The Plant Short-Chain Dehydrogenase (SDR) superfamily:genome-wide inventory and diversification patterns

Background Short-chain dehydrogenases/reductases (SDRs) form one of the largest and oldest NAD(P)(H) dependent oxidoreductase families. Despite a conserved 'Rossmann-fold' structure, members of the SDR superfamily exhibit low sequence similarities, which constituted a bottleneck in terms of identification. Recent classification methods, relying on hidden-Markov models (HMMs), improved identification and enabled the construction of a nomenclature. However, functional annotations of plant SDRs remain scarce. Results Wide-scale analyses were performed on ten plant genomes. The combination of hidden Markov model (HMM) based analyses and similarity searches led to the construction of an exhaustive inventory of plant SDR. With 68 to 315 members found in each analysed genome, the inventory confirmed the over-representation of SDRs in plants compared to animals, fungi and prokaryotes. The plant SDRs were first classified into three major types --- 'classical', 'extended' and 'divergent' --- but a minority (10 % of the predicted SDRs) could not be classified into these general types ('unknown' or 'atypical' types). In a second step, we could categorize the vast majority of land plant SDRs into a set of 49 families. Out of these 49 families, 35 appeared early during evolution since they are commonly found through all the Green Lineage. Yet, some SDR families --- tropinone reductase-like proteins (SDR65C), 'ABA2-like'-NAD dehydrogenase (SDR110C), 'salutaridine/menthone-reductase-like' proteins (SDR114C), 'dihydroflavonol 4-reductase'-like proteins (SDR108E) and 'isoflavone-reductase-like' (SDR460A) proteins --- have undergone significant functional diversification within vascular plants since they diverged from Bryophytes. Interestingly, these diversified families are either involved in the secondary metabolism routes (terpenoids, alkaloids, phenolics) or participate in developmental processes (hormone biosynthesis or catabolism, flower development), in opposition to SDR families involved in primary metabolism which are poorly diversified. Conclusion The application of HMMs to plant genomes enabled us to identify 49 families that encompass all Angiosperms ('higher plants') SDRs, each family being sufficiently conserved to enable simpler analyses based only on overall sequence similarity. The multiplicity of SDRs in plant kingdom is mainly explained by the diversification of large families involved in different secondary metabolism pathways, suggesting that the chemical diversification that accompanied the emergence of vascular plants acted as a driving force for SDR evolution

Generating and evaluating salinity and temperature resilient cyanobacteria for tropical outdoor cultivation in Australia

