Chris Bowler

Publications of Chris Bowler

• Leveraging metabolomics for functional investigations in sequenced marine diatoms.

  Authors: Alisdair R Fernie, Toshihiro Obata, Andrew E Allen, Wagner L Araújo, Chris Bowler

  Trends in plant science. 03/2012;

  Recent years have witnessed the genomic decoding of a wide range of photosynthetic organisms from the model plant Arabidopsis thaliana and the complex genomes of important crop species toRecent years have witnessed the genomic decoding of a wide range of photosynthetic organisms from the model plant Arabidopsis thaliana and the complex genomes of important crop species to single-celled marine phytoplankton. The comparative sequencing of green, red and brown algae has provided considerable insight into a number of important questions concerning their evolution, physiology and metabolism. The combinatorial application of metabolomics has further deepened our understanding both of the function of individual genes and of metabolic processes. Here we discuss the power of utilising metabolomics in conjunction with sequencing data to gain greater insight into the metabolic hierarchies underpinning the function of individual organisms, using unicellular marine diatoms as a case study to exemplify the advantages of this approach.

• A holistic approach to marine eco-systems biology.

  Authors: Eric Karsenti, Silvia G Acinas, Peer Bork, Chris Bowler, Colomban De Vargas, Jeroen Raes, Matthew Sullivan, Detlev Arendt, Francesca Benzoni, Jean-Michel Claverie [......] Fabrice Not, Hiroyuki Ogata, Stéphane Pesant, Emmanuel Georges Reynaud, Christian Sardet, Michael E Sieracki, Sabrina Speich, Didier Velayoudon, Jean Weissenbach, Patrick Wincker

  PLoS biology. 10/2011; 9(10):e1001177.

  The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizesThe structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.

• Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms.

  Authors: Andrew E Allen, Ahmed Moustafa, Anton Montsant, Angelika Eckert, Peter G Kroth, Chris Bowler

  Molecular biology and evolution. 09/2011; 29(1):367-79.

  Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked throughDiatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO(2) fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.

• Stemming epigenetics in marine stramenopiles.

  Authors: Florian Maumus, Pablo Rabinowicz, Chris Bowler, Maximo Rivarola

  Current genomics. 08/2011; 12(5):357-70.

  Epigenetics include DNA methylation, the modification of histone tails that affect chromatin states, and small RNAs that are involved in the setting and maintenance of chromatin modifications. MarineEpigenetics include DNA methylation, the modification of histone tails that affect chromatin states, and small RNAs that are involved in the setting and maintenance of chromatin modifications. Marine stramenopiles (MAS), which are a diverse assemblage of algae that acquired photosynthesis from secondary endosymbiosis, include single-celled organisms such as diatoms as well as multicellular forms such as brown algae. The recent publication of two diatom genomes that diverged ~90 million years ago (mya), as well as the one of a brown algae that diverged from diatoms ~250 Mya, provide a great system of related, yet diverged set of organisms to compare epigenetic marks and their relationships. For example, putative DNA methyltransferase homologues were found in diatoms while none could be identified in the brown algal genome. On the other hand, no canonical DICER-like protein was found in diatoms in contrast to what is observed in brown algae. A key interest relies in understanding the adaptive nature of epigenetics and its inheritability. In contrast to yeast that lack DNA methylation, homogeneous cultures of diatoms constitute an attractive system to study epigenetic changes in response to environmental conditions such as nutrient-rich to nutrient-poor transitions which is especially relevant because of their ecological importance. P. tricornutum is also of outstanding interest because it is observed as three different morphotypes and thus constitutes a simple and promising model for the study of the epigenetic phenomena that accompany cellular differentiation. In this review we focus on the insights obtained from MAS comparative genomics and epigenomic analyses.

• Physiological and molecular evidence that environmental changes elicit morphological interconversion in the model diatom Phaeodactylum tricornutum.

  Authors: Alessandra De Martino, Ana Bartual, Anusuya Willis, Agnes Meichenin, Beatriz Villazán, Uma Maheswari, Chris Bowler

  Protist. 07/2011; 162(3):462-81.

