Transcriptional and functional targets of SCHIZORIZA in root development
Liere, Sabine van - \ 2017
Wageningen University. Promotor(en): B.J.G. Scheres, co-promotor(en): R. Heidstra. - Wageningen : Wageningen University - ISBN 9789463437998 - 122
arabidopsis thaliana - biological development - root meristems - root caps - cell division - stem cells - transcriptomics - gene regulation - mutagenesis - arabidopsis thaliana - biologische ontwikkeling - wortelmeristemen - wortelmutsjes - celdeling - stamcellen - transcriptomica - genregulatie - mutagenese
In this thesis I focus on SCHIZORIZA, a gene involved in tissue specification and cell fate segregation in the Arabidopsis root. Chapter 1 describes asymmetric cell division, Arabidopdis embryo development and root meristem development. In more detail we describe the maintenance of quiescent centre and columella stem cells, the development of ground tissue and epidermis/ lateral root cap. Finally we introduce SCHIZORIZA (SCZ) as a factor involved in radial patterning and the maintenance of cortex identity.
In Chapter 2, we study the interaction between the SCZ and SHORTROOT/ SCARECROW pathways that are required in parallel during stem cell niche specification in embryogenesis for the maintenance of tissue fates. Here we investigate the strong synergy of shr and scz mutants and show that at late torpedo stage scz;shr double mutant embryos lose both ground tissue and meristem marker expression.
Chapter 3 describes the use of a transcriptomics approach to identify genes differentially regulated by SCZ. These differentially regulated genes can be divided into two distinct tissue enriched groups. Upregulated genes are enriched for root cap expression and cortex expressed genes are overrepresented in the downregulated set of genes. A subset of the upregulated genes has a HSE associated with their promoter and therefore possibly represents direct SCZ targets.
In Chapter 4 we describe a mutagenesis screen to identify functional downstream targets of SCZ. Using a cortex and lateral root cap tissue marker, we identified two suppressors of the scz mutant. Both restore the fate segregation phenotype of scz mutants. We used whole genome deep sequencing to map the causal suppressor mutations in the LBD12 gene.
The analysis of LBD12 function is described in Chapter 5. We show that the single lbd12 mutant has a QC and columella phenotype. In addition, we show that ectopic expression of LBD12 induces ectopic divisions.
Unraveling the genetics of Botrytis cinerea resistance in Gerbera hybrida
Fu, Yiqian - \ 2017
Wageningen University. Promotor(en): R.G.F. Visser, co-promotor(en): P.F.P. Arens; J.M. van Tuyl. - Wageningen : Wageningen University - ISBN 9789463431811 - 159
gerbera - plant pathogenic fungi - botrytis cinerea - disease resistance - genetic mapping - transcriptomics - quantitative trait loci - plant breeding - gerbera - plantenziekteverwekkende schimmels - botrytis cinerea - ziekteresistentie - genetische kartering - transcriptomica - loci voor kwantitatief kenmerk - plantenveredeling
Gerbera hybrida is one of the top five cut flowers. It is well-known to people for its variation in flower color and patterning. Gerbera breeding at the moment is done using conventional methods which are based on a phenotypic selection. This has drawbacks in breeding speed and efficiency, especially for complex traits like disease resistance. Gerbera gray mold, promoted by high humidity during the production in greenhouses or by an accumulation of condensate during transportation, is a considerable threat to the gerbera production. Gerbera gray mold is caused by Botrytis cinerea and plant resistance to B. cinerea is considered to be a polygenic trait that needs the contribution of multiple loci, and on top of that is highly affected by the environment. Conventional breeding might be inefficient for improving Botrytis resistance in gerbera.
In this study, the transcriptomes of four parents of two gerbera populations were sequenced using Illumina paired-end sequencing. Transcriptome data provides a resource for genetic dissection and an insight to explore gene functions for this ornamental crop. To identify the QTL regions leading to the phenotypic variation in Botrytis resistance, and establishing a relationship between marker genotype and phenotypic variation for marker assisted selection (MAS), genetic linkage maps were constructed with SNP markers in the two F1 segregating populations. A total of 20 QTLs were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflects the complex mechanism of Botrytis disease response. Narrowing down the QTL region and identifying the causal gene(s) underlying a QTL could maximize the effective use of MAS in breeding. Homologs of known functional genes involved in Botrytis resistance from other species were obtained in gerbera and SNP markers identified and mapped. Twenty-nine candidate genes were mapped and seven candidate genes could be mapped on both populations. Seven candidate genes were located in the vicinity of the QTLs detected. The co-localization of QTLs with CGs gives an indication that these candidate genes could probably be involved in resistance to Botrytis and provide a more precise possibility to use MAS in gerbera breeding in the future. A tobacco rattle virus (TRV) based gene silencing system which was used to inspect the function of two candidate genes. The two CGs are the homologs of the genes responsible for Botrytis resistance in tomato and both mapped in QTL regions related to Botrytis resistance in gerbera ray floret test. Silencing the two genes by VIGS, showed smaller lesion sizes upon Botrytis infection on gerbera ray florets compared with the controls.
The entire research went from the generation of four parental transcriptome data sets to development of SNP markers (Chapter 2), construction of genetic maps and to mapping QTLs for Botrytis resistance (Chapter 3). This was further on combined with candidate gene searching in other crops, querying and mapping homologous genes (Chapter 4) and characterizing the candidate genes which co-localized with QTLs (Chapter 5). The whole process not only helped us to unravel the genetics of Botrytis resistance in gerbera and develop genetic tools for gerbera improvement, but also could serve as guidance for developing marker-assisted selection for other ornamental plants from the beginning.
Microbiome dynamics of disease suppresive soils
Gómez Expósito, Ruth - \ 2017
Wageningen University. Promotor(en): F.P.M. Govers; J.M. Raaijmakers, co-promotor(en): J. Postma; I. de Bruijn. - Wageningen : Wageningen University - ISBN 9789463431774 - 267
suppressive soils - soil suppressiveness - plant diseases - thanatephorus cucumeris - microbial ecology - soil microbiology - rhizosphere bacteria - soil bacteria - community ecology - soil fungi - transcriptomics - taxonomy - ziektewerende gronden - bodemweerbaarheid - plantenziekten - thanatephorus cucumeris - microbiële ecologie - bodemmicrobiologie - rizosfeerbacteriën - bodembacteriën - gemeenschapsecologie - bodemschimmels - transcriptomica - taxonomie
Disease suppressive soils are soils in which plants do not get diseased from plant pathogens due to the presence (and activities) of the microbes present in the soil. Understanding which microbes contribute to confer suppression and through which mechanisms they can protect plants is crucial for a sustainable control of plant diseases. In the research conducted in this thesis, I first examined the role of Lysobacter species, previously associated with disease suppressive soils, in suppressing damping-off disease caused by the soil-borne fungal pathogen Rhizoctonia solani on sugar beet. The majority of the Lysobacter strains tested revealed a broad metabolic potential in producing a variety of enzymes and secondary metabolites able to suppress R. solani in vitro. However, any of these strains could consistently suppress damping-off disease when applied in soil bioassays. Their ability to promote plant growth was also tested for sugar beet, cauliflower, onion or Arabidopsis thaliana. Results indicated that any of the Lysobacter strains could consistently promote plant growth, neither via direct contact nor via volatile production. Second, I investigated whether the antagonistic activity of Lysobacter species could be triggered when applied as bacterial consortia, together with Pseudomonas and Streptomyces species. Although several bacterial combinations showed an increased antagonistic effect towards R. solani in vitro, no consistent effects were observed when these bacterial consortia were applied in vivo. Third, I investigated the dynamical changes in the bacterial community composition and functions occurring during the process of disease suppressiveness induction by performing whole community analyses using next-generation sequencing techniques. Results indicated that suppressiveness induction was most associated with changes in certain bacterial traits rather than changes in the bacteria community composition itself. Among the functions found as more active in suppressive soils were several ‘classic’ mechanisms of disease suppression, including competition for nutrients, iron and space and production of extracellular enzymes, indol-acetic-acid and hydrogen cyanide. Among the enzymes found in higher abundance in suppressive soil were these ones involved in the degradation of oxalic acid, a pathogenicity factor produced by pathogenic fungi to help infecting the host plant. Hence, I finally studied the role of bacteria able to produce enzymes able to degrade oxalic acid in suppressing R. solani disease. Enrichment of native oxalotrophic bacteria existing in soil, their isolation and further application into soil revealed that they could effectively suppress Rhizoctonia disease. Characterization of these oxalotrophic bacteria revealed that members within the Caulobacter and Nocardioides species could suppress R. solani disease by their own. Furthermore, the research done in this thesis highlights the importance of combining different techniques to unravel the mechanisms underlying disease suppression and the importance of studying function-over-phylogeny. Additionally, it also highlights the importance of organic amendments (such as oxalic acid) directly into soils in order to “engineer” the bacterial functions towards the control of diseases caused by R. solani.
Differences in transcriptional responses to acute and chronic dietary interventions with fatty acids
Matualatupauw, Juri C. - \ 2017
Wageningen University. Promotor(en): A.H. Kersten, co-promotor(en): L.A. Afman; J. Bouwman. - Wageningen : Wageningen University - ISBN 9789463432078 - 172
fatty acids - gene expression - genotyping - phenotypes - nutritional intervention - transcriptomics - fish oils - apolipoprotein e - adipose tissue - microarrays - polymerase chain reaction - vetzuren - genexpressie - genotyping - fenotypen - maatregel op voedingsgebied - transcriptomica - visoliën - apolipoproteïne e - vetweefsel - microarrays - polymerase-kettingreactie
Various types of dietary fatty acids have different effects on human health. The aim of this thesis was to increase our understanding of the molecular mechanisms underlying the effects of dietary fatty acids. To do this, we examined changes in whole genome gene expression profiles upon both acute as well as longer term dietary fatty acid interventions. Furthermore, from previous research, it is clear that large inter-individual differences in the response to dietary fatty acids exist. We used whole genome gene expression analyses to increase our understanding of the mechanisms underlying some of these inter-individual differences.
