Discrete changes in the amplitude, frequency, and cellular localisation of calcium ion (Ca2+) transients encode information about numerous stimuli and function to mediate stimulus-specific responses. Cytoplasmic Ca2+ (Ca2+cyt) undergoes circadian oscillations in concentration that appear to be under the control of the same endogenous oscillator that regulates expression of genes in the photoperiodic-flowering pathway. It is currently not known whether these circadian [Ca2+cyt] oscillations are biochemical artefacts or are decoded and function to transduce clock dependent responses. Calmodulin (CaM) is a primary node in Ca2+ signalling in plants and as such is a promising target for investigating the role of Ca2+ in clock-controlled processes. In this study, Arabidopsis thaliana were treated with experimentally validated concentrations of pharmacological CaM inhibitors. Under inductive photoperiods (16 h light : 8 h dark), CaM inhibition was found to increase developmental flowering time, whilst under non-inductive photoperiods no such changes were evident. Inhibition of CaM led to changes in expression of the key clock gene TIMING OF CAB EXPRESSION 1 and flowering time genes, CONSTANS and FLOWERING LOCUS T and removed repression of flowering in darkness. These observations are consistent with CaM modulating the activity of the putative clock component GIGANTEA and the proteasomal targeting protein SUPPRESSOR OF PHYA-105. Due to the unwanted side effects often generated by chemical CaM inhibitors, a peptide inhibitor of CaM comprising a green fluorescent protein / calspermin fusion and labelled smGN was developed. Surface plasmon resonance analysis and affinity chromatography showed smGN to have extremely high selectivity for, and affinity to, CaM and to function as a powerful inhibitor of CaM in vitro. Further work on the methodology used to deploy smGN as a recombinant alternative to chemical CaM inhibitors in planta is also described.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:511537 |
| Date | January 2009 |
| Creators | Murphy, Andrew James |
| Contributors | Love, John |
| Publisher | University of Exeter |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10036/99353 |
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