  Over the last decades Phaeodactylum tricornutum has become a model to study diatom biology at the molecular level. Cells have the peculiarity to be pleiomorphic and it is thought that this characterOver the last decades Phaeodactylum tricornutum has become a model to study diatom biology at the molecular level. Cells have the peculiarity to be pleiomorphic and it is thought that this character is triggered by culture conditions, although few quantitative studies have been performed and nothing is known at the molecular level. Our aim was to quantify the effect of growth conditions on cell morphology of different P. tricornutum strains by quantitative microscopy, cellular imaging, and non-targeted transcriptomics. We show that morphotype changes can be regulated by changing culture conditions, depending on the strain, and show a common trend of increased oval cell abundance as a response to stress. Examination of expressed sequence tags (ESTs) from triradiate cells infers the importance of osmoregulation in the maintenance of this morphotype, whereas ESTs derived from oval cells grown in hyposaline and low temperature conditions show a predominance of genes encoding typical components of stress pathways, especially in signaling, cell homeostasis and lipid metabolism. This work contributes to better understand the importance of the unique capability of morphotype conversion in P. tricornutum and its relevance in acclimation to changing environmental conditions.

• Integrative epigenomic mapping defines four main chromatin states in Arabidopsis.

  Authors: François Roudier, Ikhlak Ahmed, Caroline Bérard, Alexis Sarazin, Tristan Mary-Huard, Sandra Cortijo, Daniel Bouyer, Erwann Caillieux, Evelyne Duvernois-Berthet, Liza Al-Shikhley [......] Frédy Barneche, Sandra Dèrozier, Véronique Brunaud, Sébastien Aubourg, Arp Schnittger, Chris Bowler, Marie-Laure Martin-Magniette, Stéphane Robin, Michel Caboche, Vincent Colot

  The EMBO journal. 05/2011; 30(10):1928-38.

  Post-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorialPost-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ∼90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin-based regulatory mechanisms in plants.

• Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis.

  Authors: Ikhlak Ahmed, Alexis Sarazin, Chris Bowler, Vincent Colot, Hadi Quesneville

  Nucleic acids research. 05/2011; 39(16):6919-31.

  Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repressionTransposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5' to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.

• Evolution and metabolic significance of the urea cycle in photosynthetic diatoms.

  Authors: Andrew E Allen, Christopher L Dupont, Miroslav Oborník, Aleš Horák, Adriano Nunes-Nesi, John P McCrow, Hong Zheng, Daniel A Johnson, Hanhua Hu, Alisdair R Fernie, Chris Bowler

  Nature. 05/2011; 473(7346):203-7.

  Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world's most productive marine food webs. The diatom nuclear genome contains genes withDiatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world's most productive marine food webs. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont), yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline and the precursors for long-chain polyamines required for silica precipitation during cell wall formation. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.

• Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana.

  Authors: Masaaki Tachibana, Andrew E Allen, Sae Kikutani, Yuri Endo, Chris Bowler, Yusuke Matsuda

  Photosynthesis research. 03/2011; 109(1-3):205-21.

  It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in theIt is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO(2) responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.

• The conserved factor DE-ETIOLATED 1 cooperates with CUL4-DDB1DDB2 to maintain genome integrity upon UV stress.

  Authors: Enric Castells, Jean Molinier, Giovanna Benvenuto, Clara Bourbousse, Gerald Zabulon, Antoine Zalc, Stefano Cazzaniga, Pascal Genschik, Fredy Barneche, Chris Bowler

  The EMBO journal. 02/2011; 30(6):1162-72.

  Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allowsPlants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE-ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light-driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4-based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4-dependent targeted degradation of the UV-lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4-dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.

• An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light.

  Authors: Benjamin Bailleul, Alessandra Rogato, Alessandra de Martino, Sacha Coesel, Pierre Cardol, Chris Bowler, Angela Falciatore, Giovanni Finazzi

  Proceedings of the National Academy of Sciences of the United States of America. 10/2010; 107(42):18214-9.

  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 withDiatoms 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.

• Diatom cell division in an environmental context.