Many modifiable and non-modifiable factors can be the cause of these inter-individual differences. In chapter 2, we reviewed all studies that examined differences in the transcriptional response to dietary interventions based on the presence of one of these factors. These include gender, age, BMI, body composition, blood lipid levels and gut microbial composition. We conclude that transcriptome analyses are well-suited for studying the underlying mechanisms behind these differences in the response to diet. Nevertheless, the number of studies that use this approach remains limited.
Another factor that may modify the response to a dietary intervention is genetics, e.g. the apolipoprotein E4 (APOE4) variant. People who carry the APOE4 allele have an increased risk of cardiovascular disease. Fish-oil supplementation may help in the prevention of cardiovascular disease, though inter-individual differences in the response to n-3 polyunsaturated fatty acids on gene expression profiles have been observed. In chapter 3, we aimed to assess the impact of APOE4 on peripheral blood mononuclear cell (PBMC) whole genome gene expression at baseline and following a 6-month fish-oil intervention. We observed increased gene expression of IFN signaling and cholesterol biosynthesis pathways in APOE4 carriers, which might explain part of the association between APOE4 and CVD. Furthermore, fish-oil supplementation may be beneficial by decreasing interferon signalling-related gene expression in APOE4 carriers.
Another long-term dietary intervention with fatty acids was studied in chapter 4. We examined the effect of a 12-week high medium-chain saturated fatty acid diet on subcutaneous adipose tissue gene expression profiles. We observed increased expression of genes involved in oxidative energy metabolism and decreased inflammation-related gene expression due to the high medium-chain saturated fatty acid intake. Considering the role of the adipose tissue in sustaining the low-grade inflammation that is associated with obesity, these findings may be indicative of a more anti-inflammatory phenotype of the adipose tissue. We concluded that medium-chain saturated fatty acids may potentially have beneficial effects on adipose tissue functioning.
Besides studying the effects of long-term interventions with fatty acids on whole genome gene expression, we also examined the effects of acute high-fat challenges. In chapter 5, we determined the additional value of determining whole genome gene expression changes in response to a high-fat challenge compared to assessment at fasting only. In addition, we aimed to identify whether a 4 week high-fat high-calorie diet can induce a shift in gene expression profiles in healthy subjects towards a metabolic syndrome-like gene expression profile. We found that fasting whole blood whole genome gene expression profiles are highly responsive to a 4-week high-fat high-calorie diet, with changes in in the direction of a metabolic syndrome-like gene expression profile. High-fat challenge responses in healthy subjects show only minimal changes in gene expression upon the dietary intervention and a marginal shift in the direction of the metabolic syndrome. We concluded that fasting gene expression profiles are more responsive compared to high-fat challenge responses to a 4-week high-fat high-calorie diet.
Besides chapter 5, several other studies have also examined changes in whole genome gene expression in blood cells induced by high-fat challenges. In chapter 6, we combined microarray data from four high-fat challenge studies varying in study population, challenge composition and research laboratory. By performing this meta-analysis, we showed a general PBMC whole genome gene expression response to a high-fat challenge. We concluded that a meta-analysis provides added value for the discovery of consistently differentially expressed genes and pathways compared to selecting only those genes and pathways that are identified in all separate studies.
In conclusion, in this thesis we showed differences in the whole genome gene expression response to fish-oil supplementation in PBMCs of APOE4 carriers vs non-carriers. Furthermore, the effects on whole genome gene expression of the two long-term dietary interventions, i.e. the fish-oil supplementation in PBMCs of APOE4 carriers and the high medium-chain saturated fatty acid diet in adipose tissue, may be beneficial by downregulation of gene expression related to inflammation. We also showed that whole genome gene expression responses to high-fat challenges are affected by a 4-week high-fat high-calorie diet, though changes in fasting gene expression profiles are much more pronounced. Finally, we showed the value of meta-analysis of microarray data in high-fat challenge studies for identifying the general response to a high-fat challenge.
Volatile-mediated interactions in the rhizosphere
Cordovez da Cunha, Viviane - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): V.J. Carrion. - Wageningen : Wageningen University - ISBN 9789462579019 - 219
rhizosphere bacteria - rhizosphere fungi - microbial interactions - volatile compounds - suppressive soils - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - growth stimulators - biological control - defence mechanisms - genomics - transcriptomics - rizosfeerbacteriën - rizosfeerschimmels - microbiële interacties - vluchtige verbindingen - ziektewerende gronden - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - groeistimulatoren - biologische bestrijding - verdedigingsmechanismen - genomica - transcriptomica
Plants and microorganisms are constantly engaged in highly dynamic interactions both above- and belowground. Several of these interactions are mediated by volatile organic compounds (VOCs), small carbon-based compounds with high vapor pressure at ambient temperature. In the rhizosphere, VOCs have an advantage in intra- and interorganismal signaling since they can diffuse through soil pores over longer distances than other metabolites and are not dependent on water availability. The research described in this PhD thesis explored how beneficial and pathogenic microorganisms that live in the rhizosphere and endosphere modulate plant growth, development and resistance via the production of VOCs. In vitro and in vivo bioassays as well as different ‘omic’ approaches, such as volatomics, transcriptomics and genomics, were employed to investigate underlying mechanisms of VOC-mediated microbe-microbe and microbe-plant interactions.
To investigate the diversity and functions of microbial VOCs, a disease-suppressive soil was used as the source of the VOC-producing microorganisms. Previous metagenomics studies reported Actinobacteria, in particular Streptomyces and Microbacterium species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen Rhizoctonia solani. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and in addition, promoted plant growth. Coupling the Streptomyces VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also Microbacterium isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of Microbacterium sp. EC8 revealed several sulfur-containing compounds and ketones such as dimethyl disulfide, trimethyl trisulfide and 3,3,6-trimethylhepta-1,5-dien-4-one (also known as Artemisia ketone). Genome analysis of strain EC8 revealed genes involved in sulfur metabolism. Resolving the role of the identified compounds and genes in VOC-mediated plant growth promotion and induced resistance will be subject of future studies. VOC-mediated chemical warfare underground has been proposed as a key mechanism of natural disease-suppressive soils. The results presented in this thesis indeed point in that direction. However, to more conclusively determine the role of the identified Actinobacterial VOCs in soil suppressiveness to R. solani, it will be important to demonstrate that the fungicidal VOCs are actually produced in situ at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus.
In agriculture, VOCs and VOC-producing microorganisms provide a potential alternative to the use of pesticides to protect plants and to improve crop production. In the past decades, several in vitro studies have described the effects of microbial VOCs on other (micro)organisms. However, little is still known on the potential of VOCs in large-scale agriculture and horticulture. The results described in this thesis show that VOCs from Microbacterium sp. EC8 stimulate the growth of Arabidopsis, lettuce and tomato, but do not control damping-off disease of lettuce caused by R. solani. Significant biomass increases were also observed for plants exposed only shortly to the bacterial VOCs prior to transplantation of the seedlings to soil. These results indicate that VOCs from strain EC8 can prime plants for growth promotion without direct contact and prolonged colonization. Furthermore, the induction of the plant growth-promoting effects appeared to be plant tissue specific. Root exposure to the bacterial VOCs led to a significant increase in plant biomass whereas shoot exposure did not result in significant biomass increase of lettuce and tomato seedlings. Genome-wide transcriptome analysis of Arabidopsis seedlings exposed to VOCs from this bacterium showed an up-regulation of genes involved in sulfur and nitrogen metabolism and in ethylene and jasmonic acid signaling. These results suggest that the blend of VOCs of strain EC8 favors, in part, the plant’s assimilation of sulfate and nitrogen, essential nutrients for plant growth, development and also resistance.
Similar to beneficial microorganisms, plant pathogenic microorganisms have also evolved strategies to modulate growth and defense of their hosts. For instance, compounds secreted by pathogens may suppress or interfere with plant defense. In this thesis I show that R. solani produces an array of VOCs that promote growth, accelerate development, change VOC emission and reduce insect resistance of plants. Plant growth-promoting effects induced by the fungal VOCs were not transgenerational. Genome-wide transcriptome analysis of Arabidopsis seedlings revealed that exposure to fungal VOCs caused up-regulation of genes involved in auxin signaling, but down-regulation of genes involved in ethylene and jasmonic acid signaling. These findings suggest that this soil-borne pathogen uses VOCs to predispose plants for infection by stimulating lateral root formation and enhancing root biomass while suppressing defense mechanisms. Alternatively, upon perception of VOCs from soil-borne pathogens, plants may invest in root biomass while minimizing investments in defense, a trade-off that helps them to speed up growth and reproduction and to survive pathogen attack.
In conclusion, the research presented in this thesis shows that both plants and microorganisms engage via VOCs in long-distance interactions and that beneficial and pathogenic soil microorganisms can alter plant physiology in different ways. Here, I provided a first step in identifying microbial genes involved in the regulation of biologically active VOCs as well as candidate plant genes involved in VOC perception and signal transduction. How plants sense and differentiate among VOCs from beneficial and pathogenic soil microorganisms will be an intriguing subject for future studies.
