Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming

Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.

A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.

Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.

In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.

Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.

Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.

Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.

In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

Production of protein‐based polymers in Pichia pastoris
Werten, Marc W.T. - \ 2017
University. Promotor(en): Martien Cohen Stuart; Gerrit Eggink, co-promotor(en): Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789463436069 - 241
proteins - polymers - pichia pastoris - gelatin - proteolysis - biosynthesis - eiwitten - polymeren - gelatine - proteolyse - biosynthese

From a chemistry perspective, proteins can be thought of as polymers of amino acids, linked by amide bonds. They feature unsurpassed control over monomer sequence and molecular size. The amino acid sequence of proteins determines their three-dimensional folded structure, resulting in unique properties. Proteins such as collagen, elastin, and silk play a crucial structure-forming role in various tissues and animal architecture such as spider webs. These proteins are characterized by highly repetitive amino acid sequences, and can reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. These unique properties have sparked the interest of material scientists, and mimics of these proteins have been designed and produced as heterologous proteins in suitable expression systems.

The most commonly employed host for these so-called protein-based polymers, or protein polymers for short, is the bacterium Escherichia coli. In this thesis, we explored the use of an alternative platform, namely the methylotrophic yeast Pichia pastoris (Komagataella phafii). This organism is well-known for its often relatively high yields, and offers a choice between intracellular and secretory production. Secretion of the polymer into the medium provides a highly effective first purification step, and precludes the need for cell disruption procedures that are cost-prohibitive at an industrial scale.

We evaluated the secretory production in P. pastoris of various protein polymers: murine collagen fragments (gelatins), a de novo-designed highly hydrophilic gelatin, silk-like proteins, hydrogel-forming triblock copolymers with collagen-inspired end blocks, block copolymers with heterodimer-forming modules, and silk-inspired triblock copolymers that feature integrin-binding or proteoglycan-binding cell-adhesive motifs. All of these protein polymers were produced at g/L levels, and various bioprocessing and strain engineering strategies were employed to address problems such as proteolytic degradation and other undesired posttranslational modifications. The basic physicochemical properties of the polymers produced were studied, which revealed interesting characteristics. Some of these polymers show promise for further development towards biomedical applications such as tissue engineering and controlled drug release.

The ribosome regulates flavodoxin folding
Houwman, Joseline A. - \ 2017
University. Promotor(en): Willem van Berkel, co-promotor(en): Carlo van Mierlo. - Wageningen : Wageningen University - ISBN 9789463431453 - 176
proteins - flavoproteins - cofactors - ribosomes - biosynthesis - eiwitten - flavoproteinen - co-factoren - ribosomen - biosynthese

During and after their translation by the ribosome, folding of polypeptides to biologically active proteins is of vital importance for all living organisms. Gaining knowledge about nascent chain folding is required to enhance our understanding of protein folding in the cell. This in turn allows us to obtain insights into factors responsible for protein misfolding, aggregation, and, potentially, for numerous devastating pathologies.

In Chapter 1 the model protein flavodoxin is introduced. Also theories about protein folding are presented, which led to the concept of the “folding energy landscape”. Flavodoxin folds via a misfolded off-pathway intermediate, which is molten globular and forms extensively during its refolding in vitro.

In Chapter 2 we show that flavodoxin also populates an on-pathway molten globule during its folding. In the F44Y variant of apoflavodoxin, lowering the ionic strength induces the off-pathway molten globule state. By adding the cofactor FMN, we could follow aspects of the folding of this protein, as off-pathway molten globular flavodoxin first has to unfold and subsequently refold before FMN can bind. Thus, presence of the off-pathway molten globule retards FMN binding. We determined the presence of the off-pathway molten globule at decreasing ionic strengths with cofactor binding kinetics and polarized time-resolved tryptophan fluorescence spectroscopy. Comparison of both data sets revealed the presence of another, concurrently present molten globule. This species is most likely on-pathway to native protein. To our knowledge this is the first time that two concurrent molten globules have been discovered that reside on folding routes of decidedly different nature (i.e., on- and off-pathway ones).

While much work has been done on the folding of flavodoxin in vitro, the next step is to elucidate how this protein folds in vivo. In Chapter 3 the first insights into cotranslational flavodoxin folding are presented. By using ribosomal nascent chains (RNCs) we could determine that when flavodoxin is fully exposed outside the ribosome it can bind its cofactor. However, while its five C-terminal amino acids are still sequestered in the ribosomal exit tunnel, the protein is in a non-native state and cannot bind FMN. Thus the last step in production of this flavoprotein in vivo is the binding of cofactor.

Chapter 4 reveals the influence of the ribosome on formation of the off-pathway molten globule of flavodoxin. By using RNCs of the F44Y variant of apoflavodoxin, we proved that the ribosome restrains formation of this molten globule. This discovery was possible by exploiting the findings of Chapter 2 and Chapter 3, namely that cofactor binding kinetics slow down when off-pathway molten globule is present and that a fully exposed, natively folded flavodoxin nascent chain binds FMN. For F44Y RNCs no retardation in FMN binding occurs, whereas cofactor binding slows down in case of isolated, full-length F44Y in the molten globule state. Thus the ribosome restrains formation of molten globules in stalled nascent flavodoxin. This is possibly due to electrostatic repulsion of the nascent chain by the ribosomal surface, as both are negatively charged, leading to entropic stabilization of native protein at physiological ionic strength.

In Chapter 5 we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These proteins form intermediates that are off-pathway to native protein and several of these species are molten globules. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. The experimentally characterized off-pathway intermediates seem to be of α-helical nature. We discuss the probing of the cotranslational folding of flavodoxin as a first step towards a molecular description of how flavodoxin-like proteins fold in vivo.

Finally, Chapter 6 touches upon the implications of our findings and possible applications in biotechnology, health and disease. A finding that has potential application is the role FMN has as a chemical chaperone. This chemical chaperone can already work at the cotranslational level, as binding of FMN stabilizes a nascent chain and thereby protects the nascent chain against degradation.

Plant growth promotion by Pseudomonas fluorescens : mechanisms, genes and regulation
Cheng, X. - \ 2016
University. Promotor(en): Francine Govers; J.M. Raaijmakers, co-promotor(en): M. van der Voort. - Wageningen : Wageningen University - ISBN 9789462578753 - 192 p.
soil bacteria - pseudomonas fluorescens - plants - growth stimulators - soil suppressiveness - plant diseases - induced resistance - biochemistry - biosynthesis - plant-microbe interactions - transcriptomics - bodembacteriën - 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.

Thermococcus kodakarensis : the key to affordable biohydrogen production
Spaans, S.K. - \ 2016
University. Promotor(en): John van der Oost, co-promotor(en): Servé Kengen. - Wageningen : Wageningen University - ISBN 9789462577725 - 245 p.
thermococcus - thermococcus kodakarensis - hydrogen - bioenergy - canonical analysis - biosynthesis - nadph - archaea - microbiology - waterstof - bio-energie - canonische analyse - biosynthese - microbiologie
The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171 p.
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - biosynthese - peroxisomen - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - serine proteïnasen

Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.

A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.

As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.

To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.

Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.

Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.

The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.

