Search

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.

Charles Darwin is niet alleen bekend door de evolutietheorie. De negentiende eeuwse wetenschapper was namelijk de eerste die rond 1880 aantoonde dat planten reageren op de zwaartekracht. Daarna was het nog 125 jaar wachten voordat duidelijk werd dat planten de zwaartekracht ook echt nodig hebben voor een normale groei.

Auxin is a plant hormone that triggers a broad variety of responses during plant development. These responses range from correct cell division patterns during embryogenesis to formation and growth of different organs. Due to its importance for plant growth and development, many aspects of the biology of auxin have been studied. In Chapter 2, we use Arabidopsis embryogenesis as a stage to describe generalities about its biosynthesis, transport, components of its signaling pathway and transcriptional control of some know target genes. As most of the players involved in transcriptional regulation in response to auxin have been identified, the question of how the same signal can elicit so many different responses remains open. In this thesis we approach this issue by focusing on the ultimate effectors of the auxin signaling pathway: the ARF family of transcription factors. In Chapter 3 we present the crystal structure of the DNA binding Domain (DBD) of two divergent members of the family: ARF1 and ARF5. Careful observation of the structures, followed by in vitro and in vivo experiments led to the following conclusions: 1) ARF DBDs dimerize through a conserved alpha-helix, and bind cooperatively to an inverted repeat of the canonical TGTCTC AuxRE. Dimerization of this domain is important for high-affinity DNA binding and in vivo activity. 2) Monomeric ARFs have the same binding preference for the DNA sequence TGTCGG (determined by protein binding microarray). 3) DNA-contacting residues are almost completely conserved within the ARF family members. 4) The distance between the AuxREs may play a role for binding of specific ARF dimers as for example, ARF5 can accommodate and bind to different spacing (6-9 bp) compared to ARF1 which is more rigid (7-8 bp). In Chapter 4 we follow up on the observations made. First we again used structural biology to determine the reason of the high binding affinity to the TGTCGG sequence compared to the previously identified canonical TGTCTC element. We found that in complex with TGTCGG, His137 (ARF1) could rotate and make hydrogen bonds with either G5 or G6, as well as a hydrogen bond with the C opposing to G6. This rotation is not possible when in complex with TGTCTC and there the same histidine can make only one hydrogen bond with the G opposing to C6. We conclude then that this histidine plays a role in determining the strength of binding to TGTCNN elements and that this also reflects in its specific transcriptional activity as mutating the corresponding histidine in ARF5 renders a semi-functional protein in vivo (Chapter 3). The next observation we followed up in Chapter 4 is the biological meaning of ARF DBDcooperative binding to DNA. We identified AuxRE inverted repeats (IR) in the promoter of the TMO5 gene and mutated them. This brought the expression of the gene to very low levels despite the presence of other multiple single AuxREs. Thus, the single inverted AuxRE repeat in the TMO5 promoter is essential for ARF5 binding and gene regulation. Importantly, mutating only a single AuxRE element within the inverted repeat led to very pronounced loss of activity, consistent with requirement of both AuxRE sites for high-affinity ARF5 binding. We then concluded that IR AuxREs have a significant effect in gene regulation by ARFs. Next we search the genome for bipartite AuxREs that correlated to auxin response and found two main elements: inverted repeat with 8 bases of spacing (IR8) and direct repeat with 5 bases of spacing (DR5). As this kind of bipartite AuxREs are rarer to find than single AuxREs, we tested their presence in promoters as predictors of auxin responsiveness by qPCR. We found that about 75% of the selected genes containing either IR8 or DR5 responded to auxin. The expression study also show that genes containing the DR5 sequence were only up-regulated when regulated. Interestingly, Surface Plasmon Resonance study showed that only class A (activator) ARFs can bind the DR5 sequence cooperatively. As the structural differences of ARFs DBDs are subtle, we then asked if specific gene targeting is determined by this domain alone. In Chapter 5 we used a DBD swap experiment and conclude that the DBD is necessary for specific gene targeting but not sufficient and the other domains of an ARF also contribute in its specific activity. In Chapter 5 we expand our focus from the DBD to the other ARF domains, Middle Region (MR) and C-terminal (CT). As ARFs have protein-protein interaction interfaces in all three domains, we expressed the isolated domains of ARF5 and perform immuno-precipitation followed by tandem mass-spectrometry. Although the procedure needs optimization, some interactions expected for each domain could be identified. The DBD showed to interact with the general transcription machinery and the CT could interact with another ARF and 3 Aux/IAA. These interactions seem to be specific as the Aux/IAA recovered are not the most abundant in the sampled tissue. Finally, in Chapter 6 all the obtained results are put in a broader context and new questions derived from our results are proposed.

Sweet peppers arrive in waves. New fruits lure so many assimilates towards them that the flowers above them abort. This article provides various suggestions about how to flatten out the peaks and troughs. In the long term, breeding can also play an important role.

Samen met een goede fotosynthese staat voldoende vruchtzetting op de gedeelde eerste plaats bij het welslagen van de teelt van vruchtgroenten. Het zou echter veel gemakkelijker zijn als die bevruchting helemaal niet nodig was. Bij verscheidene gewassen is parthenocarpie inmiddels gewoon, maar bij een belangrijk deel blijft het zaak de juiste omstandigheden voor vruchtzetting te creëren.

Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in many plant species. These non-zygotic embryos go through analogous developmental phases and are morphologically similar to the zygotic embryo. Despite its fundamental importance and enormous application potential, the mechanisms that alter cell fate from non-embryonic to embryonic are elusive. In the past decades, a variety of different model systems have been used to identify regulators of embryo induction, but it is unclear if these act in a common network. We recently found that inhibition of auxin response in the extra-embryonic suspensor cells cell-autonomously and predictably triggers a switch towards embryo identity. In my thesis I have used the suspensor-derived embryogenesis as a uniform model system to study the crucial first reprogramming step of embryo initiation process. Through genome-wide transcriptional profiling upon local (suspensor-specific) auxin response inhibition (Chapter 2) and through testing the ability of fifteen known embryogenesis inducers to promote embryo formation in suspensor cells (Chapter 3), we suggest that suspensor to embryo transformation requires a defined set of genetic regulators. The results obtained in my thesis provide essential tools and basis for further research and are a step forward to understanding the first step of embryo initiation process and to unravel the mystery of totipotency in plants.

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.

Keywords: Somatic embryogenesis, Transcription factor, AINTEGUMENTA-LIKE, BABY BOOM, BBM, Sweet Pepper Transformation Title: Applied and Fundamental Aspects of BBM-mediated Regeneration Author: Iris Heidmann Catergories: Plant regeneration, Plant transformation, transcription factor, somatic embryogenesis Plant regeneration from tissues or single cells is essential for plant propagation. Efficient regeneration can be archieved through somatic embryogenesis using the plant growth regulator auxin or overexpression of specific transcription factors, but the underlying mechanisms are poorly understood. The potency of the BABY BOOM (BBM) AINTEGUMENTA-LIKE transcription factor to induce somatic embryogenesis in crop (sweet pepper) and model species (tobacco) was investigated. It was found that the introduction of BBM into sweet pepper, which is recalcitrant for transformation, enhanced the regeneration of transgenic plants. Exogenous cytokinin was necessary to induce somatic embryogenesis in both tobacco and sweet pepper. The mechanism underlying BBM-mediated somatic embryogenesis was studied in Arabidopsis by identifying BBM target genes (ChIPSeq). Genes controlling zygotic embryo identity and maturation (LAFL), as well as auxin biosynthesis (TAA1, YUCCA) and transport (PIN) are BBM targets. Mutant analysis and chemical inhibition studies showed that these genes play positive roles in BBM-induced somatic embryogenesis.

Het wortelstelsel van gewassen is vaak ‘de verborgen helft’ genoemd. Lange tijd bestond er een enorm gebrek aan kennis over wortelgroei. Dat maakt het moeilijk er op te veredelen of om er in de teelt anders mee om te gaan. De afgelopen jaren is het inzicht belangrijk gegroeid, met name op genetisch en moleculair niveau.

Keywords: Somatic embryogenesis, Transcription factor, AINTEGUMENTA-LIKE, BABY BOOM, BBM, Sweet Pepper Transformation Title: Applied and Fundamental Aspects of BBM-mediated Regeneration Author: Iris Heidmann Catergories: Plant regeneration, Plant transformation, transcription factor, somatic embryogenesis Plant regeneration from tissues or single cells is essential for plant propagation. Efficient regeneration can be archieved through somatic embryogenesis using the plant growth regulator auxin or overexpression of specific transcription factors, but the underlying mechanisms are poorly understood. The potency of the BABY BOOM (BBM) AINTEGUMENTA-LIKE transcription factor to induce somatic embryogenesis in crop (sweet pepper) and model species (tobacco) was investigated. It was found that the introduction of BBM into sweet pepper, which is recalcitrant for transformation, enhanced the regeneration of transgenic plants. Exogenous cytokinin was necessary to induce somatic embryogenesis in both tobacco and sweet pepper. The mechanism underlying BBM-mediated somatic embryogenesis was studied in Arabidopsis by identifying BBM target genes (ChIPSeq). Genes controlling zygotic embryo identity and maturation (LAFL), as well as auxin biosynthesis (TAA1, YUCCA) and transport (PIN) are BBM targets. Mutant analysis and chemical inhibition studies showed that these genes play positive roles in BBM-induced somatic embryogenesis.

Charles Darwin is niet alleen bekend door de evolutietheorie. De negentiende eeuwse wetenschapper was namelijk de eerste die rond 1880 aantoonde dat planten reageren op de zwaartekracht. Daarna was het nog 125 jaar wachten voordat duidelijk werd dat planten de zwaartekracht ook echt nodig hebben voor een normale groei.

Sweet peppers arrive in waves. New fruits lure so many assimilates towards them that the flowers above them abort. This article provides various suggestions about how to flatten out the peaks and troughs. In the long term, breeding can also play an important role.

Plantencellen blijken zich volgens een simpele regel in tweeën te delen; dit gebeurt in het midden, maar wel met een zo klein mogelijk deelvlak. Dit inzicht helpt te verklaren hoe planten hun definitieve vorm krijgen. Opvallend genoeg blijken cellen deze regel ook te overtreden.

Het wortelstelsel van gewassen is vaak ‘de verborgen helft’ genoemd. Lange tijd bestond er een enorm gebrek aan kennis over wortelgroei. Dat maakt het moeilijk er op te veredelen of om er in de teelt anders mee om te gaan. De afgelopen jaren is het inzicht belangrijk gegroeid, met name op genetisch en moleculair niveau.

Auxin is a phytohormone that is crucial for many aspects of plant development. The processes in which this hormone has been implicated span from embryo development to flower transition, defense, tropic responses, and many other processes during plant life. A key question in auxin biology is how this molecule is able to elicit such diverse responses. Auxin regulates the transcriptional activation or repression of genes through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. In my studies I focus in the ARF transcription factors as a likely source of variation in output specificity. We consider three levels at which ARFs differ. First, ARFs differ in their ability to interact with different Aux/IAA (antagonistic family of transcription factors), or to form homo- or heterodimers. Second, ARFs assemble into different protein complexes, transcription factors interact with other transcriptional regulators or other proteins to form transcription complexes. These, when different, may contribute to different functions of ARF complexes. Thirdly, ARFs bind to and regulate different target genes. My work offers a plausible explanation how specific auxin responses are generated and through which genes the developmental responses to auxin are generated.

Auxin is a phytohormone that is crucial for many aspects of plant development. The processes in which this hormone has been implicated span from embryo development to flower transition, defense, tropic responses, and many other processes during plant life. A key question in auxin biology is how this molecule is able to elicit such diverse responses. Auxin regulates the transcriptional activation or repression of genes through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. In my studies I focus in the ARF transcription factors as a likely source of variation in output specificity. We consider three levels at which ARFs differ. First, ARFs differ in their ability to interact with different Aux/IAA (antagonistic family of transcription factors), or to form homo- or heterodimers. Second, ARFs assemble into different protein complexes, transcription factors interact with other transcriptional regulators or other proteins to form transcription complexes. These, when different, may contribute to different functions of ARF complexes. Thirdly, ARFs bind to and regulate different target genes. My work offers a plausible explanation how specific auxin responses are generated and through which genes the developmental responses to auxin are generated.

cum laude graduation (with distinction)

cum laude graduation (with distinction)

Auxin is a structurally simple molecule, yet it elicits many different responses in plants. In Chapter 1 we have reviewed how specificity in the output of auxin signaling could be generated by distinct regulation and the unique properties of the members of the Aux/IAA and ARF transcription factor families. In Chapter 2 we further investigated the generation of specificity in auxin responses by generating a set of sensitive transcriptional reporter lines for all Arabidopsis ARFs. This facilitated a comprehensive identification of the ARF complement within a cell/tissue of interest. Our analysis of ARF expression in the root meristem revealed both ubiquitous and specific ARF expression patterns and ARF subsets that distinguished the actively dividing cells from those undergoing elongation. Moreover, a striking correlation between cell type and ARF expression patterns was revealed in the early Arabidopsis embryo, where each cell type expressed a unique ARF complement. In Chapter 3 we characterized a novel cell-autonomous auxin response is required for hypophysis specification and root meristem initiation, and identify Aux/IAA and ARF transcription factors that mediate this response. We show that auxin response components in the proembryo and the suspensor are intrinsically different, and their regulated, lineage-specific expression creates a prepattern enabling different developmental auxin responses. Surprisingly, we find that, in addition to mediating hypophysis specification, auxin response also acts to maintain suspensor cell identity. We show that auxin controlled maintenance of suspensor cell identity includes repression of the embryonic program. This finding gave us an experimental system in which to investigate suspensor cell identity and embryonic transformation. In Chapter 4 the targeted and specific inhibition of auxin response in the suspensor was coupled to new embryo dissection techniques and a microarray based approach was used to generate a unique dataset which was subsequently mined for cell identity regulators. Unexpectedly, inhibition of auxin response induced the misregulation of thousands of genes, prior to gross morphological changes, revealing a high degree of transcriptional plasticity in these cells. This complicated the identification of regulators. Moreover, the dataset also included secondary/indirect changes in embryo expressed genes, which were inevitable given the connectivity and developmental connectedness between the embryo and suspensor. One of the most striking findings from analysis of the dataset generated in Chapter 4 was the convergent regulation of members of many gene families involved in all facets of auxin homeostasis, as investigated in Chapter 5. It appears that transient auxin response inhibition is sensed as an auxin minimum and in general auxin homeostasis genes were activated or repressed in such a way that would increase cellular auxin levels (and response). Finally, many bHLH superfamily members were misregulated upon the inhibition of suspensor auxin response and subsequently found to have specific expression patterns in the embryo, the focus of in Chapter 6. Several bHLHs were shown to lose their lineage specific expression patterns upon inhibition of auxin response in the suspensor, validating further research to place these factors into the auxin response pathways controlling cell identity in the embryo.

