytes included genes involved in morphogenesis and halogen metabolism. The data herein provide evidence that cytosine methylation, although occurring at a low level, is significantly contributing to the formation of different life-cycle stages, tissue differentiation and metabolism in brown algae.

Diversity and evolution of cytochromes P450 in stramenopiles.

MAIN CONCLUSION: Comparative genomic analysis of cytochromes P450 revealed high diversification and dynamic changes in stramenopiles, associated with transcriptional responsiveness to various environmental stimuli. Comparative genomic and molecular evolution approaches were used to characterize cytochromes P450 (P450) diversity in stramenopiles. Phylogenetic analysis pointed to a high diversity of P450 in stramenopiles and identified three major clans. The CYP51 and CYP97 clans were present in brown algae, diatoms and Nannochloropsis gaditana, whereas the CYP5014 clan mainly includes oomycetes. Gene gain and loss patterns revealed that six CYP families-CYP51, CYP97, CYP5160, CYP5021, CYP5022, and CYP5165-predated the split of brown algae and diatoms. After they diverged, diatoms gained more CYP families, especially in the cold-adapted species Fragilariopsis cylindrus, in which eight new CYP families were found. Selection analysis revealed that the expanded CYP51 family in the brown alga Cladosiphon okamuranus exhibited a more relaxed selection constraint compared with those of other brown algae and diatoms. Our RNA-seq data further evidenced that most of P450s in Saccharina japonica are highly expressed in large sporophytes, which could potentially promote the large kelp formation in this developmental stage. A survey of Ectocarpus siliculosus and diatom transcriptomes showed that many P450s are responsive to stress, nutrient limitation or light quality, suggesting pivotal roles in detoxification or metabolic processes under adverse environmental conditions. The information provided in this study will be helpful in designing functional experiments and interpreting P450 roles in this particular lineage.

Ocean acidification increases iodine accumulation in kelp-based coastal food webs.

Kelp are main iodine accumulators in the ocean, and their growth and photosynthesis are likely to benefit from elevated seawater CO2 levels due to ocean acidification. However, there are currently no data on the effects of ocean acidification on iodine metabolism in kelp. As key primary producers in coastal ecosystems worldwide, any change in their iodine metabolism caused by climate change will potentially have important consequences for global geochemical cycles of iodine, including iodine levels of coastal food webs that underpin the nutrition of billions of humans around the world. Here, we found that elevated pCO2 enhanced growth and increased iodine accumulation not only in the model kelp Saccharina japonica using both short-term laboratory experiment and long-term in situ mesocosms, but also in several other edible and ecologically significant seaweeds using long-term in situ mesocosms. Transcriptomic and proteomic analysis of S. japonica revealed that most vanadium-dependent haloperoxidase genes involved in iodine efflux during oxidative stress are down-regulated under increasing pCO2 , suggesting that ocean acidification alleviates oxidative stress in kelp, which might contribute to their enhanced growth. When consumed by abalone (Haliotis discus), elevated iodine concentrations in S. japonica caused increased iodine accumulation in abalone, accompanied by reduced synthesis of thyroid hormones. Thus, our results suggest that kelp will benefit from ocean acidification by a reduction in environmental stress however; iodine levels, in kelp-based coastal food webs will increase, with potential impacts on biogeochemical cycles of iodine in coastal ecosystems.

Acclimation of bloom-forming and perennial seaweeds to elevated pCO2 conserved across levels of environmental complexity.

Macroalgae contribute approximately 15% of the primary productivity in coastal marine ecosystems, fix up to 27.4 Tg of carbon per year, and provide important structural components for life in coastal waters. Despite this ecological and commercial importance, direct measurements and comparisons of the short-term responses to elevated pCO2 in seaweeds with different life-history strategies are scarce. Here, we cultured several seaweed species (bloom forming/nonbloom forming/perennial/annual) in the laboratory, in tanks in an indoor mesocosm facility, and in coastal mesocosms under pCO2 levels ranging from 400 to 2,000 µatm. We find that, across all scales of the experimental setup, ephemeral species of the genus Ulva increase their photosynthesis and growth rates in response to elevated pCO2 the most, whereas longer-lived perennial species show a smaller increase or a decrease. These differences in short-term growth and photosynthesis rates are likely to give bloom-forming green seaweeds a competitive advantage in mixed communities, and our results thus suggest that coastal seaweed assemblages in eutrophic waters may undergo an initial shift toward communities dominated by bloom-forming, short-lived seaweeds.

