Alternative splicing in the regulation of the barley circadian clock

  • Cristiane P. G. Calixto

    Student thesis: Doctoral ThesisDoctor of Philosophy


    In the first year and a half of my PhD (October 2009 to March 2011) I studied the putative involvement of orphan snoRNAs in the regulation of alternative splicing (AS) in Arabidopsis. This project was very attractive but ran into serious and unforeseen problems with the genetic background of mutants used in the project. Despite deciding to terminate the project, it gave me more experience of molecular techniques and analysing RNA and expression. The novel work on AS in Arabidopsis clock genes coincided with availability of the barley genome sequence and the rest of my PhD was spent in examining AS in the circadian clock of barley.
    Prior to this thesis, extensive alternative splicing (AS) was shown to regulate clock genes in Arabidopsis through dynamic changes in AS transcripts, some of which are temperature-dependent and altered levels of productive mRNAs through alternative splicing/Nonsense-Mediated Decay (AS/NMD). An objective of this thesis was to determine whether clock genes and their modes of regulation are conserved in other higher plants, such as barley. By use of a robust in silico analysis and nucleotide sequence of 27 Arabidopsis core clock/clock-associated genes, 21 barley genes were identified, 60% of which are true Arabidopsis orthologues. Most of the barley clock genes have a clear daily rhythm which is maintained in constant light conditions. Mutations of the barley clock genes HvPPDH1 (orthologue of AtPRR7) and HvELF3 strongly affect flowering time and have extended the geographic range where barley is grown. We show that both mutations affect expression of clock genes: the Hvppdh1 mutation moderately affects expression levels and phase while the Hvelf3 mutation causes arrhythmicity of most of these genes, which helps to explain the early flowering phenotype. Temperature-dependent AS was identified in some of the barley core clock orthologues. The focus of this part of the analysis was HvLHY and HvPPDH1. Although specific AS events were poorly conserved, similar behaviour in terms of decreased functional mRNA was observed. This novel layer of fine clock control observed in two different species, a model plant and a crop species, might help our understanding of plant adaptation in different environments and ultimately may offer a new range of targets for plant improvement.
    Date of Award2013
    Original languageEnglish
    SupervisorJohn Brown (Supervisor)

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