Auxin is a structurally simple molecule, yet it elicits many different responses in plants. In Chapter 1 we have reviewed how specificity in the output of auxin signaling could be generated by distinct regulation and the unique properties of the members of the Aux/IAA and ARF transcription factor families. In Chapter 2 we further investigated the generation of specificity in auxin responses by generating a set of sensitive transcriptional reporter lines for all Arabidopsis ARFs. This facilitated a comprehensive identification of the ARF complement within a cell/tissue of interest. Our analysis of ARF expression in the root meristem revealed both ubiquitous and specific ARF expression patterns and ARF subsets that distinguished the actively dividing cells from those undergoing elongation. Moreover, a striking correlation between cell type and ARF expression patterns was revealed in the early Arabidopsis embryo, where each cell type expressed a unique ARF complement. In Chapter 3 we characterized a novel cell-autonomous auxin response is required for hypophysis specification and root meristem initiation, and identify Aux/IAA and ARF transcription factors that mediate this response. We show that auxin response components in the proembryo and the suspensor are intrinsically different, and their regulated, lineage-specific expression creates a prepattern enabling different developmental auxin responses. Surprisingly, we find that, in addition to mediating hypophysis specification, auxin response also acts to maintain suspensor cell identity. We show that auxin controlled maintenance of suspensor cell identity includes repression of the embryonic program. This finding gave us an experimental system in which to investigate suspensor cell identity and embryonic transformation. In Chapter 4 the targeted and specific inhibition of auxin response in the suspensor was coupled to new embryo dissection techniques and a microarray based approach was used to generate a unique dataset which was subsequently mined for cell identity regulators. Unexpectedly, inhibition of auxin response induced the misregulation of thousands of genes, prior to gross morphological changes, revealing a high degree of transcriptional plasticity in these cells. This complicated the identification of regulators. Moreover, the dataset also included secondary/indirect changes in embryo expressed genes, which were inevitable given the connectivity and developmental connectedness between the embryo and suspensor. One of the most striking findings from analysis of the dataset generated in Chapter 4 was the convergent regulation of members of many gene families involved in all facets of auxin homeostasis, as investigated in Chapter 5. It appears that transient auxin response inhibition is sensed as an auxin minimum and in general auxin homeostasis genes were activated or repressed in such a way that would increase cellular auxin levels (and response). Finally, many bHLH superfamily members were misregulated upon the inhibition of suspensor auxin response and subsequently found to have specific expression patterns in the embryo, the focus of in Chapter 6. Several bHLHs were shown to lose their lineage specific expression patterns upon inhibition of auxin response in the suspensor, validating further research to place these factors into the auxin response pathways controlling cell identity in the embryo.

Molybdeen is het sporenelement waar de plant het minst van nodig heeft. Toch is het essentieel. Het speel een cruciale rol bij de benutting van nitraatstikstof. Een molybdeengebrek leist daarom tot een stikstofgebrek. Ook de vorming van de plantenhormonen auxine an abdscisinezuur is van molybdeen afhankelijk. Over dit element is nog veel onbekend

Plants normally form one embryo per seed. Under special circumstances, such as death of the embryo, a second embryo can develop from a supportive structure called the suspensor. These suspensor cells therefore provide a reservoir of stem cells for the generation of secondary embryos. At the start of this project, the mechanisms that control the formation of secondary embryos were completely unclear. By conducting a systematic screen for cellular responses to the plant hormone auxin during embryogenesis we found that auxin prevents embryo development from suspensor cells. The detailed analysis of auxin response components allowed us to identify the auxin-dependent transcription factors that mediate auxin action in the suspensor. Furthermore, we found that the control of expression of these auxin response transcription factors contributes to early embryo pattern formation. This work identified the first molecular players in the control of suspensor-embryo transformation and provides a stepping stone for elucidating the genetic networks that control embryo identity in plants.

Molybdeen is het sporenelement waar de plant het minst van nodig heeft. Toch is het essentieel. Het speel een cruciale rol bij de benutting van nitraatstikstof. Een molybdeengebrek leist daarom tot een stikstofgebrek. Ook de vorming van de plantenhormonen auxine an abdscisinezuur is van molybdeen afhankelijk. Over dit element is nog veel onbekend

Plants normally form one embryo per seed. Under special circumstances, such as death of the embryo, a second embryo can develop from a supportive structure called the suspensor. These suspensor cells therefore provide a reservoir of stem cells for the generation of secondary embryos. At the start of this project, the mechanisms that control the formation of secondary embryos were completely unclear. By conducting a systematic screen for cellular responses to the plant hormone auxin during embryogenesis we found that auxin prevents embryo development from suspensor cells. The detailed analysis of auxin response components allowed us to identify the auxin-dependent transcription factors that mediate auxin action in the suspensor. Furthermore, we found that the control of expression of these auxin response transcription factors contributes to early embryo pattern formation. This work identified the first molecular players in the control of suspensor-embryo transformation and provides a stepping stone for elucidating the genetic networks that control embryo identity in plants.

Cyst ( Globodera spp. and Heterodera spp.) and root-knot nematodes ( Meloidogyne spp.), one of the most damaging crop pests, are a perfect example of highly adapted, sophisticated root parasites.These nematodes induces specialized feeding structures (cyst nematodes-syncytia, root-knot nematode-giant cells) within the host root and benefits from guaranteed continues supply of food and water from their host plant and protection within the cyst or gall for their progeny. This dissertation aims at elucidation of the molecular mechanisms controlling the induction and maintenance of those feeding structures. This knowledge is indispensable to make novel advances in constructing durable host plant resistance.A number of different observations suggested a primary role of the phytohormone auxin in the feeding cell development. However, it has never been directly proven. Therefore, we have used the DR5 promoter element fused to gusA reporter gene to visualize spatial and temporal changes in auxin distribution, during nematode infection in Arabidopsis thaliana roots infected with cyst or root-knot nematodes. DR5 promoter element is considered to be a specific indicator for auxin and is activation points at an increase of the perceived auxin concentration. For both, cyst and root-knot nematode species, strong GUS expression was observed at the very onset of parasitism, already at 18 hours post inoculation. This high expression level was maintained until 3-5 days post inoculation and then gradually was reduced. Semi-thin sections of infected roots were investigated microscopically, revealing the presence of GUS inside the initial cells, and in case of cyst nematodes, in cells to-be incorporated into syncytium. This implies that expansion of syncytium is marked by the regions with high gusA expression. Activation of DR5 promoter element can be explained by: an accumulation or an increased sensitivity to auxin. However, based on formerly gathered data, a local accumulation seems to be the more probable explanation. Moreover, the feeding structures of cyst and root-knot nematodes arise by distance mechanisms, hence the highly similar DR5 activation patterns in both feeding structures, is remarkable. This promptthe conclusion that notwithstanding the highly dissimilar structure and ontogeny, both nematodes interfere with similar signal transduction pathways in root morphogenesis.Apart from phytohormones, small peptide signalling plays a very important role in diverse aspects of plant growth and development, and such a molecule(s) could be potentially involved in the establishment of nematode feeding cell. Early nodulin ( ENOD ) genes have been defined as genes expressed in legumes during nodule formation. Recent studies, however, revealed a number of homologues of ENOD40 s in non-legume plants. In both plants types, ENOD40 expression associates with the development of the vascular tissue. Feeding site development by root-knot and cyst nematodes is accompanied by the distortion of the vascular tissue, therefore a local increase in ENOD40 expression could be anticipated. This holds truth in interaction between legume Medicago truncatula and root-knot nematodes. However, in non-legume tomato infected with cyst and root-knot nematodes, no change in ENOD40 transcript levels could be detected using RT-PCR. In addition, activation of the Sl-ENOD40 promoter was only observed in infected regions showing endogenous ENOD40 expression. Similar results were obtained for a legume ENOD40 promoter (soybean) in a non-legumes background ( Arabidopsis ), and - contrary to M. truncatula - over-expression of Gm-ENOD40 in Arabidopsis had no effect on nematode development. Hence, ENOD40 expression in tomato is basically unaffected by the induction of feeding sites by root-knot and cyst nematodes. This unexpected difference in nematode-induced ENOD40 is apparently plant background-dependent, and suggests the existence of a legume-specific factor that boosts the ENOD40 expression in legume hosts-nematode interaction.One of the most striking characteristic of the cyst nematode induced feeding structure (syncytium) is the extensive cell wall dissolution, as the syncytium expands towards the vascular bundle and along the xylem vessels. Endo-1,4-ß-glucanases (cellulases) reside among plant enzymes that are involved in this progressive cell wall dissolution.RT-PCR experiments in tomato revealed that the potato cyst nematode induces the expression of two out of the eight described cellulases, precisely, Sl-cel7 and Sl-cel8 . In situ hybridization and immunodetection studies showed that both cellulases are expressed inside and adjacent to nematode-induced syncytia. Transgenic potato plants carryinghpRNA-silencing constructs for cel7 and cel8 were infected with cyst nematode which resulted in a significant arrest of cyst nematodes development.Reduced development of juveniles into females (up to 60%) and increase in the fraction of females without eggs (up to 89%) was observed. This indicates that the recruitment of specific plant cellulases by potato cyst nematodes is essential for their development, and demonstrates how detailed knowledge of compatible plant-parasite relationships can lead to host plant resistance.The genomic sequence of potato, cel7 and cel8a was cloned and characterised, in order to enable the RNA interference experiments in the natural host of potato cyst nematodes. The analysis of sequences suggests the presence of multiple isoforms of cel8 in potato. That corresponding proteins consist solely of a catalytic core domain (St-CEL7) or of a catalytic domain linked to a carbohydrate binding module (CBM) (St-CEL8a). Only recently, the CBM domain of Sl-CEL8a was shown to belong to CBM family 2 and on the basis of shared features between CBM domains in Sl-CEL8 and St-CEL8a it can be concluded that St-CEL8a harbours a cellulose binding domain as well. Therefore, those findings imply that the potato cellulases recruited by the potato cyst nematode are able to hydrolyse both xyloglucan (CEL7) and crystalline cellulose (CEL8), and apart from cloning novel potato cellulases genes, provides an explanation why nematodes recruit exactly these two members of the cellulase family. Moreover, potato cellulase sequences were used to mine EST databases and this information was used for the generation of a phylogenetic tree of the Solanaceous endo-β-1,4-D-glucanase gene family. In this dissertation we take an effort to understand better the intimae relationship between the obligatory plant parasites, cyst and root-knot nematodes and host plants. We show that such a detailed knowledge can be beneficial in better understanding of plant physiology and can be used as a highly specific and bio-safe approach for pathogen management.

Cyst ( Globodera spp. and Heterodera spp.) and root-knot nematodes ( Meloidogyne spp.), one of the most damaging crop pests, are a perfect example of highly adapted, sophisticated root parasites.These nematodes induces specialized feeding structures (cyst nematodes-syncytia, root-knot nematode-giant cells) within the host root and benefits from guaranteed continues supply of food and water from their host plant and protection within the cyst or gall for their progeny. This dissertation aims at elucidation of the molecular mechanisms controlling the induction and maintenance of those feeding structures. This knowledge is indispensable to make novel advances in constructing durable host plant resistance.A number of different observations suggested a primary role of the phytohormone auxin in the feeding cell development. However, it has never been directly proven. Therefore, we have used the DR5 promoter element fused to gusA reporter gene to visualize spatial and temporal changes in auxin distribution, during nematode infection in Arabidopsis thaliana roots infected with cyst or root-knot nematodes. DR5 promoter element is considered to be a specific indicator for auxin and is activation points at an increase of the perceived auxin concentration. For both, cyst and root-knot nematode species, strong GUS expression was observed at the very onset of parasitism, already at 18 hours post inoculation. This high expression level was maintained until 3-5 days post inoculation and then gradually was reduced. Semi-thin sections of infected roots were investigated microscopically, revealing the presence of GUS inside the initial cells, and in case of cyst nematodes, in cells to-be incorporated into syncytium. This implies that expansion of syncytium is marked by the regions with high gusA expression. Activation of DR5 promoter element can be explained by: an accumulation or an increased sensitivity to auxin. However, based on formerly gathered data, a local accumulation seems to be the more probable explanation. Moreover, the feeding structures of cyst and root-knot nematodes arise by distance mechanisms, hence the highly similar DR5 activation patterns in both feeding structures, is remarkable. This promptthe conclusion that notwithstanding the highly dissimilar structure and ontogeny, both nematodes interfere with similar signal transduction pathways in root morphogenesis.Apart from phytohormones, small peptide signalling plays a very important role in diverse aspects of plant growth and development, and such a molecule(s) could be potentially involved in the establishment of nematode feeding cell. Early nodulin ( ENOD ) genes have been defined as genes expressed in legumes during nodule formation. Recent studies, however, revealed a number of homologues of ENOD40 s in non-legume plants. In both plants types, ENOD40 expression associates with the development of the vascular tissue. Feeding site development by root-knot and cyst nematodes is accompanied by the distortion of the vascular tissue, therefore a local increase in ENOD40 expression could be anticipated. This holds truth in interaction between legume Medicago truncatula and root-knot nematodes. However, in non-legume tomato infected with cyst and root-knot nematodes, no change in ENOD40 transcript levels could be detected using RT-PCR. In addition, activation of the Sl-ENOD40 promoter was only observed in infected regions showing endogenous ENOD40 expression. Similar results were obtained for a legume ENOD40 promoter (soybean) in a non-legumes background ( Arabidopsis ), and - contrary to M. truncatula - over-expression of Gm-ENOD40 in Arabidopsis had no effect on nematode development. Hence, ENOD40 expression in tomato is basically unaffected by the induction of feeding sites by root-knot and cyst nematodes. This unexpected difference in nematode-induced ENOD40 is apparently plant background-dependent, and suggests the existence of a legume-specific factor that boosts the ENOD40 expression in legume hosts-nematode interaction.One of the most striking characteristic of the cyst nematode induced feeding structure (syncytium) is the extensive cell wall dissolution, as the syncytium expands towards the vascular bundle and along the xylem vessels. Endo-1,4-ß-glucanases (cellulases) reside among plant enzymes that are involved in this progressive cell wall dissolution.RT-PCR experiments in tomato revealed that the potato cyst nematode induces the expression of two out of the eight described cellulases, precisely, Sl-cel7 and Sl-cel8 . In situ hybridization and immunodetection studies showed that both cellulases are expressed inside and adjacent to nematode-induced syncytia. Transgenic potato plants carryinghpRNA-silencing constructs for cel7 and cel8 were infected with cyst nematode which resulted in a significant arrest of cyst nematodes development.Reduced development of juveniles into females (up to 60%) and increase in the fraction of females without eggs (up to 89%) was observed. This indicates that the recruitment of specific plant cellulases by potato cyst nematodes is essential for their development, and demonstrates how detailed knowledge of compatible plant-parasite relationships can lead to host plant resistance.The genomic sequence of potato, cel7 and cel8a was cloned and characterised, in order to enable the RNA interference experiments in the natural host of potato cyst nematodes. The analysis of sequences suggests the presence of multiple isoforms of cel8 in potato. That corresponding proteins consist solely of a catalytic core domain (St-CEL7) or of a catalytic domain linked to a carbohydrate binding module (CBM) (St-CEL8a). Only recently, the CBM domain of Sl-CEL8a was shown to belong to CBM family 2 and on the basis of shared features between CBM domains in Sl-CEL8 and St-CEL8a it can be concluded that St-CEL8a harbours a cellulose binding domain as well. Therefore, those findings imply that the potato cellulases recruited by the potato cyst nematode are able to hydrolyse both xyloglucan (CEL7) and crystalline cellulose (CEL8), and apart from cloning novel potato cellulases genes, provides an explanation why nematodes recruit exactly these two members of the cellulase family. Moreover, potato cellulase sequences were used to mine EST databases and this information was used for the generation of a phylogenetic tree of the Solanaceous endo-β-1,4-D-glucanase gene family. In this dissertation we take an effort to understand better the intimae relationship between the obligatory plant parasites, cyst and root-knot nematodes and host plants. We show that such a detailed knowledge can be beneficial in better understanding of plant physiology and can be used as a highly specific and bio-safe approach for pathogen management.