Plant growth promotion by Pseudomonas fluorescens : mechanisms, genes and regulation
Cheng, X. - \ 2016
Wageningen University. Promotor(en): Francine Govers; J.M. Raaijmakers, co-promotor(en): M. van der Voort. - Wageningen : Wageningen University - ISBN 9789462578753 - 192
soil bacteria - pseudomonas fluorescens - plants - growth stimulators - soil suppressiveness - plant diseases - induced resistance - biochemistry - biosynthesis - plant-microbe interactions - transcriptomics - bodembacteriën - pseudomonas fluorescens - planten - groeistimulatoren - bodemweerbaarheid - plantenziekten - geïnduceerde resistentie - biochemie - biosynthese - plant-microbe interacties - transcriptomica
Pseudomonas fluorescens is a Gram-negative rod shaped bacterium that has a versatile metabolism and is widely spread in soil and water. P. fluorescens strain SBW25 (Pf.SBW25) is a well-known model strain to study bacterial evolution, plant colonization and biocontrol of plant diseases. It produces the biosurfactant viscosin, a lipopeptide that plays a key role in motility, biofilm formation and activity against zoospores of Phytophthora infestans and other oomycete pathogens. In addition to viscosin, Pf.SBW25 produces other metabolites with activity against plant pathogens. The production of these yet unknown metabolites appeared to be regulated by the GacS/GacA two-component regulatory system (the Gac-system). The second P. fluorescens strain SS101 (Pf.SS101) studied in this thesis is known for its plant growth-promoting activities but the underlying mechanisms and genes are largely unknown. Therefore, in this study, we aimed to identify novel metabolites and biosynthetic genes in Pf.SBW25 and Pf.SS101, and to investigate their role in plant growth promotion and biocontrol. To this end, a multidisciplinary approach involving bioinformatic analysis of the genome sequences of strains Pf.SBW25 and Pf.SS101, microarray-based expression profiling, screening of genomic libraries, bioactivity assays, mass spectrometric image analysis (MALDI-IMS) and GC/MSMS analysis was adopted. In conclusion, we showed that the GacS/GacA two-component system as a global regulator of the expression of genes play important roles in antagonism of Pseudomonas fluorescens toward plant pathogenic microbes as well as in plant growth promotion and ISR. Growth promotion by P. fluorescens is associated with alterations in auxin biosynthesis and transport, steroid biosynthesis, carbohydrate metabolism and sulfur assimilation. Moreover, advanced chemical profiling allowed us to compare the metabolite profiles of free-living P. fluorescens and P. fluorescens living in association with plant roots. A better understanding of yet unknown mechanisms exploited by the various Pseudomonas fluorescens strains will lead to new opportunities for the discovery and application of natural bioactive compounds for both industrial and agricultural purposes.
Heat stress tolerance responses in developing tomato anthers
Bita, Elena - \ 2016
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Christian Bachem. - Wageningen : Wageningen University - ISBN 9789462577701 - 109
tomatoes - solanum lycopersicum - anthers - heat stress - stress tolerance - heat tolerance - heat shock - transcriptomics - reproductive performance - gene expression profiling - meiosis - tomaten - solanum lycopersicum - helmknoppen - warmtestress - stresstolerantie - hittetolerantie - hitteshock - transcriptomica - voortplantingsvermogen - genexpressieprofilering - meiose
Global warming already has and will significantly impact crop productivity and yield in the near future. In order to meet the forecasted requirements of the future agricultural production, a proper assessment of crops environmental stress tolerance needs to be designed and implemented, from the laboratory to field. Genetic variation in the ability of tomatoes to set fruit under high temperature conditions has made selection for heat tolerance possible and multiple opportunities for improvement exist, as tolerance to high temperatures is a multi-genic character involving a complex network of chaperones and other protective proteins acting together to defend the cells from heat injury. Breeding programs involved in the development of heat tolerant cultivars should identify and make use of such tolerance traits already available in collected or wild germplasm.
The goal of this thesis was to characterize the response to high temperatures in meiotic tomato anthers with contrasting responses to heat and to identify genes that could be related to thermo-tolerance mechanisms during gamete development. Several molecular tools such as transcriptomic profiling by cDNA-AFLP and microarray analysis, RT-PCR or in situ RNA hybridisation were used to achieve this goal.
The second chapter reviews the effects of heat stress on reproductive flower development, candidate tolerance pathways and methods for production of heat tolerant crops.
The third chapter provides a general overview of the expression changes occurring in the developing anthers of a sensitive tomato genotype following exposure to a (short and) moderate high temperature stress (MHS). Using a combination of cDNA-AFLP, RT-PCR, and in situ RNA hybridisation, we characterized and verified the general transcriptional response to heat of tomato plants. Our results revealed that approximately 1% of the examined transcript-derived fragments exhibit alterations in expression pattern and the majority of these were down-regulated The putative functions associated with the genes identified by cDNA-AFLP indicated involvement of heat shock, metabolism, antioxidant and developmental processes. Based upon the observed transcriptional changes in response to MHS and on literature sources, we identified a number of candidate transcripts to be involved in heat-tolerance. The spatial expression of several such candidate genes was further examined using in situ RNA hybridisation and this showed that the investigated genes are expressed in the tapetum or/and in developing microspores. Furthermore, the expression of several candidate genes has been quantified by RT-PCR in additional genotypes with different degrees of heat tolerance. The results suggested a correlation between gene expression levels, pollen germination rates and tolerance to heat (Chapter 4).
In the fourth chapter we proceeded to profile the response to heat of meiotic anthers in a tolerant and a sensitive tomato genotype and investigated the expression of the identified candidate genes in several pairs of contrasting genotypes. Using microarray analysis (for an extensive overview of the meiotic response to heat) and RT-PCR, we were able to clearly distinguish differential responses of the tolerant genotype. After 2h of moderate heat stress, the heat-tolerant genotype exhibits fewer transcriptional changes than the heat-sensitive genotype. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression soon after MHS. Moreover, the heat-tolerant genotype also shows a different level of constitutive gene expression profiles when compared to the heat-sensitive genotype indicating a difference in genetic adaptation with regards to increased temperatures. The putative functions associated with the genes identified by microarray profiling indicate involvement of heat shock, antioxidant, metabolic, and cell development pathways. Based upon the observed differences in response to MHS we selected a number of candidate transcripts involved in heat-tolerance and confirmed their expression pattern in different tomato genotypes with contrasting responses to heat. The results suggested that the candidate genes are involved in the activation of protection mechanisms in the tomato anthers during moderate heat stress and, could therefore contribute to normal growth and development of the male gametophyte and implicitly a successful fruit set under adverse temperatures.
In the fifth chapter we tested the hypothesis that heat tolerance is associated with maintenance of organ identity, fertility and lower ABA levels during heat stress (for several tomato genotypes) and analysed the dynamics of ABA accumulation under temperature stress in several tomato genotypes with contrasting responses to heat. Furthermore, pollen germination tests were performed and additional physiological aspects of anther development for each genotype were analysed as well. The general trend observed was the accumulation of lower relative levels of ABA at the end of the experimental period compared to the initial stages in more tolerant genotypes and of higher levels in the sensitive genotypes. We concluded from these results that the morphological changes in the floral tissues and the overall changes in ABA levels are correlated with the molecular responses under increased temperature in the genotypes analysed. Whether these correlations are causally related is not clear; therefore more research is needed to resolve these issues.
The sixth chapter examines our analysis of the heat stress response in meiotic tomato anthers in a broader scientific context. I discuss the different aspects of our results and present several candidate genes involved in plant thermo-tolerance. In addition, I also discuss the potential involvement of plant growth regulators in plants´ responses to heat stress and suggest various potential follow-up experimental strategies.
Ecogenomics of plant resistance to biotic and abiotic stresses
Davila Olivas, N.H. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462576575 - 259
016-3932 - arabidopsis thaliana - defence mechanisms - drought resistance - insect pests - plant pathogenic fungi - stress - stress response - transcriptomics - genomics - genetic mapping - arabidopsis thaliana - verdedigingsmechanismen - droogteresistentie - insectenplagen - plantenziekteverwekkende schimmels - stress - stressreactie - transcriptomica - genomica - genetische kartering
In natural and agricultural ecosystems, plants are exposed to a wide diversity of abiotic and biotic stresses such as drought, salinity, pathogens and insect herbivores. Under natural conditions, these stresses do not occur in isolation but commonly occur simultaneously. However, plants have developed sophisticated mechanisms to survive and reproduce under suboptimal conditions. Genetic screenings and molecular genetic assays have shed light on the molecular players that provide resistance to single biotic and abiotic stresses. Induced defenses are attacker specific and phytohormones play an essential role in tailoring these defense responses. Because phytohormones display antagonistic and synergistic interactions, the question emerges how plants elicit an effective defense response when exposed to conflicting signals under multiple attack. Recent studies have shed light on this issue by studying the effects of combinations of stresses at the phenotypic, transcriptomic and genetic level. These studies have concluded that the responses to combined stresses can often not be predicted based on information about responses to the single stress situations or the phytohormones involved. Thus, combined stresses are starting to be regarded as a different state of stress in the plant. Studying the effects of combinations of stresses is relevant since they are more representative of the type of stresses experienced by plants in natural conditions.
