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Many Western European countries have seen strong debates about minority rights and practices such as the founding of religious schools or the wearing of religious symbols in public institutions. In these increasingly diverse societies, tolerance of one another’s practices and beliefs has often been put forward as an indispensable ingredient for harmonious intergroup coexistence. The classical notion of tolerance entails the acceptance of practices that one disapproves of – which implies that tolerance is a balance between reasons to accept and reasons to reject disapproved practices. In this dissertation, Evi Velthuis investigates those reasons for, and boundaries to, intergroup tolerance. Using large-scale surveys and experiments from the Netherlands and Germany, this dissertation presents four empirical chapters which consider three main aspects of tolerance: (1) reasons why minority practices are tolerated, (2) situational factors when practices are tolerated, and (3) individual differences in who tolerates minority practices. The findings and contributions are discussed in light of the social scientific literature on tolerance and intergroup relations.

Many Western European countries have seen strong debates about minority rights and practices such as the founding of religious schools or the wearing of religious symbols in public institutions. In these increasingly diverse societies, tolerance of one another’s practices and beliefs has often been put forward as an indispensable ingredient for harmonious intergroup coexistence. The classical notion of tolerance entails the acceptance of practices that one disapproves of – which implies that tolerance is a balance between reasons to accept and reasons to reject disapproved practices. In this dissertation, Evi Velthuis investigates those reasons for, and boundaries to, intergroup tolerance. Using large-scale surveys and experiments from the Netherlands and Germany, this dissertation presents four empirical chapters which consider three main aspects of tolerance: (1) reasons why minority practices are tolerated, (2) situational factors when practices are tolerated, and (3) individual differences in who tolerates minority practices. The findings and contributions are discussed in light of the social scientific literature on tolerance and intergroup relations.

Tolerating others' ways of life is often promoted in diverse societies as a way to live together in harmony. In this research, we examine what it is like to be tolerated from the perspective of various minorities. Being tolerated means that one's way of life is not appreciated, but that one can still live as one wishes. In the first empirical chapter, we interviewed trans people in the Netherlands and found that being tolerated can be a confusing and difficult experience which gives the impression that one is not appreciated. In the next chapter, we found that among ethnic minorities in the United States, being tolerated feels worse than being accepted, but feels better than being rejected. In the third empirical chapter, among ethnic minorities in the Netherlands, we additionally found that tolerance affects well-being due to how it influences people's sense that they are included within the larger group. In the last empirical chapter, we found that when people are tolerated within a group, their interactions are characterized by less trust and less ability to speak up for themselves compared to being accepted, but are more positive compared to being rejected. Overall, we conclude that being tolerated can bring up strong feelings of uncertainty among the tolerated; that tolerance is experienced as in between acceptance and rejection; and finally that it matters what tolerance is compared to - when compared to rejection, it is better to be tolerated, but tolerance is not as beneficial as being fully accepted.

Tolerating others' ways of life is often promoted in diverse societies as a way to live together in harmony. In this research, we examine what it is like to be tolerated from the perspective of various minorities. Being tolerated means that one's way of life is not appreciated, but that one can still live as one wishes. In the first empirical chapter, we interviewed trans people in the Netherlands and found that being tolerated can be a confusing and difficult experience which gives the impression that one is not appreciated. In the next chapter, we found that among ethnic minorities in the United States, being tolerated feels worse than being accepted, but feels better than being rejected. In the third empirical chapter, among ethnic minorities in the Netherlands, we additionally found that tolerance affects well-being due to how it influences people's sense that they are included within the larger group. In the last empirical chapter, we found that when people are tolerated within a group, their interactions are characterized by less trust and less ability to speak up for themselves compared to being accepted, but are more positive compared to being rejected. Overall, we conclude that being tolerated can bring up strong feelings of uncertainty among the tolerated; that tolerance is experienced as in between acceptance and rejection; and finally that it matters what tolerance is compared to - when compared to rejection, it is better to be tolerated, but tolerance is not as beneficial as being fully accepted.

Superdiversiteit is de nieuwe norm, in ieder geval in de grotere steden in Nederland (en Europa). De historische meerderheid is in deze context een minderheid geworden, maar er is geen nieuwe meerderheid voor in de plaats gekomen. Iedereen maakt deel uit van een minderheid. Dat geeft enerzijds ruimte voor gelijkheid: het is niet langer nodig dat verschillende minderheden zich aan de dominante meerderheid aanpassen. Iedereen staat in zekere zin op gelijke hoogte. Anderzijds maakt dit het proces niet eenvoudiger, omdat iedereen zich aan iedereen zal moeten aanpassen. In de niet-stedelijke gebieden in Nederland is de situatie behoorlijk anders. Daar is wel sprake van verscheidenheid, maar niet van superdiversiteit. Er is nog altijd een dominante meerderheid.

Superdiversiteit is de nieuwe norm, in ieder geval in de grotere steden in Nederland (en Europa). De historische meerderheid is in deze context een minderheid geworden, maar er is geen nieuwe meerderheid voor in de plaats gekomen. Iedereen maakt deel uit van een minderheid. Dat geeft enerzijds ruimte voor gelijkheid: het is niet langer nodig dat verschillende minderheden zich aan de dominante meerderheid aanpassen. Iedereen staat in zekere zin op gelijke hoogte. Anderzijds maakt dit het proces niet eenvoudiger, omdat iedereen zich aan iedereen zal moeten aanpassen. In de niet-stedelijke gebieden in Nederland is de situatie behoorlijk anders. Daar is wel sprake van verscheidenheid, maar niet van superdiversiteit. Er is nog altijd een dominante meerderheid.

Volgens Gert-Jan Veerman verdient tolerantie een duidelijke plek in het onderwijs van de toekomst. Hij pleit ervoor dat leerkrachten samen met leerlingen misvattingen over tolerantie opsporen in bijvoorbeeld nieuwsitems. En door dialogen in de les kunnen leerkrachten aandacht besteden aan misvattingen die leerlingen misschien zelf hebben over tolerantie. ‘Onderwijs is een soort minisamenleving waarin leerlingen kunnen oefenen om tolerant samen te leven,’ vindt Veerman.

Volgens Gert-Jan Veerman verdient tolerantie een duidelijke plek in het onderwijs van de toekomst. Hij pleit ervoor dat leerkrachten samen met leerlingen misvattingen over tolerantie opsporen in bijvoorbeeld nieuwsitems. En door dialogen in de les kunnen leerkrachten aandacht besteden aan misvattingen die leerlingen misschien zelf hebben over tolerantie. ‘Onderwijs is een soort minisamenleving waarin leerlingen kunnen oefenen om tolerant samen te leven,’ vindt Veerman.