De veredeling van tulp gaat traag door de lange generatietijd en door langzame vermeerdering op het veld. Een veredelingsprogramma van tulp duurt al snel 25 jaar. Dit kan aanzienlijk verkort worden door vermeerdering in weefselkweek. Er is nu een werkbaar protocol

Only a small number ofphytohormonesare sufficient to regulate virtually every aspect of plant growth and development. Hence, adequate communication betweenphytohormonesis important to coordinate cellular processes during development. In this thesis we show that the plant gene ENOD40 plays a role in the communication betweenphytohormonesignaling pathways.Mainly on the basis of its complex expression patterns, it has been suggested that ENOD40 could affect thephytohormonestatus of cells, thus participating in regulation of plant growth and development but direct evidence was lacking. We have developed bioassays to study the function of ENOD40 in relation tophytohormonesignaling, using tobacco BY-2 cell-cultures. In these filamentous cell suspensions, quantification of cell-growth and cell division is straightforward and these morphological growth parameters can be used to monitor responses tophytohormones. In wild-type cells that have no or very low levels of ENOD40 expression, both processes are by-en-large independent of each other. By quantifying the parameters cell-elongation growth and cell division frequency under variousphytohormoneregimes in transgenic, 35S: NtENOD40 containing, tobacco BY-2 cell suspensions, we showed that the relative rates of both cellular processes become coupled byoverexpressionof ENOD40 . Since elongation growth is regulated by ethylene and cell division by thecytokinin/auxinratio, our data indicate that ENOD40 is involved in cross-talk between ethylene,auxinandcytokinin.Furthermore, we present an overview of ENOD40 expression patterns and analyze the structure of ENOD40 transcripts. The transcripts are characterized by two short conserved nucleotide sequence regions, which both contain biological activity in the bio-assay, and one of the two regions encodes a remarkably shortoligopeptide.In conclusion, the observation that ENOD40 affectsphytohormoneresponses in BY-2 cells is consistent withspatio-temporal ENOD40 expression patterns, which correlates with developmental stages involving the activity ofphytohormones. We propose that coupling between cell division, cell growth and cell differentiation may be achieved by transient and local expression of plant factors like ENOD40 that are involved inphytohormonecross-talk pathways. The coupling of hormone responses may be crucial to many developmental programs, and this may explain the complexity of ENOD40 expression patterns and the variety of tissues with which ENOD40 expression is associated in different plant species.

Only a small number ofphytohormonesare sufficient to regulate virtually every aspect of plant growth and development. Hence, adequate communication betweenphytohormonesis important to coordinate cellular processes during development. In this thesis we show that the plant gene ENOD40 plays a role in the communication betweenphytohormonesignaling pathways.Mainly on the basis of its complex expression patterns, it has been suggested that ENOD40 could affect thephytohormonestatus of cells, thus participating in regulation of plant growth and development but direct evidence was lacking. We have developed bioassays to study the function of ENOD40 in relation tophytohormonesignaling, using tobacco BY-2 cell-cultures. In these filamentous cell suspensions, quantification of cell-growth and cell division is straightforward and these morphological growth parameters can be used to monitor responses tophytohormones. In wild-type cells that have no or very low levels of ENOD40 expression, both processes are by-en-large independent of each other. By quantifying the parameters cell-elongation growth and cell division frequency under variousphytohormoneregimes in transgenic, 35S: NtENOD40 containing, tobacco BY-2 cell suspensions, we showed that the relative rates of both cellular processes become coupled byoverexpressionof ENOD40 . Since elongation growth is regulated by ethylene and cell division by thecytokinin/auxinratio, our data indicate that ENOD40 is involved in cross-talk between ethylene,auxinandcytokinin.Furthermore, we present an overview of ENOD40 expression patterns and analyze the structure of ENOD40 transcripts. The transcripts are characterized by two short conserved nucleotide sequence regions, which both contain biological activity in the bio-assay, and one of the two regions encodes a remarkably shortoligopeptide.In conclusion, the observation that ENOD40 affectsphytohormoneresponses in BY-2 cells is consistent withspatio-temporal ENOD40 expression patterns, which correlates with developmental stages involving the activity ofphytohormones. We propose that coupling between cell division, cell growth and cell differentiation may be achieved by transient and local expression of plant factors like ENOD40 that are involved inphytohormonecross-talk pathways. The coupling of hormone responses may be crucial to many developmental programs, and this may explain the complexity of ENOD40 expression patterns and the variety of tissues with which ENOD40 expression is associated in different plant species.

Plantencellen blijken zich volgens een simpele regel in tweeën te delen; dit gebeurt in het midden, maar wel met een zo klein mogelijk deelvlak. Dit inzicht helpt te verklaren hoe planten hun definitieve vorm krijgen. Opvallend genoeg blijken cellen deze regel ook te overtreden.

This thesis deals with the influence of the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of callus in cultured immature embryos of Zea mays (L). In maize, two types of embryogenic callus can be induced in immature zygotic embryos.Type I callus, which is compact in appearance and hard to subculture, and type II callus, which is friable, and is well suited for subculture without loosing its capability to produce somatic embryos. The somatic embryos mature on growth regulator free medium with 6% sucrose and grow out into normal fertile plants on regeneration medium. This process of somatic embryogenesis is important for large scale manipulation in biotechnological procedures.Friable embryogenic callus can be used for the production of suspension cultures or protoplasts with regeneration capacity. It is also well suited for particle gun experiments in which foreign DNA is transferred in the cells of the callus. The influence of the 2,4-D was studied in two inbred lines.

