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Transcriptional and functional targets of SCHIZORIZA in root development
Liere, Sabine van - \ 2017
Wageningen University. Promotor(en): Ben Scheres, co-promotor(en): Renze Heidstra. - Wageningen : Wageningen University - ISBN 9789463437998 - 122
arabidopsis thaliana - biological development - root meristems - root caps - cell division - stem cells - transcriptomics - gene regulation - mutagenesis - biologische ontwikkeling - wortelmeristemen - wortelmutsjes - celdeling - stamcellen - transcriptomica - genregulatie - mutagenese
In this thesis I focus on SCHIZORIZA, a gene involved in tissue specification and cell fate segregation in the Arabidopsis root. Chapter 1 describes asymmetric cell division, Arabidopdis embryo development and root meristem development. In more detail we describe the maintenance of quiescent centre and columella stem cells, the development of ground tissue and epidermis/ lateral root cap. Finally we introduce SCHIZORIZA (SCZ) as a factor involved in radial patterning and the maintenance of cortex identity.
In Chapter 2, we study the interaction between the SCZ and SHORTROOT/ SCARECROW pathways that are required in parallel during stem cell niche specification in embryogenesis for the maintenance of tissue fates. Here we investigate the strong synergy of shr and scz mutants and show that at late torpedo stage scz;shr double mutant embryos lose both ground tissue and meristem marker expression.
Chapter 3 describes the use of a transcriptomics approach to identify genes differentially regulated by SCZ. These differentially regulated genes can be divided into two distinct tissue enriched groups. Upregulated genes are enriched for root cap expression and cortex expressed genes are overrepresented in the downregulated set of genes. A subset of the upregulated genes has a HSE associated with their promoter and therefore possibly represents direct SCZ targets.
In Chapter 4 we describe a mutagenesis screen to identify functional downstream targets of SCZ. Using a cortex and lateral root cap tissue marker, we identified two suppressors of the scz mutant. Both restore the fate segregation phenotype of scz mutants. We used whole genome deep sequencing to map the causal suppressor mutations in the LBD12 gene.
The analysis of LBD12 function is described in Chapter 5. We show that the single lbd12 mutant has a QC and columella phenotype. In addition, we show that ectopic expression of LBD12 induces ectopic divisions.
MADS specificity : Unravelling the dual function of the MADS domain protein FRUITFULL
Mourik, Hilda van - \ 2017
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kerstin Kaufmann. - Wageningen : Wageningen University - ISBN 9789463436724 - 199
transcription factors - dna binding proteins - arabidopsis thaliana - mads-box proteins - protein-protein interactions - gene regulation - transcriptiefactoren - dna-bindende eiwitten - mads-box eiwitten - eiwit-eiwit interacties - genregulatie
Encrypted in the DNA lays most information needed for the development of an organism. The transcription of this information into precise patterns of gene activity results in the development of different cell types, organs, and developmental structures. Moreover, transcriptional regulation enables an organism to respond to changing environmental conditions. Essential for the regulation of transcription are DNA-binding transcription factors (TFs). TFs bind the DNA in a sequence-specific fashion. Upon binding of a TF to its DNA binding site, TFs typically activate or repress the transcription of nearby genes. To better understand transcriptional regulation it is essential to study DNA binding specificity of TFs.
In the last decades, technological advances allowed the development of high-throughput methods to study protein-DNA interactions. Traditional in vitro methods study one or a few interactions, while new high-throughput methods can determine TF specificity by measuring relative DNA-binding affinities against a large collection or even all possible binding sites. Several high-throughput techniques to study TF-DNA interactions are discussed in Chapter 1 of this thesis. These new technologies and methods resulted in a fast growing number of studies on DNA binding specificities of TFs, expanding the knowledge about TF specificity. A review on the current knowledge of TF DNA binding specificity is described in Chapter 1.
One aspect that influences DNA binding of TFs are differences in ability to form protein-protein interactions. The aim of this thesis was to study the role of protein-protein interactions in determining DNA binding specificity of a developmental regulatory MADS domain TF in Arabidopsis thaliana. While the members of the MADS-box protein family have many, diverse in vivo functions, all members bind in vitro to a 10-bp motif called the CArG-box. Moreover, studies demonstrated that closely related MADS proteins are expressed in the same cells, therefore encountering the same DNA accessibility and DNA methylation patterns, but bind different in vivo targets. Interestingly, MADS domain proteins bind DNA obligatorily as homo- and heterodimers and the interactions between MADS domain proteins are highly protein specific. Hence, MADS domain proteins are a perfect model system to study the influence of intra-family protein interactions on DNA binding specificity.
To study the influence of protein-protein interactions on DNA-binding specificity this work focusses on one specific MADS domain protein, FRUITFULL (FUL). FUL is expressed at two stages during flower development and, in both stages FUL has highly diverse functions. In Chapter 2 we demonstrate using RNA-seq that FUL regulates different sets of target genes in the two stages. Moreover, using ChIP-seq we show that FUL genomic DNA binding is partly tissue-specific. These tissue-specifically bound and regulated genes are in line with the known dual functions of FUL during development. Interestingly, using protein complex immunoprecipitation for the two studied tissues/stages we show that the interactions of FUL with other MADS domain proteins are also tissue-specific. To determine whether the tissue-specific in vivo binding pattern are due to differences in DNA binding specificity of the FUL-MADS dimers, we studied the DNA binding specificities of the different protein complexes using SELEX-seq. The SELEX-seq results show that although all tested dimers preferably bind the canonical binding motif of MADS domain proteins, different dimers have different preferences for nucleotides within and surrounding the canonical binding site. Hence, different MADS domain dimers have different in vitro DNA binding specificities. By mapping the SELEX-seq affinities to the genome we were able to compare these results with in vivo tissue-specific ChIP-seq data. This analysis revealed a strong correlation between tissue-specific dimer affinities and tissue-specific genomic binding sites of FUL. Hence, we show that the choice of MADS dimerization partner influences DNA binding specificity, highlighting the role of intra-family protein interactions in defining DNA binding specificity.
To allow other researchers to determine genome-wide DNA binding of TFs Chapter 3 provides a step-by-step guide for ChIP-seq experiments and computational analysis. The protocol is designed for wet-lab biologists to perform ChIP-seq experiments and analyse their own ChIP-seq data.
Using the genome-wide DNA binding patterns determined by ChIP-seq, Chapter 4 and Chapter 5 take a more detailed look at some of the genes directly bound by FUL. In Chapter 4, we demonstrate a connection between developmentally and environmentally regulated growth programs. We studied a gene directly bound by FUL in pistil tissue, SMALL AUXIN UPREGULATED RNA 10 (SAUR10). SAUR10 expression is regulated by FUL in multiple tissues, among others cauline leaves, stems, and branches. The results show that the expression of SAUR10 at the abaxial side of branches is influenced by a combination of environmental and developmental regulated growth programs: hormones, light conditions, and FUL binding. This spatial regulation possibly affects the angle between the side branches and the main inflorescence stem. Additionally, we discuss several other FUL target genes involved in hormone pathways and light conditions.
Chapter 5 focusses on the putative direct targets of FUL in IM tissue. Among the putative direct targets two genes involved in flavonoid synthesis were identified, FLAVONOID SYNTHESE 1 (FLS1) and UDP-GLUCOSYL TRANSFERASE 78D3 (UGT78D3). Interestingly, similar to the ful-7 mutant, the fls1 mutant is late flowering. Moreover, expression data exposed an increased gene expression for both FLS1 and UGT78D3 in developing meristems and showed FLS1 expression to be influenced by light conditions. We report the first link between the MADS domain protein FUL and flavonoid synthesis in Arabidopsis. Moreover, our results indicate a possible link between flavonoids and flowering time.
In Chapter 6 I discuss the findings of this thesis and make suggestions for further research. Taken together, the work in this thesis shows that intra-family protein interactions can influence DNA-binding specificity of a protein. Thereby these protein-protein interactions can influence genome-wide binding patterns and, as a result, the function of a protein. Moreover, by studying several putative direct targets of FUL in more detail, we demonstrated a connection between development and environment in growth-regulated programs. Interestingly, the FUL target SAUR10 is repressed by FUL in several tissues, including cauline leaves, inflorescence stems, and branches. However, no influence of FUL on SAUR10 expression could be detected in the pistil. So, despite the binding of FUL to the promotor of SAUR10 in the pistil, this binding does not result in gene regulation. This finding reflects the complex relation between TF occupancy and gene regulation, further research is needed to better understand this relation. Moreover, besides MADS domain protein interactions, we found FUL to interact with several proteins of other families. The role of these cross-family protein interactions in cooperative gene regulation is not fully understood and will be an important research topic in the coming years.
Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - tomaten - receptoren - genen - tabak - aardappelen - katoen - transgene planten
Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.
Exploring the resistance against root parasitic plants in Arabidopsis and tomato
Cheng, Xi - \ 2017
Wageningen University. Promotor(en): H.J. Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789463437004 - 305
plants - parasitic plants - arabidopsis thaliana - solanum lycopersicum - host parasite relationships - plant growth regulators - resistance - planten - parasitaire planten - gastheer parasiet relaties - plantengroeiregulatoren - weerstand
Root parasitic plant species such as broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are notorious agricultural weeds. They cause damage to crops by depriving them of water, nutrients and assimilates via a vascular connection. The difficulty in controlling root parasitic weeds is largely due to their intricate lifecycle and partially underground lifestyle. Their life cycle includes processes such as germination of the seed, the formation of the vascular connection with the host, the growth and development of the parasite after attachment and the emergence of shoots and flowers aboveground. The germination of many parasitic plants is induced by strigolactones that were recently shown to also be signalling compounds that stimulate mycorrhizal symbiosis. In addition, in the past few years, their role in plant development and plant defense has been established revealing them as a new class of plant hormones that exert their function likely in interaction with other hormones.
Modeling spatial pattern formation in plant development
Adibi, Milad - \ 2017
Wageningen University. Promotor(en): Vitor Martins dos Santos, co-promotor(en): Christian Fleck. - Wageningen : Wageningen University - ISBN 9789462956896 - 209
plant development - mathematical models - patterns - arabidopsis thaliana - vascular system - xylem - auxins - modeling - systems biology - plantenontwikkeling - wiskundige modellen - patronen - vaatsysteem - xyleem - auxinen - modelleren - systeembiologie
Modern biological research is accumulating an ever-increasing amount of information on genes and their functions. It is apparent that biological functions can very rarely be attributed to a single genes, but rather arise from complex interaction within networks that comprise many genes. A fundamentally important challenge in contemporary biology is to extract mechanistic understanding about the complex behavior of genetic networks from the available data. The interactions within a genetic network are often exceedingly complex and no-linear in nature, and thus are not open to intuitive understanding. This situation has given rise to a host of mathematical and computational approaches aimed at in-depth analysis of genetic network topologies and dynamics. In particular these approaches focus on system level proprieties of these networks, not directly derivable from their constituent components. To a large extent the power of these theoretical approaches rely on meaningful reduction in complexity by utilizing justified simplifications and abstractions. The underlying principle is that in order to comprehend a mechanism, it is not necessary to take into account all the available information about the mechanism. Given this, Computational models that follow this approach focus on incorporating core components that are essential in answering a specific biological question, while simplifying/omitting the less relevant processes. A fundamental question is this regard is what simplifying concept should be employed when developing a theoretical model of a genetic network.
A successful approach to address this question is the notion of network motif analysis. This approach is based on the core idea that most genetic networks are not arbitrary nor unique, instead they can be categorized into common network dynamics and topologies that perform core functions. Analogous to components of an electric circuit (resistors, capacitor, etc.) these network motifs have distinct properties that are independent of the network that they are embedded in. Therefore analysis of genetic networks in terms of their constituent motifs can potentially be an effective mean in obtaining mechanistic understanding about them.
In this thesis the network motif approach is utilized to study two instances of pattern formation in plant tissues. The first study focuses on organization of stem cells within the shoot apical meristem of the model plant, Arabidopsis thaliana. The results demonstrate that three interconnected network motifs can account for a range of experimental observations regarding this system. Furthermore through an exhaustive exploration of the available data, candidate genes and interactions corresponding to these motifs are outlined, thus paving the way for future interdisciplinary investigations.
The second study explores the development of vasculature during arabidopsis embryogenesis. In contrast to shoot apical mersitem in mature plant, the cell number and arrangement of vasculature in highly dynamic during its embryonic development. To account for this feature, a computational framework was utilized that is capable of capturing the interplay between genes and cell growth and division. The outcome revealed that two interlocking networks motifs dynamically control both patterning and growth of the vascular tissue. The study revealed novel spatial features of a motif previously studies exclusively in non-spatial settings. Furthermore the study resulted in a compelling example of model-driven discovery, where theoretical analysis predicted a specific cellular arrangement to be crucial for the correct development of vasculature. Subsequent analysis of experimental data confirmed the existence of this cellular arrangement in the embryo.
The projects presented in this thesis exemplify successful applications of the network motif approach in studying spatial genetic network. In both cases the networks were successfully examined in terms of their constituent motifs, which subsequently lead to increased mechanistic understanding of them. Ultimately the work presented in this thesis demonstrates the effectiveness of studying genetic networks by a combination of careful examination of available biological data and a reductionist modeling approach guided by the concept of network motifs.
Evasion of chitin-triggered immunity by fungal plant pathogens
Rövenich, Hanna J. - \ 2017
Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463436137 - 133
plant-microbe interactions - immunity - receptors - verticillium dahliae - cladosporium - plant pathogens - chitin - arabidopsis thaliana - fungi - plant-microbe interacties - immuniteit - receptoren - plantenziekteverwekkers - chitine - schimmels
Plants establish intricate relationships with microorganisms that range from mutualistic to pathogenic. In order to prevent colonization by potentially harmful microbes, plant hosts employ surface-localized receptor molecules that perceive ligands, which are either microbe-derived or result from microbe-mediated plant manipulation. This recognition ultimately leads to the activation of host immunity. In order to circumvent recognition or suppress immune responses, microbes secrete effector proteins that deregulate host physiological processes. While the number of identified putative effectors has rapidly increased in recent years, their functions and the mechanisms governing their recognition have largely remained unexplored. To enhance our understanding of the molecular interplay between host and microbe, the work presented here was designed to identify further components involved in the recognition of the two fungal pathogens Verticillium dahliae and Cladosporium fulvum, as well as to characterize the functions of effector proteins produced by these pathogens during tomato infection.
Starch meets biotechnology : in planta modification of starch composition and functionalities
Xu, Xuan - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade. - Wageningen : Wageningen University - ISBN 9789462579200 - 169
starch - potato starch - potatoes - solanum tuberosum - plant biotechnology - biotechnology - genetic engineering - transgenic plants - modified starches - phosphate - arabidopsis thaliana - plant breeding - zetmeel - aardappelzetmeel - aardappelen - plantenbiotechnologie - biotechnologie - genetische modificatie - transgene planten - gemodificeerd zetmeel - fosfaat - plantenveredeling
Storage starch is an energy reservoir for plants and the major source of calories in the human diet. Starch is used in a broad range of industrial applications, as a cheap, abundant, renewable and biodegradable biopolymer. However, starch needs to be modified before it can fulfill the required properties for specific industrial applications. Genetic modification of starch, as a green technology with environmental and economic advantages, has attracted increasingly attention. Many achievements obtained from earlier studies have demonstrated the feasibility and potential of using this approach to produce starches with novel properties (Chapter 2).
The main objective of this research was to produce novel starches with enhanced functionalities through genetic modification, while gaining a better understanding of storage starch biosynthesis. A focus on potato was warranted as it represents a superior model system for storage starch biosynthesis studies and for the production of starches with novel properties. To this end, a number of enzymes from various sources have been expressed in potato tubers to modify starch phosphate content and polysaccharide structure, since these two characteristics have long been recognized as key features in starch properties.
To modify starch phosphate content and explore starch (de)phosphorylation, a human phosphatase enzyme named laforin, and modifications of it, were introduced into potato (Chapter 3). Interestingly, modified starches exhibited a significantly higher phosphate content rather than the expected lower phosphate content. Transcriptome analysis showed that the increase in phosphate content was a result of upregulation of starch phosphorylating genes, which revealed a compensatory response to the loss of phosphate content in potato starch. Furthermore, the increase of phosphate content in potato starch was reached to a threshold level. This was in line with the observations in the modified starches from overexpressed- Glucan water dikinase (GWD1) transgenic plants (Chapter 4). Furthermore, overexpression of two starch dikinases from Arabidopsis thaliana, glucan water dikinase 2 and 3 (AtGWD2 and AtGWD3), did not result in a significant increase in phosphate content of potato starch (Chapter 5). Taken together, these results indicated that phosphate content of potato starch is under strict control.
Morphological analysis of starch granules containing different levels of phosphate content confirmed the indispensible role of phosphate content in the normal formation of starch granules, since cracked granules were observed in the starches containing low phosphate content, while irregular bumpy shaped granules were observed in the tubers from plants containing high phosphate content. Interestingly, further analyses on the expression level of genes involved in starch metabolism and sugar-starch conversion suggested that starch phosphorylation might affect starch synthesis by controlling the carbon flux into starch while simultaneously modulating starch-synthesizing genes. Further studies are needed to confirm this finding (Chapter 4).
To produce starches with novel structures, an (engineered) 4, 6-α-glucanotransferase (GTFB) from Lactobacillus reuteri 121 was introduced into potato tubers (Chapter 6). The resulting starches showed severe changes in granule morphology, but not in starch fine structure. Transcriptome analysis revealed the existence of a self-repair mechanism to restore the regular packing of double helices in starch granules, which possibly resulted in the removal of novel glucose chains potentially introduced by the (engineered) GTFB.
This research successfully generated starches with various functionalities, including altered gelatinization characteristics (Chapter 3 and 4), improved freeze-thaw stability (Chapter 4) and higher digestibility (Chapter 6). The exploitation of relationships between starch characteristics and starch properties revealed that starch properties represent the outcome of the combined effect of many factors and are highly dependent on the genetic background in which the modification has been performed.
In conclusion, the research described in this thesis demonstrates the great potential of genetic modification in producing starches with novel properties. Meanwhile, these results revealed the presence of complex and exquisite molecular regulation mechanisms for starch biosynthesis in potato. In future research, these regulations need to be taken into account for the relational design of starch in planta. Certainly, a better understanding of the process of starch metabolism in storage organs would be a great step forward towards tailoring starch in an economically important crop such as potato.
Exploring the genetics underlying the responses to consecutive combinations of biotic stresses and drought in Arabidopsis thaliana
Huang, Pingping - \ 2016
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462578593 - 291
arabidopsis thaliana - genetic models - stress - stress response - drought - botrytis - pieris (lepidoptera) - genetics - gene expression - genetische modellen - stressreactie - droogte - genetica - genexpressie
Plants growing in natural environments are exposed to a broad range of biotic (pathogen attack, insect herbivory, etc.) and abiotic factors (drought, extreme temperatures, UV radiation, salinity, etc.) that are known to cause stress symptoms in many species (Pareek et al., 2010; Robert-Seilaniantz et al., 2010). Biotic and abiotic stress-inducing determinants often adversely impact plant growth and development, frequently leading to severe annual yield losses in agricultural production (Pierik et al., 2013; Pieterse et al., 2012; Stam et al., 2014). In the research endeavors described in this thesis, Arabidopsis thaliana was used as a model organism to study plant responses to different sequential combinations of biotic factors (infection with Botrytis or herbivory by Pieris) and drought. The main objective was to identify genes that contribute to tolerance to the aforementioned sequential stress combinations. Genome-wide association (GWA) mapping and RNA sequencing (RNA-seq) approaches were used to identify combinatorial stress responsive genes. A number of candidate genes to combinatorial stress responses were identified by GWA analysis and RNA-seq. The physiological function of some candidate genes in different stress conditions were characterized using T-DNA insertion mutants and gene expression study. However, the physiological function of many allelic variants in stress conditions remain to be discovered. The study highlights the importance of an array of genes, crucial to the underlying defense processes, as targets for breeding by allele mining, ultimately aimed at improvement of crop tolerance to frequent combinations of stress factors.
Host-plant resistance to western flower thrips in Arabidopsis
Thoen, Manus P.M. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462578807 - 191
arabidopsis thaliana - host plants - insect pests - frankliniella occidentalis - defence mechanisms - pest resistance - genomics - genome analysis - host-seeking behaviour - optical tracking - data analysis - insect plant relations - waardplanten - insectenplagen - verdedigingsmechanismen - plaagresistentie - genomica - genoomanalyse - gedrag bij zoeken van een gastheer - optisch sporen - gegevensanalyse - insect-plant relaties
Western flower thrips is a pest on a large variety of vegetable, fruit and ornamental crops. The damage these minute slender insects cause in agriculture through feeding and the transmission of tospoviruses requires a sustainable solution. Host-plant resistance is a cornerstone of Integrated Pest Management (IPM). Plants have many natural defense compounds and morphological features that aid in the protection against herbivorous insects. However, the molecular and physiological aspects that control host-plant resistance to thrips are largely unknown.
A novel and powerful tool to study host-plant resistance to insects in natural populations is genome-wide association (GWA) mapping. GWA mapping provides a comprehensive untargeted approach to explore the whole array of plant defense mechanisms. The development of high-throughput phenotyping (HTP) systems is a necessity when large plant panels need to be screened for host-plant resistance to insects. An automated video-tracking platform that could screen large plant panels for host-plant resistance to thrips, and dissect host-plant resistance to thrips in component traits related to thrips behavior, was developed. This phenotyping platform allows the screening for host-plant resistance against thrips in a parallel two-choice setup using EthoVision tracking software. The platform was used to establish host-plant preference of thrips with a large plant population of 345 wild Arabidopsis accessions (the Arabidopsis HapMap population) and the method was optimized with two extreme accessions from this population that differed in resistance to thrips. This method can be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations. EthoAnalysis, a novel software package was developed to improve the analyses of insect behavior. There were several benefits from using EthoAnalysis to analyze EthoVision data. The detailed event statistics that could be extracted from EthoAnalysis allows researchers to distinguish detailed differences in moving and feeding behavior of thrips. The potential of this additional information is discussed in the light of quantitative genetic studies.
Stress resistance was studied in the HapMap population on a total of 15 different biotic and abiotic stresses ranging from biotic stresses like insects and nematodes, to abiotic stresses like drought and salt. A multi-trait GWA study to unravel the genetic architecture underlying plant responses to the different stresses was performed. A genetic network in this study revealed little correlation between the plant responses to the different insect herbivores studied (aphids, whiteflies, thrips and caterpillars). For thrips resistance a weak positive correlation with resistance to drought stress and Botrytis, and a negative correlation with resistance to parasitic plants were observed. One of the surprising outcomes of this study was the absence of shared major QTLs for host-plant resistance and abiotic stress tolerance mechanisms. RESISTANCE METHYLATED GENE 1 (RMG1) was one of the candidate genes in this multi-trait GWA study that could be controlling shared resistance mechanisms against many different stresses in Arabidopsis. RMG1 is a nucleotide-binding site Leucine-rich repeat (NB-LRR) disease resistance protein and its potential relation to several resistance/tolerance traits was successfully demonstrated with T-DNA insertion lines.
The 15 stresses were used in a comparison with a metabolomics dataset on this Arabidopsis HapMap population. It was discovered that levels of certain aliphatic glucosinolates correlated positively with the levels of resistance to thrips. This correlation was further investigated with the screening of a RIL (Recombinant Inbred Line) population for resistance to thrips, several knockout mutants and the analysis of co-localization of GWA mapping results between glucosinolates genes and thrips resistance. In a GWA analysis, the C4 alkenyl glucosinolates that correlated the strongest with thrips resistance mapped to the genomic regions containing genes known to regulate the biosynthesis of these compounds. However, thrips resistance did not co-localize with any of the GSL genes, unless a correction for population stratification was omitted. Additional screening of a Cvi x Ler RIL population showed a QTL for thrips resistance on chromosome 2, but no co-localization with any known glucosinolate genes, nor with thrips resistance loci identified by GWA mapping. Knock-out mutants and overexpressors of glucosinolate synthesis genes could also not confirm a causal link between glucosinolates and resistance to thrips. It is possible that the crucial factors that control resistance to thrips may not have been present in sufficient quantities or in the right combinations in the mutants, RILs and NIL screened in this study. Alternatively, the correlation between thrips feeding damage and glucosinolate profiles could be based on independent geographical clines. More research should be conducted to assess which of these explanations is correct.
In the general discussion, the results from this thesis are discussed in a broader perspective. Some prototypes of new phenotyping platforms that could further aid screening for resistance to thrips in the future are presented. Natural variation in host-plant resistance to thrips is compared to the variation in host-plant resistance to aphids and caterpillars. The geographic distribution of host-plant resistance to thrips is not evident in the other insects, in line with the distribution of glucosinolate profiles and other climate factors. The chapter concludes with some suggestions for future research in the field of host-plant resistance to thrips.
Adventitious root formation in Arabidopsis : underlying mechanisms and applications
Massoumi, Mehdi - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Geert-Jan de Klerk; Frans Krens. - Wageningen : Wageningen University - ISBN 9789462578524 - 191
arabidopsis thaliana - adventitious roots - formation - plant development - quantitative traits - etiolation - auxins - explants - molecular biology - gene expression - dna methylation - rooting - ontogeny - plant breeding - adventiefwortels - formatie - plantenontwikkeling - kwantitatieve kenmerken - etiolering - auxinen - explantaten - moleculaire biologie - genexpressie - dna-methylering - beworteling - ontogenie - plantenveredeling
Adventitious root (AR) formation is indispensable in vegetative propagation and is widely used. A better understanding of the underlying mechanisms is needed to improve rooting treatments. We first established a system to study rooting in Arabidopsis, the model organism in plant biology but only occasionally used to study adventitious rooting. Inhibition of polar auxin transport reduced AR formation. The role of auxin transporter proteins (several PIN-proteins) was found to be tissue-specific. Maturation (the transition from juvenile to adult) negatively influenced AR formation. Maturation was associated with increased DNA methylation and decreased miR156 level. 5-Azacytidine, a drug that reduces DNA methylation, increased rooting. We also examined the effect of two donor plant pre-treatments, etiolation and flooding, on rooting. Both increased AR formation.
Natural genetic variation for regulation of photosynthesis response to light in Arabidopsis thaliana
Rooijen, R. van - \ 2016
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts; Jeremy Harbinson. - Wageningen : Wageningen University - ISBN 9789462578203 - 235 p.
arabidopsis thaliana - photosynthesis - genetic variation - light - efficiency - fotosynthese - genetische variatie - licht - efficiëntie
The efficiency of photosynthesis results from the composition and organization of the plant’s internal structural components as well as the capability of response to environmental fluctuations. This thesis aims at identifying the genetic loci that are regulating the (sub-) processes in photosynthetic acclimation to increased irradiance levels, in order to obtain the genetic information useful to breed for photosynthetic performance. It uses genome wide association studies (GWAS) to reveal which genetic loci are being exploited in nature for keeping good photosynthetic performances in natural conditions. Phenotypic variation among natural accessions in photosynthetic light use efficiency response to increased growth irradiance is related to its variation in genetics in order to identify the associated genetic loci. In Chapter 2 is described which light environment reveals most natural variation in photosynthetic performance and for which photosynthetic parameter this is. It shows different Arabidopsis accessions display different photosynthetic responses to various light environments, well relatable to genetic differences. A candidate gene list for the direct response to increased growth irradiance was revealed after performing genome wide association analysis. Chapter 3 elaborates on the genome wide association results by visualizing the dynamics of the associated genetic loci over the time course of the photosynthetic response to increased irradiance. It shows it is possible to simplify the complexity of photosynthetic physiology as well as the genetic analysis in such way to confirm the causal genes underlying the associated loci, by confirming this for the YELLOW SEEDLING 1 (YS1) gene, a gene encoding a Pentatrico-Peptide-Repeat (PPR) protein involved in RNA editing of plastid-encoded genes essential for photosystems I and II. Genetic variation for any trait can be on the transcriptional level or on the functional level. In Chapter 4, the gene regulation in three Arabidopsis accessions with contrasting photosynthesis efficiency responses to increased irradiance is studied. These differences in photosynthesis efficiency are associated to differences in activation extents of heat responsive genes as well as to differences in the presence of a gene activation pathway acting on membrane lipid remodelling, suggested to maintain balanced cellular phosphate concentrations. Chapter 5 confirms the significance of maintaining balanced cellular phosphate concentrations for photosynthesis efficiency responses to increased irradiance. It describes how genome wide association mapping and linkage mapping combine to reveal genetic epistatic interactions between PHOSPHATIDIC ACID PHOPSPHOHYDROLASE 2 (PAH2, phosphate metabolism gene) and ASPARAGINE SYNTHETASE 2 (ASN2, nitrogen metabolism gene), both acting in the delivery of orthophosphate in the chloroplast. In conclusion this thesis contributes new insights into the physiological and molecular pathways underlying photosynthesis responses to increased growth irradiances.
Unraveling molecular mechanisms underlying plant defense in response to dual insect attack : studying density-dependent effects
Kroes, A. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462577756 - 265 p.
016-3953 - arabidopsis thaliana - insect pests - herbivory - pest resistance - defence mechanisms - insect plant relations - molecular plant pathology - density - insectenplagen - herbivorie - plaagresistentie - verdedigingsmechanismen - insect-plant relaties - moleculaire plantenziektekunde - dichtheid
In the field, plants suffer from attack by herbivorous insects. Plants have numerous adaptations to defend against herbivory. Not only do these defense responses reduce performance of the feeding herbivore, they also result in the attraction of natural enemies of herbivores.
The majority of studies investigating plant-insect interactions addressed mainly the effects of attack by a single herbivore species on induced plant defenses. However, because plants are members of complex communities, plants are exposed to different insect attackers at the same time. Moreover, attacks by different herbivores interact at different levels of biological organization, ranging from the level of gene expression, phytohormone production and biochemical changes up to the individual level. Effects of plant responses to feeding by two or more herbivore species simultaneously might cascade through the community and thereby affect insect community composition.
The induction of plant defense responses is regulated by a network of signaling pathways that mainly involve the phytohormones jasmonic acid (JA), salicylic acid (SA) and ethylene (ET). The signaling pathways of the two phytohormones SA and JA interact antagonistically, whereas JA and ET signaling pathways can interact both synergistically and antagonistically in regulating plant defense responses. In general, JA-mediated signaling underlies defense responses against leaf-chewing herbivores, such as caterpillars, whereas phloem-feeding insects, such as aphids, mainly induce SA-regulated defenses.
When caterpillars and aphids simultaneously feed on the same host plant, crosstalk between phytohormonal signaling pathways may affect the regulation of plant defenses. Consequently, multiple insect herbivores feeding on plants interact indirectly through plant-mediated effects. Studies investigating molecular mechanisms underlying interference by multiple attacking insects with induced plant defenses will benefit studies on the ecological consequences of induced plant responses.
The aim of this thesis was to elucidate molecular mechanisms that underlie plant-mediated interactions between attacking herbivores from different feeding guilds, namely Brevicoryne brassicae aphids and Plutella xylostella caterpillars.
Because herbivore density affects the regulation of plant defense responses, it may also influence the outcome of multiple insect-plant interactions. To study if modulation of induced plant defenses in response to dual insect attack depends on insect density, plants were infested with two densities of aphids.
Responses of Arabidopsis thaliana plants to simultaneous feeding by aphids and caterpillars were investigated by combining analyses of phytohormone levels, defense gene expression, volatile emission, insect performance and behavioral responses of parasitoids. To better predict consequences of interactions between plants and multiple insect attackers for herbivore communities, the regulation of defense responses against aphids and caterpillars was also studied in the ecological model plant wild Brassica oleracea.
Transcriptomic changes of plants during multiple insect attack and their consequences for the plant’s interactions with members of the associated insect community take place at different time scales. Direct correlation of transcriptomic responses with community development is, therefore, challenging. However, detailed knowledge of subcellular mechanisms can provide tools to address this challenge.
One of the objectives of this thesis, therefore, was to investigate the involvement of phytohormonal signaling pathways and their interactions during defense responses against caterpillars or aphids at different densities, when feeding alone or simultaneously on the model plant A. thaliana. The studies show that aphids at different densities interfere in contrasting ways with caterpillar-induced defenses, which required both SA- and JA-signal-transduction pathways. Transcriptional analysis revealed that expression of the SA transcription factor gene WRKY70 was differentially affected upon infestation by aphids at low or high densities. Interestingly, the expression data indicated that a lower expression level of WRKY70 led to significantly higher MYC2 expression through SA-JA crosstalk. Based on these findings, it is proposed that by down-regulating WRKY70 expression, the plant activates JA-dependent defenses which could lead to a higher resistance against aphids and caterpillars.
Plutella xylostella caterpillars also influenced plant defense responses when feeding simultaneously with aphids. Caterpillar feeding affected aphid-induced defenses which had negative consequences for aphid performance. Induction of both ET- and JA-mediated defense responses is required for this interference. Moreover, aphid density also played an important role in the modulation by P. xylostella of aphid-induced defenses: P. xylostella caterpillars induced changes in levels of JA and its biologically active from, JA-Ile, only when feeding simultaneously with aphids at a high density.
To study the overall effect of dual herbivory on induced plant defenses, not only interference with induced direct defense, but also with induced indirect defenses was addressed in A. thaliana. We found a significant preference of the aphid parasitoid Diaeretiella rapae for volatiles from aphid-infested A. thaliana wild-type plants and ein2-1 (ET-insensitive) mutants. Interestingly, simultaneous feeding by P. xylostella caterpillars on wild-type plants increased D. rapae’s preference for odors from aphid-infested plants. However, upon disruption of the ET-signaling pathway, D. rapae did not distinguish between ein2-1 mutants infested by aphids or by both aphids and caterpillars. This showed that intact ET signaling is needed for caterpillar modulation of the attraction of D. rapae parasitoids.
On the other hand, attraction of the caterpillar parasitoid Diadegma semiclausum to volatiles emitted by A. thaliana plants simultaneously infested by caterpillars and aphids was influenced by the density of the feeding aphids. Biosynthesis and emission of the terpene (E,E)-α-farnesene could be linked to the observed preference of D. semiclausum parasitoids for the HIPV blend emitted by plants dually infested by caterpillars and aphids at a high density, compared to dually infested plants with a low aphid density.
Transcriptomic changes in the response of A. thaliana wild-type plants to simultaneous feeding by P. xylostella caterpillars and B. brassicae aphids compared to plants infested by P. xylostella caterpillars alone were assessed using a microarray analysis. I particularly addressed the question whether the transcriptomic response to simultaneously attacking aphids and caterpillars was dependent on aphid density and time since initiation of herbivory. The data show that in response to simultaneous feeding by P. xylostella caterpillars and B. brassicae aphids the number of differentially expressed genes was higher compared to plants on which caterpillars had been feeding alone. Additionally, specific genes were differentially expressed in response to aphids feeding at low or high density. Cluster analysis showed that the pattern of gene expression over the different time points in response to dual infestation was also affected by the density of the attacking aphids. These results suggest that insects attacking at a high density cause an acceleration in plant responses compared to insects attacking at low density.
