Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
Boshoven, Jordi C. - \ 2017
Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463436441 - 183
verticillium dahliae - plant pathogenic fungi - plant pathogens - disease resistance - virulence factors - virulence - immunity - host parasite relationships - plant-microbe interactions - symbiosis - mutagenesis - resistance breeding - verticillium dahliae - plantenziekteverwekkende schimmels - plantenziekteverwekkers - ziekteresistentie - virulente factoren - virulentie - immuniteit - gastheer parasiet relaties - plant-microbe interacties - symbiose - mutagenese - resistentieveredeling
Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.
Transcriptional and functional targets of SCHIZORIZA in root development
Liere, Sabine van - \ 2017
Wageningen University. Promotor(en): B.J.G. Scheres, co-promotor(en): R. Heidstra. - Wageningen : Wageningen University - ISBN 9789463437998 - 122
arabidopsis thaliana - biological development - root meristems - root caps - cell division - stem cells - transcriptomics - gene regulation - mutagenesis - arabidopsis thaliana - biologische ontwikkeling - wortelmeristemen - wortelmutsjes - celdeling - stamcellen - transcriptomica - genregulatie - mutagenese
In this thesis I focus on SCHIZORIZA, a gene involved in tissue specification and cell fate segregation in the Arabidopsis root. Chapter 1 describes asymmetric cell division, Arabidopdis embryo development and root meristem development. In more detail we describe the maintenance of quiescent centre and columella stem cells, the development of ground tissue and epidermis/ lateral root cap. Finally we introduce SCHIZORIZA (SCZ) as a factor involved in radial patterning and the maintenance of cortex identity.
In Chapter 2, we study the interaction between the SCZ and SHORTROOT/ SCARECROW pathways that are required in parallel during stem cell niche specification in embryogenesis for the maintenance of tissue fates. Here we investigate the strong synergy of shr and scz mutants and show that at late torpedo stage scz;shr double mutant embryos lose both ground tissue and meristem marker expression.
Chapter 3 describes the use of a transcriptomics approach to identify genes differentially regulated by SCZ. These differentially regulated genes can be divided into two distinct tissue enriched groups. Upregulated genes are enriched for root cap expression and cortex expressed genes are overrepresented in the downregulated set of genes. A subset of the upregulated genes has a HSE associated with their promoter and therefore possibly represents direct SCZ targets.
In Chapter 4 we describe a mutagenesis screen to identify functional downstream targets of SCZ. Using a cortex and lateral root cap tissue marker, we identified two suppressors of the scz mutant. Both restore the fate segregation phenotype of scz mutants. We used whole genome deep sequencing to map the causal suppressor mutations in the LBD12 gene.
The analysis of LBD12 function is described in Chapter 5. We show that the single lbd12 mutant has a QC and columella phenotype. In addition, we show that ectopic expression of LBD12 induces ectopic divisions.
Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): R. Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - rhizobium - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming
Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.
A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.
Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.
In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.
Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.
Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.
Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.
In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.
Opportunities of New Plant Breeding Techniques
Schaart, Jan ; Riemens, M.M. ; Wiel, C.C.M. van de; Lotz, L.A.P. ; Smulders, M.J.M. - \ 2015
Wageningen : Wageningen UR - 24
plantenveredeling - plantenveredelingsmethoden - resistentieveredeling - cisgenese - intragenic recombination - mutagenese - dna-methylering - bloei - plant breeding - plant breeding methods - resistance breeding - cisgenesis - intragenic recombination - mutagenesis - dna methylation - flowering
This brochure gives an overview of new plant breeding techniques. This overview is based on a more technical review of the scientific literature, published in a separate report. The overview presents the opportunities and limitations of these techniques from the point of view of potential applications in plant breeding with promising results for improving agricultural sustainability.
On the natural and laboratory evolution of an antibiotic resistance gene
Salverda, M.L.M. - \ 2008
Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser; John van der Oost. - [S.l.] : S.n. - ISBN 9789085049999 - 144
evolutie - bèta-lactamase - plasmiden - recombinatie - bacteriën - fenotypen - mutagenese - fylogenie - moleculaire genetica - selectie - antibioticaresistentie - evolution - beta-lactamase - plasmids - recombination - bacteria - phenotypes - mutagenesis - phylogeny - molecular genetics - selection - antibiotic resistance
TEM-1 ß-lactamase is one of the most notorious antibiotic resistance enzymes around. It exists at high frequencies in antibiotic-resistant bacteria around the world and confers resistance to ß-lactam antibiotics, including penicillins (e.g. ampicillin) and cephalosporins. The enzyme displays a remarkable phenotypic plasticity in response to the introduction of new drugs; within a few years after the clinical debut of most new ß-lactam antibiotics resistance conferring variants of TEM-1 are isolated. Such a shift in resistance phenotype is typically caused by just a few amino acid substitutions. Until today, more than 150 variants of TEM-1 with a unique amino acid sequence have been identified.
Because of the clear link between genotype and phenotype (i.e. level of resistance or fitness) and because of the ease of selecting for increased antibiotic resistance, TEM-1 has been used as a model in studies that seek new methods to optimize proteins. These studies combine the power of in vitro mutagenesis and in vivo selection and have resulted in a wealth of information about which mutations can increase resistance when the enzyme is exposed to an antibiotic that it initially hydrolyzes inefficiently. At a later stage, these techniques were adopted and used to repeat and predict the natural evolution of TEM-1 under various selective conditions. Recently, TEM-1 is increasingly being used as an experimental model for the study of fundamental evolutionary questions, particularly those that benefit from the direct relationship between genotype and phenotype.
In this thesis, both the natural and laboratory evolution of TEM-1 are studied. The aim of the laboratory work is to increase our understanding of the way in which adaptive mutations interact. For this purpose, TEM-1 is mutagenized using error-prone PCR, which creates variation in the resulting copies of the TEM-1 gene. Mutated gene-copies are placed in bacteria which are subsequently selected for increased resistance to cefotaxime (an antibiotic that TEM-1 hydrolyzes poorly). By repeating this process multiple times in independent experiments, the mutations and mutational trajectories involved in the increase of cefotaxime resistance are studied. At a fundamental level, this has lead to a better understanding of the nature of mutation interaction and its consequences for evolutionary contingency and constraint. Evidence indicating that certain ‘silent’ mutations (i.e. mutations that alter the codon sequence but not the amino acid that the respective codon encodes) can also play a role in increased resistance was found in these data as well.
A phylogenetic study of the sequences of the ~150 different TEM-alleles that have been isolated in hospitals and clinics so far indicates that recombination has played a significant role in the evolution of TEM-alleles, contrary to what is often assumed. Furthermore, amino acid substitutions present in these clinical isolates are compared to those found in laboratory evolution studies of TEM-1, in order to investigate to what extent laboratory evolution can be used as a predictive tool for the natural evolution of antibiotic resistance genes. This overview indicates that laboratory evolution very accurately repeats the natural evolution of TEM-1. Based on these findings, predictions are made about substitutions that may appear in future clinical TEM-isolates, and directions are given how laboratory evolution can be exploited as a predictive tool most efficiently.
Functional genomics strategies with transposons in rice
Greco, R. - \ 2003
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): A.B. Pereira. - Wageningen : Wageningen Universiteit - ISBN 9789058089168 - 182
rijst - oryza - genexpressie - transposons - transpositie - transcriptie - mutagenese - genexpressieanalyse - rice - oryza - gene expression - transposable elements - transposition - transcription - mutagenesis - genomics
Rice is a major staple food crop and a recognizedmonocotylenedousmodel plant from which gene function discovery is projected to contribute to improvements in a variety of cereals like wheat and maize. The recent release of rough drafts of the rice genome sequence for public research provides a vast resource of gene sequences whose functions need to be determined by reverse genetics methods.Characterisation of a mutant phenotype is one of the most promising approaches to link gene to function. Based on this assumption, mutagenesis with transposable elements was chosen as a strategy in the research described in this thesis to address gene function in rice (Chapter 2). The well characterized maize Ac/Ds and En/Spm transposon systems were employed asinsertionalmutagens based on their known ability to transpose inheterologousspecies. Transposon insertions can cause a knockout mutation by blocking the correct expression of a gene, which may result in a mutant phenotype. The mutant gene, thus "tagged" by the inserted transposon, can then be isolated by recovering the DNA flanking the insert and lead to the isolation of the wild-type gene. Constructs for knockout mutagenesis were generated which employed the autonomous Ac element and were tested in rice japonica (Chapter 3) andindica(Chapter 4) varieties. However, the utility of knockout mutations is limited, as the majority of them display no obvious phenotype. This may be due to functional redundancy, where one or more other genes can substitute for the same function, to subtle and conditional phenotypes, or to early lethality caused by the mutation. Gene detection strategies have therefore been developed in addition to classical knockout mutagenesis to address the function of genes that do not directly reveal an obvious phenotype when disrupted (Chapter 5). To utilize gene detection, japonica rice was transformed with advanced two-component enhancer trap vectors, consisting of a mobile transposon element ( Ds or I/dSpm ), and the corresponding stabletransposase( Ac or En/Spm ) source under control of theCaMV35S promoter. The mobile transposons contained in this case a GUS marker gene driven by aCaMV35S minimal promoter that could display the pattern of expression of the adjacent trapped gene and thus provide a clue for its function (Chapters 6 and 7). A large number of rice transformants were produced to test the activity of the different transposon constructs, with the final aim of identifying optimal "starter" lines for the development of tagging populations. Among the factors evaluated were the propensity for continuous transposition through successive generations, the ability to generate large numbers of independent inserts in progeny plants and the target-site specificity of insertion. The usefulness of the selectable markers incorporated in the constructs was also assessed.The results revealed high mobility of the Ac/Ds system in rice (Chapter 3, 4 and 6), although inactivation of Ds was observed in later generations (from T 2 onward). Nevertheless, the high frequency of independent transposition demonstrated to occur in early generations (T 0 and T 1 ) enabled the production of T 2 and T 3 lines with independent "stabilised insertions, which can be used directly for reverse genetics screenings without further need for selection against thetransposasesource. The autonomous Ac transposon, in contrast, does not seem to lose mobility and was shown to efficiently transpose in japonica andindicagenotypes as well, supporting its further use in the establishment of a tagging system in this economically important subspecies. Both Ac and Ds displayed amplification of copy number, which enabled the generation of lines containing multiple transposons. Pilot sequencing of genomic sites flanking the Ac and Ds inserts revealed a preferential insertion of these transposons into genes or gene-rich regions and confirmed their tendency to transpose to linked sites, which makes them suitable for targeted tagging. Preliminary testing of the Ac/Ds enhancer trap lines for their ability to function as "detectors" of gene activity, revealed a low frequency of GUS staining patterns in somatic sectors. More thorough screenings are currently under way to fully evaluate the functionality of the system.In contrast to Ac/Ds , the En/Spm system displayed a surprisingly low transposition activity in rice (Chapter 7), restricted to somatic events that were not transmitted to the next generation, in spite of being a well-established mutagenic system inheterologousdicotyledonous species such as Arabidopsis. Transcription analysis of the En/Spm maize element in rice revealed that correct splicing of the element occurs but is not sufficient for transposition ability. Rather, the relative amounts in which the differenttransposaseproducts necessary for transposition are produced seems to be critical and influenced by host factors. In addition, transposition efficiency might be further reduced by the lack of essentialcis-required sequences in the modified I/dSpm version used in this study, although similar constructs were successfully employed in Arabidopsis. Eventually, cross-talk with related endogenous transposable elements may affect the mobility of the maize transposon in rice. Indeed, an interaction of the maizetransposaseswith a rice En/Spm -homologous element was revealed, resulting in the specific suppression of an alternative transcript in the latter (Chapter 8). This finding demonstrates that interference is possible and trans-activation potentially could occur among elements belonging to the same transposon family in different species.Based on the results of these analyses, a core collection of knockout and gene detection Ac/Ds lines with active transposition could be selected as a basis for developing populations for (forward and) reverse genetic screenings. The propagation of lines containing multiple transposons and the preferential insertion into gene-rich regions will help reduce the number of plants that would have to be produced in order to saturate the genome with insertions. At present, over 10,000 stabilised T 2Ac/Ds transposon lines are being analyzed in 5 EU laboratories for transposon flanking sequences that by comparison to the complete and annotated rice sequences will reveal tagged genes of interest that can be used for reverse genetics.
