Conformational disorder analysis of the conditionally disordered protein CP12 from Arabidopsis thaliana in its different redox states

CP12 is a redox-dependent conditionally disordered protein universally distributed in oxygenic photosynthetic organisms. It is primarily known as a light-dependent redox switch regulating the reductive step of the metabolic phase of photosynthesis. In the present study, a small angle X-ray scattering (SAXS) analysis of recombinant Arabidopsis CP12 (AtCP12) in a reduced and oxidized form confirmed the highly disordered nature of this regulatory protein. However, it clearly pointed out a decrease in the average size and a lower level of conformational disorder upon oxidation. We compared the experimental data with the theoretical profiles of pools of conformers generated with different assumptions and show that the reduced form is fully disordered, whereas the oxidized form is better described by conformers comprising both the circular motif around the C-terminal disulfide bond detected in previous structural analysis and the N-terminal disulfide bond. Despite the fact that disulfide bridges are usually thought to confer rigidity to protein structures, in the oxidized AtCP12, their presence coexists with a disordered nature. Our results rule out the existence of significant amounts of structured and compact conformations of free AtCP12 in a solution, even in its oxidized form, thereby highlighting the importance of recruiting partner proteins to complete its structured final folding

Proceedings of 2010 Kentucky Water Resources Annual Symposium

This conference was planned and conducted as part of the state water resources research annual program with the support and collaboration of the Department of the Interior, U.S. Geological Survey and the University of Kentucky Research Foundation, under Grant Agreement Number 06HQGR0087. The views and conclusions contained in this document and presented at the symposium are those of the abstract authors and presenters and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Government or other symposium organizers and sponsors

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes

Abstracts of Papers, 81st Annual Meeting of the Virginia Academy of Science, May 27-30, 2003, University of Virginia, Charlottesville, Virginia

Abstracts of papers that were presented at the 81st Annual Meeting of the Virginia Academy of Science, May 27-30, 2003

Chemical profiling and biotechnological potential of marine microalgae in response to light and abiotic stress

Microalgae form the base of the aquatic food chain and have important ecological functions, including nutrient cycling and carbon capturing. These microscopic eukaryotes are incredibly diverse, with an estimated 72,000 extant species. They have been investigated for their biotechnological potential in industries such as nutraceutical, cosmetic, and biofuel. Most research has focused on specific high-value metabolites such as astaxanthin or β-carotene for human health, or classes of natural products such as polyunsaturated fatty acids for biofuels. However, a systematic untargeted approach to exploring the chemical diversity of microalgal metabolites has yet to be performed. Unlocking this chemical potential could provide further applications and incentives to the microalgal biotechnology sector. This thesis aims to fill this gap by exploring the chemical space of microalgae and the elicitation of further chemistry using abiotic stress. A comparative metabolomics study of 36 microalgal strains from both freshwater and marine environments showed that Haptophytes were a rich source of chemistry compared to the well-studied Chlorophytes. It also explored chemical diversity across strains of the same species, providing evidence that isolation environment rather than phylogenetic relationships could be used to group microalgae based on chemical profiles. To investigate the chemistry produced by three strains of marine microalgae, Dunaliella primolecta, Nannochloropsis oculata, and Phaeodactylum tricornutum were cultured under varying conditions of salinity, sodium chloride, nitrate, and pH and Global Natural Products Social (GNPS) molecular networking was used to gain insights into the effect of these stresses on metabolite production. A total of 2284 metabolites were detected across all strains and conditions, with 49% of those metabolites specific to cultures grown under stress conditions (i.e., not in the control). Salinity had the greatest effect with 22.8% of metabolites only produced under salinity stress. From comparison with over 33 libraries of mass spectral data, only five metabolites were identified, stressing the need for more open-access natural product -and specifically algal natural product - databases. Finally, we partnered with Xanthella Ltd., a marine biotechnology company in Scotland, to study the effect of 405 nm light on growth of four strains of microalgae and the production of antimicrobial metabolites. This wavelength has been shown to reduce bacterial contamination in cultures but is an expensive regimen to apply at a large scale. The production of high-value metabolites under this light regimen could enable culturing under 405 nm illumination to be economically viable. Although no bioactivity was observed from extracts or fractions, molecular networking did show that 16-25% of metabolites were either exclusively produced under 405 nm illumination or absent from the white light control condition. This thesis offers a starting point for fundamental and comparative research into microalgal growth and metabolite production and their applications in human health.Microalgae form the base of the aquatic food chain and have important ecological functions, including nutrient cycling and carbon capturing. These microscopic eukaryotes are incredibly diverse, with an estimated 72,000 extant species. They have been investigated for their biotechnological potential in industries such as nutraceutical, cosmetic, and biofuel. Most research has focused on specific high-value metabolites such as astaxanthin or β-carotene for human health, or classes of natural products such as polyunsaturated fatty acids for biofuels. However, a systematic untargeted approach to exploring the chemical diversity of microalgal metabolites has yet to be performed. Unlocking this chemical potential could provide further applications and incentives to the microalgal biotechnology sector. This thesis aims to fill this gap by exploring the chemical space of microalgae and the elicitation of further chemistry using abiotic stress. A comparative metabolomics study of 36 microalgal strains from both freshwater and marine environments showed that Haptophytes were a rich source of chemistry compared to the well-studied Chlorophytes. It also explored chemical diversity across strains of the same species, providing evidence that isolation environment rather than phylogenetic relationships could be used to group microalgae based on chemical profiles. To investigate the chemistry produced by three strains of marine microalgae, Dunaliella primolecta, Nannochloropsis oculata, and Phaeodactylum tricornutum were cultured under varying conditions of salinity, sodium chloride, nitrate, and pH and Global Natural Products Social (GNPS) molecular networking was used to gain insights into the effect of these stresses on metabolite production. A total of 2284 metabolites were detected across all strains and conditions, with 49% of those metabolites specific to cultures grown under stress conditions (i.e., not in the control). Salinity had the greatest effect with 22.8% of metabolites only produced under salinity stress. From comparison with over 33 libraries of mass spectral data, only five metabolites were identified, stressing the need for more open-access natural product -and specifically algal natural product - databases. Finally, we partnered with Xanthella Ltd., a marine biotechnology company in Scotland, to study the effect of 405 nm light on growth of four strains of microalgae and the production of antimicrobial metabolites. This wavelength has been shown to reduce bacterial contamination in cultures but is an expensive regimen to apply at a large scale. The production of high-value metabolites under this light regimen could enable culturing under 405 nm illumination to be economically viable. Although no bioactivity was observed from extracts or fractions, molecular networking did show that 16-25% of metabolites were either exclusively produced under 405 nm illumination or absent from the white light control condition. This thesis offers a starting point for fundamental and comparative research into microalgal growth and metabolite production and their applications in human health

