Caroline Dean

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Vernalization requires epigenetic silencing of FLC by histone methylation.

To ensure flowering in favourable conditions, many plants flower only after an extended period of cold, namely winter. In Arabidopsis, the acceleration of flowering by prolonged cold, a process called vernalization, involves downregulation of the protein FLC, which would otherwise prevent flowering. This lowered FLC expression is maintained through subsequent development by the activity of VERNALIZATION (VRN) genes. VRN1 encodes a DNA-binding protein whereas VRN2 encodes a homologue of one of the Polycomb group proteins, which maintain the silencing of genes during animal development. Here we show that vernalization causes changes in histone methylation in discrete domains within the FLC locus, increasing dimethylation of lysines 9 and 27 on histone H3. Such modifications identify silenced chromatin states in Drosophila and human cells. Dimethylation of H3 K27 was lost only in vrn2 mutants, but dimethylation of H3 K9 was absent from both vrn1 and vrn2, consistent with VRN1 functioning downstream of VRN2. The epigenetic memory of winter is thus mediated by a ‘histone code’ that specifies a silent chromatin state conserved between animals and plants.

An Arabidopsis Example of Association Mapping in Structured Samples

A potentially serious disadvantage of association mapping is the fact that marker-trait associations may arise from confounding population structure as well as from linkage to causative polymorphisms. Using genome-wide marker data, we have previously demonstrated that the problem can be severe in a global sample of 95 Arabidopsis thaliana accessions, and that established methods for controlling for population structure are generally insufficient. Here, we use the same sample together with a number of flowering-related phenotypes and data-perturbation simulations to evaluate a wider range of methods for controlling for population structure. We find that, in terms of reducing the false-positive rate while maintaining statistical power, a recently introduced mixed-model approach that takes genome-wide differences in relatedness into account via estimated pairwise kinship coefficients generally performs best. By combining the association results with results from linkage mapping in F2 crosses, we identify one previously known true positive and several promising new associations, but also demonstrate the existence of both false positives and false negatives. Our results illustrate the potential of genome-wide association scans as a tool for dissecting the genetics of natural variation, while at the same time highlighting the pitfalls. The importance of study design is clear; our study is severely under-powered both in terms of sample size and marker density. Our results also provide a striking demonstration of confounding by population structure. While statistical methods can be used to ameliorate this problem, they cannot always be effective and are certainly not a substitute for independent evidence, such as that obtained via crosses or transgenic experiments. Ultimately, association mapping is a powerful tool for identifying a list of candidates that is short enough to permit further genetic study.

Multiple pathways in the decision to flower : enabling, promoting, and resetting

At a certain point in their life cycle, annual plants undergo a major developmental transition and switch from vegetative to reproductive development. This process is rarely reversible, and ensuring that the timing of this transition is optimal for pollination and seed development is a major factor

The VERNALIZATION 2 Gene Mediates the Epigenetic Regulation of Vernalization in Arabidopsis

The acceleration of flowering by a long period of low temperature, vernalization, is an adaptation that ensures plants overwinter before flowering. Vernalization induces a developmental state that is mitotically stable, suggesting that it may have an epigenetic basis. The VERNALIZATION2 (VRN2) gene mediates vernalization and encodes a nuclear-localized zinc finger protein with similarity to Polycomb group (PcG) proteins of plants and animals. In wild-type Arabidopsis, vernalization results in the stable reduction of the levels of the floral repressor FLC. In vrn2 mutants, FLC expression is downregulated normally in response to vernalization, but instead of remaining low, FLC mRNA levels increase when plants are returned to normal temperatures. VRN2 function therefore stably maintains FLC repression after a cold treatment, serving as a mechanism for the cellular memory of vernalization.

FCA, a Gene Controlling Flowering Time in Arabidopsis, Encodes a Protein Containing RNA-Binding Domains

A strong promoter of the transition to flowering in Arabidopsis is encoded by FCA. FCA has been cloned and shown to encode a protein containing two RNA-binding domains and a WW protein interaction domain. This suggests that FCA functions in the posttranscriptional regulation of transcripts involved in the flowering process. The FCA transcript is alternatively spliced with only one form encoding the entire FCA protein. Plants carrying the FCA gene fused to the strong constitutive 35S promoter flowered earlier, and the ratio and abundance of the different FCA transcripts were altered. Thus, FCA appears to be a component of a posttranscriptional cascade involved in the control of flowering time.

Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target

Transcription in eukaryotic genomes generates an extensive array of non-protein-coding RNA, the functional significance of which is mostly unknown. We are investigating the link between non-coding RNA and chromatin regulation through analysis of FLC — a regulator of flowering time in Arabidopsis and a target of several chromatin pathways. Here we use an unbiased strategy to characterize non-coding transcripts of FLC and show that sense/antisense transcript levels correlate in a range of mutants and treatments, but change independently in cold-treated plants. Prolonged cold epigenetically silences FLC in a Polycomb-mediated process called vernalization. Our data indicate that upregulation of long non-coding antisense transcripts covering the entire FLC locus may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. We show that addition of the FLC antisense promoter sequences to a reporter gene is sufficient to confer cold-induced silencing of the reporter. Our data indicate that cold-induced FLC antisense transcripts have an early role in the epigenetic silencing of FLC, acting to silence FLC transcription transiently. Recruitment of the Polycomb machinery then confers the epigenetic memory. Antisense transcription events originating from 3′ ends of genes might be a general mechanism to regulate the corresponding sense transcription in a condition/stage-dependent manner.

The Transition to Flowering

The general body plan of plants is established during embryogenesis, when the undifferentiated meristematic regions of root and shoot are set aside. However, much of plant development occurs postembryonically, through the reiterative production of organ primordia at the shoot apical meristem (SAM).

A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death

Arabidopsis Isd1 mutants are hyperresponsive to cell death initiators and fail to limit the extent of cell death. Superoxide is a necessary and sufficient signal for cell death propagation. Thus, LSD1 monitors a superoxide-dependent signal and negatively regulates a plant cell death pathway. We isolated LSD1 via its map position. The predicted LSD1 protein contains three zinc finger domains, defined by CxxCxRxxLMYxxGASxVxCxxC. These domains are present in three additional Arabidopsis genes, suggesting that LSD1 defines a zinc finger protein subclass. LSD1 is constitutively expressed, consistent with the mutant phenotype. Alternate splicing gives rise to a low abundance mRNA encoding an extra five amino-terminal amino acids. We propose that LSD1 regulates transcription, via either repression of a prodeath pathway or activation of an antideath pathway, in response to signals emanating from cells undergoing pathogen-induced hypersensitive cell death.