  Authors: Chris Bowler, Alessandra De Martino, Angela Falciatore

  Current opinion in plant biology. 10/2010; 13(6):623-30.

  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. AnStudies 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.

• Transcription factor families inferred from genome sequences of photosynthetic stramenopiles.

  Authors: Edda Rayko, Florian Maumus, Uma Maheswari, Kamel Jabbari, Chris Bowler

  The New phytologist. 10/2010; 188(1):52-66.

  • By comparative analyses we identify lineage-specific diversity in transcription factors (TFs) from stramenopile (or heterokont) genome sequences. We compared a pennate (Phaeodactylum tricornutum)• By comparative analyses we identify lineage-specific diversity in transcription factors (TFs) from stramenopile (or heterokont) genome sequences. We compared a pennate (Phaeodactylum tricornutum) and a centric diatom (Thalassiosira pseudonana) with those of other stramenopiles (oomycetes, Pelagophyceae, and Phaeophyceae (Ectocarpus siliculosus)) as well as to that of Emiliania huxleyi, a haptophyte that is evolutionarily related to the stramenopiles. • We provide a detailed description of diatom TF complements and report numerous peculiarities: in both diatoms, the heat shock factor (HSF) family is overamplified and constitutes the most abundant class of TFs; Myb and C2H2-type zinc finger TFs are the two most abundant TF families encoded in all the other stramenopile genomes investigated; the presence of diatom and lineage-specific gene fusions, in particular a class of putative photoreceptors with light-sensitive Per-Arnt-Sim (PAS) and DNA-binding (basic-leucine zipper, bZIP) domains and an HSF-AP2 domain fusion. • Expression data analysis shows that many of the TFs studied are transcribed and may be involved in specific responses to environmental stimuli. • Evolutionary and functional relevance of these observations are discussed.

• Genomic insights into photosynthesis in eukaryotic phytoplankton.

  Authors: Giovanni Finazzi, Hervé Moreau, Chris Bowler

  Trends in plant science. 10/2010; 15(10):565-72.

  The evolution of photosynthesis completely altered the biogeochemistry of our planet and permitted the evolution of more complex multicellular organisms. Curiously, terrestrial photosynthesis isThe evolution of photosynthesis completely altered the biogeochemistry of our planet and permitted the evolution of more complex multicellular organisms. Curiously, terrestrial photosynthesis is carried out largely by green algae and their descendents the higher plants, whereas in the ocean the most abundant photosynthetic eukaryotes are microscopic and have red algal affiliations. Although primary productivity is approximately equal between the land and the ocean, the marine microbes represent less than 1% of the photosynthetic biomass found on land. This review focuses on this highly successful and diverse group of organisms collectively known as phytoplankton and reviews how insights from whole genome analyses have improved our understanding of the novel innovations employed by them to maximize photosynthetic efficiency in variable light environments.

• The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

  Authors: J Mark Cock, Lieven Sterck, Pierre Rouzé, Delphine Scornet, Andrew E Allen, Grigoris Amoutzias, Veronique Anthouard, François Artiguenave, Jean-Marc Aury, Jonathan H Badger [......] Declan C Schroeder, Béatrice Ségurens, Martina Strittmatter, Thierry Tonon, James W Tregear, Klaus Valentin, Peter von Dassow, Takahiro Yamagishi, Yves Van de Peer, Patrick Wincker

  Nature. 06/2010; 465(7298):617-21.

  Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominantBrown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

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• det1-1-induced UV-C hyposensitivity through UVR3 and PHR1 photolyase gene over-expression.

