Exon shuffling and alternative splicing of ROCO genes in brown algae enables a diverse repertoire of candidate immune receptors.

The ROCO family is a family of GTPases characterized by a central ROC-COR tandem domain. Interest in the structure and function of ROCO proteins has increased with the identification of their important roles in human disease. Nevertheless, the functions of most ROCO proteins are still unknown. In the present study, we characterized the structure, evolution, and expression of ROCOs in four species of brown algae. Brown algae have a larger number of ROCO proteins than other organisms reported to date. Phylogenetic analyses showed that ROCOs have an ancient origin, likely originated in prokaryotes. ROCOs in brown algae clustered into four groups and showed no strong relationship with red algae or green algae. Brown algal ROCOs retain the ancestral LRR-ROC-COR domain arrangement, which is found in prokaryotes, plants and some basal metazoans. Remarkably, individual LRR motifs in ROCO genes are each encoded by separate exons and exhibit intense exon shuffling and diversifying selection. Furthermore, the tandem LRR exons exhibit alternative splicing to generate multiple transcripts. Both exon shuffling and alternative splicing of LRR repeats may be important mechanisms for generating diverse ligand-binding specificities as immune receptors. Besides their potential immune role, expression analysis shows that many ROCO genes are responsive to other stress conditions, suggesting they could participate in multiple signal pathways, not limited to the immune response. Our results substantially enhance our understanding of the structure and function of this mysterious gene family.

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.

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.

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.

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.

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.

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.

The role of zinc in the adaptive evolution of polar phytoplankton.

Zinc is an essential trace metal for oceanic primary producers with the highest concentrations in polar oceans. However, its role in the biological functioning and adaptive evolution of polar phytoplankton remains enigmatic. Here, we have applied a combination of evolutionary genomics, quantitative proteomics, co-expression analyses and cellular physiology to suggest that model polar phytoplankton species have a higher demand for zinc because of elevated cellular levels of zinc-binding proteins. We propose that adaptive expansion of regulatory zinc-finger protein families, co-expanded and co-expressed zinc-binding proteins families involved in photosynthesis and growth in these microalgal species and their natural communities were identified to be responsible for the higher zinc demand. The expression of their encoding genes in eukaryotic phytoplankton metatranscriptomes from pole-to-pole was identified to correlate not only with dissolved zinc concentrations in the upper ocean but also with temperature, suggesting that environmental conditions of polar oceans are responsible for an increased demand of zinc. These results suggest that zinc plays an important role in supporting photosynthetic growth in eukaryotic polar phytoplankton and that this has been critical for algal colonization of low-temperature polar oceans.

Ocean acidification stimulation of phytoplankton growth depends on the extent of departure from the optimal growth temperature.

Ocean acidification and warming are two major environmental stressors; however, the generality of how warming will alter growth responses of phytoplankton to ocean acidification is less known. Here, enhancement of growth by high CO2 (HC) in Phaeodactylum tricornutum and Thalassiosira weissflogii was most prominent at optimum temperature. The extent to which growth rates in HC cultures were raised compared to low CO2 (LC) cultures tended to decrease with increasing or decreasing temperature, compared to the optimum. Further mechanistic studies in P. tricornutum revealed that cellular carbon and nitrogen content, superoxide dismutase activity, and respiration were generally higher in HC than those in LC at high and low temperatures, whereas PSII photochemical parameters were generally lower in HC than in LC at high and low temperatures. These results indicate that HC-grown cells needed to invest more energy and materials to maintain intracellular homeostasis and repair damage induced by the unsuitable temperatures.

Elevated CO2 reduces copper accumulation and toxicity in the diatom Thalassiosira pseudonana.

The projected ocean acidification (OA) associated with increasing atmospheric CO2 alters seawater chemistry and hence the bio-toxicity of metal ions. However, it is still unclear how OA might affect the long-term resilience of globally important marine microalgae to anthropogenic metal stress. To explore the effect of increasing pCO2 on copper metabolism in the diatom Thalassiosira pseudonana (CCMP 1335), we employed an integrated eco-physiological, analytical chemistry, and transcriptomic approach to clarify the effect of increasing pCO2 on copper metabolism of Thalassiosira pseudonana across different temporal (short-term vs. long-term) and spatial (indoor laboratory experiments vs. outdoor mesocosms experiments) scales. We found that increasing pCO2 (1,000 and 2,000 µatm) promoted growth and photosynthesis, but decreased copper accumulation and alleviated its bio-toxicity to T. pseudonana. Transcriptomics results indicated that T. pseudonana altered the copper detoxification strategy under OA by decreasing copper uptake and enhancing copper-thiol complexation and copper efflux. Biochemical analysis further showed that the activities of the antioxidant enzymes glutathione peroxidase (GPX), catalase (CAT), and phytochelatin synthetase (PCS) were enhanced to mitigate oxidative damage of copper stress under elevated CO2. Our results provide a basis for a better understanding of the bioremediation capacity of marine primary producers, which may have profound effect on the security of seafood quality and marine ecosystem sustainability under further climate change.

Biodiversity-based development and evolution: the emerging research systems in model and non-model organisms.

Evolutionary developmental biology, or Evo-Devo for short, has become an established field that, broadly speaking, seeks to understand how changes in development drive major transitions and innovation in organismal evolution. It does so via integrating the principles and methods of many subdisciplines of biology. Although we have gained unprecedented knowledge from the studies on model organisms in the past decades, many fundamental and crucially essential processes remain a mystery. Considering the tremendous biodiversity of our planet, the current model organisms seem insufficient for us to understand the evolutionary and physiological processes of life and its adaptation to exterior environments. The currently increasing genomic data and the recently available gene-editing tools make it possible to extend our studies to non-model organisms. In this review, we review the recent work on the regulatory signaling of developmental and regeneration processes, environmental adaptation, and evolutionary mechanisms using both the existing model animals such as zebrafish and Drosophila, and the emerging nonstandard model organisms including amphioxus, ascidian, ciliates, single-celled phytoplankton, and marine nematode. In addition, the challenging questions and new directions in these systems are outlined as well.

Integrating Transcriptomics and Metabolomics to Characterize Metabolic Regulation to Elevated CO2 in Chlamydomonas Reinhardtii.

With atmospheric CO2 increasing, a large amount of CO2 is absorbed by oceans and lakes, which changes the carbonate system and affects the survival of aquatic plants, especially microalgae. The main aim of our study was to explore the responses of Chlamydomonas reinhardtii (Chlorophyceae) to elevated CO2 by combined transcriptome and metabolome analysis under three different scenarios: control (CK, 400 ppm), short-term elevated CO2 (ST, 1000 ppm), and long-term elevated CO2 (LT, 1000 ppm). The transcriptomic data showed moderate changes between ST and CK. However, metabolic analysis indicated that fatty acids (FAs) and partial amino acids (AAs) were increased under ST. There was a global downregulation of genes involved in photosynthesis, glycolysis, lipid metabolism, and nitrogen metabolism but increase in the TCA cycle and ß-oxidation under LT. Integrated transcriptome and metabolome analyses demonstrated that the nutritional constituents (FAs, AAs) under LT were poor compared with CK, and most genes and metabolites involved in C and N metabolism were significantly downregulated. However, the growth and photosynthesis of cells under LT increased significantly. Thus, C. reinhardtii could form a specific adaptive evolution to elevated CO2, affecting future biogeochemical cycles.

The complete chloroplast genome of the brown alga Saccharina sp. ye-B (Laminariaceae: Phaeophyceae) from Sakhalin, Russia.

The complete chloroplast genome sequence of Saccharina sp. ye-B was determined using Illumina sequencing data. The chloroplast genome of Saccharina sp. ye-B is 130,587 bp in length, containing 139 protein-coding genes (PCGs), 3 ribosomal RNAs (rRNAs), and 28 transfer RNAs (tRNAs) genes. The phylogenetic reconstruction based on the chloroplast genomes of 11 brown algae resolves Saccharina sp. ye-B in a fully supported clade with S. japonica. The determination of the chloroplast genome of Saccharina sp. ye-B will benefit future algal genetics, evolution, and systematic studies in the Laminariaceae.

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.

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.

Acclimation and adaptation to elevated pCO2 increase arsenic resilience in marine diatoms.

Arsenic pollution is a widespread threat to marine life, but the ongoing rise pCO2 levels is predicted to decrease bio-toxicity of arsenic. However, the effects of arsenic toxicity on marine primary producers under elevated pCO2 are not well characterized. Here, we studied the effects of arsenic toxicity in three globally distributed diatom species (Phaeodactylum tricornutum, Thalassiosira pseudonana, and Chaetoceros mulleri) after short-term acclimation (ST, 30 days), medium-term exposure (MT, 750 days), and long-term (LT, 1460 days) selection under ambient (400 µatm) and elevated (1000 and 2000 µatm) pCO2. We found that elevated pCO2 alleviated arsenic toxicity even after short acclimation times but the magnitude of the response decreased after mid and long-term adaptation. When fed with these elevated pCO2 selected diatoms, the scallop Patinopecten yessoensis had significantly lower arsenic content (3.26-52.83%). Transcriptomic and biochemical analysis indicated that the diatoms rapidly developed arsenic detoxification strategies, which included upregulation of transporters associated with shuttling harmful compounds out of the cell to reduce arsenic accumulation, and upregulation of proteins involved in synthesizing glutathione (GSH) to chelate intracellular arsenic to reduce arsenic toxicity. Thus, our results will expand our knowledge to fully understand the ecological risk of trace metal pollution under increasing human activity induced ocean acidification.

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.

Influence of ocean acidification on thermal reaction norms of carbon metabolism in the marine diatom Phaeodactylum tricornutum.

Under the present CO2 condition, the efficiency of biological pump mediating carbon sequestration is predicted to decline in the future because respiration tends to be more sensitive to rising temperature than is photosynthesis. However, it remains unknown whether the impacts of global warming on metabolic rates of phytoplankton can be modulated by elevated CO2 induced ocean acidification. Here we show that in the model diatom species Phaeodactylum tricornutum, Ea (activation energy) of photosynthesis (~0.5 eV) was significantly lower than that of respiration (1.8 eV), while CO2 concentration had no effect on the Ea value. Eh (deactivation energy) of respiration was increased to 2.5 eV, that was equivalent to Eh of photosynthesis in high CO2-grown cells and 28.4% higher than that in low CO2-grown ones. The respiration to photosynthesis ratio (R/P) was consistently higher in high CO2 condition, which increased with temperature at the beginning and subsequently decreased in both CO2 conditions. The ratio of R/P in high CO2 to R/P in low CO2 gradually increased with temperature above the optimal temperature. Our results imply that ocean acidification will aggravate the negative impacts or offset the alleviating effects of warming on the R/P ratio depending on the temperature range in Phaeodactylum tricornutum.

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.