A genetic and molecular analysis of two genes involved in flowering initiation of Arabidopsis = [Een genetische en moleculaire analyse van twee genen die betrokken zijn bij de bloei initiatie van Arabidopsis]

W.J.J. Soppe

Research output: Thesisinternal PhD, WU

Abstract

<p>The transition from the vegetative to the reproductive phase (flowering initiation) in plants has a complex regulation which is affected by environmental and internal plant factors. The understanding of this process is not only of fundamental interest but could also lead to practical applications. The research into flowering initiation has a long history. The initial emphasis on physiological and biochemical studies led to the identification of different factors that influence flowering time. During the sixties, a genetic approach was initiated in different plant species. In Arabidopsis several late flowering mutants were isolated and genetically and physiologically characterised which revealed a complex regulation of flowering time by different pathways. These are the photoperiodic promotion pathway which promotes flowering under long day light conditions, the vernalisation promotion pathway which promotes flowering by low temperatures and the autonomous promotion pathway which promotes flowering independent of the environment. Due to its favourable genetic and molecular features, research on flowering initiation became focussed on Arabidopsis. Since the beginning of the nineties, several of the genes involved in the different pathways have been cloned, providing more information about the function of these genes in the cell and their relations with each other. Despite this increasing amount of information, the picture is still far from complete.</p><p>The aim of the work presented in this thesis is to increase our knowledge of flowering time regulation. It focussed on the genetic and molecular characterisation of the semi-dominant mutant <em>fwa</em> , which flowers late under long day light conditions and has been proposed to be part of the photoperiodic promotion pathway. One approach sought to identify additional genes that affect flowering by mutagenesis of the <em>fwa</em> mutant. In addition to three different intragenic revertants of <em>fwa</em> , this screen yielded a novel early flowering mutant.</p><p>This mutant was named <em>early flowering in short days</em> ( <em>efs</em> ). Its phenotypic characterisation has shown that the main role of the wild-type <em>EFS</em> gene is to delay flowering in plants that have entered the adult vegetative phase, which is considered to be the phase where plants are able to respond to environmental signals in order to flower. Consistent with this, <em>efs</em> mutant plants do not show an early flowering phenotype when grown under environmental conditions that lead to a shortened adult vegetative phase such as long days and vernalisation. To learn more about the role of <em>EFS</em> in relation to other genes involved in flowering initiation, double mutants were isolated. Their characterisation showed that <em>efs</em> is involved in the autonomous promotion pathway. This result, together with the lack of a vernalisation response, suggests that <em>EFS</em> is likely to represent a new element acting at a point close to the convergence of signals from the autonomous promotion pathway and the vernalisation promotion pathway.</p><p>The main topic of this thesis concerns the map based cloning of the <em>FWA</em> gene. By using plants which have a cross-over between <em>FWA</em> and surrounding markers, the <em>FWA</em> locus could be located in a region of about 60 Kb. Plant transformation experiments with cosmids spanning this region showed that the gene is located in the overlap of two cosmids. This overlap contained only one complete gene that encodes a homeodomain containing transcription factor. The altered expression of this gene in <em>fwa</em> mutants together with DNA mutations in the intragenic revertants of <em>fwa-1</em> further proved that this gene is <em>FWA</em> .</p><p>Analysis of <em>FWA</em> revealed several interesting characteristics. Surprisingly, the mutant and wild-type alleles had an identical DNA sequence in the <em>FWA</em> region, excluding DNA mutations in the gene as a cause for the mutant phenotype. Furthermore, two direct repeated sequences were found in the 5' genomic region of <em>FWA</em> . In wild-type plants these repeats were heavily methylated, whereas they were completely un-methylated in the mutant alleles. In contrast to <em>fwa</em> mutant plants, which showed a high expression of <em>FWA</em> at all developmental stages, wild-type plants showed only a low expression of <em>FWA</em> in siliques and germinating seeds. Taken together, these findings suggest that loss of methylation of the <em>FWA</em> repeats in the <em>fwa</em> mutant causes a high level of expression of the gene, leading to a late flowering phenotype. A similar correlation of late flowering, <em>FWA</em> overexpression and hypomethylation of <em>FWA</em> repeats was found in late flowering plants which were derived from the <em>ddm1</em> hypomethylation mutant. The late flowering phenotype of these plants had previously been mapped to the <em>FWA</em> region. Nevertheless, the correlation between hypomethylation of the <em>FWA</em> repeats and <em>FWA</em> expression was not found in germinating seeds of wild-type plants which showed expression of <em>FWA</em> but methylation of the repeats. Although this expression might come from residual mRNA produced earlier in developing seeds, it is possible that methylation of the repeats does not always prevent expression of <em>FWA</em> . Perhaps a different epigenetic mechanism early in development can induce expression of methylated genes.</p><p>The correlation of <em>FWA</em> expression with late flowering indicates that <em>FWA</em> is a repressor of flowering. Earlier studies had already shown that <em>FWA</em> does not only play a role in the initiation of flowering but also in flower meristems. However, the <em>FWA</em> transcript was not detected in flower buds or flowers and therefore, <em>FWA</em> might only affect this process when highly expressed in the <em>fwa</em> mutant. Possibly, <em>FWA</em> has no function in flowering initiation of wild-type. It might participate in a seed-specific process, as suggested by its expression in seeds. However, the lack of an obvious phenotype suggests that this role is minor or redundant with other genes.</p><p>The cloning of <em>FWA</em> revealed that the absence of methylation in the repeating sequences in the 5' region of the <em>FWA</em> gene leads to an enhanced expression in the <em>fwa</em> mutant. However, it did not become clear whether this correlation is direct or indirect. Also the importance of the methylation in wild-type plants is still unclear. It is possible that it has a role in the expression of the gene under specific environmental conditions.</p><p>The results discussed in this thesis have contributed to the existing knowledge of flowering initiation by the isolation of a mutant at a novel locus and the cloning of a previously known gene which are both involved in this process. In addition, the results suggest a possible role for DNA methylation in gene regulation of Arabidopsis.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Koornneef, Maarten, Promotor
Award date10 Oct 2000
Place of PublicationS.l.
Publisher
Print ISBNs9789058082893
Publication statusPublished - 2000

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Keywords

  • arabidopsis
  • arabidopsis thaliana
  • genetic analysis
  • genes
  • gene expression
  • biotechnology
  • flowering
  • photoperiodism
  • circadian rhythm
  • molecular genetics
  • mutants
  • genomes

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