Final Report Summary - DIVERSITY OF RHYTHMS (Analysis of natural-genetic variation controlling the timing of GIGANTEA expression in Arabidopsis)
Background
The circadian clock is an internal time keeping mechanism that regulates many physiological and developmental processes in plants. Several genes and proteins that participate in the regulation of the clock have been identified mainly through forward genetics strategies, but they are not sufficient to explain the observed complexity of the clock mechanism suggesting that many regulators are yet to be discovered. Mathematical and experimental data strongly suggest that Gigantea (GI) could be one of these regulators (Locke et al., 2005), as in gi mutants the rhythm of expression of key clock components is altered (Mizoguchi et al., 2005). GI is also involved in other fundamental biological programs such as photoperiodic floral induction (Suarez-Lopez et al., 2001) as well as red and blue light signalling (Huq et al., 2000; Martin-Tryon et al., 2007), suggesting a broad role for this protein in the regulation of plant development. Despite the importance of GI in these processes, its biochemical function and how its expression is regulated are still not understood.
Objectives
The main objective of the research project DIVERSITY OF RHYTHMS is to identify regulators of GI expression. The project proposes to identify new genes (or alleles) that precisely control the timing of GI transcription by advancing or delaying the time at which the peak of GI mRNA abundance occurs (GI peak time). A novel aspect of the research project resides in the use of natural genetic variation in ecotypes (accessions) of Arabidopsis thaliana to identify these genes. The use of natural variation is an interesting alternative to the classical forward genetic approach because, in addition to allowing the identification of new genes, it also provides information on the mechanisms that create variation in nature. GI expression was measured in a set of 85 ecotypes, and two ecotypes were isolated in which the GI peak time was shifted approximately two hours compared to the reference accession Col0. To establish the genetic basis underlying these differences, the two ecotypes were crossed to Col0 and the resulting F2 progenies were used to map the genomic regions (Quantitative Trait Loci) that were associated with GI peak time variations. DIVERSITY OF RHYTHMS has been dedicated to the identification of the causal gene(s) of the Quantitative Trait Loci (QTL), and to the study of the mechanism by which they regulate the expression of GI.
Results
QTL explaining variations of GI peak time between the ecotypes were detected. The genomic locations of the QTL were different between the two crosses, meaning that the genetic basis responsible for variations in GI expression was different between the ecotypes. The isolated effect of each QTL was relatively weak. Most of the QTL delayed or advanced GI expression by approximately 30 minutes, meaning that none of the QTL could explain the overall differences between the original ecotypes and Col0. In fact, overall differences between ecotypes are due to additive effects and interactions of these QTL, suggesting that variations of GI expression in natural ecotypes have a complex genetic basis. Two QTL located on chromosome II in cross1 and cross2 were confirmed in F3 families and subsequent progenies, and were chosen for further studies. In the case of the QTL detected in cross1, a candidate gene was rapidly identified based on its function. Sequencing of the candidate gene confirmed the presence of sequence variations between the ecotypes that could explain changes in GI peak time. A loss of function mutant for the candidate gene was used to more precisely determine how it was controlling GI. First, GI expression in the mutant was delayed by more than one hour, strengthening the assumption that the candidate could be the causal gene of the QTL.
The circadian clock is an internal time keeping mechanism that regulates many physiological and developmental processes in plants. Several genes and proteins that participate in the regulation of the clock have been identified mainly through forward genetics strategies, but they are not sufficient to explain the observed complexity of the clock mechanism suggesting that many regulators are yet to be discovered. Mathematical and experimental data strongly suggest that Gigantea (GI) could be one of these regulators (Locke et al., 2005), as in gi mutants the rhythm of expression of key clock components is altered (Mizoguchi et al., 2005). GI is also involved in other fundamental biological programs such as photoperiodic floral induction (Suarez-Lopez et al., 2001) as well as red and blue light signalling (Huq et al., 2000; Martin-Tryon et al., 2007), suggesting a broad role for this protein in the regulation of plant development. Despite the importance of GI in these processes, its biochemical function and how its expression is regulated are still not understood.
Objectives
The main objective of the research project DIVERSITY OF RHYTHMS is to identify regulators of GI expression. The project proposes to identify new genes (or alleles) that precisely control the timing of GI transcription by advancing or delaying the time at which the peak of GI mRNA abundance occurs (GI peak time). A novel aspect of the research project resides in the use of natural genetic variation in ecotypes (accessions) of Arabidopsis thaliana to identify these genes. The use of natural variation is an interesting alternative to the classical forward genetic approach because, in addition to allowing the identification of new genes, it also provides information on the mechanisms that create variation in nature. GI expression was measured in a set of 85 ecotypes, and two ecotypes were isolated in which the GI peak time was shifted approximately two hours compared to the reference accession Col0. To establish the genetic basis underlying these differences, the two ecotypes were crossed to Col0 and the resulting F2 progenies were used to map the genomic regions (Quantitative Trait Loci) that were associated with GI peak time variations. DIVERSITY OF RHYTHMS has been dedicated to the identification of the causal gene(s) of the Quantitative Trait Loci (QTL), and to the study of the mechanism by which they regulate the expression of GI.
Results
QTL explaining variations of GI peak time between the ecotypes were detected. The genomic locations of the QTL were different between the two crosses, meaning that the genetic basis responsible for variations in GI expression was different between the ecotypes. The isolated effect of each QTL was relatively weak. Most of the QTL delayed or advanced GI expression by approximately 30 minutes, meaning that none of the QTL could explain the overall differences between the original ecotypes and Col0. In fact, overall differences between ecotypes are due to additive effects and interactions of these QTL, suggesting that variations of GI expression in natural ecotypes have a complex genetic basis. Two QTL located on chromosome II in cross1 and cross2 were confirmed in F3 families and subsequent progenies, and were chosen for further studies. In the case of the QTL detected in cross1, a candidate gene was rapidly identified based on its function. Sequencing of the candidate gene confirmed the presence of sequence variations between the ecotypes that could explain changes in GI peak time. A loss of function mutant for the candidate gene was used to more precisely determine how it was controlling GI. First, GI expression in the mutant was delayed by more than one hour, strengthening the assumption that the candidate could be the causal gene of the QTL.