Long-term experiment on physiological responses to synergetic effects of ocean acidification and photoperiod in the Antarctic sea ice algae Chlamydomonas sp. ICE-L.

Studies on ocean acidification have mostly been based on short-term experiments of low latitude with few investigations of the long-term influence on sea ice communities. Here, the combined effects of ocean acidification and photoperiod on the physiological response of the Antarctic sea ice microalgae Chlamydomonas sp. ICE-L were examined. There was a general increase in growth, PSII photosynthetic parameters, and N and P uptake in continuous light, compared to those exposed to regular dark and light cycles. Elevated pCO2 showed no consistent effect on growth rate (p=0.8) and N uptake (p=0.38) during exponential phrase, depending on the photoperiod but had a positive effect on PSII photosynthetic capacity and P uptake. Continuous dark reduced growth, photosynthesis, and nutrient uptake. Moreover, intracellular lipid, mainly in the form of PUFA, was consumed at 80% and 63% in low and high pCO2 in darkness. However, long-term culture under high pCO2 gave a more significant inhibition of growth and Fv/Fm to high light stress. In summary, ocean acidification may have significant effects on Chlamydomonas sp. ICE-L survival in polar winter. The current study contributes to an understanding of how a sea ice algae-based community may respond to global climate change at high latitudes.

Effects of temperature and nitrogen sources on physiological performance of the coccolithophore Emiliania huxleyi.

Both temperature and nutrient levels are rising in worldwide ocean ecosystems, and they strongly influence biological responses of phytoplankton. However, few studies have addressed the interactive effects of temperature and nitrogen sources on physiological performance of the coccolithophore Emiliania huxleyi. In this study, we evaluated algal growth, photosynthesis and respiration, elemental composition, enzyme activity, and calcification under a matrix of two temperatures gradients (ambient temperature 20 °C and high temperature 24 °C) and two nitrogen sources (nitrate (NO3-) and ammonium (NH4+)). When the algae was cultured with NO3- medium, high temperature reduced algal photosynthesis and nitrate reductase activity, but it did not change other indicators significantly relative to ambient temperature. In addition, E. huxleyi preferred NO3- as the growth medium, whereas NH4+ had negative effects on physiological parameters. In the NH4+ medium, the growth rate, photosynthesis and photosynthetic rate, nitrate reductase activity, and particulate organic carbon and particulate organic nitrogen production rate of the algae decreased as temperature increased. Conversely, high temperature increased cellular particulate organic carbon, cellular particulate organic nitrogen, and particulate inorganic carbon levels. In summary, our findings indicate that the distribution and abundance of microalgae could be greatly affected under warming ocean temperature and different nutrient conditions.

Analysis of ΔpH and the xanthophyll cycle in NPQ of the Antarctic sea ice alga Chlamydomonas sp. ICE-L.

Non-photochemical fluorescence quenching (NPQ) is mainly associated with the transthylakoid proton gradient (ΔpH) and xanthophyll cycle. However, the exact mechanism of NPQ is different in different oxygenic photosynthetic organisms. In this study, several inhibitors were used to study NPQ kinetics in the sea ice alga Chlamydomonas sp. ICE-L and to determine the functions of ΔpH and the xanthophyll cycle in the NPQ process. NH4Cl and nigericin, uncouplers of ΔpH, inhibited NPQ completely and zeaxanthin (Z) was not detected in 1 mM NH4Cl-treated samples. Moreover, Z and NPQ were increased in the samples containing N,N'-dicyclohexyl-carbodiimide (DCCD) under low light conditions. We conclude that ΔpH plays a major role in NPQ, and activation of the xanthophyll cycle is related to ΔpH. In dithiothreitol (DTT)-treated samples, no Z was observed and NPQ decreased. NPQ was completely inhibited when NH4Cl was added suggesting that part of the NPQ process is related to the xanthophyll cycle and the remainder depends on ΔpH. Moreover, lutein and ß-carotene were also essential for NPQ. These results indicate that NPQ in the sea ice alga Chlamydomonas sp. ICE-L is mainly dependent on ΔpH which affects the protonation of PSII proteins and de-epoxidation of the xanthophyll cycle, and the transthylakoid proton gradient alone can induce NPQ.

Analysis of mRNA expression profiles of carotenogenesis and astaxanthin production of Haematococcus pluvialis under exogenous 2, 4-epibrassinolide (EBR).

The fresh-water green unicellular alga Haematococcus pluvialis is known to accumulate astaxanthin under stress conditions. In the present study, transcriptional expression of eight genes involved in astaxanthin biosynthesis exposed to EBR (25 and 50 mg/L) was analyzed using qRT-PCR. The results demonstrated that both 25 and 50 mg/L EBR could increase astaxanthin productivity and the eight carotenogenic genes were up-regulated by EBR with different expression profiles. Moreover, EBR25 induction had a greater influence on the transcriptional expression of ipi-1, ipi-2, crtR-B, lyc and crtO (> 5- fold up-regulation) than on psy, pds, bkt; EBR50 treatment had a greater effect on the transcriptional expression of ipi-2, pds, lyc, crtR-B, bkt and crtO than on ipi-1 and psy. Furthermore, astaxanthin biosynthesis under EBR was up-regulated mainly by ipi1Ö¾ and psy at the post-transcriptional level, pds, lyc, crtR-B, bkt and crtO at the transcriptional level and ipi-2 at both levels.

Carotenoid genes transcriptional regulation for astaxanthin accumulation in fresh water unicellular alga Haematococcus pluvialis by gibberellin A3 (GA3).

The fresh water unicellular alga Haematococcus pluvialis is a promising natural source of astaxanthin. The present study investigated the transcriptional expression of carotenoid genes for astaxanthin accumulation in H. pluvialis using real-time fluorescence quantitative PCR (qRT-PCR). With treatments of 20 and 40 mg/L of gibberllin A3 (GA3), five genes ipi-1, ipi-2, psy, pds and bkt2 were up-regulated with different expression profiles. GA20 (20 mg/L of GA3) treatment had a greater effect on transcriptional expression of bkt2 than on ipi-1 ipi-2, psy and pds (> 4-fold up-regulation). However, GA40 (40 mg/L of GA3) induced more transcriptional expression of ipi-2, psy and bkt2 than both ipi-1 and pds. The expression of lyc, crtR-B and crtO for astaxanthin biosynthesis was not affected by GA3 in H. piuvialis. In the presence of GA3, astaxanthin biosynthesis genes of ipi-1, pds and bkt2 were up-regulated at transcriptional level, psy at post-transcriptional level, whereas ipi-2 was up-regulated at both levels. The study could potentially lead to a scale application of exogenous GA3 in astaxanthin production with H. pluvialis just like GAs perform in increasing crops production and it would provide new insight about the multifunctional roles of carotenogenesis in response to GA3.

Combined effects of ocean deoxygenation, acidification, and phosphorus limitation on green tide macroalga, Ulva prolifera.