Infectious diseases in farm animals are of major concern because of animal welfare, production costs, and public health. Farms undergo huge economic losses due to infectious disease. The costs of infections in farm animals are mainly due to production losses, treatment of infected animals, and disease control strategies. Control strategies, however, are not always successful. Selective breeding for the animals that can mount a defence against infection could therefore be a promising approach. Defensive ability of an animal has two main mechanisms: resistance (ability to control the pathogen burden) and tolerance (ability to maintain performance when pathogen burden increases). When it is difficult to distinguish between resistance and tolerance, defensive ability is measured as resilience that is the ability to maintain performance during a disease outbreak regardless of pathogen burden. Studies have focused on the genetics of resistance and resilience with little known about the genetics of tolerance and its relationship with resistance and resilience. The objectives of this thesis were to: 1) estimate the genetic variation in resistance, tolerance, and resilience to infection in order to assess the amenability of these traits for selective breeding in farm animals, 2) estimate the genetic correlation between resistance, tolerance and resilience and 3) detect genomic regions associated with resistance, tolerance, and resilience. In chapter 2, we studied the variation among sows in response to porcine reproductive and respiratory syndrome (PRRS). First a statistical method was developed to detect PRRS outbreaks based on reproduction records of sows. The method showed a high sensitivity (78%) for disease phases. Then the variation of sows in response to PRRS was quantified using 2 models on the traits number of piglets born alive (NBA) and number of piglets born dead (LOSS): 1) bivariate model considering the trait in healthy and disease phases as different traits, and 2) reaction norm model modelling the response of sows as a linear regression of the trait on herd-year-week estimates of NBA. Trait correlations between healthy and disease phases deviated from unity (0.57±0.13 – 0.87±0.18). The repeatabilities ranged from 0.07±0.027 to 0.16±0.005. The reaction norm model had higher predictive ability in disease phase compared to the bivariate model. In chapter 3 we studied 1) the genetic variation in resistance and tolerance of sheep to gastrointestinal nematode infection and 2) the genetic correlation between resistance and tolerance. Sire models on faecal nematode egg count (FEC), IgA, and pepsinogen were used to study the genetic variation in resistance. Heritability for resistance traits ranged from 0.19±0.10 to 0.59±0.20. A random regression model was used to study the reaction norm of sheep body weight on FEC as an estimate of tolerance to nematode infection. We observed a significant genetic variance in tolerance (P

The interest of research groups in desiccation tolerance (DT) has increased substantially over the last decades. The emergence of germinated orthodox seeds and resurrection plants as main research models has pushed the limits of our knowledge beyond boundaries. At the same time, new questions and new challenges were posed. The work presented in this thesis aims at shedding light on some of these questions, deepening our understanding of DT and providing relevant information to improve stress resistance in crops. Chapter 2 is a survey of the literature and discusses the ecological and evolutionary significance for seeds to be able to re-acquire DT after germination. This chapter also discusses recent progress in DT studies using developing and germinated seeds of the model plants Arabidopsis thaliana and Medicago truncatula. In Chapter 3 I used microarray data from a time series of DT re-acquisition, together with network analysis of gene expression, to gain temporal resolution and identify relevant genes involved in the re-acquisition of DT in germinated A. thaliana seeds by incubation in abscisic acid (ABA). Overall, genes related to protection, response to stresses, seed development and seed dormancy were up-regulated, whereas genes related to cell growth and photosynthesis were down-regulated with time. Genes that respond early to exogenous ABA were related to wax biosynthetic processes, lipid storage, seed development and response to ABA stimulus. Genes that respond late to exogenous ABA were related to syncytium formation and response to abiotic stimulus (mainly light stimulus). The robustness of the network was confirmed by the projection of sets of genes – related to the acquisition of DT, seed dormancy, drought responses of adult plants and re-induction of DT by polyethylene glycol – on this network. In Chapter 4 the relation between DT in germinated seeds and drought resistance in adult plants is analysed, using rice (Oryza sativa) as experimental model. Considering the predictions of a future with lower availability of fresh water, efforts to increase rice drought tolerance without reducing yield are increasingly important. The results presented in this chapter suggest that the intrinsic mechanisms of drought tolerance in adult plants are part of the mechanisms used by seeds to tolerate desiccation, but the molecular nature of these mechanisms remains elusive. Chapter 5 explores the relation between DT and longevity in germinated seeds of the Brazilian tree species Sesbania virgata as experimental model. DT and longevity are acquired by orthodox seeds during the maturation phase of development and lost upon germination. DT can be re-induced in germinated seeds by an osmotic and/or ABA treatment, but there is no information on how these treatments affect seed longevity. S. virgata seeds lose DT slowly upon radicle growth. The radicle appeared to be the most sensitive organ and the cotyledons the most resistant. The ability to produce lateral roots was key for whole seedling survival. An osmotic treatment improved DT in germinated S. virgata seeds, but not longevity. This implies that DT and seed longevity can be uncoupled. Xerophyta viscosa is one of the best studied resurrection species. Despite the fact that adult plants and mature seeds display DT, young X. viscosa seedlings are sensitive to fast drying. A treatment with ABA can induce DT early in shoots of these seedlings, but not in roots. Chapter 6 addresses the changes in the transcriptome and proteome of X. viscosa seedlings during induction of DT. A draft genome sequence of X. viscosa was used to improve transcript and protein identification and annotation. Differences in ABA signalling and the cross talk between ABA and ethylene were presented as determinant for shoot and root responses. Moreover, differences in the accumulation of late embryogenesis abundant proteins were also shown as being key for DT in shoots and roots. In Chapter 7, DT-transcriptomes of distantly related organisms are compared and surveyed for a core set of genes representing the signatures of critical adaptive DT mechanisms. A shortlist of 260 genes emerged, with a significant number of genes under the control of ABI3 and related to dormancy. The results reinforced the idea that core mechanisms and key regulators involved in DT developed early in the history of life and were carried forward by diverse species and life forms in a conserved manner and in conjunction with dormancy. In Chapter 8, the findings of this thesis are integrated, showing how they can contribute to future improvement of stress tolerance in crops. The ability of germinated seeds to re-acquire DT is discussed in combination with dormancy and longevity and related to seed survival under unfavourable environmental conditions. The relationship between drought- and desiccation tolerance and the role of ABA are presented briefly. Possible approaches to mine for new genes for crop improvement, such as searching for conserved genes and analysing new genome sequences, are addressed. Finally, a new perspective of the way to consider the evolution of DT is proposed.