Metabolic engineering of biosynthesis and sequestration of artemisinin
Wang, B. - \ 2016
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789462576728 - 210 p.
artemisinin - nicotiana benthamiana - arabidopsis - biosynthesis - malaria - drugs - genetic engineering - metabolism - artemisinine - biosynthese - geneesmiddelen - genetische modificatie - metabolisme

The sesquiterpenoid artemisinin (AN) is the most important medicine for the treatment of malaria in humans. The industrial production of AN still mainly depends on extraction from the plant Artemisia annua. However, the concentration of AN in A. annua is low. Although different engineering strategies have been used in both A. annua and heterologous plant and yeast production platforms, the worldwide capacity and production costs for AN are not in balance with its demand (Chapter 1). Although the genes encoding for the entire AN biosynthesis pathway (AN-PW) of the AN precursor dihydroartemisinic acid (DHAA) have been identified, the application of these genes in pathway engineering seem to be limited by lack of control over product transport and sequestration. At the onset of this thesis project there was no information on transport in the AN-PW. However, it was known that DHAA is converted into AN outside the glandular trichome cells of A. annua. Therefore, in this thesis I tried to gain more knowledge on transport within the AN-PW and the use of different metabolic engineering strategies to improve the production of AN.

At the onset of my PhD project, the AN-PW genes from two different A. annua chemotypes were compared to understand the basis of different relative activities in the two branches of the AN-PW (Chapter 2). For these assays we used transient expression in N. benthamiana. In the AN-PW, artemisinic aldehyde (AAA) is at a branch point as it can be converted to artemisinic acid (AA) by amorphadiene oxidase (AMO), or to dehydroartemisinic aldehyde (DHAAA) by artemisinic aldehyde Δ11 (13) reductase (DBR2). AA is the precursor for arteannuin B (AB) while DHAAA may be converted by a CYP71AV1 or an ALDH1 to dehydroartemisinic acid (DHAA), the precursor for AN. In this chapter we demonstrate that the CYP71AV1 from a high AN production (HAP) chemotype has reduced activity in the AB branch of the pathway compared to the CYP71AV1 from a low AN production (LAP) chemotype. In addition, we show that the relative expression levels of DBR2 and ALDH1 also affect the AN/AB chemotype. The low catalytic efficiency of AMO from the HAP chemotype may be caused by a deletion of seven amino acids at the N-terminus of the protein compared to CYP71AV1 from LAP. Ectopic expression of the AN-PW genes in N. benthamiana showed that the bulk of the PW products are modified by glycosylation and glutathione conjugations. These side reactions therefore compete with the biosynthesis flux towards the AN precursor DHAA. At this point in my thesis the ectopic expression of AN-PW genes in N. benthamiana had not yielded any AN. At a later stage it became clear that this was due to harvest of leaves at 5-7 days post agro-infiltration (dpi), while AN in N. benthamiana leaves expressing AN-PW genes only becomes detectable after 7 dpi.

Glycosylation of the bulk of the AN-PW products in N. benthamiana stresses the need for an efficient transport of (DH)AA to the outside of cells in order to escape from the glycosylation reactions. In Chapter 3, transport and sequestration of AN precursors was investigated by studying the effect of membrane transporters (PDRs) and lipid transfer proteins (LTPs). Hereto, two membrane transporters with activity towards AN-PW products were made available by the group of Prof. Marc Boutry and we isolated three LTP genes from Artemisia annua which showed expression in the glandular trichomes. In this chapter we show that AaLTP3 displays specific activity, together with AaPDR2 towards transport of (DH)AA to the apoplast in N. benthamiana. Moreover, infiltration experiments with (DH)AA in N. benthamiana leaves revealed that these compounds are rapidly taken up by the cells and that inside the cells there is a strong reverse flux in the AN-PW by conversion of (DH)AA towards (DH)AAA and (DH)AAOH. Subsequently we demonstrated that AaLTP3 has a stronger activity in keeping products in the apoplast than the AaPDR2 membrane transporter. Therefore, I suggest that by removal of (DH)AA from the cytosol through transport over the plasma membrane by AaPDR2 and subsequent sequestration in the apoplast by AaLTP3, AaLTP3 creates sink activity which prevents reflux of (DH)AA from the apoplast back into the cells. AaLTP3 therefore contributes to a directional flux through the AN-PW towards the end product (DH)AA. Finally, in this work we could also for the first time detect AN and AB in N. benthamiana leaves by extraction of necrotic leaves at 13 dpi.

Because in A. annua glandular trichome cells both the AN sesquiterpene biosynthesis pathway and the flavonoid biosynthesis pathway are active, we explored whether there is a functional interaction between these two major secondary metabolite biosynthesis pathways. In Chapter 4 we describe how we manipulate the flavonoid biosynthesis pathway in N. benthamiana leaves using the Antirhinum majus transcription factor Rosea1 (ROS) and test coexpression of ROS with AN-PW genes. The co-expression of ROS stimulates AN-PW product accumulation. Subsequent analysis indicates that this is most likely from transcriptional activation of the enzyme Mevalonate Kinase (MVK) in the mevalonate pathway, which provides precursors for the sesquiterpene biosynthesis pathway. In addition, we demonstrate that production of flavonoids competes with AN-PW product accumulation, as co-expression of AN-PW genes with ROS, but simultaneous inhibition of chalcone synthase (CHS) by a CHSRNAi construct, results in higher AN-PW product levels. However, accumulation of the end products AN and AB was not affected significantly. Finally, the combined expression of AN-PW+ROS+AaPDR2+AaLTP3+ CHSRNAi results in highest sequestration of (DH)AA in the apoplast and highest accumulation of the end products AN and AB in N. benthamiana.

During my thesis work, in a related project it was found that expression of another sesquiterpene biosynthesis gene (caryophyllene synthase; CST) in transgenic Arabidopsis resulted in higher caryophyllene emission for a transformant expressing a genomic DNA of CST, compared with a similar transformant expressing a CST cDNA described in literature. This suggested that ectopic expression of intron containing genes is more efficient than ectopic expression of cDNAs. To test whether in the context of metabolic engineering the use of genomic (intron-containing) genes is more efficient than the use of the corresponding cDNA we generated a set of stable transformed Arabidopsis lines with either genomic CST (gCST), cDNA CST (cCST), genomic amorphadiene synthesis (gADS) and cDNA ADS (cADS). In chapter 5 we show that indeed the lines with overexpression of the genomic clones yield higher levels of the anticipated products (caryophyllene or amorphadiene) than the lines with overexpression of the corresponding cDNAs. Transcript analysis showed that for gCST the increase in caryophyllene production was higher than can be explained solely by the increase in CST transcription. In the context of transient expression in N. benthamiana leaves the intron-mediated-enhancement effect was less pronounced.

In the final discussion chapter 6 I review limitations and potential solutions to metabolic engineering of the AN-PW in plants, and I discuss the impact of our findings on AN production capacity using transient expression versus natural production in A. annua. Moreover, I discuss how the finding of this thesis go beyond just insights into the AN-PW as especially the identification of the role of LTPs in sesquestration of (sesqui)terpenes into the apoplast may have an impact on the metabolic engineering efforts of many other (sesqui)terpene pathways. Because some plant hormones are also terpenoid products the newly identified role of LTPs may also have impact on a deeper understanding of hormone signalling in plants. I have already started exploring this path by generating a set of Arabidopsis plants with overexpression of different Arabidopsis LTP genes to test whether any hormone related traits are altered (Chapter 6). Preliminary results do indeed confirm a role of LTPs in endogenous plant hormone balance, something worthwhile to be further explored in future research.

The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. - \ 2015
University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
fungi contain very large numbers of genes related to SM production. Yet these genes are typically
silent under laboratory conditions. There are now many tools and strategies available to activate
or clone silent SM genes. This thesis details our efforts to apply various methods to define and
then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
containing many silent SM genes and gene clusters.

In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
of growth conditions. Heterologous expression of silent SM genes in a production host is also
discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
source of novel biosynthetic enzymes.