Global population levels, anticipated to increase to >9 billion by 2050, present serious worldwide challenges, such as energy-, food- and freshwater security. In addition, rising greenhouse gas (GHG) emissions lead to climatic instability, reduce the availability of freshwater and challenge agricultural productivity, which is exacerbated by decreasing arable land availability. Hence, the carbon- and freshwater-constrained economy demands industries to limit freshwater usage and carbon emissions. In this context, photosynthetic microalgae or cyanobacteria offer great promise for remediating carbon-dioxide emissions and high-nutient wastewaters, which can be coupled with renewable resource production to cater for large-volume low-value markets, such as animal feed, bio-fertiliser, and energy-production. The required scale of production for these markets, however, has to date not been realised, as outdoor cultivation presents severe challenges, including access to sufficient non-arable land in close proximity to water, nutrients (inorganic fertilisers: nitrogen and phosphate) and carbon-dioxide sources. In addition, high temperature and variable salinities are major limitations to cost-effective commercial microalgal production, as these factors are challenging to control. Ectoine, a valuable osmolyte, is produced by extremophile microbes in response to variable salinities and high temperature stress. Ectoine synthesis is mediated by an ectABC gene cassette. Given this, my research aimed at engineering a de novo biosynthesis pathway for ectoine production into the freshwater cyanobacterium Synechococcus elongatus PCC 7942 - to examine: a) its effect on temperature and/or salinity tolerance and b) potential downstream effects of ectoine on fertilisation requirements and biochemical profiles of this cyanobacterium, as the latter affects bio-product potential. Synechococcus elongatus PCC7942 was chosen as a model cyanobacterium, as its genome is small and fully sequenced, commercial vectors for transformation are available and it is exempt from restriction of laboratory transformation experiments by the Office of Gene Technology Regulator (OGTR). For ectABC transformation of S. elongatus PCC7942, a codon-optimised ectABC_pSyn_6 plasmid was constructed, based on the ectABC gene nucleotide sequence from the temperature- and salinity-tolerant bacterium Halomonas elongata DSM4043. ectABC-transformed, untransformed pSyn_6 vector (lacking ectABC insert) controls and wild-type (no vector, WT) S. elongatus PCC 7942 were subjected to a three temperature (35, 40, and 45°C), three salinity (0, 18, 36 ppt) factorial design experimental challenge without acclimation. Our data confirmed that ectABC-transformed S. elongatus PCC7942 had improved temperature tolerance up to 45°C and salinity tolerance up to 18 ppt at 35°C, compared to WT and pSyn_6 empty vector controls. Limited growth was observed at 36 ppt salinity in WT, pSyn-6 and ectABC transformants, irrespective of temperature. ectABC-transformant population growth rates were highest at 35°C. High pressure liquid chromatography analysis of these ectABC transformants confirmed ectoine production, albeit minimal. Further studies are necessary at the molecular level to resolve impediments associated with the low level of ectoine expression, should ectoine be chosen as a high-value co-product for the cosmetics industry. In terms of commercial production, it is vital to assess ectABCtransformed S. elongatus PCC 7942 fertilisation requirements. Results showed that nitrogen-requirements of ectABC-transformants were higher than that of WT and pSyn_6 empty vector controls at an elevated salinity of 18 ppt, but lower at 45°C temperature stress. Phosphate uptake was lowest in ectABC-transformants at temperature and salinity stress of 45°C and 18 ppt, respectively. Fertilisation costs require serious consideration for commercial-scale cultivation of large-volume, low-value bio-products markets. Thus, the stress-induced increased nitrogen fertilisation requirements of ectABC-transformants suggest that co-location with nitrogen-rich wastewater streams would be beneficial, thereby also reducing nutrient run-off into the local river systems. Regarding the biochemical profile of hydrocarbon-based biofuel production, ectABC transformants had increased lipid and fatty acid production under both temperature (45°C) and salinity (18 ppt) stress. Thus, this research addresses an area of importance for transitioning to a bio‐economy as a whole and for implementing environmentally and economically sustainable production of renewable biofuels, animal feed, bio-fertilisers, which are perhaps best achieved through co-production of some high-value bio-products, such as ectoine or the high-value pigment - c-phycocyanin. To investigate this potential, a modelling approach using multi-criteria analysis and geographical information system analysis was adopted. ArcGIS was used to evaluate potential sites suitable for co‐locating microalgal and sugarcane production in the Great Barrier Reef (GBR) catchment region in Queensland, Australia – whilst taking into account climatic, land-use and economic factors that consider energy balances for each facility. Critical resource inputs such as land, water, CO₂, energy and climatic factors such as temperature and rainfall were considered when estimating the available resources at sugar mills in the Wet Tropics region, adjacent to the GBR. Our economic analysis revealed that co-locating microalgal biomass production with such an industry is economically feasible in the Wet Tropics, by achieving significant cost-reductions and improved economic performance. As such, this research produces valuable information for investors, policy makers, government and industry to make informed decisions about the location potential for microalgal production sites that focus on salinity and temperature-resilient microalgal cultivation for high-value compounds (e.g. the osmolyte ectoine) or low-value animal feed as their principal commodity, whilst reducing CO₂ emissions and nutrient runoff to the GBR, both of which attract tradeable credits which offer additional economic returns over and above the returns from the production and sales process

New tools and strategies for metabolic engineering of the algal chloroplast

Microalgae are attractive as cell factories for production of bioactive metabolites, therapeutic proteins and high-value metabolites. Of the several microalgae that have been explored as potential biotechnological platforms, the unicellular chlorophyte Chlamydomonas reinhardtii, is the most genetically tractable given its long history as a model species for molecular- genetic studies of cell biology. In particular, the chloroplast of C. reinhardtii represents a novel sub-cellular compartment for synthesis and accumulation of recombinant products. It possesses a small, genetically tractable genome and lacks any gene-silencing mechanisms. This allows stable and high-level expression of multiple transgenes. However, the exploitation of this microalgal platform requires further advances in the molecular tools available for metabolic engineering, together with new strategies for large-scale cultivation such as simple methods for ensuring ‘crop protection’ to reduce invasion and growth of contaminating species. Due to the increasing scarcity of phosphate reserve, phosphite is an alternative P source at economical cost and can reduce demand on non-renewable phosphate fertiliser. A key goal of my project was therefore to develop a strain improvement strategy based on the expression in the chloroplast of the bacterial gene ptxD encoding an NAD(P)-dependent phosphite oxidoreductase to allow utilization of phosphite as a source of phosphorus (P). The approach is based on the fact that most organisms cannot use phosphite, therefore, growing the transgenic microalga in phosphite provides a selective advantage over competing species. The ptxD gene was successfully introduced into chloroplast and shown to produce a functional enzyme that allowed growth on phosphite media. This allows engineered strains to be grown in non-sterile medium without significant spoilage by bacteria or fungi, thereby avoiding costly medium sterilization and culture management. Furthermore, it was demonstrated that ptxD can serve as a new non-antibiotic selectable marker for chloroplast transformation, allowing direct selection of transformants for their phosphite-utilising activity. This increases the repertoire of available selectable markers and reduces the use of antibiotics. Having developed these tools, the metabolic engineering of the chloroplast was attempted by introducing a synthetic gene encoding limonene synthase (LS). Limonene is a high-value terpenoid that has applications as a pharmaceutical, a flavour and a fragrance. Whilst the LS protein was successfully produced in the chloroplast, detailed GC-MS analysis failed to detect limonene synthesis suggesting issues with either functionality of the enzyme or availability of substrate. However, the work adds important new tools to the molecular toolbox for advancing C. reinhardtii chloroplast as an expression platform

Uso del nitrógeno en algas: desvelando piezas del rompecabezas de la asimilación del nitrógeno y su regulación en el alga modelo Chlamydomonas reinhardtii

Las algas, formando parte de la base de la cadena trófica de ecosistemas marinos y de agua dulce, son clave para la vida acuática. Estos organismos fotosintéticos, sometidos a fluctuaciones constantes de disponibilidad de nutrientes, muestran un alto nivel de adaptabilidad a estos ambientes dinámicos. Aunque el nitrógeno (N), nutriente esencial para la vida, es comúnmente usado por las algas en su forma inorgánica, algunas especies de algas pueden usar compuestos de N orgánico, los cuales pueden ser especialmente abundantes debido a la escorrentía y filtrado de áreas fertilizadas de forma intensiva. El alga modelo Chlamydomonas reinhardtii (Chlamydomonas) puede consumir fuentes de nitrógeno inorgánico (amonio, nitrato y nitrito), así como L-arginina y urea. Además, este alga presenta una Laminoácido oxidasa extracelular (LAO1) que desamina un amplio rango de aminoácidos. En este trabajo hemos estudiado el control de la señalización que da lugar a la preferencia de nitrato sobre N orgánico en Chlamydomonas, el papel clave de LAO1 en el uso de aminoácidos y péptidos, así como el establecimiento de nuevas interacciones mutualistas con bacterias que promueven el crecimiento en N orgánico. Capítulo 1 El factor de transcripción NIT2 es el regulador clave de los genes de la asimilación de nitrato en Chlamydomonas. En primer lugar, comparamos el transcriptoma de una estirpe silvestre y otra mutante nit2 de Chlamydomonas en respuesta a nitrato. Observamos que nitrato y NIT2 reprimen los genes involucrados en el uso de fuentes de N orgánicas, incluyendo LAO1. Mediante el uso de mutantes de Chlamydomonas demostramos que tanto el nitrato como el nitrito afectan negativamente el uso de aminoácidos por este alga. Capítulo 2 Las enzimas L-aminoácido oxidasa (LAAO, L-Amino Acid Oxidase) están ampliamente distribuidas en la naturaleza y se propone que su papel principal en hongos y algas es la captación de nitrógeno. Mediante búsquedas genómicas comparativas, no pudimos encontrar ningún ortólogo de LAO1 en ningún alga verde ni en plantas, pero identificamos ortólogos en 10 de otras 27 especies de algas, incluyendo Rhodophyta, Alveolata, Heterokonta, Haptophyta y Dinophyta. La construcción de un árbol filogenético de enzimas LAAO reveló que las secuencias identificadas como ortólogas de LAO1 -denominadas aquí como ALAAOs (Algal LAAOs)-, se agrupaban en la misma rama evolutiva. Observamos que en Chlamydomonas el gen LAO1 está situado adyacente a un gen que codifica una putativa proteína RidA, que resultó estar evolutivamente cercana a la de cianobacterias. Nuestro análisis filogenético apoya la idea de que las proteínas ALAAOs pueden tener un origen en el ancestro común de las algas, el cual se originó por la endosimbiosis de una cianobacteria por un protista. Mediante el uso de un mutante lao1 hemos mostrado que LAO1 era crucial para el crecimiento de Chlamydomonas en 16 de 20 aminoácidos proteinogénicos, así como para algunos di-/tri-péptidos. Además de amonio, las enzimas LAAO producen el correspondiente cetoácido y peróxido de hidrógeno. Hemos demostrado que la reacción espontánea de los productos derivados de la desaminación por LAO1 de Lalanina -ácido pirúvico y peróxido de hidrógeno- genera ácido acético. Capítulo 3 Aunque Chlamydomonas puede crecer en la mayoría de los L-aminoácidos y en algunos di-/tri-péptidos como únicas fuentes de N, este crecimiento es mucho menos eficiente que en fuentes de N inorgánicas, y además, algunos aminoácidos y péptidos no pueden ser usados por este alga. De forma fortuita descubrimos una contaminación Methylobacterium sp. que permitió el crecimiento de Chlamydomonas en un di-péptido que no puede asimilar. Las especies de Methylobacterium están incluidas en el grupo de bacterias promotoras del crecimiento de plantas (PGPB, del inglés Plant Growth- Promoting Bacteria), las cuales mejoran el crecimiento de las plantas. Tras el muestreo en campo, aislamiento e identificación de bacterias, encontramos que algunas especies salvajes, incluidas en los géneros Methylobacterium, Sphingomonas, Deinococcus y Chitinophagaceae, mejoran el crecimiento de Chlamydomonas en L-serina. Además, algunas especies de Methylobacterium permitieron el crecimiento de Chlamydomonas en algunos aminoácidos y péptidos que este alga no puede usar. Hemos demostrado un nuevo mutualismo basado en un intercambio metabólico de carbono y nitrógeno entre Chlamydomonas y M. aquaticum. Por otro lado, algunas especies de Methylobacterium mejoraron el crecimiento de Chlamydomonas en aminoácidos asimilables. Para esta mejora, la enzima LAO1 fue esencial para el crecimiento del consorcio con algunas estirpes de Methylobacterium, incluyendo M. extorquens, M. hispanicum y M. organophilum. La comunicación química en la interacción entre organismos diferentes media las relaciones simbióticas. Entre estas moléculas de señalización, el ácido indolacético es una de las más estudiadas. Descubrimos que la producción de índoles dependiente de L-triptófano por Chlamydomonas, observada aquí por primera vez, disminuyó significativamente en el mutante lao1. Además, observamos que altas concentraciones de ácido indolacético (> 30 μM) inhibe el crecimiento de Chlamydomonas y que esta inhibición se puede reducir por la presencia de especies de Methylobacterium.Algae, lying on the basis of food webs in marine and freshwater ecosystems, are key for aquatic life. These photosynthetic organisms live under continuously fluctuating nutrients availability, showing a high level of adaptability to these dynamic environments. Although the essential nutrient Nitrogen (N) is usually used by algae in its inorganic form, some algal species can use organic N compounds, which may become especially abundant due to terrestrial leaking and runoff of highly fertilized areas. The model alga Chlamydomonas reinhardtii (Chlamydomonas) uptakes inorganic N sources (i.e. ammonium, nitrate and nitrite), as well as L-arginine and urea. Moreover, this alga presents an extracellular L-amino acid oxidase (LAO1) with a broad substrate specificity that scavenges N from L-amino acids. In this work we studied the signaling control that leads the preference for nitrate over organic N in Chlamydomonas, the key role of LAO1 in the use of amino acids and peptides, as well as the establishment of new mutualistic interactions with bacteria to facilitate growth on organic N. Chapter 1 The transcription factor NIT2 is the key regulator of nitrate assimilation genes in Chlamydomonas. First, we compared the transcriptome of Chlamydomonas wild type (WT) and a nit2 mutant in response to nitrate. We observed that nitrate and NIT2 down-regulated genes involved in organic N scavenging, including LAO1. By the use of Chlamydomonas mutant strains we demonstrated that both nitrate and nitrite negatively impact the use of amino acids by this alga. Chapter 2 L-amino acid oxidase (LAAO) enzymes are widely present in nature and a major role as N scavenger has been proposed in fungal and algal LAAOs. By comparative genomic searches, we could not find any LAO1 ortholog in any green plant or plant, but we identified orthologs in 10 out of 27 other algal species, including Rhodophyta, Alveolata, Heterokonta, Haptophyta and Dinophyta algae. The construction of a LAAO phylogenetic tree revealed that algal protein sequences identified as LAO1 orthologs -named here as ALAAOs (Algal LAAOs)-, clustered on the same evolutionary branch. We observed that Chlamydomonas LAO1 gene is clustered to a putative RidA gene (LAO2), which resulted to be closely related to cyanobacterial members. Our phylogenetic analysis favoured the idea that ALAAOs may have a common origin in the archaeplastidan ancestor, originated by a protist engulfing cyanobacteria. By the use of a lao1 mutant, we showed that LAO1 was crucial for Chlamydomonas growth on 16 out of 20 proteinogenic amino acids, as well as for some di- and tripeptides. Besides ammonium, LAAO produces keto acids and hydrogen peroxide. We have demonstrated that the spontaneous reaction of the LAO1-derived products generated by L-alanine deamination, pyruvic acid and hydrogen peroxide, generates acetic acid. Chapter 3 Although Chlamydomonas can grow on most amino acids and some di-/tripeptides as the sole N sources, this growth is far less efficient than that on inorganic N, and yet, there are some amino acids and peptides that cannot be used by this alga. We serendipitously found a contaminating Methylobacterium sp. that allowed Chlamydomonas growth on a dipeptide that is not readily assimilated by this alga. Methylobacterium spp. are included in the PGPB group of bacteria (Plant Growth-Promoting Bacteria), which improve plant growth and fitness. After field sampling, isolation and identification of some bacteria, we found that some wild species, included in Methylobacterium, Sphingomonas, Deinococcus, and Chitinophagaceae genera, promoted Chlamydomonas growth on L-serine. Moreover, some Methylobacterium spp. allowed Chlamydomonas growth on amino acids and peptides that are not used by this alga. We have demonstrated a new mutualism based on carbon-nitrogen metabolic exchange between Chlamydomonas and M. aquaticum. Otherwise, some Methylobacterium spp. improved Chlamydomonas growth on assimilable amino acids. For this growth promotion, LAO1 was crucial for consortia growth with some Methylobacterium spp., including M. extorquens, M. hispanicum and M. organophilum. The chemical cross-talk between interacting organisms mediates the beneficial and pathogenic symbiotic relationships. Within these inter-kingdom signal molecules IAA (Indole-3-Acetic Acid) is one of the best studied. We found that L-tryptophan-dependent indoles production in Chlamydomonas, observed here for the first time, was significantly reduced in the lao1 mutant. Moreover, we observed that high levels of exogenously added IAA (> 30 μM) inhibits Chlamydomonas growth and that this inhibition may be relieved by the presence of Methylobacterium spp

A combined modelling and experimental characterisation of Chlamydomonas reinhardtii under monochromatic LED illumination

Industrial biotechnology is currently synonymous with heterotrophic processes that rely on bacterial, yeast, insect or mammalian cells to biosynthesise products of interest. Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites and their ability to grow under a variety of trophic strategies, including phototrophy. Despite significant process development and optimisation efforts, the full potential of these photosynthetic organisms has yet to be realised. One of the most impactful process parameters when cultivating microalgae is light. It is essential for phototrophic growth and remains highly influential on mixotrophic growth. Indoor cultivations relying on artificial light allow full control of illumination conditions. The advent of LED lights has lowered the costs and improved the flexibility of such installations. Specifically, the spectral composition of LED lights can be accurately and dynamically tailored to the needs of the culture. Spectral composition is known to exert regulatory control over the cell cycle and can affect the cell’s biochemical make up. The effects of illumination strategy on the model microalgae Chlamydomonas reinhardtii were characterised at three different levels (a) growth kinetics, (b) biochemical composition and, (c) transcriptional activity at key carbon nodes. To obtain the transcriptional data, RNA extraction protocols were compared and optimised. Additionally, a suite of candidate reference genes was validated to ensure accurate gene expression normalisation was possible in reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) studies. The growth kinetics and biochemical composition data obtained served as inputs for a previously published genome scale metabolic model. An algorithm was developed to approximate the default biomass composition in the model to experimental data in an effort to increase the fidelity of the simulations. The flux distributions obtained thereafter helped to describe the distinct metabolic fingerprints created under different trophic and illumination strategies

Molecular genetics of the immotile short tail sperm defect

v2009okDiss. : Turku : Turun yliopisto, 200

Molecular genetics of the immotile short tail sperm defect

Hedelmättömyyttä aiheuttavan siittiöiden puolihäntävian molekyyligenetiikka Suomalaisissa Yorkshire karjuissa yleistyi 1990-luvun lopulla autosomaalisesti ja resessiivisesti periytyvä hedelmättömyyttä aiheuttava siittiöiden puolihäntävika (ISTS, immotile short tail sperm). Sairaus aiheuttaa normaalia lyhyemmän ja täysin liikkumattoman siittiön hännän muodostuksen. Muita oireita sairailla karjuilla ei ole havaittu ja emakot ovat oireettomia. Tämän tutkimuksen tarkoituksena oli kartoittaa siittiöiden puolihäntävian aiheuttava geenivirhe ja kehittää DNA-testi markkeri- ja geeniavusteiseen valintaan. Koko genomin kartoituksessa vian aiheuttava alue paikannettiin sian kromosomiin 16. Paikannuksen perusteella kahden geenimerkin haplotyyppi kehitettiin käytettäväksi markkeri-avusteisessa valinnassa. Sairauteen kytkeytyneen alueen hienokartoitusta jatkettiin geenitestin kehittämiseksi kantajadiagnostiikkaan. Vertailevalla kartoituksella oireeseen kytkeytynyt alue paikannettiin 2 cM:n alueelle ihmisen kromosomiin viisi (5p13.2). Tällä alueella sijaitsevia geenejä vastaavista sian sekvensseistä löydetyn muuntelun perusteella voitiin tarkentaa sairauteen kytkeytyneitä haplotyyppejä. Haplotyyppien perusteella puolihäntäoireeseen kytkeytynyt alue rajattiin kahdeksan geenin alueelle ihmisen geenikartalla. Alueelle paikannetun kandidaattigeenin (KPL2) sekvensointi paljasti introniin liittyneen liikkuvan DNA-sekvenssin, Line-1 retroposonin. Tämä retroposoni muuttaa geenin silmikointia siten, että sitä edeltävä eksoni jätetään pois tai myös osa introni- ja inserttisekvenssiä liitetään geenin mRNA tuotteeseen. Molemmissa tapauksissa tuloksena on lyhentynyt KPL2 proteiini. Tähän retroposoni-inserttiin perustuva geenitesti on ollut sianjalostajien käytössä vuodesta 2006. KPL2 geenin ilmenemisen tarkastelu sialla ja hiirellä paljasti useita kudosspesifisiä silmikointimuotoja. KPL2 geenin pitkä muoto ilmenee pääasiassa vain kiveksessä, mikä selittää geenivirheen aiheuttamat erityisesti siittiön kehitykseen liittyvät oireet. KPL2 proteiinin ilmeneminen hiiren siittiön hännän kehityksen aikana ja mahdollinen yhteistoiminta IFT20 proteiinin kanssa viittaavat tehtävään proteiinien kuljetuksessa siittiön häntään. Mahdollisen kuljetustehtävän lisäksi KPL2 saattaa toimia myös siittiön hännän rakenneosana, koska se paikannettiin valmiin siittiön hännän keskiosaan. Lisäksi KPL2 proteiini saattaa myös toimia Golgin laitteessa sekä Sertolin solujen ja spermatidien liitoksissa, mutta nämä havainnot kuitenkin vaativat lisätutkimuksia. Tämän tutkimuksen tulokset osoittavat, että KPL2 geeni on tärkeä siittiön hännän kehitykselle ja sen rakennemuutos aiheuttaa siittiöiden puolihäntäoireen suomalaisilla Yorkshire karjuilla. KPL2 proteiinin ilmeneminen ja paikannus siittiön kehityksen aikana antaa viitteitä proteiinin toiminnasta. Koska KPL2 geenisekvenssi on erittäin konservoitunut, nämä tulokset tuovat uutta tietoa kaikkien nisäkkäiden siittiöiden kehitykseen ja urosten hedelmättömyyteen syihin.The immotile short tail sperm (ISTS) defect is an autosomal recessive disease within the Finnish Yorkshire pig population. The defect is expressed in males as a shorter sperm tail length and immotile spermatozoa. Histological examination of spermatozoa from ISTS affected boars indicates that the axonemal complex and accessory structures of the sperm tail are severely compromised. Cilia in respiratory specimens from ISTS boars are physiologically normal and no adverse effects on reproductive performance of female relatives have been observed suggesting that other ciliated cell types are not influenced. The principal aim of this study was to map the ISTS associated chromosomal region and develop a DNA-test for marker and gene assisted selection within the Finnish Yorkshire pig population. In the initial genome wide screen the disease locus was mapped on porcine chromosome 16 within a 3 cM region and a two-marker-haplotype was developed for marker-assisted selection within analyzed families. The disease associated region was further fine-mapped in order to develop a 100% specific test for carrier detection. The disease-associated area was located to a 2 cM region on human chromosome 5p13.2 and polymorphisms from orthologous porcine genes within this region defined the disease-associated haplotype to include 8 genes in the human. Sequence analysis of the most probable candidate, KPL2, revealed the presence of an inserted Line-1 retrotransposon within an intron. The insertion affects splicing of the KPL2 transcript via skipping of the upstream exon or by causing the inclusion of an intronic sequence, as well as part of the insertion in the transcript. Both changes alter the reading frame leading to premature termination of translation. Since 2006 gene assisted selection for ISTS based on this insertion sequence has been made available to pig breeders in Finland. KPL2 expression profiling revealed various tissue specific transcript variants. The long form of KPL2 including the aberrantly spliced exon is expressed predominantly in porcine testicular tissue, which explains the tissue-specificity of the ISTS defect. Localization of the KPL2 protein in the murine testis and a possible interaction with IFT20 indicate a role in the delivery of flagellar proteins. Furthermore, the presence of KPL2 in the sperm tail midpiece also suggests a structural function in the sperm tail. Possible associations of the KPL2 protein with the Golgi complex and Sertoli cell/spermatid junction require further investigation. Current results show that the KPL2 gene is important for correct sperm flagella development. Disruption of this process is responsible for the ISTS defect in Finnish Yorkshire boars. Expression and interaction studies of the KPL2 protein allowed the function of KPL2 to be elucidated. Due to the highly conserved nature of spermatogenesis these results provide novel insights into sperm tail development and male infertility disorders in all mammalian species.Siirretty Doriast

The regulatory network adjusting light-harvesting in the model green alga Chlamydomonas reinhardtii

Berger H. The regulatory network adjusting light-harvesting in the model green alga Chlamydomonas reinhardtii. Bielefeld: Bielefeld University; 2015.In photosynthetic organisms, control of light-harvesting is a key component of acclimation mechanisms that optimize photon conversion efficiencies. In this thesis, the interrelation of short- and long-term regulation of light-harvesting at photosystem II (PSII) was analyzed in the green alga Chlamydomonas reinhardtii. This model organism is able to gain carbon and energy through photosynthetic carbon dioxide fixation as well as heterotrophic feeding. A lowered inorganic or increased organic carbon supply reduces the rate of NADPH consumption by the Calvin cycle, resulting in an over-reduced photosynthetic electron transport chain and increased excitation pressure at photosystem II. A combination of molecular biology, biochemistry, chlorophyll fluorescence and physiological analyses revealed that a reduction in functional antenna size efficiently relieved excitation pressure on PSII under these conditions. Particularly, translation control on PSII-associated major light-harvesting proteins (LHCII) replaced state transitions as an initial protection mechanism in the long term. The LHCII translation repressor NAB1 emerged as key factor implicated in the acclimation to the prevailing carbon assimilation mode. The level of NAB1 was increased under carbon dioxide limitation, and expression control based on modulated promoter activity. Application of a photosynthetic electron transport inhibitor and a perturbed NAB1 accumulation in a state transition mutant suggested that chloroplast retrograde signals control nuclear NAB1 expression. To further investigate this retrograde signaling, a reporter system was developed that enables detailed promoter analyses. Systematic truncation studies identified a promoter fragment of 152 bases, which comprised essential regulatory elements and can be used as tool for the identification of cis-regulatory elements in future studies. Furthermore, chloroplast redox poise was shown to modulate the extent of LHCII translation repression in the cytosol via cysteine based redox control of NAB1. In response to moderate light intensity changes, a fine-tuning system comprising specific single cysteine nitrosylation and thioredoxin mediated re-reduction adjusted NAB1 activity to the demand for light-harvesting antenna proteins. This is the first mechanistic description of redox based translation control of nuclear encoded photosynthesis associated genes. Overall, this thesis describes regulatory circuits that adjust light-harvesting capacity over a range of time scales, involving nuclear and cytosolic expression control as well as short-term responses in the chloroplast, and provides new insights into interorganellar communication that ensures optimal photon capture

Isolation and characterisation of the cyanobacterial chlorophyll F synthase

The D1 protein encoded by the psbA gene forms the reaction centre of photosystem II (PSII) and provides most of the ligands for binding the Mn4CaO5 cluster. Although the protein is conserved among cyanobacteria, five groups of D1 (G0-G4) have been identified, some of which (G0, G1 and G2) appear from sequence comparisons to be non-functional for oxygen evolution. The G1 group of D1, which contains so-called super-rogue versions of D1 (srD1), is expressed in cyanobacteria that synthesise chlorophyll f (Chl f) during far-red light photoacclimation (FaRLiP). Ho et al. (2016) have recently suggested that the srD1 is the chlorophyll f synthase (ChlF) and might form a homodimeric complex in the membrane, but the biochemical evidence is currently lacking. In this thesis I show that heterologous expression of srD1 encoded by the chlF gene of Chroococcidiopsis thermalis PCC 7203 in Synechocystis PCC 6803 leads to the production of Chl f. Two-dimensional (2D) polyacrylamide gel electrophoresis analysis of immuno-purified FLAG-ChlF indicated that ChlF forms a heterodimer with D2 and is incorporated into a novel type of monomeric PSII complex, termed the super-rogue PSII complex. Immunoblotting experiments and mass spectrometry revealed the presence of several low-molecular-mass subunits of PSII and Psb27. 77K fluorescence spectra of the isolated FLAG-ChlF complex showed a peak at 715 nm, consistent with the presence of Chl f. Characterisation of mutants lacking PSII core protein subunits indicated that the presence of CP43, CP47, and D2 was required for the accumulation of Chl f. Analysis of PSII activity in cells showed that the PSII complex containing ChlF was not functional for water oxidation, suggesting that this particular type of complex acts as a Chl f-producing PSII. Characterisation of chimeric D1 mutants containing portions of ChlF indicated that a QD motif in ChlF is vital for the production of Chl f. In addition, the chlF gene (3XFLAG-tagged) was successfully integrated into the Chlamydomonas reinhardtii chloroplast genome. Immunoblotting experiments of thylakoid membranes separated by 2D gel electrophoresis revealed that FLAG-ChlF was present in a large complex that co-migrated with a monomeric PSII fraction. Overall, this work identifies a new type of PSII complex which plays a physiological role in Chl f biosynthesis rather than water splitting.Open Acces

Current Challenges in Modeling Cellular Metabolism

Mathematical and computational models play an essential role in understanding the cellular metabolism. They are used as platforms to integrate current knowledge on a biological system and to systematically test and predict the effect of manipulations to such systems. The recent advances in genome sequencing techniques have facilitated the reconstruction of genome-scale metabolic networks for a wide variety of organisms from microbes to human cells. These models have been successfully used in multiple biotechnological applications. Despite these advancements, modeling cellular metabolism still presents many challenges. The aim of this Research Topic is not only to expose and consolidate the state-of-the-art in metabolic modeling approaches, but also to push this frontier beyond the current edge through the introduction of innovative solutions. The articles presented in this e-book address some of the main challenges in the field, including the integration of different modeling formalisms, the integration of heterogeneous data sources into metabolic models, explicit representation of other biological processes during phenotype simulation, and standardization efforts in the representation of metabolic models and simulation results