The interest of research groups in desiccation tolerance (DT) has increased substantially over the last decades. The emergence of germinated orthodox seeds and resurrection plants as main research models has pushed the limits of our knowledge beyond boundaries. At the same time, new questions and new challenges were posed. The work presented in this thesis aims at shedding light on some of these questions, deepening our understanding of DT and providing relevant information to improve stress resistance in crops. Chapter 2 is a survey of the literature and discusses the ecological and evolutionary significance for seeds to be able to re-acquire DT after germination. This chapter also discusses recent progress in DT studies using developing and germinated seeds of the model plants Arabidopsis thaliana and Medicago truncatula. In Chapter 3 I used microarray data from a time series of DT re-acquisition, together with network analysis of gene expression, to gain temporal resolution and identify relevant genes involved in the re-acquisition of DT in germinated A. thaliana seeds by incubation in abscisic acid (ABA). Overall, genes related to protection, response to stresses, seed development and seed dormancy were up-regulated, whereas genes related to cell growth and photosynthesis were down-regulated with time. Genes that respond early to exogenous ABA were related to wax biosynthetic processes, lipid storage, seed development and response to ABA stimulus. Genes that respond late to exogenous ABA were related to syncytium formation and response to abiotic stimulus (mainly light stimulus). The robustness of the network was confirmed by the projection of sets of genes – related to the acquisition of DT, seed dormancy, drought responses of adult plants and re-induction of DT by polyethylene glycol – on this network. In Chapter 4 the relation between DT in germinated seeds and drought resistance in adult plants is analysed, using rice (Oryza sativa) as experimental model. Considering the predictions of a future with lower availability of fresh water, efforts to increase rice drought tolerance without reducing yield are increasingly important. The results presented in this chapter suggest that the intrinsic mechanisms of drought tolerance in adult plants are part of the mechanisms used by seeds to tolerate desiccation, but the molecular nature of these mechanisms remains elusive. Chapter 5 explores the relation between DT and longevity in germinated seeds of the Brazilian tree species Sesbania virgata as experimental model. DT and longevity are acquired by orthodox seeds during the maturation phase of development and lost upon germination. DT can be re-induced in germinated seeds by an osmotic and/or ABA treatment, but there is no information on how these treatments affect seed longevity. S. virgata seeds lose DT slowly upon radicle growth. The radicle appeared to be the most sensitive organ and the cotyledons the most resistant. The ability to produce lateral roots was key for whole seedling survival. An osmotic treatment improved DT in germinated S. virgata seeds, but not longevity. This implies that DT and seed longevity can be uncoupled. Xerophyta viscosa is one of the best studied resurrection species. Despite the fact that adult plants and mature seeds display DT, young X. viscosa seedlings are sensitive to fast drying. A treatment with ABA can induce DT early in shoots of these seedlings, but not in roots. Chapter 6 addresses the changes in the transcriptome and proteome of X. viscosa seedlings during induction of DT. A draft genome sequence of X. viscosa was used to improve transcript and protein identification and annotation. Differences in ABA signalling and the cross talk between ABA and ethylene were presented as determinant for shoot and root responses. Moreover, differences in the accumulation of late embryogenesis abundant proteins were also shown as being key for DT in shoots and roots. In Chapter 7, DT-transcriptomes of distantly related organisms are compared and surveyed for a core set of genes representing the signatures of critical adaptive DT mechanisms. A shortlist of 260 genes emerged, with a significant number of genes under the control of ABI3 and related to dormancy. The results reinforced the idea that core mechanisms and key regulators involved in DT developed early in the history of life and were carried forward by diverse species and life forms in a conserved manner and in conjunction with dormancy. In Chapter 8, the findings of this thesis are integrated, showing how they can contribute to future improvement of stress tolerance in crops. The ability of germinated seeds to re-acquire DT is discussed in combination with dormancy and longevity and related to seed survival under unfavourable environmental conditions. The relationship between drought- and desiccation tolerance and the role of ABA are presented briefly. Possible approaches to mine for new genes for crop improvement, such as searching for conserved genes and analysing new genome sequences, are addressed. Finally, a new perspective of the way to consider the evolution of DT is proposed.

De hygiënhypothese stelt dat de afwezigheid van infecties op de jonge kinderleeftijd gepaard gaat met een zich niet goed ontwikkelend immunoregulatienetwerk. Als gevolg hiervan zou allergische sensibilisatie toenemen en daarmee de kans op allergieën. Deze hypothese is een breed aanvaarde verklaring voor de alom geobserveerde toename van allergie in de Westerse en verwesterde samenlevingen gedurende de laatste 40 jaar. De bewijzen voor deze hypothese zijn echter zeer moeilijk te verkrijgen

In welke mate verschilt de tolerantie van homoseksualiteit in Nederland en Kroatië? Gaat dit in de toekomst veranderen? Aan de hand van voorbeelden uit de homo- en lesbische literatuur en de receptie daarvan zal ik proberen tijdens het panelgesprek antwoorden op die vragen te geven.

English summary 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.

English summary 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.

Some plants can tolerate and accumulate unusually high levels of toxic metals, and the analysis of such plants can provide insights into the ecology of environments that are polluted with heavy metals due to human industrial activities. The study of heavy metal hyperaccumulators such as Noccaea caerulescens can show how plants cope with excess metals and increase their fitness when growing in metalliferous environments. In this thesis, I compared the molecular mechanisms of zinc (Zn) homeostasis and cadmium (Cd) response in the hyperaccumulator species N. caerulescens and its non-accumulator relative Arabidopsis thalianaby investigating the regulation of the ZNT1/ZIP4gene that promotes Zn uptake and Zn/Cd tolerance.I also studied the ecological advantages of metal hyperaccumulators in nature and determined the DNA sequences of the N. caerulescens transcriptometo find candidate genes that control metal hyperaccumulation and provide an evolutionary perspective on the emergence of this trait. The functional characterization of the N. caerulescens and A. thaliana Zn-transporter genes NcZNT1 and AtZIP4 (and their promoters) showed how their differential expression pattern contributed to their role in metal tolerance and accumulation. The NcZNT1 gene is induced by Zn deficiency and the NcZNT1 protein is localized in the plasma membrane. Transgenic N. caerulescens roots containing a transgene for the green fluorescent protein (GFP) driven by the NcZNT1promoter revealed GFP fluorescence localized to pericycle and vascular tissues. This suggests that NcZNT1 contributes to metal loading into the xylem and long-distance metal transport. The overexpression of NcZNT1 in A. thaliana increased Zn and Cd tolerance and the capacity to accumulate these metals compared to wild-type plants. These results suggest that NcZNT1 plays an important role in Zn and Cd hypertolerance and hyperaccumulation in N. caerulescens, where it is expressed in both Zn-sufficient and Zn-excess conditions. The differential activity of the NcZNT1 and AtZIP4 promoters in N. caerulescens and A. thaliana implies that different cis-regulatory elements and trans-regulatory factors are present in both species. The ecological advantage of hyperaccumulators in metal-contaminated soils was investigated by studying a natural nas1mutant, in which the Nicotianamine Synthase1 gene is disrupted by a transposon insertion. This mutant allele was found in three natural N. caerulescens populations, which were compared to wild-type plants in terms of phenotype and adaptive advantage. Although the transposon disrupted the gene, the loss of NAS1 gene activity was compensated by enhanced expression of NAS3 and NAS4, resulting in increased nicotianamine (NA) production, which enhanced the Zn and Cd accumulation in the nas1 mutants. This increased their metal sensitivity compared to wild-type plants, but also made them more toxic towards Pieris rapae caterpillars, which developed more slowly and gained less weight when fed on mutant plants exposed to excess Zn or Cd. Therefore, the possible selective advantage of the nas1 mutant in nature is high metal accumulation and the protection of plants from herbivores. Differences in nas1 allele frequency among the three natural populations suggests that nas1 alleles experience different degrees of natural selection or may be at different stages on the route to fixation. Molecular evolutionary studies involve the identification of candidate genes that play a role in adaptation. Therefore, a comprehensive set of transcript sequences was obtained from N. caerulescens accession Ganges (GA) by 454 pyrosequencing. In total, the collected 23,836 isotigs (putative transcripts) were grouped into 20,378 isogroups and 93.2% of them could be matched to Brassicaceae protein sequences, which allowedtheir functional annotation. A total of 87 isogroups was annotated as metal homeostasis related genes, including metal transporter families, metal chelator biosynthesis families, and metal tolerance gene families, which are candidate genes for the molecular analysis of heavy metal homeostasis mechanisms. A group of genes required for the synthesis of glucosinolates, which are important secondary metabolites that protect plants against herbivores, were also shown to be expressed in N. caerulescens. The METALLOTHIONEIN3 (MT3) gene was found to have been duplicated in the N. caerulescens genome, when compared to related Brassicaceae. These transcript sequences will provide an important tool to annotate the genome sequence of the N. caerulescensGanges accession, which is in progress. This genome sequence will also be the basis of genome comparisons between the different N. caerulescens accessions that have different levels of metal accumulation and metal tolerance and which may therefore differ in terms of gene expression levels or modes of actions that affect metal homeostasis.