In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
of these SM genes or gene clusters were speculated, based on their similarity to those characterized
in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
fungal species. Remarkably, two predicted functional gene clusters were linked to the production
of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
findings, the majority of SM genes remained silent and only cladofulvin was detected. During
growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
another mechanism associated with a biotrophic lifestyle.

In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
of discovering new compounds. Many Ascomycete-specific global regulators of SM production
and morphological development in other fungi were identified in C. fulvum. We investigated
three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
This suggests that cladofulvin production is not required for asexual reproduction. The main
finding of this chapter is that global regulator manipulation cannot considered to be a universal
tool to discover new fungal natural products.

In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
is produced by many fungi and plants. Once synonymous only with constipation, this former
laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
forming novel dimers from existing monomers.

In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
active during early and late stages of infection of tomato, but are down-regulated during biotrophic
growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
inoculation (dpi). At each time point the infections were inspected microscopically to detect any
phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
(UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
results suggested that cladofulvin confers resistance to abiotic stress.

In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
enable discovery of homologous genes encoding enzymes with different specificities. Combining
bioinformatic and functional analyses should prove to be a powerful strategy for discovering
compounds with new biological activities, or enzymes relevant to metabolic engineering.

Regulation and natural functions of lipopeptide biosynthesis in Pseudomonas
Song, C. - \ 2015
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 - lipoproteins - biosynthesis - genetic mapping - gene regulation - genomics - transcriptomics - defence mechanisms - mutants

Summary

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.

Bridging domains : a comparison between information processing in Archaea and Eukarya
Koning, B. de - \ 2015
University. Promotor(en): John van der Oost, co-promotor(en): Stan Brouns. - Wageningen : Wageningen University - ISBN 9789462572379 - 154
archaea - transfer rna - rna-polymerase - transcriptieregulatie - biosynthese - sulfolobus solfataricus - rna polymerase - regulation of transcription - biosynthesis

Bridging Domains

A Comparison between Information Processing in Archaea and Eukarya

Studying Information Processing

Living cells evolved complex systems to handle the flow of information both accurately and efficiently. These systems are highly comparable between the three domains of life: eukaryotes, bacteria and archaea. The central components of replication, transcription, aminoacylation, and translation are found in every living cell known today, with only relatively small deviations, despite a separation of billions of years of evolution. Archaea are unicellular, do not contain organelles, and have relatively small genomes, so are, at first sight, quite similar to their far better known prokaryotic cousins: the bacteria. Nevertheless, if it comes down to information processing, archaea are, surprisingly, more related to eukaryotes than to bacteria, both at the sequence level of RNA and proteins, and at the architecture level of key complexes as well. This makes them excellent model systems to study eukaryote-like information processing. The absence of cell specialization, less cell organization, less or even no intracellular compartmentalization, and less intensive regulation, have proven to give a clearer picture of the function of conserved key elements within these complex systems. [Chapter 2]

In this thesis, we report several attempts to elucidate functional details of some very conserved factors in information processing in S. solfataricus using recently established genetic modification techniques. S. solfataricus is a thermoacidophilic crenarchaeote that grows optimally at temperatures between 70°C and 85°C and at pH values between 2 and 3. Its genome sequence is known since 2001. Best practices have become standardized between laboratories, and the genomic toolbox includes gene knockout, overexpression systems, the availability of reporter genes, and tunable promoters.

MBF1, a highly conserved activator

MBF1 (multi-protein bridging factor 1) is reported to be a transcriptional co-activator in eukaryotes. It was shown to cross the gap between transcription regulators and the transcriptional machinery itself. MBF1 was found to be highly conserved within archaea, being present in almost all species with the key exception of marine thaumarchaeotes. However, none of the associated transcription regulators were known to be present within the archaeal domain, raising the question whether a class of other regulators was overlooked, or that archaeal MBF1 might be a transcriptional activator itself, binding to DNA directly instead of indirectly via a binding partner. Additionally one study revealed a surprising dual role of this protein: in yeast it was not only associated with transcription but contributed to translation fidelity as well. A neighbourhood analysis across the archaeal domain revealed no clear preference for either transcription or translation. Elements of both systems are equally present, especially in the well conserved neighbourhood within the crenarchaeotes. [Chapter 3]

A mbf1 disruption mutant of the S. solfataricus was made using heterozygous recombination with a suicide plasmid. Under standard laboratory growth conditions mbf1 appears to be not essential for growth, and comparing growth characteristics with its parental strain did not reveal striking differences between the two. It was observed, that the Sulfolobus mbf1 disruption mutant is much more sensitive during cultivation than its parental strain, showing sudden death during growth much more often. Being hard to quantify, this behaviour was especially observed when cultures were transferred at later stages during stationary phase or unfrozen from long term storage. But the largest difference was observed in the increased sensitivity of the mbf1 disruption mutant towards paromomycin. Paromomycin is an aminoglycoside-type antibiotic that interferes with the recognition of cognate codon-anti-codon binding within the ribosomes during translation. [Chapter 4]

A more detailed study to the molecular characteristics of the archaeal MBF1 from S. solfataricus revealed hardly any associations to the transcription machinery, but strengthened the assumed association to the translation apparatus. It was found that archaeal MBF1 consists of two domains that are structurally independent: an N-terminal zinc-ribbon, which is not conserved beyond the archaeal MBF1s, and the well conserved C-terminal HTH-domain (helix-turn-helix domain). This C-terminal HTH domain was shown to bind to the small ribosomal subunit by affinity purification, and in co-purification experiments, in which we detected the presence of archaeal MBF1 in ribosomal purifications. NMR structure comparisons confirmed that archaeal MBF1 binds to the small ribosomal subunit using its C-terminal HTH domain, whereas the N-terminal zinc-ribbon might only contribute to this interaction, but does not participate directly in binding. [Chapter 5]

Altogether, these findings made us believe that MBF1s in archaea are not associated with transcription but rather with translation. Based on the observations in yeast, and more recently its binding to polyadenylated mRNAs in different eukaryotic species, and, against the backdrop that the protein domain that binds to the small ribosomal subunit in S. solfataricus is highly conserved across the archaeao-eukaryotic lineage, it is tempting to speculate that the eukaryotic MBF1 plays a comparable role in the translation process in eukaryotes as well.

TGT, a conserved dichotomy

Another well conserved element within all three domains of life, which is involved in information processing, is the TGT (tRNA-guanine transglycosylase) family of proteins. This family of proteins shows a clear dichotomy: TGT is responsible for the exchange of guanine at the wobble position (position 34) of the anti-codon of certain tRNAs with either queuosine in eukaryotes or its precursor preQ1 in bacteria, whereas, in archaea, TGT is responsible for the exchange of guanine with preQ0 at position 15 in almost, if not all, archaeal tRNAs. PreQ0 is in a later stage converted to archaeosine by another protein that belongs to the TGT family as well.

Disruption of the tgt gene, which encodes the TGT protein in S. solfataricus, revealed that it was solely responsible for this process without any redundancy present. Like mbf1, this gene appeared to be non-essential, as this mutant was also as viable as its parental strain, and showed hardly any changes in growth characteristics. In comparison to the mbf1 disruption mutant, the tgt disruption mutant was much more stable and did not reveal the sensitivity to stationary phase. It grew slightly slower than the parental strain, especially at normal temperatures (75°C), but when temperature levels were raised (87-93°C) growth returned to almost wild-type levels. [Chapter 6]

Aiding research to the basal machinery of RNAP

Beyond doubt, the best studied, element of information processing systems is the RNAP (RNA polymerase) complex. Its basal core is present in all known life forms, and is highly conserved. The surrounding, auxiliary, and regulatory elements are less conserved, but, nevertheless, the archaeal RNAP is almost identical to the eukaryotic RNAP II complex (see figure). This high resemblance already proved beneficial, as the heterologous expression of the archaeal RNAP revealed numerous functional details about the molecular characteristics of the complex as a whole, and, in addition, revealed also an unprecedented insight in the separate subunits as this provided opportunities to tamper with the subunit composition and to modify the separate subunits themselves by introducing genetic variations.

