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Functional classification of proteins from sequences alone has become a critical bottleneck in understanding the myriad of protein sequences that accumulate in our databases. The great diversity of homologous sequences hides, in many cases, a variety of functional activities that cannot be anticipated. Their identification appears critical for a fundamental understanding of the evolution of living organisms and for biotechnological applications. ProfileView is a sequence-based computational method, designed to functionally classify sets of homologous sequences. It relies on two main ideas: the use of multiple profile models whose construction explores evolutionary information in available databases, and a novel definition of a representation space in which to analyze sequences with multiple profile models combined together. ProfileView classifies protein families by enriching known functional groups with new sequences and discovering new groups and subgroups. We validate ProfileView on seven classes of widespread proteins involved in the interaction with nucleic acids, amino acids and small molecules, and in a large variety of functions and enzymatic reactions. ProfileView agrees with the large set of functional data collected for these proteins from the literature regarding the organization into functional subgroups and residues that characterize the functions. In addition, ProfileView resolves undefined functional classifications and extracts the molecular determinants underlying protein functional diversity, showing its potential to select sequences towards accurate experimental design and discovery of novel biological functions. On protein families with complex domain architecture, ProfileView functional classification reconciles domain combinations, unlike phylogenetic reconstruction. ProfileView proves to outperform the functional classification approach PANTHER, the two k-mer-based methods CUPP and eCAMI and a neural network approach based on Restricted Boltzmann Machines. It overcomes time complexity limitations of the latter., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Light harvesting is regulated by a process triggered by the acidification of the thylakoid lumen, known as nonphotochemical "energy-dependent quenching" (qE). In diatoms, qE is controlled by the light-harvesting complex (LHC) protein LHCX1, while the LHC stress-related (LHCSR) and photosystem II subunit S proteins are essential for green algae and plants, respectively. Here, we report a biochemical and molecular characterization of LHCX1 to investigate its role in qE. We found that, when grown under intermittent light, Phaeodactylum tricornutum forms very large qE, due to LHCX1 constitutive upregulation. This "super qE" is abolished in LHCX1 knockout mutants. Biochemical and spectroscopic analyses of LHCX1 reveal that this protein might differ in the character of binding pigments relative to the major pool of light-harvesting antenna proteins. The possibility of transient pigment binding or not binding pigments at all is discussed. Targeted mutagenesis of putative protonatable residues (D95 and E205) in transgenic P. tricornutum lines does not alter qE capacity, showing that they are not involved in sensing lumen pH, differently from residues conserved in LHCSR3. Our results suggest functional divergence between LHCX1 and LHCSR3 in qE modulation. We propose that LHCX1 evolved independently to facilitate dynamic tracking of light fluctuations in turbulent waters. The evolution of LHCX(-like) proteins in organisms with secondary red plastids, such as diatoms, might have conferred a selective advantage in the control of dynamic photoprotection, ultimately resulting in their ecological success., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Endogenous small noncoding RNAs (sRNAs) are a large family of essential regulators of gene expression in both eukaryotes and prokaryotes. Various types of sRNAs with different size and mapping to different genome locations have been recently identified in diatoms, a successful group of phytoplankton in the marine environment. However, their biogenesis and regulatory function are still largely unknown and unexplored in these microalgae, also due to the lack of methods for their experimental analysis. Herein, we present a point-by-point description of the protocols for detection and quantification of sRNAs by Northern-blot analysis and quantitative stem-loop RT-PCR, established in the diatom molecular model specie Phaeodactylum tricornutum., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Microalgae represent a promising resource for the production of beneficial natural compounds due to their richness in secondary metabolites and easy cultivation. Carotenoids feature among distinctive compounds of many microalgae, including diatoms, which owe their golden color to the xanthophyll fucoxanthin. Carotenoids have antioxidant, anti-obesity and anti-inflammatory properties, and there is a considerable market demand for these compounds. Here, with the aim to increase the carotenoid content in the model diatom Phaeodactylum tricornutum , we exploited genetic transformation to overexpress genes involved in the carotenoid biosynthetic pathway. We produced transgenic lines over-expressing simultaneously one, two or three carotenoid biosynthetic genes, and evaluated changes in pigment content with high-performance liquid chromatography. Two triple transformants over-expressing the genes Violaxanthin de-epoxidase ( Vde ), Vde-related ( Vdr ) and Zeaxanthin epoxidase 3 ( Zep3 ) showed an accumulation of carotenoids, with an increase in the fucoxanthin content up to four fold. Vde , Vdr and Zep3 mRNA and protein levels in the triple transformants were coherently increased. The exact role of these enzymes in the diatom carotenoid biosynthetic pathway is not completely elucidated nevertheless our strategy successfully modulated the carotenoid metabolism leading to an accumulation of valuable compounds, leading the way toward improved utilization of microalgae in the field of antioxidants.

Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms.

Periodic light-dark cycles govern the timing of basic biological processes in organisms inhabiting land as well as the sea, where life evolved. Although prominent marine phytoplanktonic organisms such as diatoms show robust diel rhythms, the mechanisms regulating these processes are still obscure. By characterizing a Phaeodactylum tricornutum bHLH-PAS nuclear protein, hereby named RITMO1, we shed light on the regulation of the daily life of diatoms. Alteration of RITMO1 expression levels and timing by ectopic overexpression results in lines with deregulated diurnal gene expression profiles compared with the wild-type cells. Reduced gene expression oscillations are also observed in these lines in continuous darkness, showing that the regulation of rhythmicity by RITMO1 is not directly dependent on light inputs. We also describe strong diurnal rhythms of cellular fluorescence in wild-type cells, which persist in continuous light conditions, indicating the existence of an endogenous circadian clock in diatoms. The altered rhythmicity observed in RITMO1 overexpression lines in continuous light supports the involvement of this protein in circadian rhythm regulation. Phylogenetic analysis reveals a wide distribution of RITMO1-like proteins in the genomes of diatoms as well as in other marine algae, which may indicate a common function in these phototrophs. This study adds elements to our understanding of diatom biology and offers perspectives to elucidate timekeeping mechanisms in marine organisms belonging to a major, but under-investigated, branch of the tree of life., (Copyright © 2019 the Author(s). Published by PNAS.)

Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.

Marine diatoms are prominent phytoplankton organisms that perform photosynthesis in extremely variable environments. Diatoms possess a strong ability to dissipate excess absorbed energy as heat via nonphotochemical quenching (NPQ). This process relies on changes in carotenoid pigment composition (xanthophyll cycle) and on specific members of the light-harvesting complex family specialized in photoprotection (LHCXs), which potentially act as NPQ effectors. However, the link between light stress, NPQ, and the existence of different LHCX isoforms is not understood in these organisms. Using picosecond fluorescence analysis, we observed two types of NPQ in the pennate diatom Phaeodactylum tricornutum that were dependent on light conditions. Short exposure of low-light-acclimated cells to high light triggers the onset of energy quenching close to the core of photosystem II, while prolonged light stress activates NPQ in the antenna. Biochemical analysis indicated a link between the changes in the NPQ site/mechanism and the induction of different LHCX isoforms, which accumulate either in the antenna complexes or in the core complex. By comparing the responses of wild-type cells and transgenic lines with a reduced expression of the major LHCX isoform, LHCX1, we conclude that core complex-associated NPQ is more effective in photoprotection than is the antenna complex. Overall, our data clarify the complex molecular scenario of light responses in diatoms and provide a rationale for the existence of a degenerate family of LHCX proteins in these algae., (© 2018 American Society of Plant Biologists. All rights reserved.)

Diatoms contribute 20% of global primary production and form the basis of many marine food webs. Although their species diversity correlates with broad diversity in cell size, there is also an intraspecific cell-size plasticity owing to sexual reproduction and varying environmental conditions. However, despite the ecological significance of the diatom cell size for food-web structure and global biogeochemical cycles, our knowledge about genes underpinning the size of diatom cells remains elusive. Here, a combination of reverse genetics, experimental evolution and comparative RNA-sequencing analyses enabled us to identify a previously unknown genetic control of cell size in the diatom Thalassiosira pseudonana. In particular, the targeted deregulation of the expression of the cell-wall protein silacidin caused a significant increase in valve diameter. Remarkably, the natural downregulation of the silacidin gene transcript due to experimental evolution under low temperature also correlated with cell-size increase. Our data give first evidence for a genetically controlled regulation of cell size in T. pseudonana and possibly other centric diatoms as they also encode the silacidin gene in their genomes.

Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use in biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the sole energy source. Some diatoms, however, can also grow in a mixotrophic mode, wherein both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum , using glycerol as the source of reduced carbon. Transcriptomics, metabolomics, metabolic modelling and physiological data combine to indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, provision of glycerol mimics typical responses of nitrogen limitation on lipid metabolism at the level of triacylglycerol accumulation and fatty acid composition. The presence of glycerol, despite provoking features reminiscent of nutrient limitation, neither diminishes photosynthetic activity nor cell growth, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'., (© 2017 The Author(s).)

Marine eukaryotic phytoplankton are major contributors to global primary production. To adapt and thrive in the oceans, phytoplankton relies on a variety of light-regulated responses and light-acclimation capacities probably driven by sophisticated photoregulatory mechanisms. A plethora of photoreceptor-like sequences from marine microalgae have been identified in omics approaches. Initial studies have revealed that some algal photoreceptors are similar to those known in plants. In addition, new variants with different spectral tuning and algal-specific light sensors have also been found, changing current views and perspectives on how photoreceptor structure and function have diversified in phototrophs experiencing different environmental conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The ability of phototrophs to colonise different environments relies on robust protection against oxidative stress, a critical requirement for the successful evolutionary transition from water to land. Photosynthetic organisms have developed numerous strategies to adapt their photosynthetic apparatus to changing light conditions in order to optimise their photosynthetic yield, which is crucial for life on Earth to exist. Photosynthetic acclimation is an excellent example of the complexity of biological systems, where highly diverse processes, ranging from electron excitation over protein protonation to enzymatic processes coupling ion gradients with biosynthetic activity, interact on drastically different timescales from picoseconds to hours. Efficient functioning of the photosynthetic apparatus and its protection is paramount for efficient downstream processes, including metabolism and growth. Modern experimental techniques can be successfully integrated with theoretical and mathematical models to promote our understanding of underlying mechanisms and principles. This review aims to provide a retrospective analysis of multidisciplinary photosynthetic acclimation research carried out by members of the Marie Curie Initial Training Project, AccliPhot, placing the results in a wider context. The review also highlights the applicability of photosynthetic organisms for industry, particularly with regards to the cultivation of microalgae. It intends to demonstrate how theoretical concepts can successfully complement experimental studies broadening our knowledge of common principles in acclimation processes in photosynthetic organisms, as well as in the field of applied microalgal biotechnology., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO 2 . Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Diatoms are phytoplanktonic organisms that grow successfully in the ocean where light conditions are highly variable. Studies of the molecular mechanisms of light acclimation in the marine diatom Phaeodactylum tricornutum show that carotenoid de-epoxidation enzymes and LHCX1, a member of the light-harvesting protein family, both contribute to dissipate excess light energy through non-photochemical quenching (NPQ). In this study, we investigate the role of the other members of the LHCX family in diatom stress responses. Our analysis of available genomic data shows that the presence of multiple LHCX genes is a conserved feature of diatom species living in different ecological niches. Moreover, an analysis of the levels of four P. tricornutum LHCX transcripts in relation to protein expression and photosynthetic activity indicates that LHCXs are differentially regulated under different light intensities and nutrient starvation, mostly modulating NPQ capacity. We conclude that multiple abiotic stress signals converge to regulate the LHCX content of cells, providing a way to fine-tune light harvesting and photoprotection. Moreover, our data indicate that the expansion of the LHCX gene family reflects functional diversification of its members which could benefit cells responding to highly variable ocean environments., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Studies of cell division in organisms derived from secondary endosymbiosis such as diatoms have revealed that the mechanisms are far from those found in more conventional model eukaryotes. An atypical acentriolar microtuble-organizing centre, centripetal cytokinesis combined with centrifugal cell wall neosynthesis, and the role of sex in relation to cell size restoration make diatoms an exciting system to re-investigate the evolution, differentiation and regulation of cell division. Such studies are further justified considering the ecological relevance of these microalgae in contemporary oceans and the need to understand the mechanisms controlling their growth and distribution in an environmental context. Recent work derived from genome-wide analyses on representative model diatoms reveals that the cell cycle is finely tuned to inputs derived from both endogenous and environmental signals., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Diatoms are prominent phytoplanktonic organisms that contribute around 40% of carbon assimilation in the oceans. They grow and perform optimally in variable environments, being able to cope with unpredictable changes in the amount and quality of light. The molecular mechanisms regulating diatom light responses are, however, still obscure. Using knockdown Phaeodactylum tricornutum transgenic lines, we reveal the key function of a member of the light-harvesting complex stress-related (LHCSR) protein family, denoted LHCX1, in modulation of excess light energy dissipation. In contrast to green algae, this gene is already maximally expressed in nonstressful light conditions and encodes a protein required for efficient light responses and growth. LHCX1 also influences natural variability in photoresponse, as evidenced in ecotypes isolated from different latitudes that display different LHCX1 protein levels. We conclude, therefore, that this gene plays a pivotal role in managing light responses in diatoms.