As a next step in the study of multiple interacting herbivores, I studied whether plant responses to dual herbivory have consequences for the performance of a subsequently arriving herbivore, Mamestra brassicae caterpillars. The ecological consequences of plant responses to dual herbivory cascading into a chain of interactions affecting other community members have remained unstudied so far. We used wild B. oleracea plants to evaluate dual herbivore-induced plant adaptations for subsequent herbivory. We found that simultaneous feeding by P. xylostella and B. brassicae resulted in different plant defense-related gene expression and differences in plant hormone levels compared to single herbivory, and this had a negative effect on subsequently arriving M. brassicae caterpillars. Differential induction of JA-regulated transcriptional responses to dual insect attack was observed which could have mediated a decrease in M. brassicae performance. The induction of plant defense signaling also affected both P. xylostella and B. brassicae performance. This study further helps to understand herbivore community build-up in the context of plant-mediated species interactions.
Altogether, findings from this thesis reveal a molecular basis underlying plant responses against multiple herbivory and provide insight in plant-mediated interactions between aphids and caterpillars feeding on plants growing in the field or used in agriculture.
Ecogenomics of plant resistance to biotic and abiotic stresses
Davila Olivas, N.H. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462576575 - 259 p.
016-3932 - arabidopsis thaliana - defence mechanisms - drought resistance - insect pests - plant pathogenic fungi - stress - stress response - transcriptomics - genomics - genetic mapping - verdedigingsmechanismen - droogteresistentie - insectenplagen - plantenziekteverwekkende schimmels - stressreactie - transcriptomica - genomica - genetische kartering
In natural and agricultural ecosystems, plants are exposed to a wide diversity of abiotic and biotic stresses such as drought, salinity, pathogens and insect herbivores. Under natural conditions, these stresses do not occur in isolation but commonly occur simultaneously. However, plants have developed sophisticated mechanisms to survive and reproduce under suboptimal conditions. Genetic screenings and molecular genetic assays have shed light on the molecular players that provide resistance to single biotic and abiotic stresses. Induced defenses are attacker specific and phytohormones play an essential role in tailoring these defense responses. Because phytohormones display antagonistic and synergistic interactions, the question emerges how plants elicit an effective defense response when exposed to conflicting signals under multiple attack. Recent studies have shed light on this issue by studying the effects of combinations of stresses at the phenotypic, transcriptomic and genetic level. These studies have concluded that the responses to combined stresses can often not be predicted based on information about responses to the single stress situations or the phytohormones involved. Thus, combined stresses are starting to be regarded as a different state of stress in the plant. Studying the effects of combinations of stresses is relevant since they are more representative of the type of stresses experienced by plants in natural conditions.
In a coordinated effort, responses of Arabidopsis thaliana to a range of abiotic and biotic stresses and stress combinations have been explored at the genetic, phenotypic, and transcriptional level. For this purpose we used an ecogenomic approach in which we integrated the assessment of phenotypic variation and Genome-Wide Association (GWA) analysis for a large number of A. thaliana accessions with an in-depth transcriptional analysis. The focus of this thesis is especially on (but not limited to) three stresses, i.e. drought, herbivory by Pieris rapae caterpillars, and infection by the necrotrophic fungal pathogen Botrytis cinerea. These stresses were chosen because the responses of A. thaliana to these three stresses are highly divergent but at the same time regulated by the plant hormones JA and/or ABA. Consequently, analysis of responses to combinatorial stresses is likely to yield information on signaling nodes that are involved in tailoring the plant’s adaptive response to combinations of these stresses. Responses of A. thaliana to other biotic and abiotic stresses are included in an integrative study (Chapter 6).
We first investigated (Chapter 2) the extent of natural variation in the response to one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips, Myzus persicae aphids) and two combined stresses (drought plus P. rapae, and B. cinerea plus P. rapae). Using 308 A. thaliana accessions originating from Europe, the native range of the species, we focused on the eco-evolutionary context of stress responses. We analyzed how the response to stress is influenced by geographical origin, genetic relatedness and life-cycle strategy, i.e. summer versus winter annual. We identified heritable genetic variation for responses to the different stresses. We found that winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars and to the combined stresses of drought followed by P. rapae and infection by the fungus B. cinerea followed by herbivory by P. rapae. Furthermore, we found differential responses to drought along a longitudinal gradient.
We further investigated, using A. thaliana accession Col-0, how phenotypic and whole-genome transcriptional responses to one stress are altered by a preceding or co-occurring stress (Chapters 3 and 4). The whole-transcriptomic profile of A. thaliana triggered by single and combined abiotic (drought) and biotic (herbivory by caterpillars of P. rapae, infection by B. cinerea) stresses was analyzed by RNA sequencing (RNA-seq). Comparative analysis of plant gene expression triggered by single and double stresses revealed a complex transcriptional reprogramming. Mathematical modelling of transcriptomic data, in combination with Gene Ontology analysis highlighted biological processes specifically affected by single and double stresses (Chapters 3). For example, ethylene (ET) biosynthetic genes were induced at 12 h by B. cinerea alone or drought followed by B. cinerea inoculation. This induction was delayed when plants were pretreated with P. rapae by inducing ET biosynthetic genes only 18 hours post inoculation. Other processes affected by combined stresses include wound response, systemic acquired resistance (SAR), water deprivation and ABA response, and camalexin biosynthesis.
In Chapter 4, we focused on the stress imposed by P. rapae herbivory alone or in combination with prior exposure to drought or infection with B. cinerea. We found that pre-exposure to drought stress or B. cinerea infection resulted in a significantly different timing of the caterpillar-induced transcriptional changes. Additionally, the combination of drought and P. rapae induced an extensive downregulation of A. thaliana genes involved in defence against pathogens. Despite the larger reduction in plant biomass observed for plants exposed to drought plus P. rapae feeding compared to P. rapae feeding alone, this did not affect weight gain of this specialist caterpillar.
In Chapter 5, we used univariate GWA to (1) understand the genetic architecture of resistance to the different stresses and (2) identify regions of the genome and possible candidate genes associated with variation in resistance to those stresses. In Chapter 5 a subset of the stresses addressed in Chapter 1 (i.e. drought, herbivory by P. rapae and P. xylostella, and the combined stresses drought plus P. rapae and B. cinerea plus P. rapae) were investigated. Results from GWA were integrated with expression data generated in Chapters 3 and 4 or available from the literature. We identified differences in genetic architecture and QTLs underlying variation in resistance to (1) P. rapae andP. xylostella and (2) resistance to P. rapae and combined stresses drought plus P. rapae and B. cinerea plus P. rapae. Furthermore, several of the QTLs identified contained genes that were differentially expressed in response to the relevant stress. For example, for P. xylostella one of the QTLs contained only two genes encoding cysteine proteases (CP1 and CP2). The expression data indicated that these genes were induced by P. rapae and P. xylostella herbivory.
In Chapter 6, the genetic architecture underlying plant resistance to 11 single stresses and some of their combinations was investigated. First, the genetic commonality underlying responses to different stresses was investigated by means of genetic correlations,, revealing that stresses that share phytohormonal signaling pathways also share part of their genetic architecture. For instance, a strong negative genetic correlation was observed between SA and JA inducers. Furthermore, multi-trait GWA identified candidate genes influencing the response to more than one stress. For example, a functional RMG1 gene seems to be associated with susceptibility to herbivory by P. rapae and osmotic stress since loss of function mutants in RMG1 displayed higher resistance to both stresses. Finally, multi-trait GWA was used to identify QTLs with contrasting and with similar effects on the response to (a) biotic or abiotic stresses and (b) belowground or aboveground stresses.
Finally, In Chapter 7, I discuss the feasibility of obtaining plants that are resistant to multiple stresses from the point of view of genetic trade-offs and experimental limitations. The ecogenomic approach for gene discovery taken in this thesis is discussed, and recommendations are especially given on the use of herbivorous insects in quantitative genetic studies of stress resistance. Furthermore, alternatives to the use of insects in quantitative genetic studies of stress resistance are discussed and proposed. Finally, I discuss the feasibility of using an ecogenomic approach to study stress responses in other plant species than the model plant of molecular genetics, A. thaliana.
A wealth of candidate genes was generated by taking an ecogenomic approach, in particular transcriptome analysis and GWA analysis. Functional characterization of these genes is a next challenge, especially in the context of multiple stress situations. These genes constitute a rich source of potential factors important for resistance to abiotic, biotic and combined stresses that in the future may be applied for crop improvement.
Environmental and physiological control of dynamic photosynthesis
Kaiser, M.E. - \ 2016
Wageningen University. Promotor(en): Leo Marcelis, co-promotor(en): Jeremy Harbinson; Ep Heuvelink. - Wageningen : Wageningen University - ISBN 9789462576346 - 248 p.
solanum lycopersicum - arabidopsis thaliana - photosynthesis - carbon dioxide - temperature - humidity - light intensity - fotosynthese - kooldioxide - temperatuur - vochtigheid - lichtsterkte
Irradiance is the main driver of photosynthesis. In natural conditions, irradiance incident on a leaf often fluctuates, due to the movement of leaves, clouds and the sun. These fluctuations force photosynthesis to respond dynamically, however with delays that are subject to rate constants of underlying processes, such as regulation of electron transport, activation states of enzymes in the Calvin cycle, and stomatal conductance (gs). For example, in leaves adapted to low irradiance that are suddenly exposed to high irradiance, photosynthesis increases slowly (within tens of minutes); this process is called photosynthetic induction. Photosynthesis in fluctuating irradiance (dynamic photosynthesis) is limited by several physiological processes, and is further modulated by environmental factors other than irradiance, such as CO2 concentration, air humidity and temperature. Studying dynamic photosynthesis and its environmental and physiological control can help to identify targets for improvements of crop growth, improve the accuracy of mathematical models of photosynthesis, and explore new, dynamic lighting strategies in greenhouses.
In this thesis, the limitations acting on dynamic photosynthesis are explored by reviewing the literature, by experimenting with a suite of environmental factors (CO2 concentration, temperature, air humidity, irradiance intensity and spectrum), genetic diversity in the form of mutants, genetic transformants and ecotypes, and by mathematical modelling. Several genotypes of tomato (Solanum lycopersicum) and the model plant Arabidopsis thaliana, all grown in climate chambers, were used in the experiments. The main findings of the thesis are that a) CO2 concentration and air humidity strongly affect the rate of change of dynamic photosynthesis through a combination of diffusional and biochemical limitations; b) Rubisco activation kinetics are pivotal in controlling rates of photosynthesis increase after a stepwise increase in irradiance, and are further affected by CO2 concentration; c) gs limits photosynthetic induction kinetics in A. thaliana but not in tomato in ambient conditions, and becomes a stronger limitation in low CO2 concentration or air humidity; and d) mesophyll conductance, non-photochemical quenching (NPQ) and sucrose synthesis do not limit dynamic photosynthesis under the conditions used.
In Chapter 1, the rationale for the research conducted is described, by introducing the concept of fluctuating irradiance and its effects on photosynthesis rates. The chapter discusses how dynamic photosynthesis is measured and described, and provides a range of possible applications of the insights gained by the research conducted in this dissertation.
In Chapter 2, the current literature is reviewed and a mechanistic framework is built to explore the effects that the environmental factors CO2 concentration, temperature and air humidity have on rates of dynamic photosynthesis. Across data from literature, higher CO2 concentration and temperature speed up photosynthetic induction and slow down its loss, thereby facilitating higher rates of dynamic photosynthesis. Major knowledge gaps exist regarding the loss of photosynthetic induction in low irradiance, dynamic changes in mesophyll conductance, and the extent of limitations imposed by gs across species and environmental conditions.
Chapter 3 is an experimental exploration of the effects of CO2 concentration, leaf temperature, air humidity and percentage of blue irradiance on rates of photosynthetic induction in dark-adapted tomato leaves. Rubisco activation, changes in stomatal and mesophyll conductance, diffusional and biochemical limitations, efficiency of electron transport through photosystem II, NPQ and transient water use efficiency, were examined to give a comprehensive overview of the environmental modulation of dynamic photosynthesis. Unlike the percentage of blue irradiance, increases in CO2 concentration, leaf temperature and air humidity all positively affected the rates of photosynthetic induction, and these effects were explained by changes in diffusional and biochemical limitations. Maximising the rates of Rubisco activation would increase CO2 assimilation by 6-10%, while maximising the rates of stomatal opening would increase assimilation by at most 1-2%, at the same time negatively affecting intrinsic water use efficiency.
In Chapter 4 it is explored whether the effects of CO2 concentration on dynamic photosynthesis are similar across various irradiance environments. Gain and loss of photosynthetic induction in several low irradiance treatments, as well as sinusoidal changes in irradiance, were studied using tomato leaves. Elevated CO2 concentration (800 ppm) enhanced the rate of photosynthetic induction by 4-12% (compared to 400 ppm) and decreased the loss of photosynthetic induction by 21-25%. Elevated CO2 concentration enhanced rates of dynamic photosynthesis regardless of initial photosynthetic induction state to a similar extent. Therefore, rising global CO2 concentration will benefit integrated assimilation throughout whole canopies, where different leaf layers experience widely differing irradiance regimes.
In Chapter 5 it is tested whether stomatal limitation exists during photosynthetic induction in tomato leaves. The abscisic acid-deficient flacca mutant and its wildtype were exposed to various CO2 concentrations to change the diffusion gradient. Despite gs being much larger in flacca, photosynthetic induction proceeded with the same speed in both genotypes in ambient CO2 concentration. This suggested that stomata did not limit photosynthetic induction in the wildtype. Using these findings, several indices of stomatal limitations were compared. Diffusional limitation, a new index, was found to be the most useful.
In Chapter 6, an exploration of some physiological limitations underlying dynamic photosynthesis is undertaken. Several mutants, transformants and ecotypes of A. thaliana, affecting rates of Rubisco activation, gs, NPQ and sucrose metabolism, were used. Next to a characterisation of their steady-state responses to CO2 concentrations and irradiance, leaves were exposed to stepwise increases and decreases in irradiance (using several intensities) and to lightflecks of several amplitudes and frequencies. Rubisco activase isoform and concentration, as well as various levels of gs, strongly affected rates of dynamic photosynthesis, while this was not the case with low NPQ or sucrose phosphate synthase concentration. This suggests Rubisco activase and gs as targets for improvement of photosynthesis in fluctuating irradiance.
Chapter 7 is a modelling exercise of dynamic photosynthesis, based on data obtained from measurements on mutants of A. thaliana (Chapter 6). This includes a goal-seeking model that allows reproducing the regulation of Rubisco by irradiance and CO2 concentration. The model also includes a full description of leaf-level NPQ, incorporates mesophyll conductance and accounts for the fundamental physics of delays introduced by open gas exchange systems on CO2 measurements. Different data sets for model calibration and validation were used. It was found that the model accurately predicted the effects of the mutants, suggesting that the assumptions of the model were sound and represented the underlying mechanisms correctly.
In Chapter 8, the findings in this thesis are synthesized. The insights gained throughout this dissertation are related to existing literature to give a comprehensive overview of the state of knowledge about the limitations of dynamic photosynthesis. The methodology of assessing transient stomatal limitations, and some aspects of using chlorophyll fluorescence measurements during photosynthetic induction, are discussed. Finally, possible applications and ideas for future research on photosynthesis in fluctuating irradiance are discussed.
Mapping moves on Arabidopsis : from natural variation to single genes affecting aphid behaviour
Kloth, K.J. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462576483 - 269 p.
016-3933 - arabidopsis thaliana - insect pests - aphidoidea - pest resistance - genetic mapping - gene expression - quantitative traits - functional genomics - feeding behaviour - insect plant relations - insectenplagen - plaagresistentie - genetische kartering - genexpressie - kwantitatieve kenmerken - functionele genomica - voedingsgedrag - insect-plant relaties
Dissecting the seed-to-seedling transition in Arabidopsis thaliana by gene co-expression networks
Silva, A.T. - \ 2015
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Henk Hilhorst; Wilco Ligterink. - Wageningen : Wageningen University - ISBN 9789462575929 - 177
arabidopsis thaliana - zaden - zaadkieming - zaailingen - genexpressie - uitdrogingstolerantie - seeds - seed germination - seedlings - gene expression - desiccation tolerance
One of the most important developmental processes in the life-cycle of higher plants is the transition from a seed to a plant and from a generative to a vegetative developmental program. The major hallmark or end-point of the transition from seed to plant is the onset of photosynthesis and the concomitant shift from a heterotrophic to an autotrophic organism. It is advantageous for a species to keep the period of seedling establishment as short as possible since young seedlings are highly sensitive to biotic and abiotic stresses. This implies that the extreme stress tolerance of seeds to i.e. desiccation is lost upon germination. If the regulatory principles of the seed-to-seedling transition are better understood it may become feasible to maintain the seed’s stress tolerance well into the seedling stage.
Despite the profound impact of seedling performance on crop establishment and yield, the seed-to-seedling transition has hardly been studied at the molecular level. This thesis aims at deciphering and understanding the molecular processes that govern this transition in Arabidopsis thaliana. A high-resolution study of the molecular events that occur during these successive transitional stages may provide clues as to the regulatory principles that drive this transition. It may also yield information about the factors that determine the (in)ability to revert to a developmental mode and which features are critical for the maintenance and loss of desiccation tolerance and other stress responses.
In Chapter 1 important processes such as abscisic acid and their regulation are described and it is discussed in what way the seed-to-seedling transition may have links to a trait such as desiccation tolerance. An overview is presented of the current knowledge of the seed-to-seedling phase transition and the existence of a temporal developmental block that can be manipulated by osmotic treatment, the carbon/nitrogen balance and by abscisic acid which results in the re-establishment of desiccation tolerance.
Chapter 2 focuses on comprehensive gene regulation by a detailed transcriptional analysis across seven developmental stages of the seed-to-seedling transition. It describes the inference of a gene co-expression network and several transcriptional modules. I show that such an approach highlights important molecular processes during seedling development, which would not likely be derived from comparative transcript profiling. Moreover, I show that a putative key regulator in one of the transcriptional modules affects late seedling establishment.
In Chapter 3 it is shown how this phase transition is expressed in the primary metabolite profiles in correlation with gene expression. A metabolite-metabolite correlation analysis suggested two profiles, which point at the metabolic preparation of seed germination and of vigorous seedling establishment. Furthermore, a linear correlation between metabolite contents and transcript abundance (Chapter 2) provides a global view of the transcriptional and metabolic changes during the seed-to-seedling transition. It creates new perspectives of the regulatory complexes underlying the seed-to-seedling transition.
Chapter 4 describes the development of a novel method to re-establish desiccation tolerance during the seed-to-seedling transition without adverse effects such as those caused by an osmotic treatment with polyethylene glycol. By using this method, named ‘Mild Air Drying Treatment’ (MADT), I show that the re-establishment of desiccation tolerance is not linked to a reduced ability to accumulate ABA in the desiccation sensitive seeds (germinated seeds at root hair stage). I also present a genetic interaction study of ABSCISIC ACID INSENSITIVE (ABI) genes in their germination response to ABA, and their response to the re-establishment of desiccation tolerance using the MADT. The interaction between ABI3 and ABI4, and between ABI4 and ABI5 act synergistically in the re-establishment of DT, as well as in the germination response to ABA.
In a more in depth study in Chapter 5 I carried out an extensive transcript analysis to infer possible mechanisms of the re-establishment of desiccation tolerance using the MADT protocol. Possible mechanisms underlying the re-establishment of desiccation tolerance were inferred by employing a time-series comparison of germinated desiccation tolerant and -sensitive seeds. Early-response genes of the re-establishment of desiccation tolerance may play a role in events that promote the initial protection to dehydration stress, whereas the late-response genes may play a role in events that help seed to respond to the changes in water dynamics. Moreover, using a gene co-expression network and transcriptional module I concluded that a crosstalk between ABA-dependent and ABA-independent transcription factors regulate the re-establishment of desiccation tolerance.
In Chapter 6 I discuss how the results presented in this thesis contribute to our knowledge of the molecular basis of the seed-to-seedling transition and the re-establishment of desiccation tolerance during its phase changes. Finally, new possibilities for further research are discussed, as well as the further use of the data sets to delineate the mechanisms underlying the seed-to-seedling transition and desiccation tolerance. Possible applications of the results for crop improvement are addressed. Thus, the generation of genetically modified plants that produce seeds with a stress tolerance that extends well into seedling stage may be feasible.
Natural genetic variation in Arabidopsis thaliana photosynthesis
Flood, P.J. - \ 2015
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts; Jeremy Harbinson. - Wageningen : Wageningen University - ISBN 9789462575004 - 278
arabidopsis thaliana - genetische variatie - fotosynthese - genomen - chlorofyl - fenotypen - genetic variation - photosynthesis - genomes - chlorophyll - phenotypes
Oxygenic photosynthesis is the gateway of the sun’s energy into the biosphere, it is where light becomes life. Genetic variation is the fuel of evolution, without it natural selection is powerless and adaptation impossible. In this thesis I have set out to study a relatively unexplored field which sits at the intersection of these two topics, namely natural genetic variation in plant photosynthesis. To begin I reviewed the available literature (Chapter 2), from this it became clear that the main bottleneck restricting progress was the lack of high-throughput phenotyping platforms for photosynthesis. To address this an automated high-throughput chlorophyll fluorescence phenotyping system was developed, which could measure 1440 plants in less than an hour for ΦPSII, a measure of photosynthetic efficiency (Chapter 3). Using this phenotyping platform I screened five populations of Arabidopsis thaliana. Three of these populations resulted from bi-parental crosses and segregated for only two genomes, using these I conducted family mapping (Chapter 4). The final two populations were composed of natural, field collected, accessions and were analysed using a genome wide association approach (Chapter 5). The family mapping approach had greater statistical power due to within population replication and the genome wide association approach had higher mapping resolution due to historical recombination. Both approaches were used to identify genomic regions (loci) which were responsible for some of the variation in photosynthesis observed. The number and average effect of these loci was used to infer the genetic architecture of photosynthesis as a highly complex polygenic trait for which there are many loci of very small effect. In addition to screening these large populations a smaller subset of 18 lines was assayed for natural variation in phosphorylation of photosystem II (PSII) proteins in response to changing light (Chapter 6). This exploratory study indicated that this process shows considerable variation and may be important for adaptation of the photosynthetic apparatus to photosynthetic extremes. The genetic mapping studies just described, focus exclusively on genetic variation in the nuclear genome, whilst this contains the majority of the plants genetic information there is also a store of genetic information in the chloroplast and mitochondria. These genetic repositories contain genes which are essential for photosynthesis and energy metabolism. Any variation in these genes could have a large impact on photosynthesis. To study natural variation in these genomes I developed a new population of reciprocal nuclear-organellar hybrids (cybrids) which could be used to study the effect of genetic variation in organelles whilst controlling for nuclear genetic variation (Chapter 7). Preliminary results indicate that this resource will be of great use in disentangling natural genetic variation in nucleo-organelle interactions. Finally I looked at one chloroplast encoded photosynthetic mutation in more detail (Chapter 8). This mutation had evolved in response to herbicide application and had spread along British railways. When studying this population of resistant plants I found empirical evidence for organelle mediated nuclear genetic hitchhiking. This is a previously undescribed evolutionary phenomenon and is likely to be quite common. In conclusion there is an abundance of genetic variation in photosynthesis which can be used to improve the trait for agriculture and provide insights into novel evolutionary phenomena in the field.
Study of natural variation for Zn deficiency tolerance in Arabidopsis thaliana
Campos, A.C.A.L. - \ 2015
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462572515 - 232
arabidopsis thaliana - voedingsstoffentekorten - sporenelementtekorten - zink - genetische variatie - tolerantie - variatie - genetica - nutrient deficiencies - trace element deficiencies - zinc - genetic variation - tolerance - variation - genetics
Zinc is an important structural component and co-factor of proteins in all living organisms. The model plant species for genetic and molecular studies, Arabidopsis thaliana, expresses more than 2,000 proteins with one or more Zn binding domains. Low Zn availability in arable soils is a widespread problem around the world which results in agricultural losses and the production of grains with low Zn content. The long-term consumption of low-Zn-content food items leads to severe health problems in humans as a result of severe or mild dietary Zn deficiency. Hence the importance of studying Zn homeostasis in plants and mechanisms involved in Zn deficiency tolerance aiming to enhance Zn concentration in plants edible parts and to develop varieties with a higher tolerance to Zn deficiency.
Plants are sessile organisms which trough evolution have developed specific traits in order to adapt to certain environmental conditions in their surroundings. As a result some plant genotypes are more tolerant to Zn deficiency and when exposed to low Zn conditions are able to perform better than others. To investigate the physiological mechanisms involved in Zn deficiency tolerance I examined natural variation present in a set of twenty diverse Arabidopsis thaliana accessions. In chapter 2, differences in shoot biomass production, Zn usage index (ZnUI), ionome (concentration of elements) and expression level of six key Zn deficiency responsive genes were studied. Accessions did not show large natural variation for shoot Zn concentration under Zn deficiency, while the decreases in shoot biomass and ZnUI were more variable. The conclusion from this is that accessions differ for the minimum Zn concentration required for growth which is associated with differences in Zn deficiency tolerance. We also found that the gene expression levels of three Zn transmembrane transporters (IRT3, ZIP3 and 4) in shoot were positively correlated with ZnUI and shoot biomass, but negatively correlated with shoot Zn concentration. This implies that a higher tolerance to Zn deficiency in A. thaliana is associated with an increased Zn translocation from root to shoot under low Zn. Furthermore, I used a logistic regression model to demonstrate that differences in the shoot ionome can be used as a biomarker to identify the plant Zn physiological state. Based on the changes in the concentrations of some elements in each of the Zn deficiency treatments it was possible to predict the Zn physiological state of the plants similarly to when Zn concentration is used alone.
The adaptive response to Zn deficiency involves physiological changes in shoots, but also in roots which play a key role in the acquisition of nutrients. In chapter 3 I used the same twenty A. thaliana accessions as described in chapter 2 to identify root system architecture traits and changes in the root ionome involved in a higher tolerance to Zn deficiency in plants. Similar to shoots, all accessions showed a strong reduction in root Zn concentration under Zn deficiency, whereas changes in other root system architecture traits were more variable between the accessions. These analyses showed that differences between the accessions in root system architecture traits and minimum Zn concentration required for growth are important for Zn deficiency tolerance. The Zn deficiency treatment also affects the formation of lateral roots and thus root system architecture. It was therefore not surprising that the Zn deficiency treatment induced changes in the concentrations of other elements which were correlated with changes in root traits.