Light essentially sustains all life on planet earth surface. Plants transform light energy into chemical energy through photosynthesis. Hence, it can be anticipated that extending the daily photoperiod, using artificial light, results in increased plant productivity. Although this premise is true for many plant species, a limit exists. For instance, the seminal work of Arthur et al. (1930) showed that tomato plants develop leaf injuries if exposed to continuous light (CL). Many studies have investigated the physiological mechanism inducing such CL-induced injury. Although important and valuable discoveries were done over the decades, by the time the present project started, a detailed and proven physiological explanation of this disorder was still missing. Here, I present the results of a 5-year effort to better understand the physiological basis of the CL-induced injury in tomato and develop the tools (genetic and conceptual) to cultivate tomatoes under CL. After an exhaustive literature search, it was found that Daskaloff and Ognjanova (1965) reported that wild tomato species are tolerant to CL. Unfortunately, this important finding was ignored by numerous studies done after its publication. Here, we used the CL-tolerance found in wild tomatoes as a fundamental resource. Hence, the specific objectives of this thesis were to (i) better understand the physiological basis of the CL-induced injuries in tomato, (ii) identify the gene(s) responsible for CL-tolerance in wild tomato species, (iii) breed a CL-tolerant tomato line and (iv) use it to cultivate a greenhouse tomato crop under CL. Chapter 1 describes how innovation efforts encountered the unsolved scientific enigma of the injuries that tomato plants develop when exposed to CL. The term CL-induced injury is defined, and a detailed description of the symptoms observed in this disorder is shown. Additionally, an overview of the most important studies, influencing the hypotheses postulated and/or tested in this dissertation, is presented. Finally, a description and motivation of the main questions that this dissertation pursued to answer is presented alongside a short description of the strategy chosen to answer them. Chapter 2 reviews the literature, published over the last 80 years, on CL-induced injury using modern knowledge of plant physiology. By doing so, new hypotheses aiming to explain this disorder are postulated in addition to the ones collected from literature. Additionally, we highlight that CL is an essential tool for understanding the plant circadian clock, but using CL in research has its challenges. For instance, most of the circadian-clock-oriented experiments are performed under CL; consequently, interactions between the circadian clock and the light signalling pathway are overlooked. This chapter is published here. Chapter 3 explores the benefits and challenges of cultivating CL-tolerant tomato under CL. Considering that current commercial tomato varieties need six hours of darkness per day for optimal growth, photosynthesis does not take place during a quarter of the day. Hence, if tomatoes could be grown under CL, a substantial increase in production is anticipated. A simulation study is presented, which shows that if an ideal continuous-light-tolerant tomato genotype is used and no crop adaptations to CL are assumed, greenhouse tomato production could be 26% higher when supplementing light to 24 h day-1 in comparison with a photoperiod (including supplementary lighting) of only 18 h day-1. In addition, the expected changes in greenhouse energy budgets and alterations in crop physiological responses that might arise from cultivating tomatoes under continuous light are discussed. This chapter is published here. Chapter 4 maps the locus conferring CL-tolerance in wild tomatoes to chromosome seven, and shows that its introgression into modern tomato cultivars enhances yield by 20%, when grown under CL. In addition, genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III Light-Harvesting Chlorophyll a/b Binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In Arabidopsis thaliana this protein is thought to have a regulatory role in balancing light harvesting by photosystems I and II. The likely mechanisms that link CAB-13 with CL-tolerance are discussed. This chapter is published here. Chapter 5 investigates from which part of the plant CL-tolerance originates and whether this trait acts systemically. By exposing grafted plants bearing both tolerant and sensitive shoots to CL, the trait was functionally located to the shoot rather than the roots. Additionally, an increase in continuous-light tolerance was observed in sensitive plants when a continuous-light-tolerant shoot was grafted on it. Our results show that in order to increase yield in greenhouse tomato production by using CL, the trait should be bred into scion rather than rootstock lines. Chapter 6 discusses the factors that differ between injurious and non-injurious light regimes. Each of these factors may potentially be responsible for triggering the injury in CL-grown tomato and was experimentally tested here. In short, these factors include (i) differences in the light spectral distribution between sunlight and artificial light, (ii) continuous signalling to the photoreceptors, (iii) constant supply of light for photosynthesis, (iv) constant photo-oxidative pressure, and (v) circadian asynchrony — a mismatch between the internal circadian clock frequency and the external light/dark cycles. The evidence presented here suggests that the continuous-light-induced injury does not result from the unnatural spectral distribution of artificial light or the continuity of the light per se. Instead, circadian asynchrony seems to be the factor inducing the injury. As the discovered diurnal fluctuations in photoinhibition sensitivity of tomato seedlings are not under circadian control, it seems that circadian asynchrony does not directly induce injury via photoinhibition as it has been proposed. Chapter 7 investigates a possible role for phytochromes (PHY) in CL-induced injury in tomato. Mutant and transgenic tomato plants lacking or over-expressing phytochromes were exposed to CL, with and without far-red light enrichment, to test the role of individual phytochromes on the induction and/or prevention of injury. PHYA over-expression confers complete tolerance to CL regardless the light spectrum. Under CL with low far-red content, PHYB1 and PHYB2 diminished and enhanced the injury, respectively, yet the effects were small. These results confirm that phytochrome signaling networks are involved in the injury induction under CL. The link between CAB-13 and PHYA is discussed. Chapter 8 investigates the role of carbohydrate accumulation in the induction of CL-induced injury in tomato by using untargeted metabolomics and transcriptomics data. These data reveal a clear effect of CL on sugar metabolism and photosynthesis. A strong negative correlation between sucrose and starch with the maximum quantum efficiency of photosystem II (Fv /Fm) was found across several abnormal light/dark cycles, supporting the hypothesis that carbohydrates play an important role in CL-induced injury. I suggest that CL-induced injury in tomato is caused by a photosynthetic down-regulation showing characteristics of both cytokinin-regulated senescence and light-modulated retrograde signaling. Molecular mechanisms linking carbohydrate accumulation with photosynthetic down-regulation are discussed. Chapter 9 provides a synthesis of the most important findings and proposes a generic model of CL-induced injury in tomato. I propose that CL-induced injury in tomato arises from retrograde signals that counteract signals derived from the cellular developmental program that promote chloroplast development, such that chloroplast development cannot be completed, resulting in the chlorotic phenotype. Finally, perspectives on what future directions to take to further elucidate the physiological basis of this trait and successfully implement it in greenhouses are presented.