Members of the cryptochrome/photolyase family (CPF) are widely distributed throughout all kingdoms, and encode photosensitive proteins that typically show either photoreceptor or DNA repair activity. Animal and plant cryptochromes have lost DNA repair activity and now perform specialized photoperceptory functions, for example, plant cryptochromes regulate growth and circadian rhythms, whereas mammalian and insect cryptochromes act as transcriptional repressors that control the circadian clock. However, the functional differentiation between photolyases and cryptochromes is now being questioned. Here, we show that the PtCPF1 protein from the marine diatom Phaeodactylum tricornutum shows 6-4 photoproduct repair activity and can act as a transcriptional repressor of the circadian clock in a heterologous mammalian cell system. Conversely, it seems to have a wide role in blue-light-regulated gene expression in diatoms. The protein might therefore represent a missing link in the evolution of CPFs, and act as a novel ultraviolet/blue light sensor in marine environments.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

Photoreceptors allow living organisms to optimize perception of light in the natural environment and thus to gain information about their external world. In this review, we describe blue and red light photoreceptors in bacteria, plants, and animals in relation to their evolution. Analyses performed in different organisms have revealed wonderful examples of structural modifications of the light-sensing proteins themselves, as well as diversification of the signal transduction pathways they use in relation with their evolutionary history and function. In different organisms, the same photoreceptor may have a very conserved role (convergent evolution of function) or may modulate different responses (acquisition of new function). Multiple photoreceptors of the same family in the same organism indicate gene duplication events during evolution, with a consequent enhanced sensitivity to variations in ambient light. Conversely, two different photoreceptors may be involved in the control of the same physiological response. Genomic analysis in marine diatoms, combined with phylogenetic studies, has also revealed the presence of blue and red light photoreceptors in the marine environment. This discovery has intriguing implications for the understanding of light perception and its evolution in photosynthetic organisms. In addition, the characterization of these photoreceptors likely will add to our understanding of photoreceptor diversity as an adaptation to different habitats.

The gene encoding the chloroplast ribosomal protein S1 from Chlamydomonas reinhardtii, CreS1, was cloned and the RNA binding properties and the expression patterns were studied. Gel-shift analysis revealed that CreS1 binds AU-rich 5'-untranslated regions (5'-UTR) of chloroplast mRNAs with higher affinity than the corresponding sequence of a GC-rich nuclear transcript. The binding affinity of CreS1 for a mutant form of the psbD 5'-UTR with a deletion of a U-rich stretch that is required for translation decreases 4-fold as compared to the wild-type 5'-UTR. Our results suggest that CreS1 protein interacts with U-rich sequences. Most of CreS1 is bound to high-molecular-weight complexes which co-migrate with the 30S small ribosomal subunit, and only a small fraction of CreS1 exists in its free form. CreS1 is localized mainly to the chloroplast stroma albeit a significant fraction is associated with chloroplast membranes. The results suggest that most of CreS1 is associated with the 30S ribosomal subunit throughout the translation process. Upon a shift of cells from the dark to the light, the mRNA levels of CreS1 and Psrp-7, both components of the 30S ribosomal subunit, increase transiently and return to the dark levels after 8 h. However, during this dark-to-light transition the levels of CreS1 and of other components of the 30S subunit remain the same suggesting that either protein synthesis or degradation is regulated. The possible implications of these findings are discussed.

The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor expression of a gene encoding a fucoxanthin, chlorophyll a/c-binding protein (FCP) in the centric planktonic diatom Thalassiosira weissflogii. Analysis of FCP gene expression in dark-adapted diatom cultures revealed that mRNA levels increase 5- to 6-fold in response to white light irradiation and peak around 6 to 8 h. To determine the photoreceptors involved in this response action spectra of FCP gene expression were determined using the Okazaki large spectrograph. Responses consistent with the presence of cryptochrome-, rhodopsin- and phytochrome-type receptors could be detected. The apparent presence of phytochrome-mediated responses is of particular interest given the low fluences of red and far-red light wavelengths in the marine environment.

: We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms.