Genetic control of photoperiod responsiveness in pea

The change from vegetative to reproductive growth is one of the most important transitions in the life of a plant, and the precise timing of flowering is critical to maximise reproductive success. Flowering time is therefore regulated by many different environmental cues, including daylength and temperature, and responsiveness to these factors determines the geographic and seasonal range of many crop species. The genetic control of flowering time has been well-characterised in the model species, Arabidopsis thaliana and this has identified many of the molecular mechanisms by which flowering time is regulated by environmental and endogenous signals. More recently, research on other species has begun to show how these mechanisms differ in different species and varieties that respond differently to photoperiod changes. The legumes are a large and agriculturally important plant group that includes crop species such as lentils, beans, chickpea, peanut, clover, alfalfa, and soybean. In several of these species, genetic loci have been identified with a function in regulation of flowering time and photoperiod responsiveness and in many cases, natural allelic variation at these loci are important in classical breeding programs to improve crop performance. In garden pea (Pisum sativum L.), recessive alleles at several different loci result in early flowering and a loss of photoperiod sensitivity. These include two loci, STERILE NODE (SN) and HIGH RESPONSE (HR) first defined in studies of naturally-occurring variation, and two others, DIE NEUTRALIS (DNE) and PHOTOPERIOD (PPD), defined by studies of induced mutants. Phenotypic comparisons with Arabidopsis photoperiod-response mutants suggested that all four loci might participate in circadian clock function, a conclusion supported by a preliminary analysis of diurnal rhythms of gene expression. The overall aim of the research in this thesis was to discover the molecular identity of DNE, SN, PPD, and HR and hence to better understand their roles in the circadian clock and photoperiodic control of flowering. A ‚ÄövÑvªpositional candidate‚ÄövÑvº approach was taken, in which pea homologs of Arabidopsis circadian clock-related genes were isolated and assessed as candidates on the basis of map position and through phenotypic comparisons with Arabidopsis. The availability of extensive Medicago truncatula genome sequence and the close synteny between Medicago and pea greatly assisted the identification process of pea clock gene isolation, and the use of primers directly based on Medicago sequence proved to be highly successful for pea gene isolation. Using this approach, pea sequence of several clock-related genes were isolated or extended, including EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), LUX ARRHYTHMO (LUX), and TIME FOR COFFEE (TIC), and components of gene families of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) /ELONGATED HYPOCOTYL (LHY) and PSEUDO RESPONSE REGULATOR (PRR). This demonstrates that the majority of Arabidopsis clock-related genes are present in pea, although in some cases gene families differ in size. Most pea genes show diurnal expression patterns similar to their Arabidopsis homologs suggesting they may function in a similar way. Map positions ruled out several genes as candidates for the photoperiod loci, but identified several others as strong candidates. Further analysis resulted in identification of DNE, SN, and HR as orthologs of Arabidopsis clock genes, ELF4, LUX, and ELF3 respectively. The HR locus in particular is considered to be to be one of the most important loci controlling photoperiod responsiveness in cultivated pea germplasm, and sequence diversity at HR was examined across a range of accessions. A single-nucleotide indel in HR was correlated with photoperiod responsiveness and the difference between spring and winter pea cultivars. Analysis of circadian rhythms of clock gene expression in our standard wild-type, NGB5839 revealed damped circadian rhythms under constant light but not constant dark. This line, likes many other spring cultivars carries loss-of-function alleles at HR/PsELF3. This demonstrates that other flowering mutants in the NGB5839 background, including dne, sn, and ppd, are all effectively double mutants, which is likely to explain the subtle effects on diurnal and circadian rhythms of clock gene expression in these mutants. Finally, the pathway through which DNE, SN, PPD and HR act to control flowering was also investigated by grafting experiments and expression analysis of genes in the CO and FT families. In dne, sn, and ppd mutants, the early flowering phenotypes were found to be associated with elevated expression of several FT genes, but these mutants showed no alterations on diurnal expression of CO-like genes. Collectively, these detailed physiological, genetic and molecular analyses of DNE, SN, PPD, and HR have revealed the importance of the circadian clock for photoperiodic responsiveness in pea.