Unfortunately, purification of homologously expressed complexes, which are expressed in archaeal systems itself, are, in contrast to ones heterologously expressed in bacterial hosts, hard to obtain, and involve a number of purification steps and therefore a substantial amount of biomass. To enable easier purification, a method was developed in which a purification tag was inserted in the genome of S. solfataricus after a gene that encodes an RNAP subunit, avoiding artificial overproduction by viral infections or heterologous expression in other less adapted hosts. In a proof of principle experiment, the enrichment an RNAP core component was proven, whereas an auxiliary element was tagged using this novel method. [Chapter 7]

Biosynthesis, regulation and biological role of strigolactones in rice
Moura Luis Cardoso, C.S. De - \ 2014
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789462570917 - 166
oryza sativa - rijst - striga - parasitaire planten - rizosfeer - lactonen - gigaspora rosea - biosynthese - rice - parasitic plants - rhizosphere - lactones - biosynthesis

In her thesis Catarina Cardoso studied strigolactone biosynthesis in rice. Strigolactones are multifunctional compounds produced by plants. They are plant hormones that regulate plant architecture, but in addition plants release strigolactones into the soil to communicate and initiate beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. Parasitic plants of the genera Striga, Orobanche and Phelipanche take advantage of this communication to also recognize their hosts and infest them. These parasites infect crops and cause significant economic losses in Mediterranean regions and especially in Sub Saharan Africa where they put food security at risk. Catarina found there is large variation in strigolactone biosynthesis between the two major rice groups (indica and japonica) and located the genes responsible for this. She also showed that the different strigolactones produced by rice have a differential impact on AM fungi and seeds of parasitic plants. These findings suggest that it is possible to select crop varieties that can interact with AM fungi, without inducing parasitism. The knowledge generated in this study can contribute to the urgent need to control the worldwide parasitic weed problems. At the same time strigolactones also control plant development and the results of this study may resuylt in tools to develop better yielding and sustainable crops.

Elucidation of strigolactone biosynthesis in the host plant rice and the signal perception in the parasitic plant Striga hermonthica
Zhang, Y. - \ 2014
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789462570191 - 208
striga hermonthica - parasitaire planten - biosynthese - signaaltransductie - oryza sativa - rijst - enzymen - plantenfysiologie - parasitic plants - biosynthesis - signal transduction - rice - enzymes - plant physiology

Strigolactones (SLs) are a newly identified class of plant hormones regulating plant architecture, including shoot and root branching. Plants also secrete blends of SLs into the rhizosphere, where they stimulate colonisation of the host roots by arbuscular mycorrhizal (AM) fungi, beneficial organisms for the host. But SLs also induce the seed germination of root parasitic plants, such as Striga, which can have a big negative impact on crop yield. A better insight in how the different SLs are synthesized by the host and how the parasitic plant Striga perceives them could help to develop crops with proper AM colonisation and Striga resistance at the same time. In this thesis, two cytochrome P450 enzymes responsible for the last step in SL formation and SL structural diversification in rice were identified. In addition, the F-Box protein MAX2 of Striga (ShMAX2), a SL signalling component, was characterised, representing the first example from a root parasitic plant species, which is paving the way for furthering our understanding of how SLs are perceived by these parasites. The knowledge gained in this thesis brings us a significant step closer to the possibility to improve crop breeding strategies for parasitic weed resistance.

Biosynthesis and transport of terpenes
Ting, H.M. - \ 2014
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789461738929 - 183
nicotiana benthamiana - artemisia annua - planten - plantensamenstelling - terpenen - biosynthese - genen - genisolatie - plants - plant composition - terpenoids - biosynthesis - genes - gene isolation

Terpenoids are the largest class of natural product that are produced by plants, with functions that range from a role in plant development to direct defence against pathogens and indirect defence against insects through the attraction of natural enemies. While terpene biosynthesis genes have been well studied, there is still only limited knowledge on how terpenes are transported within the cell and from the cell to the apoplast. In this thesis, different aspects of transport of terpenes in plants were addressed. Firstly, the issue of intermediate transport between enzymes was studied, focussing on the regulation of intermediate flux between two different biosynthesis enzymes (CYP71AV1 and DBR2) that determines the resulting Artemisia annua, low or high artemisinin, chemotype. We also investigated the role of Lipid Transfer Proteins and vesicles in the transport of terpenes. Some LTP mutants were indeed shown to emit lower terpene levels, but the exact mechanism could not be resolved. Inhibition of vesicle transport increased terpene levels, most likely due to an effect on protein stability. I conclude that there are multiple transport mechanisms involved in terpene transport which complicates the analysis of a single transport pathway.

Discovery and reconstitution of the secoiridoid pathway of Catharanthus roseus
Dong, L. - \ 2014
University. Promotor(en): Richard Immink, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789461738462 - 201
catharanthus roseus - secoïridoïden - secundaire metabolieten - medicinale eigenschappen - genen - biosynthese - secoiridoids - secondary metabolites - medicinal properties - genes - biosynthesis

Terpene indole alkaloids (TIAs) are important plant-produced secondary metabolites for

humans, because of their anti-cancer properties. The production of TIAs still fully relies on

extraction from medicinal plants like Catharanthus roseus, which only contains extreme low

amounts of these compounds and new ways need to be found to efficiently produce these

anticancer drugs at low cost. The common precursor for TIAs is strictosidine and in my PhD

project I tried to produce strictosidine in fast-growing tobacco by transferring the genes of the

whole biosynthesis pathway into tobacco. At the onset of my project 6 out of the presumed

12 genes of the pathway in C. roseus had not been discovered yet. My thesis tells the story of

discovery and characterization of the missing genes and reconstruction of the full strictosidine

pathway in tobacco.

1

Elucidation of the sesquiterpene lactone biosynthetic pathway in feverfew (Tanacetum parthenium)
Liu, Q. - \ 2013
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen UR - ISBN 9789461737564 - 134
tanacetum parthenium - sesquiterpenen - biosynthese - bioactieve verbindingen - medicinale eigenschappen - plantenfysiologie - fytochemie - sesquiterpenes - biosynthesis - bioactive compounds - medicinal properties - plant physiology - phytochemistry

Parthenolide is the major bioactive compound of feverfew and has anti-inflammatory and anti-cancer activity. Chapter 1gives an overview of the history and current status of research on parthenolide in feverfew. As a promising anti-cancer drug, parthenolide has attracted a lot of attention from medical institutes and companies. A search with ‘parthenolide’ in Google patents yields more than 2000 hits on extraction of parthenolide or its use in treating cancer or other diseases. However, information on the parthenolide biosynthetic pathway is scarce. Elucidation of the full pathway to parthenolide would open up new opportunities for production of this compound in heterologous, more efficient production platforms.

To elucidate the biosynthetic pathway of parthenolide, knowledge on the tissue(s) in which parthenolide is produced and stored is important. In Chapter 2, parthenolide was found to highly accumulate particularly in floral trichomes, suggesting that this is also the preferred site of biosynthesis. These floral trichomes were subsequently used to isolate germacrene A synthase (TpGAS), the gene encoding the first dedicated step in parthenolide biosynthesis, using a degenerate primer PCR approach. The transcript level of TpGASwas indeed highest in glandular trichomes. The high expression of TpGASin glandular trichomes which also contain the highest concentration of parthenolide, supports the assumption that glandular trichomes are the organ where parthenolide biosynthesis and accumulation occur.

During my work on Chapter 2, a Canadian group reported a germacrene A oxidase (GAO) from lettuce. As the 454 cDNA library of feverfew trichomes was not available yet, we decided to use a 454 cDNA library of chicory (which also produces costunolide) to continue screening candidate genes involved in the next step of the parthenolide biosynthetic pathway, costunolide synthase (COS). In Chapter 3, four P450s (belonging to the CYP71 group) were selected from the chicory cDNA library for functional characterisation in yeast. One of them, named CYP71BL3, was found to be costunolide synthase, and can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. The biosynthetic pathway of costunolide was reconstituted in Nicotiana benthamiana by transient expression (agro-infiltration) ofTpGAS, CiGAO(which we also identified in the chicory library) and CiCOS, whichresulted in costunolide production of up to 60 ng.g-1FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

When the 454 sequences of the feverfew trichome library became available, we continued to identify additional genes involved in the biosynthetic pathway of parthenolide. In Chapter 4, the parthenolide biosynthetic pathway was elucidated by isolating all the structural genes from feverfew, TpGAS, TpGAO, TpCOS and TpPTS. Moreover, the whole pathway was reconstituted in N. benthamiana, through transient expression. In the agro-infiltrated plants, parthenolide as well as a number of conjugates (with cysteine and glutathione) were produced. In an anti-cancer bioassay, these relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. Finally, also a gene encoding a costunolide and parthenolide 3β-hydroxylase was identified, which could potentially be used in biotechnological applications to produce hydroxylated parthenolide. The conjugation and hydroxylation of parthenolide open up new options to improve the water solubility of parthenolide and therefore its potential as a drug.

Besides genes involved in the biosynthetic pathway of parthenolide, we also identified two other P450 genes that can utilize costunolide as substrate. In Chapter 5, Tp8879 is identified. Tp8879 can cyclise the monocyclic germacranolide sesquiterpene lactone costunolide to form the bicyclic guaianolide sesquiterpene lactone kauniolide, and is hence called kauniolide synthase. The biosynthetic pathway of kauniolide was reconstituted in N. benthamiana, through transient expression.

This thesis combines a series of existing and new technologies for gene discovery – transcriptomics and metabolomics - as well as optimisation of plant metabolic engineering – using transient expression in N. benthamiana- and reports on novel combinatorial biochemistry occurring in metabolic engineering of heterologous plant hosts, resulting in novel sesquiterpene lactone derivatives with the potential to be new drug leads. The use of transient expression and metabolomics for unexpected product identification are technologies that will be of great value to others working in the field of metabolic engineering. The strategies for identification and characterization of candidate genes, the strategies and tools for metabolic engineering and the possibilities to further improve pathway metabolic engineering are discussed in Chapter 6.

Dual responsive physical networks from asymmetric biosynthetic triblock copolymers
Pham, T.H.T. - \ 2013
University. Promotor(en): Martien Cohen Stuart; Jasper van der Gucht, co-promotor(en): Frits de Wolf. - Wageningen : Wageningen UR - ISBN 9789461737359 - 163
polymeren - gels - biopolymeren - biosynthese - elastine - collageen - polymers - biopolymers - biosynthesis - elastin - collagen

The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random coil-like connector, and B is a second self-assembling block. A and B have to be chosen such that they do not cross-assemble. With these molecules it should be possible to fabricate hydrogels in which direct ‘loops’ are excluded. We exploited genetic engineering to design proper genes encoding asymmetric triblock protein polymer and fermentation to produce monodisperse protein polymers. There different asymmetric triblock protein polymers were produced and characterized.

The first molecule, silk-elastin hybrid molecule (SCE), was inspired by natural silk and elastin. The silk-like block (S) forms a pH-sensitive beta-roll (beta-sheet like) structure that further stacks into long fibrils. The elastin-like block(E) has thermo-responsive properties; above the lower critical solution temperature (LCST), it forms aggregates. We find that polymers that have both silk and elastin-like domains show temperature dependent fibril formation. At high temperature, the elastin blocks irreversibly induce bundling and aggregation of fibrils. The presence of the elastin-like block also changes the kinetics of fibril formation. Whereas silk-like protein without elastin forms monodisperse fibrils, the presence of elastin results in polydisperse fibrils due to homogenous nucleation.

The self-assembly of silk-elastin hybrid molecule is further analysed in the presence of NaCl. We find that the thermo-responsive behaviors of elastin-like block are strongly dependent on salt concentration. At high salt concentration, the aggregation transition is much more pronounced. At high pH, where the S block does not self-assemble, the polymer forms micellar aggregates upon heating in the presence of NaCl. At low temperature, lowering the pH leads to fibril formation. When both blocks are induced to self-assemble, the final structure reveals a pathway-dependence. Heating the solution of fibrils formed at low temperature results in fibril aggregates which do not dissociate upon cooling. The pH-triggered fibril formation of preheated protein solutions leads to the formation of large objects, which likely cause sedimentation. The structural difference is also demonstrated clearly in the morphology of gels formed at high protein concentration: whereas the gel formed in the first pathway (first lower the pH, then increase the temperature) is transparent, the gel formed in the latter pathway (first increase the temperature, then lower the pH) is milky and has a higher elastic modulus.

The second type of asymmetric triblock copolymer (TR4H or TR4K) has a collagen-like, triple-helix-forming motif at one end, and a poly cationic block at the other. The collagen-like end-block T consists of 9 (PGP) repeats and forms thermo-responsive triple helices upon cooling. Such helices are reversibly disrupted when the temperature is raised above the melting temperature. The other end-block has 6 positively charged amino acids (histidine-H or lysine-K) and forms micelles when a negatively charged polymer is added. The charge-driven complexation of this block depends on its degree of deprotonation, which is determined by the pKa and the pH. The additives used in this study are a flexible polyanion (polystyrene sulfonate, PSS) and a semi-flexible polyanion (xanthan). We find micelle-to-network transition of the triblock TR4H in complexation with PSS. First, the self-assembly of each end-block is studied separately. As expected, the collagen-like block reversibly forms triple helices upon cooling. The cationic H block forms charge-driven complexes upon adding PSS, leading to micelles with an aggregation number that depends on ionic strength. At high concentration, the micellar TR4H/PSS solutions form a viscoelastic gel upon cooling, which melts at high temperature, indicating the formation of helical junctions between the micelles. Liquid-liquid phase separation is observed when the concentration is below the gelation point (around 90 g/L). This leads to a dilute phase on top of a concentrated gel phase. The phase separation is driven by the attraction between charge-driven micelles caused by the triple helices. It disappears when the solution is heated or when the ionic strength is increased.

The charge-driven complexation of TR4K with xanthan, a negatively charged polysaccharide is also studied. At high temperature and at very low xanthan concentration, the TR4K binds to the xanthan backbone via the charged block K, leading to charge-driven bottle brushes, as indicated by a significant increase in light scattering intensity due to the increased mass. This interaction is dependent on the pH, due to protonation of the cationic K block. The xanthan/TR4K complex shows thermo-sensitivity due to the helical interaction of the collagen-like blocks. At a xanthan concentration around the overlap concentration (~7 g/L), the presence of the triblock results in an increase in elastic modulus of xanthan gels. At high temperature, the elastic modulus increases by 3 times after adding the triblock. As triple helices do not form, this must be due to changes in the entanglement of the bottle brushes. Also the non-linear rheology of the xanthan/TR4K gels differs significantly from that of xanthan alone. At low temperatures when the helical junctions are formed, the elastic modulus increases even further approximately two times compared with the corresponding value at high temperature. This is ascribed to the formation of crosslinks induced by the proteins between the xanthan molecules. The triblock protein modifies the properties of the xanthan hydrogels in three ways: (1) a significant increase in storage modulus, (2) thermo-sensitivity and (3) a two-step strain softening, where the first step is probably due to unbinding of the proteins from the xanthan backbones.

