In the model plant Arabidopsis thaliana, the circadian clock is believed to be composed of a number of coupled transcriptional negative feedback loops. The LATE ELONGATED HYPOCOTYL (LHY) gene is thought to form part of at least two of these transcriptional feedback loops, as well as playing a role in the perception of light signals by the clock. To better understand how multiple transcriptional feedback loops might be integrated in the transcriptional regulation of LHY, we have performed an analysis of the cis-regulation of this gene. Through deletion analysis of reporter gene constructs, we have identified a 957 basepair region of the LHY promoter which contains sufficient sequence to direct the characteristic expression profile of LHY. Furthermore, we provide evidence that at least two circadian signals converge on this region. Electrophoretic mobility shift assays identified four classes of candidate cis-elements within the LHY promoter including a poly-CTT tract, an AAAAA motif, a candidate MYB-binding site and a G-box motif. Through mutational analysis of these elements, we have been able to determine aspects of their in vivo regulatory function. We report that a G-box motif and the previously uncharacterized AAAAA element are implicated in the regulation of LHY transcription by light signals. In etiolated seedlings, the region of the LHY promoter containing the MYB-binding site motif and multiple copies of the poly-CTT motif mediates regulation of LHY by both light-responsive and circadian signals.

The aim of the work presented in this thesis was to assess the genetic variation present within the wild tomato species Solanum pennellii that can be adapted to improve the texture of the domesticated tomato species. Using a population of S. pennellii introgression lines, 23 significant QTL supporting intervals were identified. Nine of the QTL were significant in two growing seasons. Three QTL were identified for pericarp firmness. Lines containing the firmness QTL F-Sp 2.1 were used to create BC1 populations. Sensory analyses were used to correlate the instrumental texture measurements to those perceived during mastication. Repeated texture measurements were conducted on lines representing similar chromosomal regions from another wild tomato species Solanum habrochaites introgression line population. To better understand the genetic basis of one of the firmness QTL identified, the Syngenta tomato Affymetrix GeneChip was used to quantify the differential expression of S. pennellii genes within the QTL-introgression line through development in comparison to the recurrent parent S. lycopersicum L. cv M82. The microarray analyses were extended to the ripening mutants Cnr, nor and rin. Differential gene expression between the ripening mutants and the wild type Ailsa Craig were compared through development. Candidate genes for the firmness QTL and fruit development were nominated.

Meiotic recombination in eukaryotes requires two orthologues of the E. coli RecA proteins, Rad51 and Dmc1. Both genes play an important role in the binding of single strand DNA, homology search, strand invasion and strand exchange resulting in Holliday junctions which are resolved into crossovers or non-crossovers events. Even though both genes are well characterized in a variety of organisms including plants, very little information is available from hexaploid wheat. In most diploid plant species, deletion of either the RAD51 or DMC1 orthologues leads to sterility but wheat being a polyploid, offers a unique opportunity to examine the effects of the deletion of specific homoeologue, while maintaining a degree of fertility. The transcript expression profiling of RAD51 and DMC1 genes in Arabidopsis, rice and wheat using available microarray databases indicated higher levels of expression in mitotically and meiotically active tissues compared to other tissues. However, the possible function of the DMC1 gene in mitotic-active tissues needs to be investigated further. Previously cDNA sequences of TaRAD51 and TaDMCl of hexaploid wheat were cloned and reported. In this study, it has been demonstrated that the reported TaRAD51A1 and TaRAD51A2 cDNA sequences are (D) and (A) homoeologues of TaRAD51 respectively and TaDMCl cDNA sequence is (D) homoeologue of the TaDMC1. This study also found that the amino acid sequences and evolutionary relationships of RAD51 and DMC1 cDNA homoeologues are highly conserved across eukaryotes. Functional characterization of TaRAD51 and TaDMCl gene homoeologues was undertaken in planta using Forward Genetics, Reverse Genetics and Complementation methods. Forward and Reverse Genetic screening of a subset of a Highbury mutant population could not identify any mutants that have deletions in TaRAD51 and TaDMC1 genes. However, Reverse Genetics screening of Paragon mutant population identified mutant lines that tested as having deletions for all the three homoeologues of TaRAD51 and TaDMCl. However, most likely due to high mutational load and a deleterious phenotype, only a few mutant lines survived. Phenotypic and cytogenetic analysis indicated the probable functional redundancy of TaRAD51 (B) homoeologue in meiosis, although the unknown size of the deletion and limited phenotype makes it impossible to completely certain of this. The single mutants for TaDMC1 (B) and (D) indicated a reduction in pollen viability and ear fertility compared to wild-type. The cytological examination of these mutants indicated low levels of abnormal diakinesis, resulting in the formation of dyads. However, the single mutants were still able to produce normal tetrads. This suggests that there is a possible dosage effect of these homoeologues in hexaploid wheat. Unless deletion lines for the (A) and (D) homoeologues of TaRAD51 and (B) homoeologue of TaDMC1 can be recovered and characterized the above assumptions will remain inconclusive. The results of complementation assays using over-expressing CaMV35S::TaRAD51(D)±GFP constructs demonstrated a very low (-14% and -2%, respectively, with +GFP and -OFP constructs) functional complementation in terms of seed set compared to 0% in homozygous Atrad51 mutants. One explanation of these results is that the wheat genes are not complete functional orthologues for the inactivated Arabidopsis genes. The functional complementation experiments could not be performed for TaDMC1 gene because of time limitation, although the transformants were produced in AtDMC1/atdmc1 background. Finally, overexpression of the TaRAD51 gene suggests 2-fold increase in genetic distances in Arabidopsis using CaMV35S::TaRAD51(D) construct. This was done by crossing the appropriate transformant with fluorescent tetrad lines. However the results need to be confirmed by a large scale analysis.