Ocean deoxygenation, acidification, and decreased phosphorus availability are predicted to increase in coastal ecosystems under future climate change. However, little is known regarding the combined effects of such environmental variables on the green tide macroalga Ulva prolifera. Here, we provide quantitative and mechanistic understanding of the acclimation mechanisms of U. prolifera to ocean deoxygenation, acidification, and phosphorus limitation under both laboratory and semi-natural (mesocosms) conditions. We found that there were significant interactions between these global environmental conditions on algal physiological performance. Although algal growth rate and photosynthesis reduced when the nitrogen-to­phosphorus (N/P) ratio increased from 16:1 to 35:1 under ambient CO2 and O2 condition, they remained constant with further increasing N/P ratios of 105:1, 350:1, and 1050:1. However, the increasing alkaline phosphatase activities at high N/P ratios suggests that U. prolifera could use organic P to support its growth under phosphorus limitation. Deoxygenation had no effect on specific growth rate (SGR) but decreased photosynthesis under low N/P ratios of 16:1, 35:1, and 105:1, with reduced activities of several enzymes involved in N assimilation pathway being observed. Elevated CO2 promoted algal growth and alleviated the negative effect of deoxygenation on algal photosynthesis. The patterns of responses to high CO2 and low O2 treatments in in situ experiments were generally consistent with those observed in laboratory experiments. Our results generally found that the strong physiological acclimation capacity to elevated CO2, low O2, and high N/P could contribute to its large-scale blooming in coastal ecosystem.

Elevated pCO2 alleviates the toxic effects of polystyrene nanoparticles on the marine microalga Nannochloropsis oceanica.

Concerns about the environmental effects of nanoplastics on marine ecosystems are increasing. Ocean acidification (OA) has also become a global environmental problem. Plastic pollution occurs concomitantly with anthropogenic climate stressors such as OA. However, the combined effects of NP and OA on marine phytoplankton are still not well understood. Therefore, we have investigated the behavior of ammonia (NH2) polystyrene nanoparticles (PS NP) in f/2 medium under 1000 µatm pCO2 and discussed the toxicity of PS NP (100 nm; 0.5 and 1.5 mg/L) on Nannochloropsis oceanica under long and short-term acidification (LA and SA; pCO2 ~ 1000 µatm). We observed PS NP suspended in pCO2 1000 µatm f/2 medium aggregated to a size greater than nanoscale (1339.00 ± 76.10 nm). In addition, we found that PS NP significantly inhibited the growth of N. oceanica at two concentrations, which also produced oxidative stress. Whereas, the growth of algal cells under the coupling of acidification and PS NP was significantly better than that of single PS NP exposure. This indicated that acidification significantly alleviated the toxic effects of PS NP on N. oceanica, and long-term acidification can even promote the growth of N. oceanica under low-density NP. To further understand the mechanism, we analyzed a comparative transcriptome. The results showed that PS NP exposure inhibited the expression of genes involved in the TCA cycle. The acidification was possibly reflected in ribosomes and corresponding processes, which alleviated the negative effects of PS NP on N. oceanica by promoting the synthesis of related enzymes and proteins. This study provided a theoretical basis for assessing the damage of NP to marine phytoplankton under OA. We propose that future studies evaluating the toxicology of NP to marine ecology should consider the changing ocean climate.

Gene duplication and functional divergence of new genes contributed to the polar acclimation of Antarctic green algae.

Psychrophilic microalgae successfully survive in the extreme and highly variable polar ecosystems, which represent the energy base of most food webs and play a fundamental role in nutrient cycling. The success of microalgae is rooted in their adaptive evolution. Revealing how they have evolved to thrive in extreme polar environments will help us better understand the origin of life in polar ecosystems. We isolated a psychrophilic unicellular green alga, Microglena sp. YARC, from Antarctic sea ice which has a huge genome. Therefore, we predicted that gene replication may play an important role in its polar adaptive evolution. We found that its protein-coding gene number significantly increased and the duplication time was dated between 37 and 48 million years ago, which is consistent with the formation of the circumpolar Southern Ocean. Most duplicated paralogous genes were enriched in pathways related to photosynthesis, DNA repair, and fatty acid metabolism. Moreover, there were a total of 657 Microglena-specific families, including collagen-like proteins. The divergence in the expression patterns of the duplicated and species-specific genes reflects sub- and neo-functionalization during stress acclimation. Overall, key findings from this study provide new information on how gene duplication and their functional novelty contributed to polar algae adaptation to the highly variable polar environmental conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00203-z.