Some plants can tolerate and accumulate unusually high levels of toxic metals, and the analysis of such plants can provide insights into the ecology of environments that are polluted with heavy metals due to human industrial activities. The study of heavy metal hyperaccumulators such as Noccaea caerulescens can show how plants cope with excess metals and increase their fitness when growing in metalliferous environments. In this thesis, I compared the molecular mechanisms of zinc (Zn) homeostasis and cadmium (Cd) response in the hyperaccumulator species N. caerulescens and its non-accumulator relative Arabidopsis thalianaby investigating the regulation of the ZNT1/ZIP4gene that promotes Zn uptake and Zn/Cd tolerance.I also studied the ecological advantages of metal hyperaccumulators in nature and determined the DNA sequences of the N. caerulescens transcriptometo find candidate genes that control metal hyperaccumulation and provide an evolutionary perspective on the emergence of this trait. The functional characterization of the N. caerulescens and A. thaliana Zn-transporter genes NcZNT1 and AtZIP4 (and their promoters) showed how their differential expression pattern contributed to their role in metal tolerance and accumulation. The NcZNT1 gene is induced by Zn deficiency and the NcZNT1 protein is localized in the plasma membrane. Transgenic N. caerulescens roots containing a transgene for the green fluorescent protein (GFP) driven by the NcZNT1promoter revealed GFP fluorescence localized to pericycle and vascular tissues. This suggests that NcZNT1 contributes to metal loading into the xylem and long-distance metal transport. The overexpression of NcZNT1 in A. thaliana increased Zn and Cd tolerance and the capacity to accumulate these metals compared to wild-type plants. These results suggest that NcZNT1 plays an important role in Zn and Cd hypertolerance and hyperaccumulation in N. caerulescens, where it is expressed in both Zn-sufficient and Zn-excess conditions. The differential activity of the NcZNT1 and AtZIP4 promoters in N. caerulescens and A. thaliana implies that different cis-regulatory elements and trans-regulatory factors are present in both species. The ecological advantage of hyperaccumulators in metal-contaminated soils was investigated by studying a natural nas1mutant, in which the Nicotianamine Synthase1 gene is disrupted by a transposon insertion. This mutant allele was found in three natural N. caerulescens populations, which were compared to wild-type plants in terms of phenotype and adaptive advantage. Although the transposon disrupted the gene, the loss of NAS1 gene activity was compensated by enhanced expression of NAS3 and NAS4, resulting in increased nicotianamine (NA) production, which enhanced the Zn and Cd accumulation in the nas1 mutants. This increased their metal sensitivity compared to wild-type plants, but also made them more toxic towards Pieris rapae caterpillars, which developed more slowly and gained less weight when fed on mutant plants exposed to excess Zn or Cd. Therefore, the possible selective advantage of the nas1 mutant in nature is high metal accumulation and the protection of plants from herbivores. Differences in nas1 allele frequency among the three natural populations suggests that nas1 alleles experience different degrees of natural selection or may be at different stages on the route to fixation. Molecular evolutionary studies involve the identification of candidate genes that play a role in adaptation. Therefore, a comprehensive set of transcript sequences was obtained from N. caerulescens accession Ganges (GA) by 454 pyrosequencing. In total, the collected 23,836 isotigs (putative transcripts) were grouped into 20,378 isogroups and 93.2% of them could be matched to Brassicaceae protein sequences, which allowedtheir functional annotation. A total of 87 isogroups was annotated as metal homeostasis related genes, including metal transporter families, metal chelator biosynthesis families, and metal tolerance gene families, which are candidate genes for the molecular analysis of heavy metal homeostasis mechanisms. A group of genes required for the synthesis of glucosinolates, which are important secondary metabolites that protect plants against herbivores, were also shown to be expressed in N. caerulescens. The METALLOTHIONEIN3 (MT3) gene was found to have been duplicated in the N. caerulescens genome, when compared to related Brassicaceae. These transcript sequences will provide an important tool to annotate the genome sequence of the N. caerulescensGanges accession, which is in progress. This genome sequence will also be the basis of genome comparisons between the different N. caerulescens accessions that have different levels of metal accumulation and metal tolerance and which may therefore differ in terms of gene expression levels or modes of actions that affect metal homeostasis.

Light essentially sustains all life on planet earth surface. Plants transform light energy into chemical energy through photosynthesis. Hence, it can be anticipated that extending the daily photoperiod, using artificial light, results in increased plant productivity. Although this premise is true for many plant species, a limit exists. For instance, the seminal work of Arthur et al. (1930) showed that tomato plants develop leaf injuries if exposed to continuous light (CL). Many studies have investigated the physiological mechanism inducing such CL-induced injury. Although important and valuable discoveries were done over the decades, by the time the present project started, a detailed and proven physiological explanation of this disorder was still missing. Here, I present the results of a 5-year effort to better understand the physiological basis of the CL-induced injury in tomato and develop the tools (genetic and conceptual) to cultivate tomatoes under CL. After an exhaustive literature search, it was found that Daskaloff and Ognjanova (1965) reported that wild tomato species are tolerant to CL. Unfortunately, this important finding was ignored by numerous studies done after its publication. Here, we used the CL-tolerance found in wild tomatoes as a fundamental resource. Hence, the specific objectives of this thesis were to (i) better understand the physiological basis of the CL-induced injuries in tomato, (ii) identify the gene(s) responsible for CL-tolerance in wild tomato species, (iii) breed a CL-tolerant tomato line and (iv) use it to cultivate a greenhouse tomato crop under CL. Chapter 1 describes how innovation efforts encountered the unsolved scientific enigma of the injuries that tomato plants develop when exposed to CL. The term CL-induced injury is defined, and a detailed description of the symptoms observed in this disorder is shown. Additionally, an overview of the most important studies, influencing the hypotheses postulated and/or tested in this dissertation, is presented. Finally, a description and motivation of the main questions that this dissertation pursued to answer is presented alongside a short description of the strategy chosen to answer them. Chapter 2 reviews the literature, published over the last 80 years, on CL-induced injury using modern knowledge of plant physiology. By doing so, new hypotheses aiming to explain this disorder are postulated in addition to the ones collected from literature. Additionally, we highlight that CL is an essential tool for understanding the plant circadian clock, but using CL in research has its challenges. For instance, most of the circadian-clock-oriented experiments are performed under CL; consequently, interactions between the circadian clock and the light signalling pathway are overlooked. This chapter is published here. Chapter 3 explores the benefits and challenges of cultivating CL-tolerant tomato under CL. Considering that current commercial tomato varieties need six hours of darkness per day for optimal growth, photosynthesis does not take place during a quarter of the day. Hence, if tomatoes could be grown under CL, a substantial increase in production is anticipated. A simulation study is presented, which shows that if an ideal continuous-light-tolerant tomato genotype is used and no crop adaptations to CL are assumed, greenhouse tomato production could be 26% higher when supplementing light to 24 h day-1 in comparison with a photoperiod (including supplementary lighting) of only 18 h day-1. In addition, the expected changes in greenhouse energy budgets and alterations in crop physiological responses that might arise from cultivating tomatoes under continuous light are discussed. This chapter is published here. Chapter 4 maps the locus conferring CL-tolerance in wild tomatoes to chromosome seven, and shows that its introgression into modern tomato cultivars enhances yield by 20%, when grown under CL. In addition, genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III Light-Harvesting Chlorophyll a/b Binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In Arabidopsis thaliana this protein is thought to have a regulatory role in balancing light harvesting by photosystems I and II. The likely mechanisms that link CAB-13 with CL-tolerance are discussed. This chapter is published here. Chapter 5 investigates from which part of the plant CL-tolerance originates and whether this trait acts systemically. By exposing grafted plants bearing both tolerant and sensitive shoots to CL, the trait was functionally located to the shoot rather than the roots. Additionally, an increase in continuous-light tolerance was observed in sensitive plants when a continuous-light-tolerant shoot was grafted on it. Our results show that in order to increase yield in greenhouse tomato production by using CL, the trait should be bred into scion rather than rootstock lines. Chapter 6 discusses the factors that differ between injurious and non-injurious light regimes. Each of these factors may potentially be responsible for triggering the injury in CL-grown tomato and was experimentally tested here. In short, these factors include (i) differences in the light spectral distribution between sunlight and artificial light, (ii) continuous signalling to the photoreceptors, (iii) constant supply of light for photosynthesis, (iv) constant photo-oxidative pressure, and (v) circadian asynchrony — a mismatch between the internal circadian clock frequency and the external light/dark cycles. The evidence presented here suggests that the continuous-light-induced injury does not result from the unnatural spectral distribution of artificial light or the continuity of the light per se. Instead, circadian asynchrony seems to be the factor inducing the injury. As the discovered diurnal fluctuations in photoinhibition sensitivity of tomato seedlings are not under circadian control, it seems that circadian asynchrony does not directly induce injury via photoinhibition as it has been proposed. Chapter 7 investigates a possible role for phytochromes (PHY) in CL-induced injury in tomato. Mutant and transgenic tomato plants lacking or over-expressing phytochromes were exposed to CL, with and without far-red light enrichment, to test the role of individual phytochromes on the induction and/or prevention of injury. PHYA over-expression confers complete tolerance to CL regardless the light spectrum. Under CL with low far-red content, PHYB1 and PHYB2 diminished and enhanced the injury, respectively, yet the effects were small. These results confirm that phytochrome signaling networks are involved in the injury induction under CL. The link between CAB-13 and PHYA is discussed. Chapter 8 investigates the role of carbohydrate accumulation in the induction of CL-induced injury in tomato by using untargeted metabolomics and transcriptomics data. These data reveal a clear effect of CL on sugar metabolism and photosynthesis. A strong negative correlation between sucrose and starch with the maximum quantum efficiency of photosystem II (Fv /Fm) was found across several abnormal light/dark cycles, supporting the hypothesis that carbohydrates play an important role in CL-induced injury. I suggest that CL-induced injury in tomato is caused by a photosynthetic down-regulation showing characteristics of both cytokinin-regulated senescence and light-modulated retrograde signaling. Molecular mechanisms linking carbohydrate accumulation with photosynthetic down-regulation are discussed. Chapter 9 provides a synthesis of the most important findings and proposes a generic model of CL-induced injury in tomato. I propose that CL-induced injury in tomato arises from retrograde signals that counteract signals derived from the cellular developmental program that promote chloroplast development, such that chloroplast development cannot be completed, resulting in the chlorotic phenotype. Finally, perspectives on what future directions to take to further elucidate the physiological basis of this trait and successfully implement it in greenhouses are presented.

Onderzoek werd verricht naar het voorkomen van aflatoxine M1 in 25 monsters babyvoedingsmiddel, bereid op basis van melk(produkten), 3 monsters lactose, 6 monsters caseinaat en 1 monster weipoeder, alle in poedervorm. In 91% van alle monsters kon de aanwezigheid van aflatoxine M1 worden vastgesteld (> 0,02 mug/kg). De gevonden gehalten varieerden van 0,04 - 1,39 mug/kg poedervormig produkt; gemiddeld werd 0,22 mug/kg aangetroffen. Vergeleken met de studies, uitgevoerd in 1977 en 1980 bevindt de contaminatie met M1 zich, na een veelbelovende verbetering in 1980, thans weer op het niveau van 1977. Vermoedelijk is de oorzaak hiervan gelegen in het feit dat de gehalten aan aflatoxine in melk de laatste jaren zijn toegenomen.

Onderzoek werd verricht naar het voorkomen van aflatoxine M1 in 25 monsters babyvoedingsmiddel, bereid op basis van melk(produkten), 3 monsters lactose, 6 monsters caseinaat en 1 monster weipoeder, alle in poedervorm. In 91% van alle monsters kon de aanwezigheid van aflatoxine M1 worden vastgesteld (> 0,02 mug/kg). De gevonden gehalten varieerden van 0,04 - 1,39 mug/kg poedervormig produkt; gemiddeld werd 0,22 mug/kg aangetroffen. Vergeleken met de studies, uitgevoerd in 1977 en 1980 bevindt de contaminatie met M1 zich, na een veelbelovende verbetering in 1980, thans weer op het niveau van 1977. Vermoedelijk is de oorzaak hiervan gelegen in het feit dat de gehalten aan aflatoxine in melk de laatste jaren zijn toegenomen.

Schaduwminnende potplanten kunnen veel meer licht verdragen dan ze in de praktijk krijgen, blijkt onderzoek van WUR Glastuinbouw. Daarmee is de teelt te versnellen en energie te besparen. Maar hoe ver kan een teler daarmee gaan?

In general, it can be concluded that very few clearly unexpected effects were observed during the large scale post-release growing of herbicide-tolerant crops and Bt crops. Of course, one has to keep in mind Applied Plant Research (Praktijkonderzoek Plant & Omgeving ) 14 that there will always be an element of subjectivity in assessing effects as “unexpected”. Furthermore, it is also possible that certain effects are becoming visible only after a longer period than 10 – 15 years, but no indications for such effects were found in the literature. Besides the reports about the Farm Scale Evaluations no other reports were found with information about effects of GM crops on biodiversity in ecosysystems surrounding the production fields. Unexpected effects caused directly by the genetic modification were not found. Only in herbicide-tolerant crops, we concluded to some indirect unexpected effects: the reduced uptake of micro-nutrients and some positive and negative effects on susceptibility to diseases. These effects were specific to herbicide use with glyphosate-tolerant crops. Based on this, it was not possible to draw general conclusions for developing protocols for post-release monitoring of environmental effects

Ancient Greece is often seen as the cradle of democracy, of free thought and of free speech. This article presents a number of historical cases of intellectual repression in Greece and Rome, including examples even of book-burning. It discusses the political and philosophical concept of ‘parrhèsia’ and analyses the importance of religious restrictions through well known charges of ‘asebeia’ (Anaxagoras, Protagoras and Socrates). In the second section of the article the philosophical arguments are put forward developed by Plato in favour of preventive and repressive censorship and it ends with a short discussion of the Stoic attitude towards censorship.

In general, it can be concluded that very few clearly unexpected effects were observed during the large scale post-release growing of herbicide-tolerant crops and Bt crops. Of course, one has to keep in mind Applied Plant Research (Praktijkonderzoek Plant & Omgeving ) 14 that there will always be an element of subjectivity in assessing effects as “unexpected”. Furthermore, it is also possible that certain effects are becoming visible only after a longer period than 10 – 15 years, but no indications for such effects were found in the literature. Besides the reports about the Farm Scale Evaluations no other reports were found with information about effects of GM crops on biodiversity in ecosysystems surrounding the production fields. Unexpected effects caused directly by the genetic modification were not found. Only in herbicide-tolerant crops, we concluded to some indirect unexpected effects: the reduced uptake of micro-nutrients and some positive and negative effects on susceptibility to diseases. These effects were specific to herbicide use with glyphosate-tolerant crops. Based on this, it was not possible to draw general conclusions for developing protocols for post-release monitoring of environmental effects In het algemeen kan gesteld worden dat er in de grootschalige commerciële teelten geen grote onverwachte milieueffecten van herbicidetolerante gewassen en gewassen met insectenresistentie zijn gevonden. Het is mogelijk dat bepaalde effecten pas na een periode die langer is dan 10 -15 jaar, zichtbaar worden. Tot nu toe zijn hiervoor echter geen aanwijzingen. Naast de rapportage van de Farm Scale Evaluations (FSE) zijn er geen andere rapporten gevonden van onderzoek waarin monitoring van effecten van genetisch gemodificeerde gewassen op de biodiversiteit in akkerranden en omringende ecosystemen heeft plaats gevonden. Directe onverwachte effecten van de genetische modificatie zijn zowel bij de herbicidetolerante gewassen als bij de Bt-gewassen niet gevonden. Indirecte onverwachte effecten zijn wel geconstateerd bij de herbicidentolerante gewassen, nl. een effect op de opname van micronutriënten en een effect op de ziektegevoeligheid. Deze effecten zijn echter zeer specifiek voor glyfosaattolerante gewassen. Algemene conclusies die bruikbaar zijn voor het opstellen van monitoringsprotocollen zijn hieruit niet te trekken