Classical mutagenesis in higher plants
Koornneef, M. - \ 2002
In: Molecular Plant Biology / Gilmartin, P.M., Bowler, C., Oxford, GB : Oxford University Press - p. 1 - 10.
genetica - mutagenese - mutanten - plantenveredeling - mutagenen - genetics - mutagenesis - mutants - plant breeding - mutagens
For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power of mutant approaches in combination with molecular techniques to investigate the molecular nature of the genes became fully appreciated
Identification of potato genes involved in Phytophthora infestans resistance by transposon mutagenesis
Enckevort, L.J.G. van - \ 2000
Agricultural University. Promotor(en): E. Jacobsen; A. Pereira. - S.l. : S.n. - ISBN 9789058083432 - 144
aardappelen - solanum tuberosum - phytophthora infestans - plantenziekteverwekkende schimmels - ziekteresistentie - transposons - merken van genen - selectie - plantenveredeling - transpositie - mutagenese - potatoes - solanum tuberosum - phytophthora infestans - plant pathogenic fungi - disease resistance - transposable elements - transposition - mutagenesis - gene tagging - selection - plant breeding
The late blight disease, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is a serious threat to the potato crop every growing season. This has, for example, led to the disastrous Irish famine in the middle of the 19 th century, and continued in the 20 th century to remain a serious problem for potato growers. Since the early 1980s P. infestans populations changed more rapidly and epidemics even increased in their severity. Resistance breeding stimulated the introduction of resistance genes ( R genes) from wild Solanum species into cultivated potato, Solanum tuberosum , but newly occurring virulent races of P. infestans circumvented these R gene mediated resistances and no cultivars with durable resistance were obtained. At the moment, methods using fungicides supervised by spraying control via decision support systems are the only available control measures.
Characteristic for R gene type mediated resistance reactions is the hypersensitive response (HR) leading to local cell death causing necrotic spots at the site of attempted infection. Genetic analysis of HR mediated resistances showed that activation of HR is highly specific and induced upon recognition between a specific R gene in the plant and a corresponding avirulence gene ( Avr gene) in the pathogen. Insights in the molecular mechanisms underlying this HR resistance reaction in Solanum species might facilitate the development of potato cultivars that are more durable in maintaining a resistance phenotype.
A two component Ac-Ds transposon tagging strategy in diploidised potato was developed to identify and isolate genes involved in the R1 gene mediated resistance response to P. infestans . Transposable elements are molecular genetic tools to mutate and identify genes. The transposable elements Ac and Ds were first characterised in maize and their molecular isolation led to the identification of maize genes that were tagged by these elements. The autonomous Ac element is able to transpose by itself and also to induce transposition of the non-autonomous Ds element that is transposase defective. Introduction of these elements in heterologous species demonstrated their utility for isolating genes in self-fertilising plant species. Also in the highly heterozygous and tetraploid potato, the Ac and Ds transposable elements were shown to be functional. A cell autonomous visual marker gene for potato, the granule bound starch synthase gene ( GBSS gene), enabled a refined characterisation of Ac transposition in potato. Further molecular characterisation showed high levels of Ac-Ds transposition both somatically and germinally, so that suitable populations could be generated for tagging purposes.
The production of clones homozygous for the gene of interest that are normally required for efficient tagging strategies, turned out to be time consuming in potato due to self-incompatibility at the diploid level. Therefore, an alternative method based on somatic transposition was developed for the direct selection of transposition events instead of recovering germinally transmitted transpositions. Highly chimaeric Ac-Ds seedlings with active Ds transposition linked to the R1 resistance gene on chromosome 5 of potato were selected. Protoplasts were isolated from actively transposing seedlings and using the hygromycin excision selection marker, regenerants could be selected with new independent Ds insertions. The resulting R1 resistant transposon mutagenised population of almost 2000 hygromycin resistant regenerants formed an ideal start for the identification of an R1 tagged mutant, or other Ds insertional mutants with an altered R1 resistance response.
The somatically regenerated tagging population was analysed for the P. infestansR1 type HR resistance response, using a detached leaf assay for P.infestans inoculation. In a primary screening, 33 potential R1 resistance variants showing partial susceptibility to P. infestans race 0 were identified. These results were further quantified using stringent inoculation conditions on replicate samples leading to the identification of four putative mutants with a distinctly altered R1 resistance response. In these putative mutants less than 50% of the inoculated leaves showed the R1 type HR response and clear colonisation with sporulation of P. infestans was observed. The flanking sequences of the Ds insertion sites in these putative R1 mutants were analysed and in two cases a potential biological correlation between the insertion sequences and the phenotype was evident. One putative mutant contained a Ds insertion in a region with auxin and abscisic acid response cis-elements homologous to a specific region (TAPIR) of the tomato defence related genes TAPG2 and TAP1 .
The second P. infestansR1 resistance mutant, mutant 1000 with a striking susceptible phenotype was characterised in more detail. Two Ds insertions were identified and the insertion site flanking sequences both showed high homology to serine/threonine protein kinases. The Ds insertion sites turned out to be homologous but not identical, indicating two independent Ds insertions in homologous but not identical genes. Both sequences showed protein identity to all the conserved regions of serine/threonine protein kinases and they contained a conserved intron position. The closest homology was to the serine/threonine protein kinase domain of the Xanthomonas resistance gene Xa21 , which is involved in the induction of a HR resistance response in rice. This indicates that the isolated Solanum tuberosum protein kinase (StPK) homologs are candidate genes involved in resistance gene activity in potato. Further specific molecular analyses identified at least 11 homologs by sequence, which probably belong to a large family of serine/threonine protein kinases in potato. Both homologs in which the Ds transposons are inserted were present in susceptible parental potato clones. Therefore, it is unlikely that the isolated sequences represent the R1 gene itself. The mutated StPKs were designated rpr1 and rpr2 , r equired for P hytophthora infestansr esistance gene 1 and 2. Studying these mutants and the StPKs involved might help in understanding the pathway leading to HR resistance in potato.
Coenzyme recognition in para-hydroxybenzoate hydroxylase
Eppink, M.H.M. - \ 1999
Agricultural University. Promotor(en): N.C.M. Laane; W.J.H. van Berkel; H.A. Schreuder. - S.l. : Eppink - ISBN 9789058080653 - 167
oxygenasen - benzoaten - mutagenese - co-enzymen - oxygenases - benzoates - mutagenesis - coenzymes
Biochemistry is the science that studies the chemistry of life. This 'biological' chemistry includes growth, differentiation, movement, conductivity, immunity, transport and storage. During these processes proteins play an important role. The building blocks of proteins are amino acids, of which twenty are known. With these building blocks at hand it is possible to construct numerous proteins with many specific functions. A protein is not an elongated chain of amino acid residues but a compact very well defined three-dimensional structure. Two basic substructures are known in a protein, a cylindricalα-helix and an elongatedβ-strand. A number of theseα-helices and/orβ-strands connected by loop regions form a protein domain and a protein is built up of one or more domains. Furthermore, proteins can contain certain motifs (folds), structural conserved patterns. A large group of proteins with similar function and/or structure are called a protein family.
A special group of proteins, called enzymes or biocatalysts, are able to increase the rate of a chemical reaction by lowering the activation energy of that reaction. Enzymes are highly specific, because they influence the reactivity of the substrate in such a way that the substrate is quickly and efficiently converted into a product. Moreover, flexible/dynamic movements in enzymes may play an important role during catalysis, because enzymes are not always rigid bodies. To control the reaction, enzymes often need cofactors. Some examples are the already mentioned dinucleotides NAD(P)H and FAD, that play a role in electron transfer (redox) reactions. Generally speaking, these cofactors bind very specific to a protein. A well-known binding motif for NAD(P)H and FAD in different enzyme families is the Rossmann fold (Chapter 1), discovered by Michael Rossmann in 1974.
The NAD(P)H cofactor binds to the enzyme, electron transfer takes place and finally, the oxidized cofactor is released. In some proteins, the mode of NADPH binding is unknown. One example is p -hydroxybenzoate hydroxylase (PHBH), a flavoprotein monooxygenase that belongs to the family of FAD-dependent aromatic hydroxylases.