Plants respond to different concentrations of Zn supply by changing the expression levels of genes involved in the Zn homeostasis network. This is important for the control of the Zn concentration and sequestration in plant cells, tissues and organs and involves the uptake, accumulation, transport and redistribution of Zn within the plant. Based on the work described in chapter 2, three A. thaliana accessions were selected with contrasting tolerance to Zn deficiency, and used for a whole genome transcription profiling analysis using RNA sequencing. Chapter 4 describes the identification of sets of general and core genes used by A. thaliana in its response to Zn deficiency. The purpose of using three accessions was to complement previous studies, which used only one accession, and identify new candidate genes involved in the general response to Zn deficiency in A. thaliana. General transcriptional changes were observed in the regulation of carbohydrate metabolism, glucosinolate biosynthesis and the circadian clock. As the transcriptional changes were recorded at two time points, it was also possible to distinguish early and late responses to Zn deficiency. The early response to Zn deficiency was stronger in roots with the induction of several Zn homeostasis genes and repression of Fe uptake genes. The late response to Zn deficiency comprised of the strong induction of several Zn uptake, transport and remobilization genes in both roots and shoots. These analysis confirmed several genes previously identified in Col-0 to have a general role in the Zn deficiency response, but it also led to the identification of new candidate genes, such as defensins and defensin-like genes, as very promising new actors in the A. thaliana Zn deficiency homeostasis network.
Chapter 5 describes the A. thaliana accession-specific Zn deficiency responsive transcript profiles, comparing Tsu-0, Pa-2 and Col-0, with the aim to identify biological processes involved in the observed differences in Zn deficiency tolerance between these three accessions. Tsu-0 displayed a high tolerance to Zn deficiency in shoot, Col-0 (reference accession) showed a high tolerance to Zn deficiency in both root and shoot, whereas Pa-2 root and shoot were more sensitive to Zn deficiency. Some of the accession-specific Zn deficiency responsive transcripts were involved in similar biological processes, such as defence response, programmed cell death and carbohydrates and glucosinolates metabolism. The differential regulation of these processes between the three accessions may reflect their differences in Zn deficiency tolerance. Among the Col-0 specific transcripts were several genes encoding proteins kinases which may play a role in a more specific separation of the abiotic and biotic stress responses in this accession and possibly involved in its higher tolerance to Zn deficiency in both shoots and roots. Tsu-0 specifically changes the expression of a set of shoot transcripts encoding ethylene responsive transcription factors which are involved in the regulation of shoot growth and plant tolerance to abiotic and biotic stresses, corresponding well with the observed shoot Zn deficiency tolerance. Accession Pa-2 down-regulated transcripts involved in cell wall organization in roots which correlates with its high sensitivity to Zn deficiency in this organ. Finally, the accessions specific response to Zn deficiency also resulted in the differential regulation of transcripts encoding transposases which may reflect large scale chromatin reorganization or demethylation in response to the stress condition.
The main findings of the research described in this thesis and their implications are described in the General Discussion (chapter 6). By investigating the response to Zn deficiency in a diverse set of A. thaliana accessions both at the physiological and transcriptional level important mechanisms involved in Zn deficiency tolerance were identified. Furthermore, several key candidate genes among the accessions general and accession-specific Zn deficiency responsive transcripts were identified. The further functional characterization of these genes is expected to reveal important new steps in the regulation of Zn homeostasis and Zn deficiency tolerance in A. thaliana.
Numerical and structural chromosome aberrations in cauliflower (Brassica oleracea var. botrytis) and Arabidopsis thaliana
Ji, X. - \ 2014
Wageningen University. Promotor(en): Hans de Jong, co-promotor(en): Erik Wijnker. - Wageningen : Wageningen University - ISBN 9789462571600 - 137
brassica oleracea var. botrytis - arabidopsis thaliana - cytogenetica - chromosomen - chromosoomafwijkingen - chromosome banding - repetitief dna - meiose - aneuploïdie - karyotypen - cytogenetics - chromosomes - chromosome aberrations - repetitive dna - meiosis - aneuploidy - karyotypes
Numerical and structural chromosome aberrations in cauliflower (Brassica oleracea var. botrytis) and Arabidopsis thaliana.
I studied numerical and structural chromosome aberrations in cauliflower (Brassica oleracea var. botrytis) and Arabidopsis thaliana. The large genomic changes are important for gene balance control, gene expression and regulation, and may affect the plant’s phenotype. Moreover, chromosome changes, in particular polyploidy, inversions and translocations play a significant role in evolution. In this thesis, several cytogenetic tools are described to study numerical and structural chromosomal aberrations of cauliflower (Brassica oleracea var. botrytus) and Arabidopsis thaliana. I focus on some of such changes, their origin and implications for genetic, genomic and plant breeding research. The technologies that I used for my study are advanced karyotype analysis based on Fluorescent in situ Hybridization (FISH) with repetitive and single copy sequences as probes, chromosome identification in aneuploids in cauliflower, analysis of meiosis elucidating the cause of meiotic disturbances responsible for aneuploid gametes, and characterization of an inversion using PCR, chromosome painting and immunofluorescence of meiotic proteins.
Signal transduction pathway(s) in guard cells after prolonged exposure to low vapour pressure deficit
Ali Niaei Fard, S. - \ 2014
Wageningen University. Promotor(en): Ernst Woltering, co-promotor(en): Uulke van Meeteren. - Wageningen : Wageningen University - ISBN 9789462570627 - 167
arabidopsis thaliana - vicia faba - dampdruk - verdroging - huidmondjes - abscisinezuur - signaaltransductie - plantenfysiologie - vapour pressure - desiccation - stomata - abscisic acid - signal transduction - plant physiology
Keywords: Abscisic acid, Arabidopsis thaliana, calcium, CYP707As, desiccation, environmental factors, guard cells’ signalling pathway, hydrogen peroxide, natural variation, nitric oxide, photosystem II efﬁciency, RD29A, relative water content, secondary messengers, stomata, vapour pressure deficit, Vicia faba
In short-term, guard cells close stomata in response to an increase in vapour pressure deficit (VPD) and they open the stomata after exposure to low VPDs. However, in long-term responses to low VPD, adaptation processes occur which make stomata less sensitive to stimuli which usually induce stomatal closure (stomatal malfunctioning). Cellular mechanism(s) leading to occurrence of stomatal malfunctioning is (are) still unknown. The aim of this project was to elucidate the processes that are involved in the malfunctioning of stomata after long-term exposure to low VPD. To elucidate whether the problem of stomatal malfunctioning is due to alterations in stomatal morphology and leaf anatomy or in the ABA signalling pathway, fava bean plants were grown at low or moderate VPDs and some plants that developed their leaves at moderate VPD were then transferred for four days to low VPD. Leaf anatomical and stomatal morphological alterations due to low VPD were not the main reason of stomatal malfunctioning in response to ABA and desiccation. Within one day exposure to low VPD, the level of foliar ABA decreased to the same level as in low VPD-grown plants, while the level of ABA-glucose ester was not affected. Spraying ABA during a 4-day exposure to low VPD maintained closing ability of the stomata after 4-day low VPD-exposure. Therefore, alteration in the signalling pathways due to low foliar ABA level was recognized as the main reason for stomatal malfunctioning after long-term low VPD-exposure. Coincidence in changes of Ca2+, ABA receptors, and positive and negative regulators of ABA signalling are proposed as early steps for stomatal malfunctioning induced by low VPD-exposure. Transcriptional activators, transcriptional repressors as well as E3 ligases are proposed for long-term adaptation of cellular processes which consequently cause decreased stomatal response to closing stimuli afterwards. In order to find the molecular mechanism(s) of stomatal malfunctioning, possible variation in stomatal response to closing stimuli was studied among Arabidopsis thaliana accessions after a 4-day low VPD-exposure. Accessions could be grouped to very sensitive, moderately sensitive and less sensitive to closing stimuli using principle component analysis. A positive correlation was found between foliar ABA level (before desiccation) and stomatal closure response to ABA (but not to desiccation) after exposure to different VPDs. Stomatal response to desiccation was positively correlated with the foliar ABA level after desiccation. In order to elucidate the molecular network underlying stomatal malfunctioning in response to ABA due to long-term low VPD-exposure, two groups of Arabidopsis accessions were used as accessions that maintained responsiveness to ABA after low VPD-exposure and accessions with low VPD induced non-ABA-responsive stomata. The foliar ABA content in all accessions correlated with the stomatal response to ABA: only when the ABA level was above a threshold value, stomata responded to ABA. After low VPD-exposure, mainly due to catabolism of ABA, the foliar ABA content decreased. This decrease in ABA level resulted in down regulation of RD29A, which caused decreased stomatal responsiveness to ABA.