Acclimation and adaptation to low-iron conditions in the marine diatoms Phaeodactylum tricornutum and Thalassiosira oceanica

In the open ocean phytoplankton growth is widely limited by the availability of iron, an essential element of the photosynthetic electron transport system. Survival under these conditions requires sophisti-cated strategies to maintain growth, e.g. lowering iron requirements and enhancing cellular iron affinity. In this work we used genomic and transcriptomic data to unravel acclimation (transcriptomics) and adaptation (genomics) to low-iron conditions in the two diatoms Thalassiosira oceanica and Phaeodactylum tricornutum, who both are highly tolerant to iron limitation. The acclimation response to low ambient iron concentrations is very similar in the two diatoms. Both undergo an extensive cellular retrenchment, best visible from chloroplast reduction and from the concomitant pigment loss that imposes a chlorotic phenotype on the cells. Growth rates are very low with a high degree of photosynthetic energy dissipation. The differential regulation of the genes for ferre-doxin (PETF) and flavodoxin (FLDA) and for class II and class I fructose-bisphosphate aldolases (FBA) indicates that cellular iron requirements are lowered by replacing abundant iron-rich proteins with iron-free substitutes. Genetically fixed features of T. oceanica and P. tricornutum that may represent beneficial adaptations to low-iron are the small cell sizes found for these species and the possession of class I FBA genes not present in the coastal diatom species Thalassiosira pseudonana. As adaptations specific to T. oceanica we observe the constitutive expres-sion of the plastocyanin gene (PCY) that encodes for a substitute of iron-containing cytochrome c6. The ferredoxin gene (PETF) has been transferred from the chloroplast to the nuclear genome in this species, facilitating its co-regulation with the flavodoxin gene (FLDA). The acclimation of diatoms to low-iron resembles the processes running off in green plants and algae upon low-iron stress. Thus, the response to low-iron represents an ancient cellular mechanism com-mon to most, if not all photosynthetic groups. Genetic adaptation to a persistent shortage of iron such as in the open ocean likely occurs by exploring strategies to optionally or permanently replace abundant iron-rich proteins by iron-free substitutes, thereby approximating the cellular iron requirements to a lowest possible level. The adaptive significance of substitution strategies is strengthened by their absence in coastal diatoms like T. pseudonana which is adapted to iron-rich coastal waters and is highly sensitive to low ambient iron concen-trations

Akklimatisierung und Anpassung an Eisenmangelbedingungen in den marinen Kieselalgen Phaeodactylum tricornutum und Thalassiosira oceanica