FAD-dependent Aromatic Hydroxylases
FAD-dependent aromatic hydroxylases play a role in the biodegradation of aromatic compounds. In nature, these compounds occur in plant polymers (lignin) as well as in proteins, steroïds and terpenes. During this century, the natural pool of aromatic compounds has been extended with products of industrial origin. Many of these synthetic compounds (pesticides, herbicides, fungicides and detergents) place a heavy burden on the environment and accumulate in soil and sludge. Microbial FAD-dependent aromatic hydroxylases catalyze the conversion of natural and synthetic aromatic substrates into products that can be further degraded to carbon dioxide and water. Recently, it was found that these enzymes are also involved in the biosynthesis of steroïds, plant hormones and antibiotics. PHBH is the archetype (prototype) of the family of FAD-dependent aromatic hydroxylases. In Wageningen, research on PHBH and related enzymes is embedded in the Wageningen Graduate School of Environmental Chemistry & Toxicology.
p -Hydroxybenzoate Hydroxylase
p -Hydroxybenzoate hydroxylase is isolated from the soil bacterium Pseudomonas fluorescens . This microbe can grow on 4-hydroxybenzoate (POHB) and other aromatic compounds as sole carbon source. PHBH catalyzes the conversion of POHB into 3,4-dihydroxybenzoate (DOHB) in the presence of NADPH and molecular oxygen. DOHB is a common intermediate in the aerobic degradation of plant material. After ring cleavage of DOHB and further degradation, the final products acetyl coenzyme A and succinate are fed into the citric acid cycle to provide energy for the cell.
p -Hydroxybenzoate hydroxylase has been subject to detailed kinetic and structural studies. The three-dimensional structure of PHBH is built up of three domains (Chapter 1). The first domain is the FAD-binding domain with the specific Rossmann fold for binding the ADP part of FAD. The second domain is the substrate-binding domain and the third domain (interface domain) is important for the interaction with another PHBH subunit, because PHBH exists as a dimer.
The structure of the enzyme-substrate complex is known in atomic detail. Recently, it was found that the flavin ring is able to move between an "open" and "closed" conformation. This flavin mobility is important for substrate binding and product release. However, unknown is the NADPH-binding site and where the reaction between NADPH and FAD takes place. Related questions are:
-Which amino acids play a role in cofactor binding?
-Is there a particular sequence motif for cofactor binding?
Another very important question concerns the effector role of the substrate. Upon binding of the aromatic substrate the flow of electrons from NADPH to FAD is 105 times enhanced. However, the molecular principles of this control are poorly understood. In this thesis we have tried to shed more light on the coenzyme recognition by PHBH.
Flavin ring mobility
In Chapter 2 the FAD in PHBH is substituted by a modified FAD, normally present in alcohol oxidase from methylotrophic yeasts. The crystal structure of p -hydroxybenzoate hydroxylase with this flavin analog not only represents the first crystal structure of an enzyme reconstituted with a modified flavin, but also provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The reconstituted enzyme-substrate complex shows that the flavin ring attains the "open" conformation. In the native enzyme-substrate complex the flavin ring is located in the "closed" conformation. The rate of flavin reduction by NADPH is much more rapid as compared to the native enzyme-substrate complex, suggesting that the mobility of the flavin ring is essential for the efficient reduction of the enzyme/substrate complex.
Amino acids involved in NADPH binding
To investigate the mode of NADPH binding, several amino acid residues were replaced by site-directed mutagenesis. The amino acids were selected on the basis of earlier results from chemical modification, crystallographic and modeling studies. Chapters 3, 4, 6 and 8 describe the properties of single mutants. It is concluded that Arg33, Gln34, Tyr38, Arg42, Arg44, His162 and Arg269 are involved in NADPH binding.
Structural motif for NADPH binding
PHBH contains two conserved sequence motifs, both involved in FAD binding. Chapter 5 describes a new unique sequence motif for the family of FAD-dependent aromatic hydroxylases, putatively involved in both FAD and NAD(P)H binding. From the recently determined crystal structure of phenol hydroxylase it is deduced that this sequence motif is also structurally conserved. Chapter 6 and 7 show that only His162 of this novel motif is directly important for the binding of NADPH.
Chapter 8 describes the cloning, purification and characterization of PHBH from Pseudomonas species CBS3. This is the first PHBH enzyme with known sequence that is active with NADH. Based on sequence analysis and homology modelling it is proposed that the helix H2 region is important for the binding of the 2'-phosphate moiety of NADPH. In Chapter 9 , the coenzyme specificity of PHBH from Pseudomonas fluorescens was addressed in further detail. Multiple replacements in helix H2 showed that Arg33 and Tyr38 are crucially involved in determining the coenzyme specificity. For the first time, a PHBH enzyme was constructed, which is more efficient with NADH.
Substrate binding is essential for a rapid reduction of FAD. This allows the subsequent attack of oxygen and the formation of the flavinhydroperoxide hydroxylating species. The question arises whether the stimulating effect of substrate binding on flavin reduction is caused by a large conformational change or merely due to subtle rearrangements in the active site. Chapter 10 describes the crystal structure of the substrate-free enzyme. This study shows that no large conformational changes take place upon substrate (analog) binding. The stimulating role of POHB is probably caused by several subtle effects. Stabilisation of the phenolate form of the substrate results in distribution of the electronic charges in the active site. These charge distributions influence the dynamic equilibrium between the "open" and "closed" conformation of FAD in such a way that the nicotinamide ring of NADPH and the isoalloxazine ring of FAD become optimally oriented for efficient reduction.
Genetic analysis of seed development in Arabidopsis thaliana = [Genetische analyse van de zaadontwikkeling in Arabidopsis thaliana]
Leon - Kloosterziel, K. - \ 1997
Agricultural University. Promotor(en): M. Koornneef. - S.l. : Leon-Kloosterziel - ISBN 9789054857709 - 119
genen - genomen - mutaties - mutagenese - mutagenen - plantenfysiologie - plantenontwikkeling - vruchten - rijp worden - brassicaceae - zaadzetting - zaden - formatie - kieming - zaadkieming - kiemrust - genes - genomes - mutations - mutagenesis - mutagens - plant physiology - plant development - fruits - ripening - brassicaceae - seed set - seeds - formation - germination - seed germination - seed dormancy
This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection procedures. The mutants have been analyzed genetically, physiologically, and morphologically. Some of the mutants are impaired in the biosynthesis or sensitivity to the plant hormone, abscisic acid (ABA). All ABA-related mutants show reduced seed dormancy, indicating the important role of this hormone in the establishment of dormancy. In a direct screen for reduced dormancy, two mutants (rdo) with reduced dormancy were found. These were not ABA-deficient and showed the same sensitivity to ABA, ethylene, auxin, and cytokinin as the wild-type. In contrast to this embryo-determined reduced dormancy, reduced dormancy can also originate in an altered seed coat (testa), like in the altered testa shape ( ats ) mutant. Here, the altered testa shape is caused by a defect in the development of the integuments. Extreme ABA-insensitive mutants ( abi3 ) have green seeds that fail to complete many other aspects of seed maturation, including the induction of dormancy and desiccation tolerance, and the accumulation of seed storage proteins and lipids. In addition to abi3 mutants, lec and fus mutants exhibit such a severely disturbed seed maturation as well, with dark purple seeds due to anthocyanin accumulation. The fus3 mutant shows normal ABA-sensitivity. These various seed maturation mutants indicate that specific genes, some acting dependently and some acting independently from ABA, are responsible for seed maturation programs. The seed maturation mutants were subjected to a physiological and biochemical analysis. A GA-deficient mutant was combined with these mutants. Analysis of these double mutants indicated that seeds of the abi3 and lec mutants did not require GA for germination, in contrast to fus3 seeds. This correlates with ABA-sensitivity for germination. The composition of storage proteins and carbohydrates in abi3 , lec, and fus3 mutant seeds has been compared. The abi3 , lec, and fus3 mutants all showed severely reduced storage proteins. The desiccation intolerance of these seed maturation mutants was not correlated with the lack of specific carbohydrates. Furthermore, the mutants had a higher total content of carbohydrates. This is probably a consequence of the lower levels of storage lipids and proteins.
Genetic (in)stability in tomato
Wisman, E. - \ 1993
Agricultural University. Promotor(en): A. van Kammen; W.J.T. Zabel; M.S. Ramanna. - S.l. : Wisman - ISBN 9789054851530 - 141
genetische variatie - mutaties - solanum lycopersicum - tomaten - genen - mutagenese - genetic variation - mutations - solanum lycopersicum - tomatoes - genes - mutagenesis
In the present study tomato lines carrying unstable alleles of the loci yv or sulfurea were characterized. In addition, we aimed at the isolation of an endogenous transposable element supposedly active in the unstable lines. Since the unstable loci were not cloned, we developed a transposon target system that enabled us to select for putative insertion mutants of the Adh-1 gene. While investigating the genetic characteristics of the Yv msand Sulfurea line it became apparent that the instability of their marker loci was not necessarily related to the activity of transposition elements. Also, the instability could not be related to the activity of tomato sequences homologous to transposable elements from related species. This invited speculation about the stability of DNA sequences in tomato. It is proposed that mechanisms which are known to underlie genetic instability in other organisms, have a role in the unstable expression of yv and sulfurea . These alternative mechanisms have been summarized in Chapter 1. Chapter 2 describes the isolation of an EMS induced Adh-1 null mutant and its use in crosses with the unstable tomato lines. In the resulting F 1 populations additional Adh-1 null mutants were selected through the resistance of seeds or pollen grains to allyl alcohol (Chapter 4). The cloning of the entire Adh-1 gene allowed the molecular analysis of the Adh-1 null mutants (Chapter 3). Chapter 5 describes the cytogenetic and genetic characterization of the tomato line Yvms. 'Me characteristics of a newly isolated unstable allele of the sulfurea locus are presented in chapter 6. Finally, chapter 7 describes the hybridization of tomato DNA to alien transposable element probes. These experiments emphasize the stability of transposable element-like sequences in the tomato genome.
Photomorphogenetic mutants of higher plants
Peters, J.L. - \ 1992
Agricultural University. Promotor(en): W.J. Vredenberg; R.E. Kendrick. - S.l. : Peters - ISBN 9789090051031 - 117
genetische variatie - mutaties - licht - fotoperiodiciteit - mutagenese - solanum lycopersicum - tomaten - groei - plantenontwikkeling - plantenveredeling - genetic variation - mutations - light - photoperiodism - mutagenesis - solanum lycopersicum - tomatoes - growth - plant development - plant breeding
In this thesis the hypothesis that different molecular types of phytochrome have different physiological roles has been elaborated and an attempt has been made to assign functions to these phytochrome types. For this purpose a genetic approach was chosen and the following potential phytochrome mutants were used: the tomato aurea ( au ) mutant; the cucumber long-hypocotyl ( lh ) mutant and the Arabidopsis long-hypocotyl ( hy3 ) mutant. Comparing the response of the mutant affected in the abundance or function of a particular phytochrome type with that of its isogenic wild type shows directly the relevance of the phytochrome type concerned. The au mutant is proposed to lack the light-labile phytochrome type (phy A), while normal levels of the lightstable phytochrome type (phy B) are present. This results in a reduction of. (i) the inhibition of the hypocotyl growth; (ii) the anthocyanin synthesis; (iii) the chlorophyll content, but a normal end-of-day far-red (EOD FR) response. In the lh and hy3 mutants the opposite situation appears to be found: normal levels of phy A, but a lack of phy B. Both the lh and hy3 mutant do not show the EOD FR response, while the hypocotyl growth in continuous R and B is less inhibited when compared to the wild type. Therefore it is proposed that phy B plays a role in the EOD FR response, while both phy A and phy B are important for the inhibition of the hypocotyl growth. With the help of other mutants described in the literature a more detailed assignment of functions to the different phytochrome types is proposed.
In addition, a potential phytochrome signal transduction mutant, the tomato high- pigment ( hp ) mutant, was characterized. This mutant exhibits exaggerated phytochrome responses, e.g. high anthocyanin synthesis and short hypocotyl length when compared to wild type. It. is proposed that the hp mutation affects an amplification step in the phytochrome transduction chain.
Desynapsis and FDR 2N-megaspore formation in diploid potato : potentials and limitations for breeding and for the induction of diplosporic apomixis
Jongedijk, E. - \ 1991
Agricultural University. Promotor(en): J.G.T. Hermsen; J. Sybenga. - S.l. : Jongedijk - 111
solanum tuberosum - aardappelen - cytogenetica - meiose - mutaties - mutagenese - mutagenen - apomixis - parthenogenese - solanum tuberosum - potatoes - cytogenetics - meiosis - mutations - mutagenesis - mutagens - apomixis - parthenogenesis - cum laude
The cultivated potato, Solanum tuberosum L., is a highly heterozygous autotetraploid (2n=4x=48) plant species, which after its introduction into Europe in the 16th century has become one of the world's major food crops. The potato has traditionally been grown from tubers. However, as tubers are an excellent substrate for many pathogens and parasites, it is extremely difficult and expensive to produce healthy seed tubers. Most developing countries lack both the knowledge and infrastructure required for the efficient production of healthy seed tubers and the currency for its importation and distribution. As a consequence small farmers in developing countries are generally forced to use diseased tubers from a previous harvest, which may result in dramatic yield losses. In response to the urgent need of cheap but healthy plant material the International Potato Center (CIP) in Peru has, since the Planning Conference on this subject in 1979, propagated the new technology of growing potatoes from true seeds, True potato seeds are relatively easy and cheap to produce and even when harvested from heavily diseased plants generally do not carry any diseases. However one of the major problems in breeding potato varieties that can be maintained and grown from true seeds is the lack of uniformity. True seed progeny of existing varieties or inter-varietal hybrids is mostly highly heterogeneous due to the extreme heterozygosity of the potato.
Several methods to synthesize sufficiently uniform true potato seed varieties have been proposed. One of these methods takes advantage of the frequent occurrence of numerically unreduced (2n) gametes in wild and cultivated diploid potato species, which enables the production of hybrid tetraploid progeny from tetraploid-diploid (unilateral sexual polyploidization) or diploid-diploid (bilateral sexual polyploidization) matings. The vigour and uniformity of tetraploid populations produced in this way, largely depends on the mode of 2n-gamete formation in the selected diploid parents. Depending on the genetic consequences of meiotic abnormalities that result into 2n-gamete formation, two distinct modes, viz. first division restitution (FDR) and second division restitution (SDR), can be distinguished. In general FDR is considered superior to SDR because of its abilitity to preserve a relatively large amount of favourable parental heterozygosity, including complex types of epistasis. In this respect the combination of FDR 2n-gamete formation with mutant synaptic genes that substantially reduce gene recombination, is of particular significance as it would provide a means to enhance the ability of FDR 2n-gametes to pick up the genetic constitution of the selected diploid parents with a minimum amount of reassortment. Using synaptic mutants with a virtually complete lack of gene recombination maximum performance and nearly complete uniformity may thus be expected from 2xFDR-2xFDR crosses.
Complete or nearly complete uniformity might also be achieved by the induction of apomictic seed formation. Apomixis sensu stricto is the asexual development of maternal embryo's and seeds and thus combines the advantages of both vegetative propagation (uniformity) and generative propagation (disease free plant material). Apomictic embryos may arise either directly from somatic cells outside the embryosac (adventitious embryony) or from unreduced and unfertilized (parthenogenesis) cells, usually egg cells, of the embryosac (gametophytic apomixis). In the latter case unreduced embryosacs are formed that may be of either aposporic or diplosporic origin. In apospory it develops directly from a somatic, mostly nucellar cell of the ovule. In diplospory the unreduced embryosac derives from a generative archesporial cell of the ovule, either directly by omission of meiosis or indirectly by modified meiosis in which neither reduction in chromosome number nor (substantial) gene recombination takes place. Fertilization of the secondary embryosac: nucleus may or may not be required as a stimulus for endosperm formation and subsequent parthenogenetic development of the unreduced egg cells into mature embryos and seeds (pseudogamous and autonomous apomixis respectively).
Although apomictic seed formation has never been observed in Solanaceae its importance for growing potatoes from true seeds justified an attempt to breed for apomictic reproduction in potato. The research described in this thesis focussed on the perspectives for inducing gametophytic apomixis, in particular pseudogamous diplosporic apomixis, because:
An accurate knowledge of the normal pattern of female meiosis and embryosac formation is essential for deciding whether or not an unreduced embryosac is of diplosporic origin and for recognizing abnormalities in female meiosis that are associated with the expression of mutant synaptic genes and the formation of 2n-eggs. Therefore, normal meiosis and embryosac formation were studied in several diploid potato clones (Chapter 1). In contrast to results reported in the literature, this study indicated that the archesporium of potato cannot be delimited to a single cell. A surplus of archespores sometimes developed into normal sexual embryosacs. So the occurrence of multiple embryosacs within a single ovule need not necessarily be due to apospory as had previously been suggested by some potato researchers. On the basis of the normal sequence of female meiosis it was inferred that 2n-megaspores, if formed by meiotic abnormalities in normal synaptic plants, are likely to be of exclusive SDR origin, whereas meiotic abnormalities resulting in consistent FDR 2n-megaspore formation, and thus the induction of diplospory, would actually require mutant synaptic conditions. Subsequent research was therefore primarily focussed on (1) the identification and characterization of mutant synaptic genes that express in female meiosis, (ii) the identification of synaptic mutants with consistent FDR 2n-megaspore formation and (iii) the elucidation of the cytological mechanisms of FDR 2n-megaspore formation.
For this purpose, first quick routine methods allowing for large scale screening and detailed studies of female meiosis had to be developed, because conventional embedding-sectioning techniques are very laborious and hamper quantitative interpretation of meiotic processes. Two techniques were developed. One enabled the screening of female meiosis and embryosac development in intact methyl salicylate cleared ovules and permitted bulk preparation of fixed ovaries within 2 hours (Chapter 2). The other, a 30 minute enzyme-squash procedure, allowed for detailed studies on the effect of mutant synaptic genes and genes for 2n-megaspore formation on chromosome behaviour (Chapter 3).
Using these techniques it was established that (at least) three of the six mutant synaptic genes that had been reported for potato were allelic and similarly expressed in both male and female meiosis, whereas a mutant that had been claimed to express in female meiosis only proved to be non- existent. The former three mutants were characterized by normal chromosome behaviour throughout pachytene and a falling apart of bivalent chromosomes at diakinesis and thus displayed a typical desynaptic behaviour. They were therefore reassigned the gene symbol ds -1 (Chapter 4). Asynaptic mutants with a virtually complete absence of chromosome pairing and hence gene recombination are the most attractive candidates for engineering diplosporic apomixis. However, as cogent cases of asynapsis have not yet been reported for potato, desynaptic mutants are the best alternative that is currently available. Therefore the following questions were raised:
In chapter 6 the first two questions are addressed. The level of 2n-megaspore formation was determined in 126 ds -1 mutants using seed set from 2x.4x and 2x.2xFDR testcrosses as a criterion. Although the majority formed on the average less than 5 seeds/fruit, 14% of all ds-1 mutants produced 2n-megaspores in frequencies that resulted in consistent seed set within the 5-25 seeds/fruit range and allowed for routine production of nearly exclusive tetraploid progeny from 2x.2xFDR crosses. Subsequent cytological analyses revealed that 2n-megaspore formation in ds-1 mutants resulted from a direct equational division of univalent chromosomes in the first meiotic division (pseudo-homotypic division), an FDR mechanism that had previously been reported to occur in some of the diplosporic apomictic plant species. Additional data on SDR 2n-megaspore formation in full-sib normal synaptic plants indicated that both SDR and FDR 2n-megaspore formation are likely to be caused by common genes for precocious chromosome division. Depending on the relative timing of cell cycle and chromosome division this precocious chromosome division may impose post-reductional (SDR) or pre-reductional (FDR) 'restitution' of the somatic chromosome number under normal and mutant synaptic conditions respectively.
Simply inherited marker traits are required for the analysis of genetic recombination in ds -1 mutants and had to be identified first. Some of the genetic markers used are characterized in chapter 8. On the basis of extensive analysis of chiasma formation and gene-centromere mapping of a number of simply inherited marker genes in normal synaptic plants and desynaptic mutants it could be concluded that the ds -1 gene substantially reduced the overall frequency of crossing-over and thus gene recombination in both male and female meiosis (Chapters 5 and 7). In addition, the genetic analyses revealed that FDR 2n-megaspores and FDR 2n-pollen from ds-1 mutants preserve approximately 94.1 % of the overall parental heterozygosity as opposed to 79.5 % that is preserved by FDR 2n-pollen from normal synaptic plants. The ds -1 gene was further demonstrated to particularly enhance the ability of FDR 2n-megaspores, and 2n-pollen to pick up the genetic constitution of the parental clone, including complex types of favourable epistasis, with a minimum amount of reassortment.
Summarizing, it may be stated that the identification of diploid desynaptic mutants with consistent FDR 2n-megaspore formation extends the opportunities for direct transfer of enhanced diploid germplasm to tetraploids by means of sexual polyploidization and, since FDR 2n-megaspores and 2n-pollen from ds -1 mutants are relatively efficient in preserving the genetic constitution of selected diploid parents particularly demonstrates the feasibility of 2x( ds -1;FDR)-2x( ds -1;FDR) crosses for the production of relatively uniform and vigorous true potato seed varieties.
Because of the potential for limited genetic recombination the use of the ds -1 gene in the development of diplosporic apomixis seems less obvious. However, as genes for asynapsis have not been identified in potato so far, it may be considered the best alternative that is currently available. Moreover some genetic diversity in apomictic progeny of diplosporic plant species as a consequence of autosegregation is quite common. The finding that some desynaptic clones formed FDR 2n-eggs through pseudo-homotypic division (≈diplospory) strongly supports the hypothesis that gametophytic apomixis consists of a number of distinct and genetically controlled elements which may be combined to attain approximately identical reproduction in largely sexual plant species. The application of this approach to produce completely uniform true potato seed varieties obviously requires breeding for increased levels of FDR 2n-egg formation in synaptic mutants completely suppressing genetic recombination, and in addition either introduction of genes for pseudogamous seed development in such clones or the development of an efficient system for pseudogamous seed production.
Finally, it should be recognized that mutant synaptic genes may impose certain limitations. Because they are generally expressed in both male and female meiosis and thus are either largely sterile or produce only functional FDR 2n-gametes; resulting in polyploidization upon crossing, they have to be manipulated in heterozygous condition. Breeding schemes that consist of (i) introducing mutant synaptic genes and genes for FDR 2n-gamete formation in advanced diploids through backcrossing and (ii) subsequent selection of improved mutant synaptic segregants with FDR 2n-gamete formation following intercrossing of advanced heterozygotes, would be appropriate but laborious. As to FDR 2n-megaspore formation it should moreover be realized that mutant synaptic conditions are actually required. Since heterozygous diploid clones are normal synaptic and thus at best form SDR 2n-megaspores the question remains how to predict whether or not such clones carry genes that bring about substantial FDR 2n-megaspore formation in derived synaptic mutants. If the hypothesis holds true, that SDR and FDR 2n-megaspore formation are caused by common genes for division precocity (Chapter 6), the occurrence of SDR 2n-megaspore formation through postreductional precocious chromosome division in normal synaptic heterozygotes, might be a helpful criterion.
Occurrence of indole compounds in some vegetables : toxicological implications of nitrosation with emphasis on mutagenicity
Tiedink, H.G.M. - \ 1991
Agricultural University. Promotor(en): J.H. Koeman; L.W. van Broekhoven; W.M.F. Jongen. - S.l. : Tiedink - 157
groenten - groenteteelt - carcinogenese - carcinogenen - toxicologie - mutaties - mutagenese - mutagenen - vegetables - vegetable growing - carcinogenesis - carcinogens - toxicology - mutations - mutagenesis - mutagens
From the introduction to the chemistry, occurrence and formation of N-nitroso compounds (NOC) (Chapter 1), it can be concluded that human exposure to these potent carcinogenic compounds is mainly through the endogenous nitrosation of dietary precursors. Vegetables are the major source of nitrosating compounds, while also nitrosatable substrates can occur in vegetables. Therefore the first aim of the present study was to screen Dutch vegetables for the presence of nitrosatable compounds by measuring their potential to form directly mutagenic NOC upon nitrite treatment. The second aim was to study the identity and mutagenic properties of the NOC formed. Since it was not feasible to cover all vegetables in connection to the second aim of the study, the efforts were concentrated on two of them, brassicas and fava beans. The latter were chosen because they were already known to contain precursors of directly mutagenic NOC and consequently the study was divided into two parts: Part 1 dealing with brassicas and Part 2 with fava beans.
In Chapter 4 experiments are described in which extracts of 31 Dutch vegetables were screened for their ability to form directly mutagenic NOC upon nitrite treatment, irrespective of their nitrate content. All vegetables tested formed NOC upon nitrosation and induced Salmonella ( S. ) typhimurium revertants; Brassica vegetables were high responders on both of these parameters. Moreover, a significant correlation was found between their glucosinolate content (both alkyl/aryland indolylglucosinolate) and the amounts of NOC formed in extracts of these vegetables upon nitrosation. This suggests that glucosinolates are involved in the formation of NOC. Therefore purified glucosinolates were tested for their potential to form NOC (Chapter 6). Only indolylglucosinolates and their hydrolysis products formed NOC upon nitrosation. Mutagenicity was restricted to the nitrosated hydrolysis products. Since upon hydrolysis of indolylglucosinolates indole compounds are formed, which are nitrosatable substrates, the kinetics of the formation of NOC from indole compounds were investigated, as well as the stability of the nitrosated products formed (Chapter 5). Indole-3-acetonitrile (I 3 A), indole-3-carbinol (I 3 C) and indole, the hydrolysis products of the most commonly indoyglucosinolate, glucobrassicin, immediately reacted with nitrite to form directly mutagenic NOC and after an incubation time of about 15 min. maximal amounts of NOC were formed. The formed NOC were stable at both pH 2 and 8, but only when nitrite was present.
In order to determine the contribution of indole compounds to the total mutagenicity of nitrite treated brassicas, the presence of several known indole compounds in green cabbage was investigated (Chapter 6). Only indole-3-carboxaldehyde and I 3 A could be detected. Both were not found to be important precursors of directly mutagenic NOC in green cabbage. The former did not form NOC at all and the second, although it occurred in considerable amounts (12 mg/kg fresh weight), only contributed for 2% to the total mutagenicity of nitrite treated green cabbage.
Since in brassicas other precursors of directly mutagenic NOC occur, further studies are recommended to elucidate their identity and the implications of their potential endogenous nitrosation.
4-Chloro-6-methoxyindole (4C6MI), the naturally occurring indole compound in fava beans, was evaluated for its genotoxic and tumour promoting potential after nitrite treatment (Chapter g). Remarkably, nitrosated 4C6MI appeared to have both genotoxic and tumour promoting potentials. The initiation effects were measured in bacteria and mammalian cells, the tumour promoting effects were measured by inhibition of gap junctional intercellular communication of V79 Chinese hamster cells. Both effects were observed at concentrations, which were in the same order of magnitude as the estimated daily intake of 4C6MI in a Colombian population. Hence, the results support the model for gastric cancer etiology, proposed by Correa et al. (1976, 1983). In this model the formation of NOC out of fava beans was supposed to be a causative factor of gastric cancer of the intestinal type, endemic in Colombia.
In the study described in Chapter 10 the occurrence of 4C6MI in Dutch fava beans was investigated. An improved purification method was developed because the procedures described in the literature proved to be inadequate. Although the new purification method requires further improvement, a reasonable estimate of the levels of 4C6MI could be made. The levels ranged from about 3 to 7.5 mg/kg dry weight, which is one to two orders of magnitude higher than those reported for Colombian beans. However, in Chapter 12 It was assumed that the levels of 4C6MI in Colombian beans may be much higher than those reported. Further studies are required to investigate this aspect. From the results described in Chapter 10 it was concluded that almost all mutagenicity of nitrite treated Dutch fava beans can be attributed to 4C6MI.
Although in previous studies a difference was observed in the mutagenicity of nitrite treated white and brown cooking cultivars after nitrosation, this was not found in the present study (Chapter 10 & 11). Moreover, all cultivars induced much more S. typhimurium revertants after nitrosation than in previous studies. No explanation can be given for these equivocal results and therefore studies are recommended to investigate the influence of environmental factors on the levels of 4C6MI in fava beans (Chapter 10).
The mutagenicity of nitrite treated fava beans could be inhibited for 80-100% by addition of casein, indicating binding of nitrosated 4C6MI to casein (Chapter 11). This binding is independent of pH, in a range of pH 2-6 and appeared to be reversible, since mutagens could be released from the casein. It was estimated that about 25% of nitrosated 4C6MI formed in nitrite treated fava beans binds to proteins present in fava beans. This binding is reversible too. Fava bean mutagens also bind to wheat bran. Although the binding efficiency to wheat bran is less than to casein and the reversibility of this binding has not been studied, the binding of fava bean mutagens to wheat bran can be an important observation. Wheat bran fibres will not be digested and therefore might serve as vehicles for mutagens to leave the body without harmful effects. Further investigation of this is recommended.
The results of the present study are further support for the hypothesis that the consumption of fava beans is causally related to the etiology of gastric cancer in Colombia. But the risks involved in the consumption of fava beans due to the endogenous nitrosation of 4C6MI for the population in The Netherlands is considered to be low, because of: (1) The low consumption of fava beans. (2) The lower levels of nitrate in Dutch drinking water. (3) Differences in food patterns between the Colombian and the Dutch population (the levels of consumption of vegetables and dairy products in particular). However, it can not be excluded that there are groups in The Netherlands with consumption habits deviating from that of the average population, who can in principle be assigned as persons at risk.
It is recommended to investigate the in vivo nitrosation of 4C6MI and the carcinogenicity of its nitrosated product in further detail by using an appropriate animal study.
Indoor and outdoor airborne particles : an in vitro study on mutagenic potential and toxicological implications
Houdt, J.J. van - \ 1988
Agricultural University. Promotor(en): J.H. Koeman; J.S.M. Boleij. - S.l. : van Houdt - 120
toxicologie - luchtverontreiniging - stof - blootstelling - atmosfeer - aërosolen - samenstelling - luchtkwaliteit - mutaties - mutagenese - mutagenen - toxicology - air pollution - dust - exposure - atmosphere - aerosols - composition - air quality - mutations - mutagenesis - mutagens
Air pollution components are present as gases and as particulate matter. As particle deposition takes place in various parts of the respiratory system particulate matter may have other toxicological implications than gaseous pollutants, which all may penetrate in the lower part of the respiratory tract. In addition, suspended particulate matter represents a group of pollutants of variable physical as well as chemical composition. Therefore airborne particulate matter cannot be regarded as a single, pure pollutant.
Mutagenic activity of extracts of airborne particulate matter from outdoor and indoor environments
Chapter 1 provides a summary of literature data on physical and chemical characteristics of airborne particulate matter.
Exposure is a function of concentration and time. In The Netherlands people spend most of the time at home. As all indoor samples show a strong indirect mutagenic activity, it may be concluded that exposure to genotoxins will be determined to a large extent by the level of pollution inside homes, which implies that exposure to indirect acting mutagens is quantitatively of far greater concern than exposure to direct acting mutagens.
Biological availability of particle bound organic compounds
Chapter 5 reviews a number of studies which deal with some aspects of the biological fate of mutagenic compounds and their 'carrier' particles. Respirable airborne particles to which most potential mutagenic compounds, detected in the Ames assay, are adsorbed may deposit in various parts of the respiratory tract. one of the defence mechanisms with regard to possible harmful action of these particles is clearance, as it reduces residence time on potentially sensitive epithelial surfaces. Alveolar macrophages provide the initial defence of the lower respiratory tract towards particulate matter.
|Verslag van een dienstreis naar Belgie en Frankrijk, van 23 - 26 april 1985
Lange, W. - \ 1985
Wageningen : SVP - 14
genetica - mutaties - mutagenese - mutagenen - onderzoek - frankrijk - genetics - mutations - mutagenesis - mutagens - research - france
Development and standardization of a mutagenicity test system using Aspergillus nidulans as test organism
Boschloo, J.G. - \ 1985
Landbouwhogeschool Wageningen. Promotor(en): J.H. van der Veen. - Wageningen : Boschloo - 115
aspergillus - biologische technieken - uitrusting - experimenten - methodologie - mutagenese - aspergillus - biological techniques - equipment - experiments - methodology - mutagenesis
The suitability of Aspergillusnidulans as a test organism for mutagenicity testing has been investigated. Several test systems have been tried. Those systems were preferred in which the mutants or recombinants could be selected without the need of adding a compound to the medium which itself is genetically active.
Four incubation methods have been compared. The plate incorporation assay (method 1) is the easiest method for testing chemical compounds, but it is often difficult to obtain reliable quantitative data. Moreover, complications may arise when the compound is toxic or causes a selective advantage of the mutants. In the liquid suspension test (method 2) the conditions can be controlled more accurately, so this method is more suitable for obtaining reliable quantitative data. For testing germinating conidia, the suspension test (method 3) turned out to be less suitable, since some conidia germinated directly from the start causing - in spite of the presence of 0.2% agar - big clumps of conidia at the end of the incubation period. Germinating conidia (and growing mycelium) can better be tested with the media mediated assay (method 4). However, an extra control experiment must be performed in order to discriminate between a real mutagenic activity and a selective advantage.
Strain 110, which carries the methGl allele, proved to be very suitable for measuring the point mutation frequency when methionine independent suppressor mutants are scored. The instability of a duplication strain can be also used as an indication for the genetic activity of a chemical compound. The construction of a duplication strain heterozygous for the recessive sorA2 allele (strain 007) reduced the number of plates needed for the scoring of segregants. The exact process by which the sectors arise is not known. Some crossing-over products are scored as deletion products when no special attention is
The system for measuring the frequency of recessive lethals developed by Morpurgo et al. (1978) has also been investigated, but the background in the control was relatively high. This was most likely caused by incomplete haploidization on FPA medium. Changing the medium did hardly improve the results. This implies that one can only measure larger effects on the frequency of recessive lethals.
Organic mutagens and drinking water in The Netherlands : a study on mutagenicity of organic constituents in drinking water in The Netherlands and their possible carcinogenic effects
Kool, H.J. - \ 1983
Landbouwhogeschool Wageningen. Promotor(en): J.H. Koeman. - Wageningen : Kool - 116
carcinoom - chemicaliën - schade - drinkwater - koolwaterstoffen - mutagenese - mutagenen - mutaties - neoplasma's - verontreinigingsbeheersing - waterverontreiniging - waterzuivering - organische scheikunde - carcinoma - chemicals - damage - drinking water - hydrocarbons - mutagenesis - mutagens - mutations - neoplasms - pollution control - water pollution - water treatment - organic chemistry
Several mutagenic and carcinogenic organic compounds have been detected in Dutch surface waters and in drinking water prepared from these surface waters. Although the levels of these compounds in drinking- and surface water are relatively low, in general below μg per litre, it appeared that organic concentrates tested in the Ames/microsome assay, showed mutagenic activity in 50 ml surface- and 500 ml drinking water.Such a result however was not expected based on the concentration of organic mutagens identified in these waters. Therefore the conclusion had been drawn that a number of unknown organic mutagens in combination with the identified mutagens were responsable for the level of mutagenic activity. With this in mind, an extensive investigation was carried out in an attempt to answer the following questions :
- do drinking waters prepared from surface water, groundwater or a mixture of both show mutagenic activity;
- do different water treatment processes during the preparation of drinking water influence the mutagenic activity;
- what kind of physical-chemical properties have the organic compounds in drinking water concentrates showing mutagenic activity in the Ames test;
- do mutagenic organic concentrates prepared from drinking water show carcinogenic properties?Against this background an inventory study was made on the presence of mutagenic activity in drinking water of 18 cities in The Netherlands.Besides this study, the influence of different water treatment processes on the mutagenic activity was examined in a number of water works. Furthermore, an attempt was made to characterize the organic compounds which are responsable for the mutagenic activity. Finally, a carcinogenicity study was carried out to see whether mutagenic drinking water concentrates induce carcinogenic effects in rats.Chapter 1, describes the aim of this investigation and the factors which have led to the present concern regarding the possible toxic effects of organic (micro)pollutants in drinking water in The Netherlands.Furthermore a summary is given of the literature on the identification of individual compounds and some data are given of the mutagenic and (suspect) carcinogenic organic compounds which are detected in surface- and drinking water in The Netherlands.In Chapter 2, materials and methods are described which were used for the inventory study on the presence of mutagenic activity in drinking water of 18 cities (Chapter 3), the characterization of organic mutagens (Chapter 4) and for a carcinogenicity study with mutagenic drinking water concentrates (Chapter 5).This chapter describes the XAD resins which have been used for concentrating organic mutagens from water, the concentration procedures with the aid of XAD resins and freeze drying, a mutagenicity assay viz. the Ames Salmonella/microsome assay (Ames test). The methods for chemical analysis which are used in the 18 city survey, fractionating techniques like thinlayer chromatography (TLC), high performance liquid chromatography (HPLC) and gelfiltration are described. The treatment, dose levels and experimental design and conduct which are used in a carcinogenicity study are explained. Finally, statistical procedures which have been used for analyzing tumour incidence in the carcinogenicity experiment and relating chemical parameters in drinking water to mutagenic activity in organic concentrates prepared from these drinking waters are described.In Chapter 3, the need for concentrating organic mutagens is explained. Mutagenicity results (Ames test) of five surface waters in The Netherlands are presented, in which the organic mutagens are concentrated with the XAD procedures. Depending on the concentration factor applied (103-4.103) it appeared that in all five surface waters mutagenic activity could be detected. The river Rhine showed the highest mutagenic activity viz. a doubling of revertants in 50 ml water.To be certain that the XAD procedure is a selective concentration method for organic mutagens, this procedure was compared with a freeze drying technique. The results of this comparative investigation showed that the XAD procedure is a reliable method for concentrating organic mutagens from surface water. on the basis of the surface water results, the XAD procedure was also applied for concentrating organic mutagens from drinking water. It was found that drinking water of four out of six cities showed mutagenic activity in volumes varying from 0.5 to 3 litre.To see whether the mutagenic results obtained in the six cities are representive for drinking water in The Netherlands, an extensive inventory study on the presence of mutagenic activity in several types of drinking water was carried out. In this study eighteen cities (twenty drinking waters) were investigated three times for the presence of mutagenic activity and the following chemical parameters : AOCL, EOCl, THM, VOCl, TOC, Tot. N, over a period of 2 years. The chemical parameters were measured to see whether a relationship could be found between one or more of these parameters and mutagenic activity.The selection of the drinking water was based on the water source (ground water, surface water) the storage facility (dune filtration, bankfiltration, storage reservoir) and the application of a chlorine treatment during the preparation of drinking water. The results of this study showed that in fourteen of the twenty drinking waters, mutagenic activity could be detected in volumes varying from 0.5 to 3 litre. When the cities were classified according to their water source, storage facility and type of treatment, it appeared that only three of the fifteen cities which prepare their drinking water from surface water or a mixture of surface- and groundwater, did not show mutagenic activity. Two of the five cities which use groundwater as a drinking water source showed mutagenic activity although in one city the activity was marginal.Correlating the chemical parameters with the mutagenic activity, it was demonstrated that AOCL showed the highest correlation with the direct mutagenic activity in strain TA 98 and TA 100. It was also shown that a chlorine treatment applied during drinking water preparation correlated well with the direct mutagenic activity in strain TA 98 and TA 100. In addition, chemical parameters which showed a significant difference (p< 0.01) in concentration between mutagenic and non mutagenic samples (Mann-Whitney) and a significant correlation (p< 0.01, Kendall tau) with mutagenic activity were used for straight curve fitting. From these fitted regressions, concentration levels were calculated above which a doubling of revertants may be expected.In the second part of Chapter 3 (3.4) the influence of different treatment processes on the mutagenic activity and some chemical parameters were investigated in three waterworks. Application of a chlorine treatment, generally increased the direct and promutagenic activity, but the amount of increase proved to be dependent on the type of water chlorinated. The use of ozone in the preparation of drinking water decreased the mutagenic activity in the water. The amount of reduction was dependent on the type of water ozonated. Dune filtration greatly reduced the mutagenic activity.Slow sand filtration could not be evaluated, because of the toxicity of the organic concentrates for the bacterial strains. Filtration over active carbon filters which operated for about 1 year, reduced the mutagenic activity below the detection level. Carbon filters which operated more than 1.5 years in a pilot plant, showed a break-through of mutagenic activity. This result suggests that carbon filters are able to remove organic mutagens only for a short time.From the results of the chemical parameters before and after the different treatment processes it appeared that the level of AOCL, behaved very similar with the mutagenic activity in the neutral fraction. These results confirm those obtained in the eighteen city survey and support the idea that A0C1 might be a useful indicator for mutagenic activity in drinking water.The physical-chemical characterization of organic mutagens in drinking water concentrates is described in Chapter 4. The first approach was to examine the influence of the pH on the adsorption behaviour of organic mutagens on the XAD resins. Lowering the original pH of drinking water with HCl to pH = 2- 3, revealed the presence of another class of organic mutagens the so-called acid fraction which hardly adsorb on the XAD resins at pH = 7.5.Further it was investigated whether different organic solvents are able to eluate the organic mutagens from the XAD column. it was found that diethylether only eluted a minor part of the mutagenic activity from the XAD resins and subsequent elution with acetone eluted the major part of the activity.This result showed that it is not likely that the organic mutagens in the acetone fraction are identical with the already identified organics in the ether fraction in this drinking water. Furthermore it appeared that the organic mutagens arenot only less volatile but also resistent to boiling. Another approach to characterize the organic mutagens was to apply fractionation techniques. Using TLC and HPLC, it was found with HPLC analysis that the mutagenic activity was present predominantly in two fractions. Finally, gelfiltration on Sephadex LH20, showed that organic mutagens which demonstrated mutagenic activity with strain TA 98, had a molecular weight in the order of 200.Chapter 5, presents the results of a carcinogenicity study, in which Wistar SSP TOX rats were exposed to mutagenic drinking water concentrates of one city in The Netherlands. Drinking water concentrates were prepared every week using the XAD concentration procedure and the organic concentrates (DMSO) were mixed with the non mutagenic drinking water of the Wistar SSP TOX rats.Dosage levels were based on multiples of expected human exposure levels. In the calculation the average human exposure was assumed to be approximately 29 ml/kg bw./day. Furthermore it was assumed that a rat of 250 gram would consume about 30 ml water per day. In the experiment rats were divided into four groups (50 males and 50 females per group), a control group and groups which received respectively 10, 30 and 90 times the human exposure level in their drinking water.During the experiment (106 weeks) the water consumption of the rats was measured weekly. Body weights were recorded weekly in the first two months of the experiment and once a month thereafter. The mutagenic activity of concentrates which were mixed with non mutagenic drinking water were measured after a week. It was found that the mutagenic activity with strain TA 98 could be recovered for 60-90%, while the TA 100 activity hardly could be recovered. The latter was explained by the observed partial decrease of TA 100 activity after mixing the mutagenic drinking water concentrate with drinking water.During the experiment it appeared that the assumed water consumption of 30 ml per day per rat, on which the dose level was based, not was reached.Moreover, it appeared that Wistar SSP TOX rats became much heavier than was expected and therefore the actual dose levels in ml/kg bw. of the three exposed groups were not 10, 30 and 90 times the expected human exposure levels, but 4.5, 14 and 40 for male rats respectively and 7, 22 and 68 for female rats. Exposing Wistar SSP TOX rats to these dose levels for 106 weeks, did not result in a significant increase (p <0.05) in tumour induction.Furthermore it was shown, that the development and types of tumours were similar in the treated and control groups. The number of animals with tumours and the animals which died of tumours in the exposed groups was not significantly different (p < 0.05) from the control group. The negative results in this carcinogenicity study indicate that mutagenic drinking water concentrates did not contain very potent carcinogens in effective concentrations like for instance N-Nitrosodiethylamine because when these kind of compounds are present, the carcinogenicity should be positive at the dose levels tested.On the other hand one cannot exclude the presence of weak carcinogens in the mutagenic concentrates, because one cannot detect a carcinogenic effect at the dosis tested. Based on the carcinogenicity results, an estimation of a risk factor was made for the people who consumed this mutagenic drinking water.For the estimation the following three assumptions had been made:
- when organic carcinogens had been present in drinking water, they were present solely in the mutagenic drinking water concentrates
- results obtained in the present carcinogenicity study with rats may be extrapolated to man
- linear extrapolation of high dosis to low dosis give correct results.When all these assumption are correct it appeared after statistical analysis that less than 356 of 110.000 people might be at risk. On this basis the contribution of drinking water, if there is a contribution at all, seems relatively small viz. less than 1. 1 % in comparison to the expected tumour incidence of 33.000. The latter value is based on an average tumour incidence of 30 per 100, due to background processes.To improve, however the reliability of an estimation of the risk factor for people who consume polluted drinking water, organic concentratres of drinking water which have not been included in the present carcinogenicity study should be examined for complete carcinogenicity in combination with higher doses of concentrates of organic mutagens as used in this study.Besides this investigation it is recommended that both organic concentrates will be tested for tumour promotion and initiation activity as well as identification of the organic responsable for these effects should receive priority. Finally Chapter 6 describes a number of conclusions emerging from this investigation. The data presented show that mutagenic activity was detectable in drinking water of many cities in The Netherlands. The number of cities which showed mutagenic activity in their drinking water was the least where groundwater was used as a drinking water source. Therefore it is recommended that groundwater is preferable over other sources of drinking water and the quality of the groundwater should be protected very carefully so that oxidation and disinfection with a chlorine treatment will not be necessary. This recommendation is not only based on the eighteen city survey but also from data obtained in waterworks which prepare their drinking water mainly from surface water, because mutagenic activity is significantly increased by a chlorine treatment which may often result in mutagenic activity in the end product. It is recommended further that the organic mutagens should be identified in order to evalute them with respect to their possible toxic properties. Also higher dosis of mutagenic drinking water concentrates in combination with concentrates prepared from the rest of the organics in drinking water, should be tested for carcinogenicity and chronic toxicity for a more reliable risk estimation. Besides, this investigation it is recommended that both organic concentrates should be investigated for tumour promotion and initiation activity.
The genetics of some planthormones and photoreceptors in Arabidopsis thaliana (L.) Heynh.
Koornneef, M. - \ 1982
Landbouwhogeschool Wageningen. Promotor(en): J.H. van der Veen. - Wageningen : Koornneef - 157
brassicaceae - genetische variatie - genetica - heritability - overerving - mutagenese - mutagenen - mutaties - fytochroom - plantengroeiregulatoren - plantenpigmenten - brassicaceae - genetic variation - genetics - heritability - inheritance - mutagenesis - mutagens - mutations - phytochrome - plant growth regulators - plant pigments - cum laude
This thesis describes the isolation and characterization in Arabidopsis thaliana (L.) Heynh. of induced mutants, deficient for gibberellins (GA's), abscisic acid (ABA) and photoreceptors.These compounds are known to regulate various facets of plant growth and differentiation, so mutants lacking one of these substances are expected to be affected in several aspects of their physiology. It is shown in this thesis that the earliest expression of these mutants occurs during seed development and seed germination. Therefore these processes form an excellent phase to screen for these mutants.Planthormone and photoreceptor mutants in relation to seed physiology.In general three major periods may be distinguished in the history of a seed: 1) Seed development and maturation, 2) developmental arrest of the mature seed, characterized either by a dormant state in which seeds even do not germinate under favourable environmental conditions, or by a quiescent state in which seeds only require rehydration, and 3) germination, starting with water uptake and often requiring breaking of dormancy, which is triggered by specific environmental factors such as light and temperature. Planthormones may play a regulatory role in all three phases.Non-germinating GA-responsive mutants as described in Chapter 1 have a strongly reduced gibberellin biosynthesis (Barendse, pers.comm.) which may lead to an increased level of dormancy and/or to the inability of the seeds to
break dormancy after imbibition of mature seeds. Clearly the presence of GA's, either by de novo GA synthesis, or by hydrolysis of bound forms, is not always a prerequisite for seed germination: genotypes that combine GA- and ABA deficiency like the revertants of non-germinating ga-1 mutants described in Chapter 2 do readily germinate.Apart from the absence of endogenous factors such as gibberellins, also the lack of receptors for environmental factors that normally break dormancy might prevent germination. An example are the hy-1 and hy-2 mutants (Chapter 4), which are characterized by an increased hypocotyl length in white light and the absence of detectable phytochrome in dark grown hypocotyls. It was shown by Spruit et al. (1980), that these mutants hardly show any germination and correspondingly, have strongly reduced levels of phytochrome in their seeds. Their reduced germination capacity is restored by (relatively high) concentrations of exogeneously applied GA 4+7 (Koornneef et al., 1981). Consequently one might expect such phytochrome deficient mutants to occur among the GA responsive non-germination mutants in Arabidopsis, like van der Veen and Bosma actually found for a tomato mutant (see Koornneef et al., 1981). Remarkably this was not the case in Arabidopsis. The reason for this seems to be the absence of a light requirement in the hy mutants from the M 2 populations screened for non-germinating mutants of Arabidopsis. It happened that these M 2 seeds in all cases were harvested from M 1 plants grown in winter, in contrast to the seeds studied by Spruit et al. (1980) which were harvested in summer. We have observed during a number of years that seeds (including wild-type seeds), which developed in winter (natural daylight with additional continuous light by Philips TL 57) were less dormant than seeds from summer grown mother plants (long days, high light intensity, no additional light). Relevant environmental factors in this respect may be light intensity, light quality (McCullough and Shropshire, 1970) and daylength (Karssen, 1970; Luiten, 1982). The effect of light quality (McCullough and Shropshire, 1970) indicates that phytochrome may be involved in the determination of the level of dormancy.To select mutants with a reduced or absent seed dormancy, one may choose those conditions, where the wild-type is clearly dormant. However, the high and probably complex environmental variability of this character and the relatively rapid change in the level of dormancy during dry storage of the seeds makes this selection system less attractive.Selection for revertants in the progeny of mutagen treated non-germinating ga-1 mutants proved to be an effective procedure to isolate mutants with a reduced dormancy (Chapter 2). As the reverting effect (restored germination) was caused by a mutation at a different locus, the ga-1 allele could be replaced by its wild-type allele by crossing the revertant with the wild-type parent followed by selection in F 2 . These newly selected monogenic recessive mutants had a reduced level of ABA in the leaves and in both the developing and ripe seeds. Correspondingly the mutant allele was called aba (ABA-types are aba/aba plants).The germination of seeds collected at different stages of their development on both ABA- and wild-type plants showed that dormancy developed during the last part of seed maturation in wild-type, but not in the aba -mutant. This shows that the function of ABA is dormancy induction. ABA determinations in unripe siliquae showed a peak level of ABA at about 10-12 days after anthesis, followed subsequently by a decrease, a short period at a constant level and a further decrease (Chapter 3). In addition to ABA-type mothers with ABA-type embryo's and wild-type mothers with wild-type embryo's, one can also obtain by means of the appropriate reciprocal crosses ABA-type mothers with wild-type embryo's and wild-type mothers with (50%) ABA type embryo's. So the effects of maternal and embryonic genotype can be separated. It was found (Chapter 3) that the genotype of the mother plant regulated the sharp rise in ABA content halfway seed development (maternal ABA). The genotype of the embryo and endosperm was responsible for a second ABA fraction (embryonic ABA), which reached lower levels; but persisted for some time after the maximum in maternal ABA. The onset of dormancy showed a good correlation with the presence of the embryonic ABA fraction and not with the maternal ABA.Another category of mutants which also may give some understanding of the role of ABA in seed germination are the ABA tolerant mutants recently isolated by us in Arabidopsis. Compared to wild-type these mutants require an upto 20 fold higher concentration of exogeneously applied ABA to inhibit seed germination. These mutants too are characterized by a reduced seed dormancy.Other genetically determined factors than those mentioned above are certainly also involved in seed development and seed germination. Thus in Arabidopsis mutations leading to the absence of seed coat pigments (transparent testa) and simultaneously to the absence of a mucilage layer around the seed have a reduced dormancy (Koornneef, 1981). The latter seedcoat characters are determined purely by the maternal genotype.Planthormone and photoreceptor mutants in relation to other physiological effects.Non-germinating mutants at the loci ga-1, ga-2 and ga-3, when made to germinate by adding gibberellin, initially develop into normal looking seedlings. Later on they become dark green bushy dwarfs with reduced petals and stamens. Regular GA-spraying from the seedling stage onwards maintains the wild-type phenotype completely or nearly so (Chapter 1). The strong and quick response of the dwarfs to GA sprays (the elongation of the petals of older dwarfs becomes visible within two days) clearly demonstrates the essential role of gibberellin in elongation growth.Recently the non-germinating ga alleles were shown to have a strongly re duced kaurene synthetase activity in young siliquae compared to wild type. These analyses were performed by Dr. G.W.M. Barendse (pers.comm.). This indicates that these genes control some early step(s) in GA biosynthesis.Apart from mutants that do not germinate without GA, also more or less normally germinating GA responsive dwarfs were isolated. Half of these were found to be allelic to the non-germinating ga-1 , ga-2 and ga-3 mutants. These mutant alleles behave like so called "leaky alleles", i.e. the alleles are only partly defective and produce sufficient GA for seed germination, but not enough to give normal elongation growth.GA sensitive dwarfs were also found at two other loci ( ga-4 , ga-5 )of which no non-germinating alleles have been isolated so far (Chapter 1). These mutants have normal or slightly reduced kaurene synthetase activity (Barendse, pers.comm.), which indicates that these genes regulate steps beyond kaurene, or affect GA metabolism in another way. It is also possible that in the mutants cell elonga tion factors are blocked for which the relatively high concentration of exogeneously applied GA may substitute. Locus ga-4 seems to control interconversions between GA's, which is suggested by the insensitivity of ga-4 dwarfs to GA 9 . which gibberellin is effective with mutants at the other 4 loci.Abscisic acid (ABA) deficient mutants are characterized not only by reduced seed dormancy but also by disturbed water relations (wiltiness, withering), probably as a result of failure to close the stomata upon conditions of water stress (Chapter 2). This is characteristic for ABA deficient mutants in tomato (Tal and Nevo, 1973) and potato (Quarrie, 1982). ABA deficient mutants in maize are in addition to a reduced seed dormancy (viviparous mutants, gene symbol op) characterized by the failure to synthesize carotenoids and they accumulate precursors of these pigments (Robichaud et al., 1980). As ABA deficient mutants in Arabidopsis, tomato and potato have normal pigments, it is suggested that in the latter species the ABA biosynthesis may be blocked in the last part of the pathway, whilst in the maize mutants it is blocked at an earlier stage, i.e. where ABA and carotenoids still have a common pathway.Some of the photoreceptor mutants are affected in their germination behaviour as discussed above. However, the most conspicuous effect observed is the partial lack of light induced inhibition of hypocotyl elongation (Chapter 4). Mutants in Arabidopsis, and also in tomato and cucumber (Koornneef et al., 1981; Koornneef et al., unpublished), that have an elongated hypocotyl when grown in white light, were shown to have locus-specific alterations in the spectra of light inhibition when grown in light of restricted spectra] regions. In these "colour blind" mutants at two loci (hy-1 and hy-2) little or no spectrophotometrically detectable phytochrome was present in dark grown hypocotyls, nor was it in the seeds. In these mutants the inhibitory effect of red and farred was almost absent. Mutants of other genes were characterized by the absence only of red inhibition (hy-3) or by a decreased sensitivity to the shorter wavelengths of the spectrum (hy-4, hy-5). Hy-5 also showed a reduced inhibitory effect of far-red light. The differential sensitivity of the genotypes to specific spectral regions strongly suggests the involvement of more than one pigment in the inhibition by light of hypocotyl elongation and probably also in other photomorphogenetic processes. Some authors ascribed this role solely to phytochrome (Schäfer, 1976).Since under specific conditions phytochrome could nevertheless be detected in so called phytochrome deficient mutants (Koornneef and Spruit, unpublished) the genes hy-1 and hy-2 probably do not represent the structural genes of the phytochrome protein or the phytochrome chromophore, but instead may play a role in the regulation of phytochrome metabolism.Further genetic aspects of plant hormone and light receptor mutants.Mutation frequencies for the different groups of loci were estimated for ethylmethanesulphonate (EMS), fast neutrons and X-rays (Chapter 5). Average mutation frequencies calculated per diploid cell, per locus and per MM EMS during 1 hr at 24 °C, were for ga-1, ga-2, ga-3 8.0 ± 1.8 x 10 -6, for hy-1, hy-2, hy-3 4.2 ± 1.4 x 10 -6and for the aba locus about 27 x 10 -6. These mutation frequencies are relatively high compared to other loci studied by us and others. It is not excluded that in these categories loci escaped detection simply because of a low mutation frequency.It is a good custum to locate newly induced mutations on the organisms gene map, especially when they are the basis of extensive research like our ga, aba and hy mutants. Unfortunately, the gene map of Arabidopsis was rather fragmentary, and contradictory or wrong conclusions about linkage relations could be found in literature. Since we had gradually built up the complete set of 5 primary trisomics supplemented with a number of telotrisomics (one chromosome arm extra) and also made a collection of mutations at many loci, induced in the course of various experiments at our department and supplemented with mutants described in literature, we had a good starting point to construct a more representative gene map for Arabidopsis. The required scale of operations was only feasable thanks to the accurate assistance of many students who performed trisomic analysis and gene mapping as part of their university training program. Important further additional data were obtained from the department of Genetics of Groningen University and from literature.The trisomic analysis aimed at assigning linkage groups (via representative markers) to the different chromosomes is described in Chapter 6. The gene maps in centimorgans for the five Arabidopsis chromosomes is presented in Chapter 7. On the basis of 76 loci mapped the genetic length of the Arabidopsis chromosomes now compares well with that of individual chromosomes in e.g. tomato and maize. This notwithstanding the small size of the Arabidopsis chromosomes.Genes with a similar mutant phenotype (and probably comparable functions) seem to be distributed at random over the Arabidopsis genome.Our set of mutants at the ga-1 locus of Arabidopsis provides an excellent opportunity for fine structure analysis of the gene. The system has a very high resolving power, for the intragenic recombinants are found as the rare wild-type seedlings among thousands of non-germinating seeds per petri dish. The results show (Chapter 8) that 8 different alleles could be arranged into an internally consistent map on the basis of the frequencies of intragenic recombinants. One fast neutron induced allele behaved as an intragenic deletion. The order of the sites with respect to other genes on chromosome 4 could be established.
Chimerism and diplontic selection
Balkema, G.H. - \ 1971
Wageningen University. Promotor(en): J.H. van der Veen. - Rotterdam : A.A. Balkema - 173
plantkunde - mutagenese - mutaties - plantenveredeling - botany - mutagenesis - mutations - plant breeding
Chimerism is the concurrence of genotypically different tissues in one individual and usually results from a mutation early in the development of that individual. With the possibility to induce mutations came the problem of chimerism which gives heterogeneous plants and, allegedly, loss of mutations by diplontic selection, i.e. selection between genetically different tissues within an individual where the mutated tissue is assumed to be at a disadvantage.Literature on anatomy, morphology and development, and on the. mutation process gives the explanation for various aspects of chimerism: The appearance of chimerism depends on the constitution of the material treated and on the action of the mutagen used. Periclinal and sectorial chimerism are related to the structure of the apex i.e. the existence of independent cell layers and of a few central (apical) initial cells. The development of chimerism after a mutagenic treatment is determined by the differentiation already present in the material treated (usually seed), in which the destiny of most of the cells is already fixed.The presented experiments (on arabidopsis and sunflower) indicate that the mutagenic action of the mutagens used (EMS and colchicine applied separately or simultaneously) was mainly confined to the duration of the treatment. In addition the mutagens influenced development, stimulating it at first and later (especially colchicine) retarding it, which has consequences for sensitivity with combined treatments, and which may affect chimerism. Differences in average distribution of mutations (from seed versus seedling treatment, "first" versus "second" mutation) were ascribed to differential mutagen (EMS) sensitivity of cells destined to form the various parts of the plant (main inflorescence, side shoots). Chimerism in the sporogenic tissue (pollen and M 2 were scored) occurred as sectors (usually 2 in the inflorescence and up to 4 in the stem of arabidopsis) which often seemed twisted probably due to the (twisted) growth of the plants. Generally chimerism was lost, apparently at random with regard to the observed mutations (polypIoidy and M 2 chlorophyll, mutants).Although diplontic selection is often mentioned in the literature, most of the cases reported can be explained equally well, and often better, as aspects of normal development or as the result of differential (mutagen) sensitivity. Environmental conditions influence the development of the plant and also the (observed) mutation rate, but this influence may be indirect i.e. through the effect on chimerism. Chimerism depends on the stability of the apical initial cells which will be related to the stability of the apex and therefore to plant development and thus indirectly to environmental conditions.Plants from experiments with various growth conditions (greenhouse vs field, daylength) differed in development and vigour, and simultaneously, in degree and persistence of chimerism, vigorous plants showing more chimerism. Treatments which retard apical development at an early stage (etiolation, vernalization) decreased chimerism. In both cases effects on the stability of the apex were probably responsible.This loss of chimerism, at random with regard to the genotype, may be called diplontic drift. It depends on conditions affecting plant growth and may offer an opportunity to manipulate chimerism. Chimerism decreases the chance to detect a mutation but increases the number of (different) mutations that can be obtained from one plant, so that the degree of chimerism desired, may vary.