The third molecule is an asymmetric triblock copolymer (TR4T-Cys), which has two triple helix forming end-blocks (T), with a cysteine residue (Cys) added to one of these. Under oxidizing conditions, the cysteine residues can form disulfide bonds between two polymers whereas reducing conditions restore the thiol groups. Since cysteine can form only one S-S bridge, intramolecular loops are prevented. The presence of S-S bonds significantly enhances the thermal stability of the triple helical network. This results in the appearance of two melting temperatures, of which the higher one is due to the S-S stabilized triple helices. The elastic modulus of the physical gels in the presence of S-S bonds is almost 2 times higher than that of the physical gels in the absence of S-S bonds. The relaxation time also triples under oxidizing conditions, which indicates that triple helical knots are also kinetically stabilized by S-S bonds.

In summary, the design of S-C-S (S: functional end-block, C: connector) network-forming components might meet the increasing demands of high performance biomaterials that must be able to build a physical gel under narrowly defined conditions. Such class of telechelic polymer might display various interesting dynamic behaviors including shear banding, self-assembly, rheochaos, and phase-separation. Another aspect is the functionality of the end-block which self-assembles upon triggering. However, connectors often return to the same nodes, resulting in loop formation. Loop formation is a structural imperfection that weakens network connectivity and lowers the material’s elasticity. The asymmetric triblock with two different end-blocks is designed in order to: (1) prevent unimolecular loops and improve mechanical properties (2) achieve multi-responsiveness: this allows us to observe different assembling pathways. In this work, with respect to (1), we indeed observed the decrease in loop formation in physical gels formed by TR4T-Cys due to the formation of S-S bridges. With respect to (2), we indeed obtained multi-responsive hydrogels with all three asymmetric triblock proteins. However, we have only scratched the surface as understanding kinetics of self-assembly and pathway dependent processes. Further investigations are needed to get more insights into how to manipulate various parameters in controlling the final structures.

Fructan biosynthesis in crop plants : the molecular regulation of fructan biosynthesis in chicory (Cichorium intybus L.)
Arkel, J. van - \ 2013
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Ingrid van der Meer. - S.l. : s.n. - ISBN 9789461736635 - 158
cichorium intybus - gewassen - zea mays - solanum tuberosum - transgene planten - koolhydraten - fructanen - biosynthese - inuline - polymerisatie - crops - transgenic plants - carbohydrates - fructans - biosynthesis - inulin - polymerization

Fructan is a polymer of fructose produced by plants and microorganisms. Within the plant kingdom about 45.000 species accumulate fructan as storage carbohydrate in addition to, or instead of, starch. Fructan accumulating species are mainly found in temperate and sub-tropical regions with seasonal or sporadic rainfall. During the last decades, the use of fructan in the (food) industry has rapidly evolved, because of its health promoting characteristics and interesting functional properties.Chicory (Cichorium intybus L.) is a biennial taproot-bearing crop plant that is grown for the production of inulin on an industrial scale. Inulin, a ß(2,1) linked linear fructan with a terminal glucose residue, is stored in the chicory taproots. The degree of polymerisation (DP) determines the application of the inulin and hence the value of the crop. This leads us to the central question of this thesis:

What regulates the fructan yield and the degree of polymerisation, and how can we modify this?

The DP is highly dependent on the field conditions and harvest time, and therefore the first step in answering this question was tostudy the regulation of fructan (inulin) metabolism throughout the growing season. This is described in Chapter 2. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. We determined the concentrations of soluble carbohydrates, the inulin mean degree of polymerisation (mDP), inulin yield, gene expression and activity of enzymes involved in inulin metabolism in the taproots. Inulin biosynthesis - catalysed by sucrose: sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan: fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT) - started at the onset of taproot development. Inulin yield increased with time following a sigmoid curve reaching a maximum in November. The maximum inulin mDP of 15 was reached in September and then gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three phases in the growing season and analysed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST is not regulated by day length and temperature. From mid-September onwards the mDP decreased gradually although inulin yield still increased. This is most probably the result from back-transfer activity of 1-FFT and fructan exohydrolase activity (EC 3.2.1.153) (1-FEH). In plants 1-FEH catalyses the breakdown of fructan in order to release the stored carbohydrates necessary in periods of stress, like cold or drought periodsor flowering. This information was used to design two strategies to obtain the desired, increased inulin DP and yield. Overexpression of 1-SSTwas performed to increase the mDP and to keep the sucrose concentration low, to prevent 1-FFT from depolymerizing inulin. The result was a higher mDP during the growing season. Unfortunately, no effect on the mDP was seen at the end of the growing season, most probably due to activity of FEH. Secondly, anFEH I antisense fragment was introduced into chicory in order to block depolymerisation at the end of the growing season. This resulted in a reduction in FEH Iexpression upon cold induction, but had only minor effects on the mDP. The degradation of inulin was most probably caused by the remaining 1-FEH activity. Overall this study showed that inulin metabolism in chicory is tightly regulated, but also revealed options to further steer inulin metabolism in chicory.

The next step in answering the central question was to study the regulation of the genes involved in fructan biosynthesis. In Chapter 3this was studied at three different levels. Firstly, fructan gene expression and carbohydrate concentrations were studied in axial sections of mature chicory root, revealing the highest expression levels and carbohydrate levels in the phloem. Correlations were found between the gene expression patterns of 1-SST, 1-FFT and the carbohydrate levels, suggesting a possible involvement of sugars in the regulation of 1-SSTand 1-FFTgene expression. Secondly, the induction of 1-SSTand 1-FFTexpression was studied in excised chicory leaves. Expression of both 1-SSTand 1-FFTwas induced upon sucrose and glucose feeding, suggesting that both genes are at least partly regulated in the same way. Upon fructose feeding, the induction of fructan biosynthesis was less pronounced than with sucrose. The expression of 1-SSTwas induced by fructose but this resulted in only low amounts of 1-kestose. The expression of 1-FFTwas not induced upon fructose feeding.Thirdly, to further unravel the mechanism of induction, the promoters of 1-SSTand 1-FFTfrom chicory were isolated and characterized through in silicoand in planta(only 1-FFT) analysis. Computational analysis of fructosyltransferase (FT) promoters revealed elements that are common in fructan biosynthesis-promoters among different species and also occur in Arabidopsis promoter sequences. One of these elements is predominantly present in genes involved in sugar metabolism and transport. This element did also contain a core sequence involved in MYB transcription factor binding important for fructan biosynthesis activation in wheat, as was published recently. An 1100bp 1-FFTpromoter fragment was shown to be functional in transgenic chicory and in the non-fructan accumulating plants species, Arabidopsis and potato. Application of carbohydrates resulted in the expression of the reporter gene GUS comparable to 1-FFTexpression upon carbohydrate feeding in chicory. This study provides information on the regulation of inulin biosynthesis, suggestions for studies on transcription factors, and provides a promoter for steering the expression of fructan biosynthetic genes in transgenic plants. An alternative way for the production of inulin with the desired DP and yield, circumventing the problems in chicory rather than trying to solve them, is the introduction of the fructan biosynthetic pathway in non-fructan metabolizing and catabolizing plant species.

Towards this end we have expressed the inulin synthesizing enzymes, 1-SST and 1-FFT from Jerusalem artichoke, in maize and potato, as described inChapter 4. Transgenic maize plants produced inulin type fructan (at 3.2 milligram per gram kernel) and kernel development was not affected. Potato tubers expressing 1-SSTaccumulated 1.8 milligram inulin per gram tuber while tubers with a combined expression of 1-SSTand 1-FFTaccumulated 2.6 milligram inulin per gram tuber. Inulin accumulation in maize kernels was modulated by kernel development, first peaking in young seeds and then decreasing again through degradation during late kernel development. In potato, inulin mDP was relatively stable throughout tuber development and little evidence of degradation was observed. The accumulation of 1-kestose in transgenic maize correlated positively with kernel sucrose concentration and introduction of the fructan biosynthetic pathway in a high-sucrose maize background increased inulin accumulation to 41 milligram per gram kernel kernel. This study shows the importance of sugar availability and the absence of degradation mechanisms in platform crops for tailor-made fructan production.

Further evaluation of the production of tailor-made inulin and putative platform crops is discussed in Chapter5.Here we come to the conclusion that the mDP, the distribution and yield depend on the origin of the fructan biosynthesis genes and the availability of sucrose in the host. The combination of genes from different origins could result in new types and different lengths of fructan molecules resulting in (new) specific properties of fructan. Limitations for the production of tailor-made fructan in chicory are not seen in putative new platform crops, such as sugar beet, sugarcane and rice.

The work described in this thesis on fructan biosynthesis in chicory and in new platform crops has resulted in new insights that will lead new applied and fundamental research in this field.

Unravelling the bruising discoloration of Agaricus bisporus, the button mushroom
Weijn, A. - \ 2013
University. Promotor(en): Harry Wichers, co-promotor(en): Jurriaan Mes. - [S.l.] : s.n. - ISBN 9789461735638 - 262
agaricus bisporus - paddestoelen - kneuzen - verkleuring - melaninen - fenolverbindingen - biosynthese - genetische analyse - mushrooms - bruising - discoloration - melanins - phenolic compounds - biosynthesis - genetic analysis

In this research the browning-discoloration caused by bruising of button mushrooms was analysed. Brown-discoloration of mushrooms can amongst others be caused by the picking and storage of mushrooms. Current day commercial hybrids can not be used for mechanical harvesting because mushrooms are sensitive for discoloration. Mechanical harvesting can be used to lower the production costs of mushrooms. To make this possible new hybrids should be available that have a higher tolerance for bruising-discoloration. To breed for new hybrids the cause of bruising-discoloration needs to be analysed. This was done by analysing the compounds (substrates) involved in brown-discoloration and to look at the genes involved. These genes code for the enzymes involved in the conversion of the substrates into the dark brown pigment melanin. The research was performed with commercial and wild strains and the offspring of a segregating population.

Pyrethrum secondary metabolism: biosynthesis, localization and ecology of defence compounds
Ramirez, A.M. - \ 2013
University. Promotor(en): Harro Bouwmeester; Marcel Dicke, co-promotor(en): Maarten Jongsma. - [S.l.] : s.n. - ISBN 9789461735171 - 187
tanacetum cinerariifolium - pyrethrinen - metabolisme - biosynthese - verdedigingsmechanismen - plantenfysiologie - afweersecreties - secreties - pyrethrins - metabolism - biosynthesis - defence mechanisms - plant physiology - defensive secretions - secretions

The use of botanical insecticides is in today’s world an attractive alternative to the less safe and environmentally malign synthetic chemicals, whose overall longer persistence in the environment does not only make them more contaminant, but also increases their chances of causing a rapid development of resistance in the target pest and having negative-side effects on beneficial organisms. Yet, at present only a handful of botanical products are in commercial use for insect control on crops. Among the most important botanical insecticides (pyrethrins, rotenone, neem and essential oils), pyrethrins have the longest history of effective use against a wide range of insects and best record of low toxicity to mammals. Although pyrethrins were relegated from their once prominent position in the mid-1930’s, the recent market trends towards “reduced risk” pesticides and host plant resistance have brought pyrethrins back to the attention and initiated the generation of knowledge around them. Pyrethrins refer to an oleoresin extracted from the dried daisy-like flowers of pyrethrum (Tanacetum cinerariifolium). The active constituents are six esters formed by a combination of two acids (chrysanthemic acid and pyrethric acid) and three alcohols (pyrethrolone, cinerolone and jasmolone, collectively called rethrolones).The esters of chrysanthemic acid with the rethrolones constitute type I pyrethrins, whereas the esters of pyrethric acid are collectively known as type II pyrethrins. Apart from being the source of pyrethrins, pyrethrum also produces a range of other defense compounds collectively known as sesquiterpene lactones, which have also been implicated in plant defense against herbivores, pathogens, and competing plant species. Although pyrethrins and sesquiterpene lactones are found throughout the whole plant, the highest concentrations of both types of compounds are found in the achenes of the flowers, which are densely covered with glandular trichomes. GC-MS analysis revealed that trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. Although glandular trichomes were known to participate in the production of mono- and sesquiterpene compounds that were stored in or emitted from the subcuticular cavity just outside the apical cells, here we demonstrate that basipetal secretion can also occur. In pyrethrum, the monoterpene-derived portion of pyrethrins, chrysanthemic acid (CA), is translocated from the trichomes to the pericarp, where it is esterified into pyrethrins that accumulate in the intercellular space. We also show that during seed maturation, pyrethrins stored in the pericarp are absorbed by the developing embryo, and that during seed germination these embryo-stored pyrethrins are recruited by the germinating seedling, which, due to the lack of trichomes, cannot produce defense compounds themselves. At early stages, not only sesquiterpene lactones that diffuse to the soil from the seed coat, but also the pyrethrins found in the seedlings, seem to play a more important role as antimicrobials than as insecticides.

Although there has been considerable progress on the chemistry of pyrethrins, the molecular/biochemical basis of their biosynthesis was largely unknown. The acid and alcohol moieties of pyrethrins derive from distinct pathways. Whereas the alcohol portion is believed to be derived from linolenic acid and share a large part of its biosynthetic pathway with jasmonic acid, the acid moieties are monoterpenes with a cyclopropane ring that are supposed to be derived from an irregular monoterpene pathway. Before the start of this project, only one enzyme, chrysanthemyl diphosphate synthase (TcCDS), had been isolated and demonstrated to catalyze the first step in the biosynthesis of the acid portion of pyrethrins, which consist of the condensation of two molecules of DMAPP to produce chrysanthemol (COH) via chrysanthemyl diphosphate. During this project an additional acyltransferase enzyme (TcGLIP) was isolated by another group and demonstrated to be responsible for the esterification of (1R,3R)-chrysanthemoyl-CoA and (S)-pyrethrolone, one of the last steps in the biosynthesis of pyrethrins, which was demonstrated in this thesis to likely take place in the pericarp.

To identify additional genes of the pyrethrin biosynthetic pathway, we generated three EST libraries derived from ovaries, trichomes and leaves. Gene candidates were obtained either by keyword interrogation of the annotated contigs or by blasting the libraries with known genes catalyzing similar reactions in other plants. Given the likelihood of cytochrome P450s as potential candidates to catalyze the missing steps in the biosynthesis of the acid moiety of pyrethrins, the pyrethrum EST libraries, were first interrogated for genes encoding cytochrome P450 (CYP) enzymes with a developmental expression pattern similar to TcCDS, and a specific expression in CA-producing glandular trichomes. Experiments with yeast microsomes allowed the selection of two enzymes capable of converting COH into chrysanthemal. Although after agro-infiltration none of these enzymes affected the background level of CA, one of them (Ct21854) resulted in a strong reduction of COH emission, which correlates with a significantly higher amounts of a CA conjugate, confirming that Ct21854 qualifies as a chrysanthemic acid synthase efficiently converting COH into CA.

Rethrolones have been proposed to originate from linolenic acid and share part of the oxylipin pathway with jasmonic acid, which in turns implies that one of the first committed steps should involve a lipoxygenase enzyme, catalyzing the hydroperoxidation of linolenic acid at position 13 of the hydrocarbon chain. Based on this assumption the pyrethrum EST libraries were interrogated for genes encoding LOX enzymes. The expression patterns of twenty-five lipoxygenase EST contigs were characterized, and the ones with a developmental regulation similar to TcCDS and TcGLIP were selected. Subsequently, the molecular cloning of a lipoxygenase, TcLOX1, was carried out. Recombinant TcLOX1was demonstrated to catalyze the peroxidation of the linolenic acid substrate at the C13

position. The gene shares the developmental and trichome-specific expression pattern with TcCDS, suggesting that a trichome production and translocation could be operating for the alcohol moiety of pyrethrins as well.

Finally, besides pyrethrins, pyrethrum plants are also a source of sesquiterpene lactones. Even though considerable information on bioactivity and industrial significance of pyrethrum sesquiterpene lactones is available, the localization and biosynthetic origins were largely unknown. Like in other species of the Asteraceae family, pyrethrum sesquiterpene lactones are exclusively stored in trichomes and it is shown that germacratrien-12-oic acid (GAA) is most likely the central precursor of all known sesquiterpene lactones found in pyrethrum. Candidate genes implicated in the first two committed steps leading from farnesyl diphosphate to GAA were retrieved from the pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. Furthermore, a gene encoding an enzyme capable of catalyzing the C6 hydroxylation of GAA was characterized. This hydroxylation results in spontaneous lactonization likely resulting in the putatively identified C6-C7-costunolide-derived STLs in pyrethrum. However, the enzyme may also catalyze the hydroxylation at the C6 position of the lactonized precursor of all reported C7-C8-type STLs.

In conclusion, the work compiled in this thesis presents new insights into the participation of pyrethrum trichomes in the biosynthesis and selective trafficking of pyrethrins precursors and STLs in opposite directions, and contributes to understanding the role of these flower-stored secondary metabolites in the immunization of the next generation against insect and fungal pathogens. Moreover, this study makes an important contribution towards understanding the biochemical and molecular bases of pyrethrins and STLs, the two most relevant bioactive compounds found in pyrethrum.

Biosynthesis of monoterpene alcohols, derivatives and conjugates in plants : roles in resistance to western flower thrips
Yang, T. - \ 2013
University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - S.l. : s.n. - ISBN 9789461735317 - 116
planten - plaagresistentie - verdedigingsmechanismen - insectenplagen - frankliniella occidentalis - monoterpenen - vluchtige verbindingen - biosynthese - pyrethrinen - plants - pest resistance - defence mechanisms - insect pests - monoterpenes - volatile compounds - biosynthesis - pyrethrins

Western flower thrips (WFT), Frankliniella occidentalis, is one of the most serious pests in several vegetable and flower crops worldwide. It is a highly polyphagous insect and a vector of several plant viruses of which the Tomato Spotted Wilt Virus and the Impatiens Necrotic Spot Virus are the most important. Feeding by WFT causes light coloured patches on leaves, petals and fruits, stunted plant growth, and flower and fruit deformation. Synthetic pesticides has been widely used to control WFT. However, the frequent use of these pesticides leads to rapid resistance in WFT, and they are a threat to the environment. Therefore, it is desirable to identify natural sources of resistance effective against WFT to allow breeders to improve resistance in crop species.

Monoterpenes, as constituents of floral scents and plant resins, play an important role in pollinator attraction and in direct and indirect plant defence against pest insects and pathogens. For example, linalool is a common floral scent constituent and found to be emitted from the leaves by many plant species after herbivore attack. In earlier work, linalool-overexpressing Arabidopsis has been tested for resistance to the pest aphid, Myzus persicae, in dual-choice assays, and transgenic plants significantly repelled or deterred the aphids. A linalool synthase (LIS) was overexpressed in chrysanthemum plants and studied the effect of transgenic plants on WFT (Chapter 2). The volatiles from leaves of transgenic plants were significantly attractive to WFT, however, WFT were significantly deterred by the content of leaf discs from transgenic plants. The headspace analysis showed that the volatiles of LIS chrysanthemum leaves were strongly dominated by linalool,but, they also emitted small amounts of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. In addition, LC-MS analysis showed that several non-volatile linalool glycosides were significantly increased in the leaves of LIS chrysanthemum compared with leaves of wild-type plants. A geraniol synthase (GES) was overexpressed in maize to see whether WFT could be affected by geraniol or its derivatives (Chapter 3). However, geraniol produced in transgenic maize was all efficiently converted to non-volatile glycoside, geranoyl-6-O-malonyl-β-D-glucopyranoside, and GES maize had no effect on WFT behaviour. These studies demonstrate complex effects of terpene engineering on the metabolic changes in transgenic plants. These results suggest that the release/glycosylation of terpenes should be controlled to improve plant resistance against WFT upon metabolic engineering with terpene synthases.

The research subsequently focused on a well-known natural pesticide—pyrethrins. Pyrethrins comprise a group of six closely related esters, derived from the monoterpene alcohol chrysanthemol. Pyrethrins are the economically most important natural insecticide with broad uses in homes, agriculture and stored products for more than 150 years. The effect of pyrethrins against WFT was evaluated on its survival, feeding behaviour, and reproduction both in vitro and in planta (infiltrated chrysanthemum leaves) (Chapter 4). Pyrethrins at 0.1% (w/v) and 1% (w/v) exhibited a significantly negative effect on feeding, and the effects of natural concentrations of pyrethrins in pyrethrum leaves can explain the observed high mortality of WFT feeding on pyrethrum leaves. After the finding of this strong effect of pyrethrins on WFT, the study on the biosynthetic pathway of pyrethrins was continued in order to introduce pyrethrin biosynthesis in transgenic plants. A second function of the published enzyme, chrysanthemyl diphosphate synthase (CDS) was identified (Chapter 5). CDS has been reported to catalyse the formation of chrysanthemyl diphosphate (CPP). However, CDS was demonstrated to also catalyse the next step of CPP into chrysanthemol both in vitro and in vivo. CDS was proposed to be renamed as a chrysanthemol synthase (CHS) using DMAPP as substrate. The gene involved in the next step converting chrysanthemol to chrysanthemic acid has also been characterized (Ramirez, 2013). A chrysanthemic acid:CoA

ligase, which is involved in the final stage of pyrethrin biosynthesis was also studied (Chapter 6). The function of this enzyme was confirmed in vitro and the encoding gene showed a similar expression pattern as CHS in several different tissues and flower developmental stages. The gene responsible for making the final esters is a GDSL-lipase-like acyltransferase (Kikuta et al., 2012). We assume still three to four enzymes are required for the biosynthesis of the basic one of the six pyrethrin esters, jasmolin I, from the precursors DMAPP and jasmonic acid which are universal in plants. And four to five extra genes are required for the complete biosynthesis of all six pyrethrin esters.

In this study, new insights were gained for the biosynthesis of monoterpenes and their derivatives and conjugates, as well as for plant resistance to WFT mediated by these compounds. The characterization of genes involved in pyrethrin biosynthesis paves the way for metabolic engineering of this natural pesticide in other crops.

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