In a coordinated effort, responses of Arabidopsis thaliana to a range of abiotic and biotic stresses and stress combinations have been explored at the genetic, phenotypic, and transcriptional level. For this purpose we used an ecogenomic approach in which we integrated the assessment of phenotypic variation and Genome-Wide Association (GWA) analysis for a large number of A. thaliana accessions with an in-depth transcriptional analysis. The focus of this thesis is especially on (but not limited to) three stresses, i.e. drought, herbivory by Pieris rapae caterpillars, and infection by the necrotrophic fungal pathogen Botrytis cinerea. These stresses were chosen because the responses of A. thaliana to these three stresses are highly divergent but at the same time regulated by the plant hormones JA and/or ABA. Consequently, analysis of responses to combinatorial stresses is likely to yield information on signaling nodes that are involved in tailoring the plant’s adaptive response to combinations of these stresses. Responses of A. thaliana to other biotic and abiotic stresses are included in an integrative study (Chapter 6).
We first investigated (Chapter 2) the extent of natural variation in the response to one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips, Myzus persicae aphids) and two combined stresses (drought plus P. rapae, and B. cinerea plus P. rapae). Using 308 A. thaliana accessions originating from Europe, the native range of the species, we focused on the eco-evolutionary context of stress responses. We analyzed how the response to stress is influenced by geographical origin, genetic relatedness and life-cycle strategy, i.e. summer versus winter annual. We identified heritable genetic variation for responses to the different stresses. We found that winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars and to the combined stresses of drought followed by P. rapae and infection by the fungus B. cinerea followed by herbivory by P. rapae. Furthermore, we found differential responses to drought along a longitudinal gradient.
We further investigated, using A. thaliana accession Col-0, how phenotypic and whole-genome transcriptional responses to one stress are altered by a preceding or co-occurring stress (Chapters 3 and 4). The whole-transcriptomic profile of A. thaliana triggered by single and combined abiotic (drought) and biotic (herbivory by caterpillars of P. rapae, infection by B. cinerea) stresses was analyzed by RNA sequencing (RNA-seq). Comparative analysis of plant gene expression triggered by single and double stresses revealed a complex transcriptional reprogramming. Mathematical modelling of transcriptomic data, in combination with Gene Ontology analysis highlighted biological processes specifically affected by single and double stresses (Chapters 3). For example, ethylene (ET) biosynthetic genes were induced at 12 h by B. cinerea alone or drought followed by B. cinerea inoculation. This induction was delayed when plants were pretreated with P. rapae by inducing ET biosynthetic genes only 18 hours post inoculation. Other processes affected by combined stresses include wound response, systemic acquired resistance (SAR), water deprivation and ABA response, and camalexin biosynthesis.
In Chapter 4, we focused on the stress imposed by P. rapae herbivory alone or in combination with prior exposure to drought or infection with B. cinerea. We found that pre-exposure to drought stress or B. cinerea infection resulted in a significantly different timing of the caterpillar-induced transcriptional changes. Additionally, the combination of drought and P. rapae induced an extensive downregulation of A. thaliana genes involved in defence against pathogens. Despite the larger reduction in plant biomass observed for plants exposed to drought plus P. rapae feeding compared to P. rapae feeding alone, this did not affect weight gain of this specialist caterpillar.
In Chapter 5, we used univariate GWA to (1) understand the genetic architecture of resistance to the different stresses and (2) identify regions of the genome and possible candidate genes associated with variation in resistance to those stresses. In Chapter 5 a subset of the stresses addressed in Chapter 1 (i.e. drought, herbivory by P. rapae and P. xylostella, and the combined stresses drought plus P. rapae and B. cinerea plus P. rapae) were investigated. Results from GWA were integrated with expression data generated in Chapters 3 and 4 or available from the literature. We identified differences in genetic architecture and QTLs underlying variation in resistance to (1) P. rapae andP. xylostella and (2) resistance to P. rapae and combined stresses drought plus P. rapae and B. cinerea plus P. rapae. Furthermore, several of the QTLs identified contained genes that were differentially expressed in response to the relevant stress. For example, for P. xylostella one of the QTLs contained only two genes encoding cysteine proteases (CP1 and CP2). The expression data indicated that these genes were induced by P. rapae and P. xylostella herbivory.
In Chapter 6, the genetic architecture underlying plant resistance to 11 single stresses and some of their combinations was investigated. First, the genetic commonality underlying responses to different stresses was investigated by means of genetic correlations,, revealing that stresses that share phytohormonal signaling pathways also share part of their genetic architecture. For instance, a strong negative genetic correlation was observed between SA and JA inducers. Furthermore, multi-trait GWA identified candidate genes influencing the response to more than one stress. For example, a functional RMG1 gene seems to be associated with susceptibility to herbivory by P. rapae and osmotic stress since loss of function mutants in RMG1 displayed higher resistance to both stresses. Finally, multi-trait GWA was used to identify QTLs with contrasting and with similar effects on the response to (a) biotic or abiotic stresses and (b) belowground or aboveground stresses.
Finally, In Chapter 7, I discuss the feasibility of obtaining plants that are resistant to multiple stresses from the point of view of genetic trade-offs and experimental limitations. The ecogenomic approach for gene discovery taken in this thesis is discussed, and recommendations are especially given on the use of herbivorous insects in quantitative genetic studies of stress resistance. Furthermore, alternatives to the use of insects in quantitative genetic studies of stress resistance are discussed and proposed. Finally, I discuss the feasibility of using an ecogenomic approach to study stress responses in other plant species than the model plant of molecular genetics, A. thaliana.
A wealth of candidate genes was generated by taking an ecogenomic approach, in particular transcriptome analysis and GWA analysis. Functional characterization of these genes is a next challenge, especially in the context of multiple stress situations. These genes constitute a rich source of potential factors important for resistance to abiotic, biotic and combined stresses that in the future may be applied for crop improvement.
Big data dringen door in de tuinbouw. Naar een volledig begrip van plantgedrag en productkwaliteit
Kierkels, T. ; Zedde, H.J. van de - \ 2015
Onder Glas 12 (2015)11. - p. 24 - 25.
tuinbouw - glastuinbouw - innovaties - landbouwkundig onderzoek - kunstmatige intelligentie - gewaskwaliteit - plantenveredeling - sorteren - kwaliteit - teeltsystemen - 3d analyse - metabolomica - genomica - transcriptomica - horticulture - greenhouse horticulture - innovations - agricultural research - artificial intelligence - crop quality - plant breeding - sorting - quality - cropping systems - 3d analysis - metabolomics - genomics - transcriptomics
Vijftien onderzoeksgroepen van Wageningen UR hebben de handen ineengeslagen om te komen tot het beter meten, begrijpen en voorspellen van plantgedrag en productkwaliteit. Het vakgebied heet ‘Plant phenomics’ en maakt gebruik van innovatieve technieken en sensoren. Het doel is een betere beheersing van teelt- en veredelingsproces en productkwaliteit. Toepassingen voor de praktijk liggen nog vooral op het terrein van sorteren en automatische kwaliteitsbeoordeling.
Strain improvement of oleaginous microalgae
Jaeger, L. de - \ 2015
Wageningen University. Promotor(en): Gerrit Eggink; Rene Wijffels, co-promotor(en): Dirk Martens. - Wageningen : Wageningen University - ISBN 9789462574847 - 200
algen - biomassa - oliën - productiviteit - opbrengsten - transcriptomica - triacylglycerol lipase - bioreactoren - transformatie - mutanten - algenteelt - biomassa productie - algae - biomass - oils - productivity - yields - transcriptomics - triacylglycerol lipase - bioreactors - transformation - mutants - algae culture - biomass production
The increasing world population and living standards have enlarged the demand for food, feed, and for chemicals. Traditional fossil fuel based commodities need to be replaced, not only because these resources are finite, but also to relieve the impact of carbon emission and pollution, resulting from fossil fuel derived processes. Much attention is on using plants to produce sustainable, renewable alternatives to petrochemical based processes. Palm oil is the crop with the highest lipid yield known today, but the production of palm oil causes deforestation on a large scale. Microalgae are a promising platform for the production of sustainable commodity products. A commodity product that can be produced in microalgae is triacylglycerol (TAG). The TAG molecules that are accumulated in microalgae are comparable to the TAG profiles of commonly used vegetable oils, and can directly be applied for edible oil as well as for biodiesel production. Currently, microalgae derived products have proven to be functional and a potential replacement for conventional crops. However, microalgae derived products, especially TAGs, are not economically feasible yet. In order to make microalgal derived products a reality we need to decrease the production costs by smart technological solutions, biological understanding and metabolic engineering.
To get more insight in the lipid accumulation mechanism of microalgae, and to define targets for future strain improvement strategies, transcriptome sequencing of the oleaginous microalgae Neochloris oleoabundans was done. This oleaginous microalga can be cultivated in fresh water as well as salt water. The possibility to use salt water gives opportunities for reducing production costs and fresh water footprint for large scale cultivation.
In chapter 2 the lipid accumulation pathway was studied to gain insight in the gene regulation 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen depleted conditions in a comparable way in both fresh and salt water. The transcriptome sequencing revealed a number of genes, such as glycerol-3-phosphate acyltransferase and via glycerol-3-phosphate dehydrogenase, that are of special interest and can be targeted to increase TAG accumulation in microalgae. NMR spectroscopy revealed an increase in proline content in saline adapted cells, which was supported by up regulation of the genes involved in proline biosynthesis. In addition to proline, the ascorbate-glutathione cycle seems to be of importance for successful osmoregulation by removal of reactive oxygen species in N. oleoabundans, because multiple genes in this pathway were upregulated under salt conditions. The mechanism behind the biosynthesis of compatible osmolytes in N. oleoabundans can be used to improve salt resistance in other industrially relevant microalgal strains.
Another very promising candidate for TAG production is the oleaginous green microalga Scenedesmus obliquus.
In chapter 3, UV mutagenesis was used to create starchless mutants, since no transformation approach was available for this species, due to its rigid and robust cell wall. All five starchless mutants that were isolated from over 3500 screened mutants, showed an increased triacylglycerol productivity. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant (Slm1) showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion and reached a TAG content of 49.4% (%CDW).
In chapter 4 the Slm1 strain was compared to the wild type strain using photobioreactors. In the wild type, TAG and starch accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The Slm1 did not produce starch and the carbon and energy acquired from photosynthesis was partitioned towards TAG synthesis. This resulted in an increase of the maximum TAG content in Slm1 to 57% (%CDW) compared to 45% (%CDW) in the wild type. Furthermore, it increased the maximum yield of TAG on light by 51%, from 0.144 in the wild type to 0.217 g TAG mol-1 photon-1 in the Slm1 mutant. No differences in photosynthetic efficiency between the Slm1 mutant and the wild type were observed, indicating that the mutation specifically improved carbon partitioning towards TAG and the photosynthetic capacity was not affected.
To identify the mutation that caused the starchless phenotype of Slm1 the transcriptome of both the wild type and the Slm1 mutant was sequenced as described in chapter 5. A single nucleotide polymorphism (SNP) was discovered in the small subunit of the starch biosynthesis rate-controlling enzyme ADP-glucose pyrophosphorylase, which resulted in the introduction of a STOP codon in the messenger RNA of the enzyme. The characterization of the mutation increases the understanding of carbon partitioning in oleaginous microalgae, leading to a promising target for future genetic engineering approaches to increase TAG accumulation in microalgae.
To use the insight that is gained in chapters 2-5 for metabolic engineering of TAG accumulation and carbon partitioning, a metabolic engineering toolbox is required. However, the development of transformation protocols for new and less well studied industrially relevant microalgae is challenging. In chapter 6, a simple and effective tool for the optimization of transformation protocols is proposed. Optimal voltage settings were determined for five microalgae: C. reinhardtii, Chlorella vulgaris, N. oleoabundans, S. obliquus, and Nannochloropsis sp. This method can be used to speed up the screening process for species that are susceptible for transformation and to successfully develop transformation strategies for industrially relevant microalgae, which lack an efficient transformation protocol.
In addition to the increase in productivity, improving the quality in terms of fatty acid composition of TAG molecules would be desired as well. For example, the accumulation of stearic acid rich TAG molecules is of special interest, because of the improved structural properties. The lipid accumulating starchless mutant of the model species C. reinhardtii BAFJ5 was used as model species in chapter 7, since genetic toolbox is well established for this species. In this chapter, stearoyl-ACP desaturase (SAD), is silenced by artificial microRNA. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that increasing the fraction of stearic acid in TAG is possible. Furthermore, we hypothesize that in addition to direct conversion in the chloroplast, C. reinhardtii is able to redirect stearic acid from the chloroplast to the cytosol and convert it to oleic acid in the endoplasmic reticulum by stearoyl-CoA desaturase.
In chapter 8, an outlook is given on microalgal strain improvement strategies for the future, reflecting on the results obtained in this thesis. Also a roadmap is suggested to get genetically modified microalgal derived products on the market. The results presented in this thesis, provide a significant improvement in the understanding of TAG accumulation and carbon partitioning in oleaginous microalgae. Furthermore, improved microalgal strains with increased TAG accumulation or improved TAG fatty acid composition under nitrogen depleted conditions were generated. In addition, an outlook is presented in which the major bottlenecks are presented in future industrial applications of microalgae.
Sexual development of Botrytis species
Terhem, R.B. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - Wageningen : Wageningen University - ISBN 9789462574144 - 188
botrytis - plantenziekteverwekkende schimmels - geslachtsontwikkeling - fylogenie - genomica - transcriptomica - paarsystemen - schimmelmorfologie - nieuwe soorten - botrytis - plant pathogenic fungi - sexual development - phylogeny - genomics - transcriptomics - mating systems - fungal morphology - new species
Sexual Development of Botrytis Species
Razak bin Terhem
The fruiting bodies of species in the genus Botrytis are called apothecia. Apothecia are ascomas with an open cup shape on top of a stipe. Currently there is little information on processes occurring during apothecium development in Botrytis species. The aims of the research described in this thesis were to study the mechanisms involved in apothecium development of Botrytis cinerea, and to describe the morphology of Botrytis species and their fruiting bodies. Chapter 2 describes a genome-wide transcriptome analysis of different stages of apothecium development and a study on the function of MAT genes in apothecium development of B. cinerea. Functional analyses by targeted knockout mutagenesis revealed that the MAT1-1-1 gene and the MAT1-2-1 gene are both required for the initiation of sexual development. By contrast, mutants in the MAT1-1-5 gene and the MAT1-2-4 resulted in normal development of stipes which, however, were defective in the formation of an apothecial disk, asci and ascospores. Chapter 3 describes the functional analysis of three hydrophobin genes in sclerotium and apothecium development of B. cinerea. All three genes contribute to sclerotium and apothecium development. Chapter 4 describes the structure of the MAT1-1 and MAT1-2 locus in Botrytis elliptica and the morphology of apothecia of B. elliptica. Chapter 5 provides a morphological and phylogenetic description of Botrytis deweyae, the only species within the genus that behaves as an endophyte and in certain conditions is able to cause disease on Hemerocallis plants. Chapter 6 discusses the results presented in this thesis and puts them in a broader perspective. A model of processes and mechanisms involved in apothecium development is proposed.
Regulation and natural functions of lipopeptide biosynthesis in Pseudomonas
Song, C. - \ 2015
Wageningen University. Promotor(en): Francine Govers, co-promotor(en): Jos Raaijmakers. - Wageningen : Wageningen University - ISBN 9789462572690 - 173
pseudomonas fluorescens - lipoproteïnen - biosynthese - genetische kartering - genregulatie - genomica - transcriptomica - verdedigingsmechanismen - protozoa - mutanten - pseudomonas fluorescens - lipoproteins - biosynthesis - genetic mapping - gene regulation - genomics - transcriptomics - defence mechanisms - protozoa - mutants
Lipopeptides (LPs) are surface-active, antimicrobial compounds composed of a lipid moiety linked to a short linear or cyclic oligopeptide. In bacteria, LPs are synthesized by large nonribosomal peptide synthetases (NRPSs) via a thiotemplate process. Compared to the understanding of LP biosynthesis, little is known about the genetic regulation.
The aims of this PhD thesis were to identify new regulatory genes of LP biosynthesis and to unravel the natural functions of LPs in plant-associated Pseudomonas species. Using a combination of various ‘omics’-based technologies, we identified two small RNAs, designated RsmY and RsmZ, that, together with the repressor proteins RsmA and RsmE, regulate the biosynthesis of the LP massetolide in the rhizosphere bacterium Pseudomonas fluorescens SS101. Four other regulatory genes (phgdh, dnaK, prtR and clpA) of massetolide biosynthesis were identified via random mutagenesis. Mutations in each of these four genes caused a deficiency in massetolide production, swarming motility and biofilm formation, two natural functions associated with the production of LPs in Pseudomonas. Results further indicated that the ClpAP protease complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.
LPs exhibit broad-spectrum antimicrobial activities and have diverse natural functions for the producing bacteria. LPs of P. fluorescens were shown to play an important role in defense against protozoan predation. Genome-wide transcriptome analysis revealed that 55 and 73 genes were up- and down-regulated respectively in P. fluorescens strain SS101 upon grazing by the protozoan predator Naeglaria americana. The up-regulated genes included the LP biosynthesis genes massABC, but also genes involved in alkane degradation and in putrescine catalysis. Putrescine induced encystment of the protozoa, possibly providing a second line of defense against predation. MALDI imaging mass spectrometry (IMS) and live colony NanoDesi mass spectrometry further revealed, in real time, site-specific LP production at the interface of Pseudomonas-protozoa interactions. When the closely related strain P. fluorescens SBW25 was exposed to N. americana, similar overall transcriptional and metabolic responses were observed as found for strain SS101, but also strain-specific responses were apparent. These results indicate that closely related bacterial strains exhibit common and unique transcriptomic and metabolic responses to protozoan predation. Next to defense against competitors and predators, LPs are well-known for their role in swarming motility, a flagella-driven multicellular behavior of bacteria. Orfamide-deficient mutants of P. protegens Pf-5, either with deletions in the biosynthesis gene ofaA or in the regulatory gene gacA, cannot swarm on their own but ‘hitch-hike’ with parental strain Pf-5. However, distinctly different spatial distributions in co-swarming colonies were observed for these two mutants, with the ofaA mutant moving behind the wild type and the gacA mutant predominating on the edge of the swarming colony. Subsequent experimental evolution assays showed that repeated swarming cycles of strain Pf-5 drives parallel evolution toward fixation of spontaneous gacS/gacA mutants on the edge, ultimately causing colony collapse. Transcriptome analyses revealed that genes associated with resource acquisition, motility, chemotaxis and efflux were significantly upregulated in these regulatory mutants. Moreover, microscopic analysis showed that gacA mutant cells were longer and more flagellated than wild type and ofaA mutant cells, which may explain their predominance on the edge of co-swarming colonies. Collectively, these results indicated that adaptive convergent evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these spontaneous mutations during dispersal of bacteria into new territories are frequency-dependent.
Molecular mechanisms underlying the effects of dietary fiber in the large intestine
Lange, K. - \ 2015
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Guido Hooiveld. - Wageningen : Wageningen University - ISBN 9789462572706 - 201
voedingsvezels - voeding en gezondheid - darmfysiologie - dikke darm - transcriptomica - microbiota van het spijsverteringskanaal - dietary fibres - nutrition and health - intestinal physiology - large intestine - transcriptomics - gastrointestinal microbiota
Interactions between diet, microbiota and host response are important for intestinal health. Dietary fibers are known to promote intestinal health. Dietary fibers are edible plant-derived food components that encompass complex carbohydrates and lignin, resist the digestion in the small intestine of which some are degraded and fermented by gut microbiota in the large intestine, i.e. cecum and colon. The beneficial health effects of dietary fiber are suggested to be mediated by short-chain fatty acids (SCFA), which are produced by gut microbial fermentation. The underlying mechanisms of the interaction between dietary fiber, SCFA, and the host, however, are not in detail known.
The objective of the research described in this thesis was to investigate the molecular effects and mechanisms underlying the effects of dietary fiber and its fermentation products, SCFA, in the large intestine.
Firstly, the colonic transcriptional response to the main SCFA, acetate, propionate and butyrate, was investigated. SCFA were administered by rectal infusion in C57BL/6 mice fed a low fat/high carbohydrate (LFD) or high fat/low carbohydrate diet (HFD) and whole-genome gene expression analysis was performed on colonic scrapings by microarray technology. The analysis revealed specific and overlapping genes regulated between acetate, propionate and butyrate. In addition, gene response to SCFA was dependent on the diet, in particular for propionate. A set of propionate-regulated genes was activated on LFD while suppressed on a HFD and vice versa, indicating that diet composition is important factor in colonic response to SCFA.
Secondly, the molecular effects of different dietary fibers and a control diet on the large intestine were investigated. Five different dietary fibers (inulin, fructo-oligosaccharide, arabinoxylan, guar gum, resistant starch) and a control diet were fed to C57BL/6 mice (10 days). The transcriptional response to the fermentable fibers was comparable in gene expression, microbiota composition, and luminal SCFA level in colon. In common for all fermented dietary fibers, the transcriptional regulator Pparg was identified as potential upstream regulator for the mucosal gene expression response. Moreover, bacteria mainly belonging to Clostridium cluster XIVa were found to correlate with mucosal genes related to metabolic, energy-generating processes.
Next to common responses, analysis of the transcriptome revealed distinct responses of different dietary fibers. With respect to the cecal metatranscriptome, we identified distinct activities of bacterial families in the fermentation of dietary fiber. Moreover, using multivariate statistical analysis, we found correlations of the mucosal transcriptome with both the microbiota composition and metatranscriptome.
In addition, we showed that SCFA, particularly butyrate and to a lesser extend propionate, transactivate PPARg and regulate the PPARg target gene Angptl4 in colonic cells.
Thirdly, we tested the hypothesis that epithelial Pparg plays an important role in the fermentation of dietary fibers in the gut. Mice with an intestine-specific knock out (KO) of Pparg (cre-villin) and wild type (WT) mice were fed inulin (10 days). Whole-genome gene expression analysis of the colon revealed that diet had a larger effect than genotype on colonic, luminal microbiota composition, metabolome and mucosal transcriptome. We identified genes that were regulated by inulin in Pparg-dependent manner. In addition, we also identified genes regulated by butyrate in Pparg-dependent manner in organoids grown from colonic crypt cells derived from KO or WT mice.
In conclusion, we identified distinct mucosal gene expression responses to the main fermentation products of dietary fiber, SCFA, on both low fat/high carbohydrate and high fat/low carbohydrate diet backgrounds. Dietary fibers induce common and specific effects in colon. Epithelial Pparg partially governs the response to fermentation of dietary fiber in colon. Next to the commonalties of dietary fiber for intestinal physiology, specific and differential effects were identified for microbial gene activity and composition as well as mucosal transcriptome response indicating that omics tools are useful in elucidating and dissecting effects of dietary fiber.
Physiological and molecular adaptations of Lactococcus lactis to near-zero growth conditions
Ercan, O. - \ 2014
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Eddy Smid. - Wageningen : Wageningen University - ISBN 9789462570719 - 206
lactococcus lactis - adaptatiefysiologie - voedselmicrobiologie - groeitempo - groeispanning - transcriptomica - metabolomica - lactococcus lactis - adaptation physiology - food microbiology - growth rate - growth stress - transcriptomics - metabolomics
Lactococcus lactis is an important lactic acid bacteria (LAB) species that is used for the manufacture of dairy products, such as cheese, buttermilk, and other fermented products. The predominant function of this bacterium in dairy fermentation is the production of lactic acid, as its major fermentation end-product that contributes to preservation and microbial safety of the product. Moreover, L. lactis is frequently encountered in natural ecosystems such as in (rotting) plant material.
Due to restricted energy source availability, natural microbial communities commonly live in a situation that can be characterized as ‘hunger’, which is different from strict nutrient-starvation. As a consequence, environmental microbes commonly grow at very low-growth rates as compared to laboratory cultures. Analogously, microorganisms can experience such nutrient-poor conditions in diverse industrial fermentation applications. For example, LAB encounter extreme low or no energy source availability during the extended ripening process of cheeses or dry sausages, which can take months. Despite these harsh environmental conditions, many LAB are able to remain viable in these processes for months and sustain a low-level metabolic activity, which plays an important role in their contribution to flavor and aroma formation in the product matrix.
In this thesis, the quantitative physiology of L. lactis at near-zero specific growth rates was studies, employing both metabolic and genome-wide transcriptome studies in an experimental set-up of carbon-limited retentostat cultivation. Chapter 2 describes how retentostat cultivation enables uncoupling of growth and non-growth related processes in L. lactis, allowing the quantitative analysis of the physiological adaptations of this bacterium to near-zero growth rates. In chapter 3, transcriptome and metabolome analyses were integrated to understand the molecular adaptation of L. lactis to near-zero specific growth rate, and expand the studies in chapter 2 towards gene regulations patterns that play a profound role in zero-growth adaptation. Chapter 4 describes the enhanced robustness to several stress conditions of L. lactis after its adaptation to extremely low-specific growth rate by carbon-limited retentostat cultivation. In this chapter correlations were modelled that quantitatively and accurately describe the relationships between growth-rate, stress-robustness, and stress-gene expression levels, revealing correlation coefficients for each of the varieties involved. Chapter 5 evaluates the distinction between the transcriptome responses to extended carbon-limited growth and severe starvation conditions, where the latter condition was elicited by switching off the medium supply of the retentostat cultures described in chapter 1. Chapter 6 highlights the comparison of the physiological and molecular adaptations of industrially important microorganisms towards carbon-limited retentostat conditions. In conclusion, this thesis describes the quantitative physiological, metabolic, and genome-wide transcriptional adaptations of L. lactis at near-zero specific growth rates induced by carbon source limited retentostat cultivation, and compares these molecular adaptations to those elicited by strict carbon-starvation conditions.
Nutritional Systems Biology of Fat : integration and modeling of transcriptomics datasets related to lipid homeostasis
Ohid Ullah, M. - \ 2012
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Guido Hooiveld. - S.l. : s.n. - ISBN 9789461733818 - 158
vetzuren - genexpressie - lipidenmetabolisme - obesitas - transcriptomica - statistische analyse - wiskundige modellen - fatty acids - gene expression - lipid metabolism - obesity - transcriptomics - statistical analysis - mathematical models
Fatty acids, in the form of triglycerides, are the main constituent of the class of dietary lipids. They not only serve as a source of energy but can also act as potent regulators of gene transcription. It is well accepted that an energy rich diet characterized by high intakes of dietary fat is linked to the dramatic increase in the prevalence of obesity in both developed and developing countries in the last several decades. Obese individuals are at increased risk of developing the metabolic syndrome, a cluster of metabolic abnormalities that ultimately increase the risk of developing vascular diseases and type 2 diabetes. Many studies have been performed to uncover the role of fatty acids on gene expression in different organs, but integrative studies in different organs over time driven by high throughput data are lacking. Therefore, we first aimed to develop integrative approaches on the level of individual genes but also pathways using genome-wide transcriptomics datasets of mouse liver and small intestine that are related to fatty acid sensing transcription factor peroxisome proliferator activated receptor alpha (PPARα). We also aimed to uncover the behavior of PPARαtarget genes and their corresponding biological functions in a short time series experiment, and integrated and modeled the influence of different levels of dietary fat and the time dependency on transcriptomics datasets obtained from several organs by developing system level approaches.
We developed an integrative statistical approach that properly adjusted for multiple testing while integrating data from two experiments, and was driven by biological inference. By quantifying pathway activities in different mouse tissues over time and subsequent integration by partial least squares path model, we found that the induced pathways at early time points are the main drivers for the induced pathways at late time points. In addition, using a time course microarray study of rat hepatocytes, we found that most of the PPARα target genes at early stage are involved in lipid metabolism-related processes and their expression level could be modeled using a quadratic regression function. In this study, we also found that the transcription factorsNR2F, CREB, EREF and RXR might work together with PPARα in the regulation of genes involved in lipid metabolism. By integrating time and dose dependent gene expression data of mouse liver and white adipose tissue (WAT), we found a set of time-dose dependent genes in liver and WAT including potential signaling proteinssecreted from WAT that may induce metabolic changes in liver, thereby contributing to the pathogenesis of obesity.
Taken together, in this thesis integrative statistical approaches are presented that were applied to a variety of datasets related to metabolism of fatty acids. Results that were obtained provide a better understanding of the function of the fatty acid-sensor PPARa, and identified a set of secreted proteins that may be important for organ cross talk during the development of diet induced obesity.
Development of genomic resources for ornamental lilies (Lilium L.)
Shahin, A. - \ 2012
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jaap van Tuyl; Paul Arens. - S.l. : s.n. - ISBN 9789461733009 - 169
lilium - sierplanten - plantenveredeling - transcriptomica - genetische kartering - genotyping - genetische merkers - nucleotidenvolgordes - genomica - lilium - ornamental plants - plant breeding - transcriptomics - genetic mapping - genotyping - genetic markers - nucleotide sequences - genomics
Lily (Lilium L.) is a perennial bulbous ornamental, belonging to subclass Monocotyledonae and family Liliaceae. Lily, according to statistics of Dutch auctions, is the fifth most important cut flower and the second in flower bulbs based on acreage. This species has been extensively used for cytogenetic studies, but molecular genetic studies are limited. The heterogenic nature and the very complex and huge genome (36 Gb) of lily might be the reason for this. To improve the efficiency of breeding and selection in this species, and set up the basis for genetic studies in Lilium, genomic resources are needed.
Next generation sequencing (NGS) technology (454 pyro-sequencing) was used to sequence the transcriptomes (RNA-seq) of four lily cultivars: ‘Connecticut King’, ‘White Fox’, ‘Star Gazer’, and Trumpet that belong to the four most important hybrid groups: Asiatic, Longiflorum, Oriental, and Trumpet respectively. Successfully, 52,172 unigenes with an average length of 555 bp were developed and used for a wide range of genetic and genomic studies: SNP marker identification for genetic mapping, gene annotation, and comparative genomic studies.
Combining NGS with SNP genotyping techniques to accelerate genetic studies is of considerable interest in different species. In this study, thousands of SNPs out of the 52,172 lily unigenes were identified. Genotyping technique KASPar (KBiosciences competitive Allele Specific PCR) was used to genotype two lily mapping populations: ‘LA (L. longiflorum ‘White Fox’ x Asiatic hybrid ‘Connecticut King’) and AA (‘Connecticut King’ x ‘Orlito’) using 225 SNP markers selected from ‘Connecticut King’ unigenes. Genotyping success rate was 75.5% (170 SNP markers worked), polymorphic SNP rate was 45% (102 SNP markers), and mapped SNP marker rate was 42% (94 SNP mapped) in LA population and 38% (85 SNP mapped) in AA population. Thus, we validated a subset of the putative SNP makers and showed the usability of this type of markers to improve genetic maps for complex genomes like that of lily.
The SNP markers together with the available AFLP (amplified fragment length polymorphisms), DArT (diversity arrays technology), and NBS (nucleotide binding site) markers were used to build reference genetic maps for these two lily populations. These maps represent the first reasonably saturated maps that cover 89% of the lily genome with an average marker density of one marker per 4 cM. The availability of more SNP markers for genotyping, opens the door for further enriching these genetic maps and thus improve the marker density.
The genetic maps were used to map and understand the genetic of several horticultural traits in Lilium. Fusarium oxysporum and lily mottle virus (LMoV) are considered as very serious diseases in Lilium and as such present important targets for breeding. Six putative QTLs (quantitive trait loci) were identified for Fusarium resistance in AA population, from which QTL1 was the strongest (explains ~25 % of phenotypic variation). In LA population, QTL1 was also confirmed. Thus, QTL1 is a strong and reliable QTL in both populations and it can be used to develop markers for most of the Fusarium resistance for molecular assisted breeding (MAB) applications. The LMoV was mapped as a marker on the AA genetic maps, however, no close markers to this trait (i.e. distance of the closest marker to LMoV was 9 cM) were identified yet. Several ornamental traits: lily flower color ‘carotene’ (LFCc), flower spots (lfs), stem color (LSC), antherless phenotype (lal), and flower direction (up-side facing, lfd) were phenotyped and mapped. Some of these traits showed to be recessive traits (spots, antherless, and flower direction) and controlled by a single gene. Developing markers for recessive traits is valuable since such markers allow the identification of suitable breeding parents so the presence of the recessive trait can be either enhanced or repressed. A more complex trait is flower longevity because it is a function of: the number of buds per inflorescence, the expansion and opening of the buds, the life span of the individual flowers, and also the life-span of the leaves. Moreover, senescence in Lilium is ethylene-insensitive and the regulator(s) of its vase life is not known yet. Our study showed that vase life of individual lily flowers increased significantly by the exogenous application of sugars. Abscisic acid (ABA) level, furthermore, increased dramatically in lily flowers at senescence compared with anthesis. This indicates that ABA might be the main regulator of vase life in lily. However, more experiments should be conducted to prove this conclusion.
The genomic resources developed for lily together with genomic resources developed for Tulipa L., in the same way, offered a valuable source of information to conduct comparative genomic studies within and between these two genera. We initiated the first step towards linking molecular genetic maps of Lilium and Tulipa using transcriptome sequences generated by 454 pyro-sequencing. Orthologous genes between lily and tulip were identified (10,913 unigenes) based on sequence data of four lily cultivars and five tulip cultivars. Next, common SNP and EST-SSR markers between the parents of lily mapping populations (AA and LA population) and the parents of tulip mapping population (‘Kees Nelis’ (T. gesneriana) x ‘Cantata’ (T. fosteriana)) based on these orthologous sequences were generated. A total of 229 common SNP and 140 common EST-SSR markers were identified. Genotyping and mapping these markers in the populations of both genera will link the genetic maps of Lilium and Tulipa and thus allow insight into the preservation of gene order, structure, and ‘putative’ functional homology in addition to evolutionary processes.
Also, these genomic resources can be used to increase the resolution of, and support for, phylogenetic trees. We selected a set of orthologous genes of Lilium (19 genes, 11,766 bp containing 433 polymorphic sites), of Tulipa (20 genes, 10,347 bp containing 216 polymorphic sites), and of the orthologous genes between the two genera (7 genes, 5,790 bp containing 587 polymorphic sites). These sets are uniquely present in the sequences and informative in estimating the genetic divergence of the two genera, thus they can be used to genotypes more species per genera to build genera and maybe family trees later on. The nucleotide polymorphism rate of Lilium was twice as high as that of Tulipa, on average one substitution per 26 bp for Lilium compared with one substitution per 48 bp for Tulipa. NGS provide a valuable source for large numbers of phylogenetic informative substitutions that might revolutionize the phylogenetic, population genetic, and biodiversity studies. However, the use of bi-allelic information from multiple loci in phylogenetic studies is still challenging and it needs to be studied further.
Moreover, having such high numbers of sequence data, allows us to test some evolutionary hypotheses such as positive selection: selection during domestication/breeding processes might be imprinted in the species genome, which can be examined based on omega (dn/ds) values. The higher the omega value the stronger the indication of positive selection. Positive selection was recorded in Lilium and Tulipa genomes when this small subset of gene contigs (46) of the two genera was tested. Our hypothesis could not be confirmed, however to draw final conclusions on this matter, omega values for many more genes of the two genera have to be measured.
Finally, a wealth of putative molecular markers (SNPs and SSRs) has become available that can have direct applications for breeding in these genera. SNP markers are important since they are user friendly, efficient, transferable, and co-dominant markers. Applying high throughput genotyping technology to genotype the two lily populations improved the coverage of the two genetic maps. Also, genotyping the same SNP markers in the two populations facilitated the comparisons between the linkage groups of the two populations and will allow the construction of a consensus map. Consequently, exchange of genetic knowledge (mainly QTLs) between the two populations will be easier. The thousands of SNPs identified in the genome of the four lily cultivars opens the door for combining the current linkage mapping studies with association studies which will have a direct impact on improving the resolution of mapping and on MAB applications in Lilium.
Chicken intestinal development in health and disease : transcriptomic and modeling approaches
Schokker, D.J. - \ 2012
Wageningen University. Promotor(en): Mari Smits, co-promotor(en): Annemarie Rebel. - S.l. : s.n. - ISBN 9789462575325 - 225
fowls - intestines - biological development - gene expression - transcriptomics - animal health - poultry diseases - intestinal physiology - immunology - mathematical models - kippen - darmen - biologische ontwikkeling - genexpressie - transcriptomica - diergezondheid - pluimveeziekten - darmfysiologie - immunologie - wiskundige modellen
Intestinal health is an important condition for sustainable animal production. Since it is known that there is significant variation in intestinal health and functionality, there is much to gain in this respect. However, to fully exploit the biological potential of the animal’s gastro-intestinal tract, the mechanism and regulation of major intestinal processes need to be unraveled first. In addition, identification of key components and processes involved in intestinal adaptation mechanisms may help to identify internal and external factors that influence the health and functioning of the gut. Improved knowledge in this area may contribute in defining rational strategies to improve sustainable animal production.