  Authors: Enric Castells, Jean Molinier, Stephanie Drevensek, Pascal Genschik, Fredy Barneche, Chris Bowler

  The Plant journal : for cell and molecular biology. 05/2010;

  Summary Obligate photoautotrophs such as plants must capture energy from sunlight and are therefore exposed to the damaging collateral effects of ultraviolet (UV) irradiation, especially on DNA. HereSummary Obligate photoautotrophs such as plants must capture energy from sunlight and are therefore exposed to the damaging collateral effects of ultraviolet (UV) irradiation, especially on DNA. Here we investigated the interconnection between light signaling and DNA repair, two concomitant pathways during photomorphogenesis, the developmental transition associated with the first light exposure. It is shown that combination of an enhanced sunscreen effect and photoreactivation confers a greater level of tolerance to damaging UV-C doses in the constitutive photomorphogenic de-etiolated1-1 (det1--1) Arabidopsis mutant. In darkness, expression of the PHR1 and UVR3 photolyase genes, responsible for photoreactivation, is maintained at a basal level through the positive action of HY5 and HYH photomorphogenesis-promoting transcription factors and the repressive effects of DET1 and COP1. Upon light exposure, HY5 and HYH activate PHR1 gene expression while the constitutively expressed nuclear-localized DET1 protein exerts a strong inhibitory effect. Altogether, the data presented indicate a dual role for DET1 in controlling expression of light-responsive and DNA repair genes, and describe more precisely the contribution of photomorphogenic regulators in the control of light-dependent DNA repair.

• Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 downregulated tomato fruit.

  Authors: Eugenia M A Enfissi, Fredy Barneche, Ikhlak Ahmed, Christiane Lichtlé, Christopher Gerrish, Ryan P McQuinn, James J Giovannoni, Enrique Lopez-Juez, Chris Bowler, Peter M Bramley, Paul D Fraser

  The Plant cell. 04/2010; 22(4):1190-215.

  Fruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects onFruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects on yield. In an attempt to further our understanding of how modulation of this gene leads to improved quality traits, detailed targeted and multilevel omic characterization has been performed. Metabolite profiling revealed quantitative increases in carotenoid, tocopherol, phenylpropanoids, flavonoids, and anthocyanidins. Qualitative differences could also be identified within the phenolics, including unique formation in fruit pericarp tissues. These changes resulted in increased total antioxidant content both in the polar and nonpolar fractions. Increased transcription of key biosynthetic genes is a likely mechanism producing elevated phenolic-based metabolites. By contrast, high levels of isoprenoids do not appear to result from transcriptional regulation but are more likely related to plastid-based parameters, such as increased plastid volume per cell. Parallel metabolomic and transcriptomic analyses reveal the widespread effects of DET1 downregulation on diverse sectors of metabolism and sites of synthesis. Correlation analysis of transcripts and metabolites independently indicated strong coresponses within and between related pathways/processes. Interestingly, despite the fact that secondary metabolites were the most severely affected in ripe tomato fruit, our integrative analyses suggest that the coordinated activation of core metabolic processes in cell types amenable to plastid biogenesis is the main effect of DET1 loss of function.

• Characterization of two members of the cryptochrome/photolyase family from Ostreococcus tauri provides insights into the origin and evolution of cryptochromes.

  Authors: Marc Heijde, Gérald Zabulon, Florence Corellou, Tomoko Ishikawa, Johanna Brazard, Anwar Usman, Frédéric Sanchez, Pascal Plaza, Monique Martin, Angela Falciatore, Takeshi Todo, François-Yves Bouget, Chris Bowler

  Plant, cell & environment. 04/2010; 33(10):1614-26.

  Cryptochromes (Crys) are blue light receptors believed to have evolved from the DNA photolyase protein family, implying that light control and light protection share a common ancient origin. In thisCryptochromes (Crys) are blue light receptors believed to have evolved from the DNA photolyase protein family, implying that light control and light protection share a common ancient origin. In this paper, we report the identification of five genes of the Cry/photolyase family (CPF) in two green algae of the Ostreococcus genus. Phylogenetic analyses were used to confidently assign three of these sequences to cyclobutane pyrimidine dimer (CPD) photolyases, one of them to a DASH-type Cry, and a third CPF gene has high homology with the recently described diatom CPF1 that displays a bifunctional activity. Both purified OtCPF1 and OtCPF2 proteins show non-covalent binding to flavin adenine dinucleotide (FAD), and additionally to 5,10-methenyl-tetrahydrofolate (MTHF) for OtCPF2. Expression analyses revealed that all five CPF members of Ostreococcus tauri are regulated by light. Furthermore, we show that OtCPF1 and OtCPF2 display photolyase activity and that OtCPF1 is able to interact with the CLOCK:BMAL heterodimer, transcription factors regulating circadian clock function in other organisms. Finally, we provide evidence for the involvement of OtCPF1 in the maintenance of the Ostreococcus circadian clock. This work improves our understanding of the evolutionary transition between photolyases and Crys.