Plastic responses lead to increased neurotoxin production in the diatom Pseudo-nitzschia under ocean warming and acidification.

Ocean warming (OW) and acidification (OA) are recognized as two major climatic conditions influencing phytoplankton growth and nutritional or toxin content. However, there is limited knowledge on the responses of harmful algal bloom species that produce toxins. Here, the study provides quantitative and mechanistic understanding of the acclimation and adaptation responses of the domoic acid (DA) producing diatom Pseudo-nitzschia multiseries to rising temperature and pCO2 using both a one-year in situ bulk culture experiment, and an 800-day laboratory acclimation experiment. Ocean warming showed larger selective effects on growth and DA metabolism than ocean acidification. In a bulk culture experiment, increasing temperature +4 °C above ambient seawater temperature significantly increased DA concentration by up to 11-fold. In laboratory when the long-term warming acclimated samples were assayed under low temperatures, changes in growth rates and DA concentrations indicated that P. multiseries did not adapt to elevated temperature, but could instead rapidly and reversibly acclimate to temperature shifts. However, the warming-acclimated lines showed evidence of adaptation to elevated temperatures in the transcriptome data. Here the core gene expression was not reversed when warming-acclimated lines were moved back to the low temperature environment, which suggested that P. multiseries cells might adapt to rising temperature over longer timescales. The distinct strategies of phenotypic plasticity to rising temperature and pCO2 demonstrate a strong acclimation capacity for this bloom-forming toxic diatom in the future ocean.

De novo sequencing and analysis of the Ulva linza transcriptome to discover putative mechanisms associated with its successful colonization of coastal ecosystems.

BACKGROUND: The green algal genus Ulva Linnaeus (Ulvaceae, Ulvales, Chlorophyta) is well known for its wide distribution in marine, freshwater, and brackish environments throughout the world. The Ulva species are also highly tolerant of variations in salinity, temperature, and irradiance and are the main cause of green tides, which can have deleterious ecological effects. However, limited genomic information is currently available in this non-model and ecologically important species. Ulva linza is a species that inhabits bedrock in the mid to low intertidal zone, and it is a major contributor to biofouling. Here, we presented the global characterization of the U. linza transcriptome using the Roche GS FLX Titanium platform, with the aim of uncovering the genomic mechanisms underlying rapid and successful colonization of the coastal ecosystems. RESULTS: De novo assembly of 382,884 reads generated 13,426 contigs with an average length of 1,000 bases. Contiguous sequences were further assembled into 10,784 isotigs with an average length of 1,515 bases. A total of 304,101 reads were nominally identified by BLAST; 4,368 isotigs were functionally annotated with 13,550 GO terms, and 2,404 isotigs having enzyme commission (EC) numbers were assigned to 262 KEGG pathways. When compared with four other full sequenced green algae, 3,457 unique isotigs were found in U. linza and 18 conserved in land plants. In addition, a specific photoprotective mechanism based on both LhcSR and PsbS proteins and a C4-like carbon-concentrating mechanism were found, which may help U. linza survive stress conditions. At least 19 transporters for essential inorganic nutrients (i.e., nitrogen, phosphorus, and sulphur) were responsible for its ability to take up inorganic nutrients, and at least 25 eukaryotic cytochrome P450s, which is a higher number than that found in other algae, may be related to their strong allelopathy. Multi-origination of the stress related proteins, such as glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase and heat-shock proteins, may also contribute to colonization of U. linza under stress conditions. CONCLUSIONS: The transcriptome of U. linza uncovers some potential genomic mechanisms that might explain its ability to rapidly and successfully colonize coastal ecosystems, including the land-specific genes; special photoprotective mechanism based on both LhcSR and PsbS; development of C4-like carbon-concentrating mechanisms; muti-origin transporters for essential inorganic nutrients; multiple and complex P450s; and glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase, and heat-shock proteins that are related to stress resistance.

Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR.

Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis.