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]

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.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 fwa , 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 fwa mutant. In addition to three different intragenic revertants of fwa , this screen yielded a novel early flowering mutant.This mutant was named early flowering in short days ( efs ). Its phenotypic characterisation has shown that the main role of the wild-type EFS 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, efs 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 EFS in relation to other genes involved in flowering initiation, double mutants were isolated. Their characterisation showed that efs is involved in the autonomous promotion pathway. This result, together with the lack of a vernalisation response, suggests that EFS 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.The main topic of this thesis concerns the map based cloning of the FWA gene. By using plants which have a cross-over between FWA and surrounding markers, the FWA 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 fwa mutants together with DNA mutations in the intragenic revertants of fwa-1 further proved that this gene is FWA .Analysis of FWA revealed several interesting characteristics. Surprisingly, the mutant and wild-type alleles had an identical DNA sequence in the FWA 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 FWA . In wild-type plants these repeats were heavily methylated, whereas they were completely un-methylated in the mutant alleles. In contrast to fwa mutant plants, which showed a high expression of FWA at all developmental stages, wild-type plants showed only a low expression of FWA in siliques and germinating seeds. Taken together, these findings suggest that loss of methylation of the FWA repeats in the fwa mutant causes a high level of expression of the gene, leading to a late flowering phenotype. A similar correlation of late flowering, FWA overexpression and hypomethylation of FWA repeats was found in late flowering plants which were derived from the ddm1 hypomethylation mutant. The late flowering phenotype of these plants had previously been mapped to the FWA region. Nevertheless, the correlation between hypomethylation of the FWA repeats and FWA expression was not found in germinating seeds of wild-type plants which showed expression of FWA 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 FWA . Perhaps a different epigenetic mechanism early in development can induce expression of methylated genes.The correlation of FWA expression with late flowering indicates that FWA is a repressor of flowering. Earlier studies had already shown that FWA does not only play a role in the initiation of flowering but also in flower meristems. However, the FWA transcript was not detected in flower buds or flowers and therefore, FWA might only affect this process when highly expressed in the fwa mutant. Possibly, FWA 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.The cloning of FWA revealed that the absence of methylation in the repeating sequences in the 5' region of the FWA gene leads to an enhanced expression in the fwa 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.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

Genetic control of flowering using the FWA gene

This invention relates to the determination, cloning and expression of the flowering time gene FWA and the use of this gene to delay or accelerate flowering in a large variety of plant species. Specifically the gene that was determined is that of Arabidopsis thaliana. Naturally the invention extends to other plants as well

Natural modifiers of seed longevity in the Arabidopsis mutants abscisic acid insensitive3-5 (abi3-5) and leafy cotyledon1-3 (lec1-3)

• Seed longevity is an important trait in many crops and is essential for the success of most land plant species. Current knowledge of its molecular regulation is limited. The Arabidopsis mutants abscisic acid insensitive3-5 (abi3-5) and leafy cotyledon1-3 (lec1-3) have impaired seed maturation and quickly lose seed viability. abi3-5 and lec1-3 were used as sensitized genetic backgrounds for the study of seed longevity. • We exploited the natural variation of Arabidopsis to create introgression lines from the Seis am Schlern and Shahdara accessions in, respectively, the abi3-5 and lec1-3 backgrounds. These lines carry natural modifiers of the abi3 and lec1 phenotypes. Longevity tests and a proteomic analysis were conducted to describe the seed physiology of each line. • The modifier lines showed improved seed longevity. The Shahdara modifiers can partially re-establish the seed developmental programs controlled by LEC1 and restore the accumulation of seed storage proteins that are reduced in abi3-5 and lec1-3. • The isolation and characterization of natural modifiers of the seed maturation mutants abi3-5 and lec1-3, and the analysis of their seed proteomes, advance our current understanding of seed longevit

Chromatin dynamics during seed dormancy

The chromatin structure determines gene expression and thereby regulates developmental processes in the plant. The molecular mechanisms regulating the induction and release of seed dormancy are still largely unknown and the underlying changes in chromatin organization have hardly been analyzed. Most chromatin studies in plants have been performed on vegetative tissues and have focused on seedlings. The composition of seeds hampers molecular analyses and requires adaptation of the methods that are used for other tissues. Here, we give an overview of the current methods that are used to study different aspects of chromatin organization in seeds. Cytogenetic methods, like fluorescence in situ hybridization and immunolocalization, are used to study chromatin at the microscopic level. Changes in DNA methylation and histone modifications can be studied with molecular methods, like bisulfite sequencing, immunoblotting, and chromatin immunoprecipitation