• Overexpressing tagged proteins in plants using a modified gateway cloning strategy.

  Authors: Manu J Dubin, Chris Bowler, Giovanna Benvenuto

  Cold Spring Harbor protocols. 03/2010; 2010(3):pdb.prot5401.

  In recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations inIn recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations in many different organisms, including plants. Because of their versatility and ease of use, these methods have gained favor in low- and medium-throughput investigations as well. However, due to the recombination step, the resulting fusion proteins contain long and often highly charged polylinker sequences that can interfere with their physiological function. Furthermore, in some cases the gene of interest must be cloned twice (once with and once without a stop codon) for N- and C-terminal tagging. Here, we present a hybrid combinatorial cloning strategy that overcomes many of these limitations. In the first step, the gene of interest is cloned into an entry vector containing standardized cloning sites with the desired N- or C-terminal tag and an optimized polylinker sequence. A Gateway recombination reaction is used to transfer the protein-tag fusion from the entry clone to a Gateway destination vector with the desired promoter and selectable marker for the organism of interest. As experimental requirements evolve, constructs for expressing the protein of interest with the desired tag, promoter, and selectable marker or other features can rapidly and easily be created.

• Spectro-temporal characterization of the photoactivation mechanism of two new oxidized cryptochrome/photolyase photoreceptors.

  Authors: Johanna Brazard, Anwar Usman, Fabien Lacombat, Christian Ley, Monique M Martin, Pascal Plaza, Laetitia Mony, Marc Heijde, Gérald Zabulon, Chris Bowler

  Journal of the American Chemical Society. 03/2010; 132(13):4935-45.

  The photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochrome photolyase family (CPF), belonging to the green alga Ostreococcus tauri , was studied by broadband UV-visThe photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochrome photolyase family (CPF), belonging to the green alga Ostreococcus tauri , was studied by broadband UV-vis femtosecond absorption spectroscopy. Upon excitation of the protein chromophoric cofactor, flavin adenine dinucleotide in its oxidized form (FAD(ox)), we observed in both cases the ultrafast photoreduction of FAD(ox): in 390 fs for OtCPF1 and 590 fs for OtCPF2. Although such ultrafast electron transfer has already been reported for other flavoproteins and CPF members, the present result is the first demonstration with full spectral characterization of the mechanism. Analysis of the photoproduct spectra allowed identifying tryptophan as the primary electron donor. This residue is found to be oxidized to its protonated radical cation form (WH(*+)), while FAD(ox) is reduced to FAD(*-). Subsequent kinetics were observed in the picosecond and subnanosecond regime, mostly described by a biexponential partial decay of the photoproduct transient signal (9 and 81 ps for OtCPF1, and 13 and 340 ps for OtCPF2), with reduced spectral changes, while a long-lived photoproduct remains in the nanosecond time scale. We interpret these observations within the model proposed by the groups of Brettel and Vos, which describes the photoreduction of FADH(*) within E. coli CPD photolyase (EcCPD) as a sequential electron transfer along a chain of three tryptophan residues, although in that case the rate limiting step was the primary photoreduction in 30 ps. In the present study, excitation of FAD(ox) permitted to reveal the following steps and spectroscopically assign them to the hole-hopping process along the tryptophan chain, accompanied by partial charge recombination at each step. In addition, structural analysis performed by homology modeling allowed us to propose a tentative structure of the relative orientations of FAD and the conserved tryptophan triad. The results of preliminary transient anisotropy measurements performed on OtCPF2 finally showed good compatibility with the oxidation of the distal tryptophan residue (WH(351)) in 340 ps, hence, with the overall Brettel-Vos mechanism.