In the open ocean phytoplankton growth is widely limited by the availability of iron, an essential element of the photosynthetic electron transport system. Survival under these conditions requires sophisti-cated strategies to maintain growth, e.g. lowering iron requirements and enhancing cellular iron affinity. In this work we used genomic and transcriptomic data to unravel acclimation (transcriptomics) and adaptation (genomics) to low-iron conditions in the two diatoms Thalassiosira oceanica and Phaeodactylum tricornutum, who both are highly tolerant to iron limitation. The acclimation response to low ambient iron concentrations is very similar in the two diatoms. Both undergo an extensive cellular retrenchment, best visible from chloroplast reduction and from the concomitant pigment loss that imposes a chlorotic phenotype on the cells. Growth rates are very low with a high degree of photosynthetic energy dissipation. The differential regulation of the genes for ferre-doxin (PETF) and flavodoxin (FLDA) and for class II and class I fructose-bisphosphate aldolases (FBA) indicates that cellular iron requirements are lowered by replacing abundant iron-rich proteins with iron-free substitutes. Genetically fixed features of T. oceanica and P. tricornutum that may represent beneficial adaptations to low-iron are the small cell sizes found for these species and the possession of class I FBA genes not present in the coastal diatom species Thalassiosira pseudonana. As adaptations specific to T. oceanica we observe the constitutive expres-sion of the plastocyanin gene (PCY) that encodes for a substitute of iron-containing cytochrome c6. The ferredoxin gene (PETF) has been transferred from the chloroplast to the nuclear genome in this species, facilitating its co-regulation with the flavodoxin gene (FLDA). The acclimation of diatoms to low-iron resembles the processes running off in green plants and algae upon low-iron stress. Thus, the response to low-iron represents an ancient cellular mechanism com-mon to most, if not all photosynthetic groups. Genetic adaptation to a persistent shortage of iron such as in the open ocean likely occurs by exploring strategies to optionally or permanently replace abundant iron-rich proteins by iron-free substitutes, thereby approximating the cellular iron requirements to a lowest possible level. The adaptive significance of substitution strategies is strengthened by their absence in coastal diatoms like T. pseudonana which is adapted to iron-rich coastal waters and is highly sensitive to low ambient iron concen-trations.Im offenen Ozean ist das Phytoplankton-Wachstum häufig limitiert durch die Verfügbarkeit von Eisen, einem wesentlichen Bestandteil der photosynthetischen Elektronen-Transportkette. Damit Wachstum und Vermehrung aufrechterhalten werden können, muss der zelluläre Eisenbedarf gesenkt bzw. das Binde- und Aufnahmevermögens für Eisen erhöht werden. In der vorliegenden Arbeit werden Genom- und Transkriptomdaten genutzt, um Akklimatisierung (Transkriptomics) sowie Adaptation (Genomics) der Kieselalgen Thalassiosira oceanica und Phaeodactylum tricornutum an eisenarme Bedingungen zu unter-suchen. Beide Spezies sind sehr tolerant gegenüber Eisenmangel. Die Akklimatisierung an eisenarme Bedingungen verläuft in beiden Spezies sehr ähnlich. Die gesamte Zell-Biomasse wird stark reduziert, erkennbar an der Reduktion der Chloroplasten und dem damit einher-gehenden Pigmentverlust, welcher der Zelle einen ausgeblichenen, chlorotischen Phänotyp verleiht. Wachstumsraten sind extrem niedrig bei gleichzeitig hohem Anteil photosynthetisch nichtverwertbarer Lichtenergie, die ungenutzt abgeleitet wird. Die differentielle Regu-lation der Gene für Ferredoxin (PETF) und Flavodoxin (FLDA) sowie von Genen für Klasse II und Klasse I Fructosebisphosphataldolasen (FBA) weist darauf hin, daß der zelluläre Eisenbedarf gesenkt wird, indem eisenreiche durch eisenfreie Proteine ersetzt werden. Genetisch manifestierte Merkmale von T. oceanica und P. tricornu-tum, welche vorteilhafte Adaptationen an Eisenmangelbedingungen darstellen könnten, sind sowohl die geringen Zellgrößen beider Arten, als auch der Besitz von Klasse I FBA-Genen, welche nicht in der küstennah beheimateten Art Thalassiosira pseudonana vorkommen. Als für T. oceanica spezifische Adaptationen beobachten wir die konsti-tutive Expression des Plastocyanin-Gens (PCY), das ein Substitut für das eisenhaltige Cytochrom c6 kodiert. Das Ferredoxin-Gen (PETF) wurde in dieser Spezies vom Chloroplasten- zum Kerngenom verlagert, was seine Koregulation mit dem Flavodoxin-Gen (FLDA) erleichert. Die Akklimatisierung von Kieselalgen an Eisenmangelbedingungen ähnelt den Reaktionen von grünen Pflanzen und Algen auf Eisenstress und repräsentiert einen evolutionär alten Mechanismus, der den mei-sten, wenn nicht allen, photosynthetischen Organismen gemein ist. Genetische Adaptation an dauerhaften Eisenmangel, wie er im offenen Ozean vorherrscht, geschieht wahrscheinlich vorwiegend durch fortgesetztes Ausloten neuer Möglichkeiten, wie eisenreiche durch eisenarme Proteine ersetzt werden können, um somit den zellulären Eisenbedarf auf ein Minimum zu reduzieren. Die mögliche Bedeutung solcher Ersetzungsstrategien für evolutionäre Anpassung an Eisen-mangel wird unterstrichen durch deren Fehlen in Küsten-Spezies wie T. pseudonana, die an eisenreiche Standorte angepasst ist