Minerals are essential for humans, plants and animals and have an important micronutrient role in physiological and metabolic processes of plants. Next to this essential role of minerals, they can also be very toxic when available to the plant in elevated amounts. Plants therefore need to keep very tight control over the intracellular mineral concentrations in a process called metal homeostasis. Although the metal homeostasis mechanisms are supposed to be universal within plants, there are plant species that can tolerate and even accumulate large amounts of metals without any sign of toxicity. Thlaspi caerulescens J. & C. Presl (Brassicaceae), a close relative of the plant reference species Arabidopsis thaliana (Arabidopsis), is one of these natural metal hyperaccumulator species. The overall aim of this project is to unravel the molecular genetic mechanism of heavy metal tolerance and hyperaccumulation of the metal hyperaccumulating plant species T. caerulescens. To achieve this goal heterologous transcript profiling experiments were performed, which involved comparative microarray hybridization experiments of the hyperaccumulator T. caerulescens and Arabidopsis. Arabidopsis is used as the reference species for heterologous transcript profiling because of the availability of genetic resources and the complete genome sequence. The micronutrient zinc has an essential role in physiological and metabolic processes in plants as a cofactor or structural element. Thlaspi caerulescens can accumulate up to 3% of zinc on a dry weight basis without any sign of toxicity. The question postulated here is if this has drastic effects on the zinc homeostasis mechanism. We examined in detail the transcription profiles of roots of Arabidopsis and T. caerulescens plants grown under deficient, sufficient and excess supply of zinc (Chapter 2). A total of 608 genes were detected in Arabidopsis and 352 in T. caerulescens that responded transcriptionally to changes in zinc supply. Only 14% of these genes were also zinc-responsive in Arabidopsis. When comparing Arabidopsis and T. caerulescens at comparable zinc exposures, over 2200 genes were significantly differentially expressed. While a large fraction of these genes are of yet unknown function, many genes with a different expression between Arabidopsis and T. caerulescens appear to function in metal homeostasis, in abiotic stress response and in lignin biosynthesis. The high expression of lignin biosynthesis genes corresponds to the deposition of lignin in the endodermis. Contrary to Arabidopsis roots, which have one endodermal cell layer, we found there are two endodermal layers in T. caerulescens roots. This extra physical barrier could enhance the control of metal fluxes in the plant, in addition to the higher expression of metal transporters in the root. Cadmium is a widespread, naturally occurring non-essential element that is toxic for plants in higher concentrations. In chapter 3 we compare between and within species transcript profiles of Arabidopsis and T. caerulescens roots exposed to cadmium, with the aim to establish which genes are most likely to be relevant for the tolerance to cadmium exposure of T. caerulescens. The comparative transcriptional analysis of the cadmium response of roots of the T. caerulescens and Arabidopsis emphasizes the role of genes involved in lignin-, glutathione- and sulfate metabolism. Furthermore two transcription factors, MYB72 and bHLH100, with an altered expression after exposure to cadmium, are studied for their involvement in metal homeostasis. Analysis of a myb72 knock-out mutant showed enhanced sensitivity to excess zinc or iron deficiency. Rather than controlling Cd tolerance, this gene appears to be involved in iron homeostasis, affecting the response to Cd indirectly. Arabidopsis transformants overexpressing the transcription factor bHLH100 showed enhanced zinc and nickel tolerance, and although the exact role of this gene still needs to be resolved, the genes appears to have a role in metal homeostasis in Arabidopsis. T. caerulescens accessions exhibit distinct metal accumulation, translocation and tolerance characteristics. T. caerulescens accession Ganges can accumulate high amounts of cadmium and is extremely tolerant to cadmium, whereas the La Calamine accession is also tolerant to cadmium but accumulates much less cadmium compared to Ganges. The transcription profiles of leaves and roots of T. caerulescens accessions Ganges and La Calamine plants grown with and without cadmium were examined using the Qiagen-Operon Arabidopsis Genome Array and results are described in chapter 4. A total of 161 genes were differentially expressed between the two T. caerulescens accessions in response to changes in cadmium supply and 38 genes were differentially expressed in T. caerulescens accession Ganges leaves in response to cadmium. The comparative transcriptional analysis emphasizes that there are just minor differences between the two accessions but the genes which are differentially expressed could play an important role in the hyperaccumulation of cadmium in Ganges. The microarray data suggest that especially genes involved in cell wall modification and stress response relate to the major difference between the two accessions in cadmium hyperaccumulation. Plants have evolved a complex network of homeostatic mechanisms that serve to control the uptake, accumulation, trafficking and detoxification of metals. One potential mechanism for heavy metal detoxification in plants is the chelation of metal ions to ligands like organic acids, amino acids, peptides and polypeptides. This mechanism is important for the distribution of metal ions by keeping metal ions mobile within the plant. In plants metals are often found to be chelated to nicotianamine. Nicotianamine is formed by trimerization of S-adenosylmethionine, which is catalyzed by the enzyme nicotianamine synthase. Arabidopsis contains four nicotianamine synthase (NAS) genes. Also in T. caerulescens four full-length cDNAs encoding nicotianamine synthase members were identified (Chapter 5). The four genes were named TcNAS1-TcNAS4, analogous to the corresponding closest homologue in Arabidopsis. Arabidopsis plants over-expressing TcNAS1, TcNAS2, TcNAS3 or TcNAS4 that were tested for their response to growth on media with different zinc, iron, nickel or cadmium supply, provided evidence that the Thlaspi genes all have a genuine NAS function because they complement the NAS deficiency in specific triple knock-out Arabidopsis mutants. Evidence for a functional role in metal homeostasis was sought in studying the Arabidopsis single, double, triple and a quadruple nicotianamine synthase T-DNA insertion mutants. The combination of null mutations in three or four AtNAS genes, results in a severe phenotype that includes interveinal chlorosis and altered metal concentrations in leaves, roots and seeds. Arabidopsis transformants overexpressing TcNAS3 or TcNAS4 showed enhanced zinc and nickel tolerance compared to wild type plants. The research described in this thesis does contribute to a better understanding of heavy metal hyperaccumulation in T. caerulescens and it can be concluded that it seems unlikely that altered regulation and overexpression of single genes will be sufficient to convert metal nonaccumulators into hyperaccumulators. However, the possibility that overexpression of one or two key regulatory loci have this effect remains.

Minerals are essential for humans, plants and animals and have an important micronutrient role in physiological and metabolic processes of plants. Next to this essential role of minerals, they can also be very toxic when available to the plant in elevated amounts. Plants therefore need to keep very tight control over the intracellular mineral concentrations in a process called metal homeostasis. Although the metal homeostasis mechanisms are supposed to be universal within plants, there are plant species that can tolerate and even accumulate large amounts of metals without any sign of toxicity. Thlaspi caerulescens J. & C. Presl (Brassicaceae), a close relative of the plant reference species Arabidopsis thaliana (Arabidopsis), is one of these natural metal hyperaccumulator species. The overall aim of this project is to unravel the molecular genetic mechanism of heavy metal tolerance and hyperaccumulation of the metal hyperaccumulating plant species T. caerulescens. To achieve this goal heterologous transcript profiling experiments were performed, which involved comparative microarray hybridization experiments of the hyperaccumulator T. caerulescens and Arabidopsis. Arabidopsis is used as the reference species for heterologous transcript profiling because of the availability of genetic resources and the complete genome sequence. The micronutrient zinc has an essential role in physiological and metabolic processes in plants as a cofactor or structural element. Thlaspi caerulescens can accumulate up to 3% of zinc on a dry weight basis without any sign of toxicity. The question postulated here is if this has drastic effects on the zinc homeostasis mechanism. We examined in detail the transcription profiles of roots of Arabidopsis and T. caerulescens plants grown under deficient, sufficient and excess supply of zinc (Chapter 2). A total of 608 genes were detected in Arabidopsis and 352 in T. caerulescens that responded transcriptionally to changes in zinc supply. Only 14% of these genes were also zinc-responsive in Arabidopsis. When comparing Arabidopsis and T. caerulescens at comparable zinc exposures, over 2200 genes were significantly differentially expressed. While a large fraction of these genes are of yet unknown function, many genes with a different expression between Arabidopsis and T. caerulescens appear to function in metal homeostasis, in abiotic stress response and in lignin biosynthesis. The high expression of lignin biosynthesis genes corresponds to the deposition of lignin in the endodermis. Contrary to Arabidopsis roots, which have one endodermal cell layer, we found there are two endodermal layers in T. caerulescens roots. This extra physical barrier could enhance the control of metal fluxes in the plant, in addition to the higher expression of metal transporters in the root. Cadmium is a widespread, naturally occurring non-essential element that is toxic for plants in higher concentrations. In chapter 3 we compare between and within species transcript profiles of Arabidopsis and T. caerulescens roots exposed to cadmium, with the aim to establish which genes are most likely to be relevant for the tolerance to cadmium exposure of T. caerulescens. The comparative transcriptional analysis of the cadmium response of roots of the T. caerulescens and Arabidopsis emphasizes the role of genes involved in lignin-, glutathione- and sulfate metabolism. Furthermore two transcription factors, MYB72 and bHLH100, with an altered expression after exposure to cadmium, are studied for their involvement in metal homeostasis. Analysis of a myb72 knock-out mutant showed enhanced sensitivity to excess zinc or iron deficiency. Rather than controlling Cd tolerance, this gene appears to be involved in iron homeostasis, affecting the response to Cd indirectly. Arabidopsis transformants overexpressing the transcription factor bHLH100 showed enhanced zinc and nickel tolerance, and although the exact role of this gene still needs to be resolved, the genes appears to have a role in metal homeostasis in Arabidopsis. T. caerulescens accessions exhibit distinct metal accumulation, translocation and tolerance characteristics. T. caerulescens accession Ganges can accumulate high amounts of cadmium and is extremely tolerant to cadmium, whereas the La Calamine accession is also tolerant to cadmium but accumulates much less cadmium compared to Ganges. The transcription profiles of leaves and roots of T. caerulescens accessions Ganges and La Calamine plants grown with and without cadmium were examined using the Qiagen-Operon Arabidopsis Genome Array and results are described in chapter 4. A total of 161 genes were differentially expressed between the two T. caerulescens accessions in response to changes in cadmium supply and 38 genes were differentially expressed in T. caerulescens accession Ganges leaves in response to cadmium. The comparative transcriptional analysis emphasizes that there are just minor differences between the two accessions but the genes which are differentially expressed could play an important role in the hyperaccumulation of cadmium in Ganges. The microarray data suggest that especially genes involved in cell wall modification and stress response relate to the major difference between the two accessions in cadmium hyperaccumulation. Plants have evolved a complex network of homeostatic mechanisms that serve to control the uptake, accumulation, trafficking and detoxification of metals. One potential mechanism for heavy metal detoxification in plants is the chelation of metal ions to ligands like organic acids, amino acids, peptides and polypeptides. This mechanism is important for the distribution of metal ions by keeping metal ions mobile within the plant. In plants metals are often found to be chelated to nicotianamine. Nicotianamine is formed by trimerization of S-adenosylmethionine, which is catalyzed by the enzyme nicotianamine synthase. Arabidopsis contains four nicotianamine synthase (NAS) genes. Also in T. caerulescens four full-length cDNAs encoding nicotianamine synthase members were identified (Chapter 5). The four genes were named TcNAS1-TcNAS4, analogous to the corresponding closest homologue in Arabidopsis. Arabidopsis plants over-expressing TcNAS1, TcNAS2, TcNAS3 or TcNAS4 that were tested for their response to growth on media with different zinc, iron, nickel or cadmium supply, provided evidence that the Thlaspi genes all have a genuine NAS function because they complement the NAS deficiency in specific triple knock-out Arabidopsis mutants. Evidence for a functional role in metal homeostasis was sought in studying the Arabidopsis single, double, triple and a quadruple nicotianamine synthase T-DNA insertion mutants. The combination of null mutations in three or four AtNAS genes, results in a severe phenotype that includes interveinal chlorosis and altered metal concentrations in leaves, roots and seeds. Arabidopsis transformants overexpressing TcNAS3 or TcNAS4 showed enhanced zinc and nickel tolerance compared to wild type plants. The research described in this thesis does contribute to a better understanding of heavy metal hyperaccumulation in T. caerulescens and it can be concluded that it seems unlikely that altered regulation and overexpression of single genes will be sufficient to convert metal nonaccumulators into hyperaccumulators. However, the possibility that overexpression of one or two key regulatory loci have this effect remains.

De hygiënhypothese stelt dat de afwezigheid van infecties op de jonge kinderleeftijd gepaard gaat met een zich niet goed ontwikkelend immunoregulatienetwerk. Als gevolg hiervan zou allergische sensibilisatie toenemen en daarmee de kans op allergieën. Deze hypothese is een breed aanvaarde verklaring voor de alom geobserveerde toename van allergie in de Westerse en verwesterde samenlevingen gedurende de laatste 40 jaar. De bewijzen voor deze hypothese zijn echter zeer moeilijk te verkrijgen

Praktijkonderzoek Plant & Omgeving heeft een tweetal proeven uitgevoerd in twee jaren (2002 en 2003): een veldproef met rassen met verschillende tolerantieniveaus en een biotoets met daarin de bepaling van de bodembesmetting vóór en na de veldproef en "uitzieking" van de grond door de jaren heen bij verschillende telers. De uitzieking wordt afgeleid door de besmettingsgraad van diverse percelen van één teler onderling te vergelijken. Er zijn percelen bij waar onlangs vatbare uien hebben gestaan en percelen waar 6 tot 8 jaar geleden vatbare uien hebben gestaan

Praktijkonderzoek Plant & Omgeving heeft een tweetal proeven uitgevoerd in twee jaren (2002 en 2003): een veldproef met rassen met verschillende tolerantieniveaus en een biotoets met daarin de bepaling van de bodembesmetting vóór en na de veldproef en "uitzieking" van de grond door de jaren heen bij verschillende telers. De uitzieking wordt afgeleid door de besmettingsgraad van diverse percelen van één teler onderling te vergelijken. Er zijn percelen bij waar onlangs vatbare uien hebben gestaan en percelen waar 6 tot 8 jaar geleden vatbare uien hebben gestaan