In the Netherlands, the rose is the most important glasshouse cut flower. Approximately 50% of the glasshouse roses are combination plants, consisting of a scion cultivar and a rootstock of a different genotype. Rootstocks inducing differences in the vigour of the scion are suggested to differ in cytokinin production and export to the shoot, thereby affecting bud break and outgrowth of the scion.In the present study, the course of endogenous cytokinins in rose combination plants was determined. The high contribution of isopentenyladenine-type cytokinins in young leaves indicated that these leaves, as the roots, were capable of de novo synthesis of cytokinins. Export of cytokinins from the roots was estimated based on the assumption that the cytokinin concentration in bleeding sap is representative for the concentration in xylem sap in situ , which was experimentally verified. The concentration of zeatin riboside (ZR) in bleeding sap was shown to be correlated with bud break of axillary shoots and bottom breaks, it increased before bud break and decreased thereafter. This suggests a quantitative relationship between cytokinin export from the roots and shoot development. Growth of the scion was also influenced by environmental factors, since bud break was advanced at higher root temperatures in the range of 11 to 26°C.However, this effect could not be correlated reliably with the cytokinin export from roots to shoot. Grafting the scion Madelon on rootstocks varying in vigour revealed that the rootstock that induces earlier bud break of the scion supplies larger quantities of cytokinins to the shoot. As a consequence, the concentrations of cytokinins in bleeding sap may be used as an early selection criterion for rose rootstocks.An attempt was made to describe correlative inhibition in rose. The apex of the primary shoot is thought tot exert apical dominance over the axillary buds along the shoot, until the terminal flower is visible. As at that time, the auxin export from the apex decreases, the inhibition of the uppermost 2-4 axillary buds is released, resulting in bud break. Later, the young axillary shoots will take over apical dominance and inhibit bud break further down the stem by their auxin production. When the terminal flower buds of the axillary shoots are visible, apical dominance is reduced, leading, following the same reasoning as above, to basal bud break.The effectivity of ZR in side-shoot formation, as was shown in in vitro experiments, combined with the fact that ZR is the major translocation form of cytokinins in the xylem points at a key role for ZR or its immediate metabolite, which might be zeatin, in growth and development of rose plants.

In the Netherlands, the rose is the most important glasshouse cut flower. Approximately 50% of the glasshouse roses are combination plants, consisting of a scion cultivar and a rootstock of a different genotype. Rootstocks inducing differences in the vigour of the scion are suggested to differ in cytokinin production and export to the shoot, thereby affecting bud break and outgrowth of the scion.In the present study, the course of endogenous cytokinins in rose combination plants was determined. The high contribution of isopentenyladenine-type cytokinins in young leaves indicated that these leaves, as the roots, were capable of de novo synthesis of cytokinins. Export of cytokinins from the roots was estimated based on the assumption that the cytokinin concentration in bleeding sap is representative for the concentration in xylem sap in situ , which was experimentally verified. The concentration of zeatin riboside (ZR) in bleeding sap was shown to be correlated with bud break of axillary shoots and bottom breaks, it increased before bud break and decreased thereafter. This suggests a quantitative relationship between cytokinin export from the roots and shoot development. Growth of the scion was also influenced by environmental factors, since bud break was advanced at higher root temperatures in the range of 11 to 26°C.However, this effect could not be correlated reliably with the cytokinin export from roots to shoot. Grafting the scion Madelon on rootstocks varying in vigour revealed that the rootstock that induces earlier bud break of the scion supplies larger quantities of cytokinins to the shoot. As a consequence, the concentrations of cytokinins in bleeding sap may be used as an early selection criterion for rose rootstocks.An attempt was made to describe correlative inhibition in rose. The apex of the primary shoot is thought tot exert apical dominance over the axillary buds along the shoot, until the terminal flower is visible. As at that time, the auxin export from the apex decreases, the inhibition of the uppermost 2-4 axillary buds is released, resulting in bud break. Later, the young axillary shoots will take over apical dominance and inhibit bud break further down the stem by their auxin production. When the terminal flower buds of the axillary shoots are visible, apical dominance is reduced, leading, following the same reasoning as above, to basal bud break.The effectivity of ZR in side-shoot formation, as was shown in in vitro experiments, combined with the fact that ZR is the major translocation form of cytokinins in the xylem points at a key role for ZR or its immediate metabolite, which might be zeatin, in growth and development of rose plants.

This thesis deals with the influence of the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of callus in cultured immature embryos of Zea mays (L). In maize, two types of embryogenic callus can be induced in immature zygotic embryos.Type I callus, which is compact in appearance and hard to subculture, and type II callus, which is friable, and is well suited for subculture without loosing its capability to produce somatic embryos. The somatic embryos mature on growth regulator free medium with 6% sucrose and grow out into normal fertile plants on regeneration medium. This process of somatic embryogenesis is important for large scale manipulation in biotechnological procedures.Friable embryogenic callus can be used for the production of suspension cultures or protoplasts with regeneration capacity. It is also well suited for particle gun experiments in which foreign DNA is transferred in the cells of the callus. The influence of the 2,4-D was studied in two inbred lines.

De veredeling van tulp gaat traag door de lange generatietijd en door langzame vermeerdering op het veld. Een veredelingsprogramma van tulp duurt al snel 25 jaar. Dit kan aanzienlijk verkort worden door vermeerdering in weefselkweek. Er is nu een werkbaar protocol