Functional genomics of chilo iridescent virus: a transcriptoproteomic approach
Ince, I.A. - \ 2012
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - S.l. : s.n. - ISBN 9789461731449 - 114
invertebrate iridescent virus 6 - iridovirus - insectenvirussen - functionele genomica - transcriptomica - eiwitexpressieanalyse - dierenvirussen - biologische bestrijding - virale insecticiden - invertebrate iridescent virus 6 - iridovirus - insect viruses - functional genomics - transcriptomics - proteomics - animal viruses - biological control - viral insecticides
Iridoviruses are disease causing agents in (pest) insects, fishes and amphibians with serious ecological and economic impacts. Insight in the composition of the virions and the transcriptional regulation of the virion protein genes is crucial to unravel the biology of this lesser known family of viruses. In this thesis, the virions of Chilo iridescent virus (CIV) (genus Iridovirus) were analyzed by mass spectrometry, revealing 54 virion proteins. A novel transcriptomic approach for non-polyadenylated RNA transcripts, called LACE, was developed and applied to unravel the temporal class of the virion protein genes. This showed that many virion protein genes were expressed as early genes. Another intriguing finding is that an infected cell-specific 100 kDa protein interacted with a crucial delayed-early promoter motif in the DNA polymerase gene and it turned out that this motif was conserved in other (putative) delayed early genes in CIV and other iridoviruses. The hypothesis is that this 100 kDa protein is responsible for transcriptional activation of delayed-early genes. CIV is an example of an invertebrate iridoviruses that deals with induction and inhibition of apoptosis during infection. In this study, a gene for a functional inhibitor of apoptosis (193R), unique for an iridovirus, was identified. In addition, several candidates for pro-apoptotic proteins were found in the virion. In this dissertation fundamental knowledge was obtained on the proteome of CIV virions and the regulation of CIV gene expression. Due to the development and application of novel technics, this thesis provides new venues to answer remaining questions concerning the infection cycle of this interesting iridovirus.
Iridovirus, transcriptomics, proteomics, virus-host interaction
The dynamic interplay of microbiota and mucosa drives establishment of homeostasis in conventionalized mice
Aidy, S.F. El - \ 2012
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Peter van Baarlen; Erwin Zoetendal. - S.l. : s.n. - ISBN 9789461731951 - 168
kiemvrije dieren - muizen - darmmicro-organismen - homeostase - slijmvlies van het spijsverteringskanaal - transcriptomica - metabolomica - germfree animals - mice - intestinal microorganisms - homeostasis - digestive tract mucosa - transcriptomics - metabolomics
The intimate interplay between gut microbiota, host, and nutrient flow is crucial in defining the health status of the host. During microbial conventionalization of germfree mice, tightly regulated molecular responses assure the establishment of homeostasis and immune tolerance towards the microbiota. To decipher the temporal and regional dynamics of host-microbiota communication during the process of conventionalization, a combination of transcriptomics, (immune-)histology, metabonomics (tissue, urine, and plasma), as well as MITchip (Mouse Intestinal Tract chip) based microbiota profiling was employed. To this end, C57/B6 J germfree mice were conventionalized with mouse fecal microbiota and responses were followed in a time-resolved manner for thirty days. The colonizing microbiota was characterized by a shift from low towards higher diversity of its composition, over the period of conventionalization. Microbial colonization was rapidly (after one day) reflected by increased concentrations of specific urine and jejunal metabolites as well as by biologically relevant changes in jejunal tissue transcriptome profiles. Conversely, ileal and colonic transcriptome responses could be measured later, after four days post-conventionalization, and led towards stable molecular profiles at sixteen and thirty days of conventionalization, albeit with region-specific differences. The major molecular responses included strong induction of innate immune response followed by stimulation of adaptive and regulatory immune functions, as well as modulation of metabolic pathways involved in lipid, carbohydrate, and anabolic metabolism. Conventionalization was characterized by two stages separated by one stage of a single day which, particularly in the colon, resembled a transient stage of inflammation, based on transcriptomes, histology and transiently elevated levels of specific plasma markers. This state coincided with temporal domination of specific microbial groups that have previously been identified as “pathobionts”, suggestive of a transient state of dysbiosis. Extensive transcriptome profile analyses throughout the GI tract enabled the identification of central gene regulatory networks that govern the molecular responses during conventionalization and are proposed to serve as genetic signatures for the control of intestinal homeostasis in mice. Nearly all genes in these regulatory networks have human orthologues, suggesting that the biological findings of this study is also relevant for human intestinal biology. In support of this hypothesis, in the jejunum, the identified gene regulatory network appeared to be strongly associated with human metabolic disorders. This notion also suggests that at least in mice, possibly also in human, there is a prominent role of the proximal small intestine in systemic metabolic control.
This thesis exemplifies the pivotal role of the dynamic molecular interactions between the microbiota and the intestinal mucosa, in the establishment and maintenance of mucosal homeostasis in healthy mice. The molecular signatures obtained from these studies in mice may provide novel diagnostic tools and/or therapeutic targets in humans for specific disorders associated with intestinal dysbiosis and loss of mucosal homeostasis.
Keywords: C57/BL6 J mice, conventionalization, transcriptomics, (immune-)histology, metabonomics, microbiota
Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus
Walther, J. - \ 2012
Wageningen University. Promotor(en): John van der Oost; Willem de Vos. - S.l. : s.n. - ISBN 9789461732033 - 162
sulfolobus solfataricus - biochemische omzettingen - koolstofpathways - transcriptomica - bioreactoren - sulfolobus solfataricus - biochemical pathways - carbon pathways - transcriptomics - bioreactors
The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and specific metabolic potential, the ease of cultivation of this organism, and the relative large amount of knowledge about this heat-loving acidophilic organism. We selected a systems approach to study the behaviour of this organism trying to make steps forward into the unknown, whenever possible trying to link exploration to exploitation. The cultivation of S.solfataricus is an essential element in all systems approaches that link genotype to phenotype. Hence, specific attention is given to the advanced culturing systems for this extremophile that have been used in all experimental studies described here (Chapters 3-6).
Systems analysis includes the integration of all available omics data and is increasingly used in the analysis of Archaea (Chapters 3 and 4). However, most attention has been given to archaeal transcriptome analysis and hence the most important literature on heat-loving Archaea is summarized (Chapter 2).
In the experimental chapters (Chapters 3-6) various systems approaches are applied to gain understanding of metabolic pathways in Sulfolobus. Chapter 3 describes the study of the central carbon pathways, consisting of the (non-) phosphorilated Entner-Douderoff (ED) pathway and the citric acid cycle. Different functional genomic approaches were applied on the model organism Sulfolobus solfataricus to study the response of growth on different carbon sources, D-Glucose vs. Tryptone and Yeast Extract. The complete transcriptome was studied using PCR-based microarrays. In addition the proteome was studied using 2D-electrophoresis map in combination with 13N- labelling technique to determine protein fluctuations. Despite the large difference in medium, very few significant differences on protein or RNA level were observed for the two conditions. Therefore regulation of these pathways does in all probability not occur through changes in protein abundance but presumably rather by direct changes in enzyme activity. This is unlike two thermophilic Euryarchaea: Thermococcus kodaaraensis (Kanai, Akerboom et al. 2007)and Pyrococcus furiosus (Schut, Brehm et al. 2003)where extensive regulation of glycolytic genes was observed in a similar situation.
Chapter 4describes the study of the degradation of D-arabinose through a similar approach as was described in chapter 3. S. solfataricus was grown on either D-arabinose or D-glucose and a comprehensive transcriptome and proteome study was carried out. The result of these studies was not only elucidation of the D-arabinose degradation route, but also a general prokaryotic pentose, hexaric acids and hydroxyproline degradation route, which supports the theory of metabolic pathway genesis by enzyme recruitment. Also this study predicted a cis-regulatory element to induce the arabinose degrading pathway when needed. The enzymes involved in the proposed pathway were cloned, expressed and their function was biochemically measured. This showed that using these enzymes, D-arabinose can be degraded to 2-oxogluterate, one of the metabolites that are part of the citric acid cycle.
Chapter 5reports on the effects of different oxygen concentrations on the behaviour of Sulfolobus solfataricus. The oxygen amount can be controlled relatively easily in a bioreactor, which is crucial for rapid and reproducible growth. Based on growth experiments in microcosms, different types of behaviour could be seen. At 35% (v/v gas phase) the cultures did not grow, indicating that S. solfa-taricus experiences a lethal dose of oxygen. At 26-32% growth was impaired, suggesting a moderate toxicity compared to the reference (21%). In the ranges 16-24% of oxygen, standard growth was observed, suggesting that S. solfataricus is comfortable in these oxygen ranges. For the lower amounts of oxygen (1.5-15%), the growth was comparable to the reference, but the respiratoryefficiency was increased. To get some more insight into this behaviour, we looked at the transcriptome. It showed differential expression of several genes, including genes encoding terminal oxidases, indicating that the organism adapts to lower oxygen concentrations by adapting its respiratory machinery.
Chapter 6 describes the zeaxanthin pathway in the Sulfolobus species. Zeaxanthin is a colorant and of vital importance for the function of the human eye. In this chapter the genes responsible for zeaxanthin production are presented. For this, DNA microarrays, bioinformatics as well as molecular genetics techniques were used. A crtx-like gene is operational in most of the known Sulfolobus species that is able to attach sugar-like molecules to zeaxanthin, which improves its solubility in water, which is very important in many food uses. We have cloned this crtx-like gene of S. solfataricus, S. shibatae, and S. acidocaldarius in a zeaxanthin overproducing E. coli strain. It has been demonstrated that the gene products of S. shibatae and S. acidocaldarius were responsible for attaching sugar-like molecules to zeaxanthin. The ctrx-like gene of S. solfataricus was not operating in E. coli. This is probably due to the fact that the gene is truncated. This chapter has further improved the understanding of archaeal carotenoid pathways and it has shown that the Sulfolobus species are able to modify zeaxanthin, although each species produces different zeaxanthin modifications.