Analysis of the regulation of GIGANTEA transcript and protein accumulation in Arabidopsis and recombinant expression

Abstract

A plant's ability to correctly synchronise its flowering to coincide with the most favourable time of the year is crucial for its reproductive success, and many plants use day length as a main seasonal cue. One of the major players in flowering time regulation in Arabidopsis thaliana (Arabidopsis) is a component of the photoperiod pathway called GIGANTEA (GI). During the day, circadian clock control of GI transcription together with post-translational control of GI protein levels results in accumulation of GI, which is then degraded in the dark. Despite recent discoveries of GI interactors that might explain how GI influences some of the processes it is involved in, the exact biochemical function of GI is still unknown. This thesis aimed to clarify GI‘s role in flowering time control by biochemical characterisation of recombinantly expressed GI protein, and examination of GI protein cycling in planta. Expression and purification of a predicted ryegrass GI domain in Escherichia coli (E. coli) revealed that the protein aggregated and was unstable. Attempts were made to refine the construct to match the exact GI domain boundaries, and an Arabidopsis GI construct was tested for its suitability for biochemical studies. Full-length Arabidopsis GI was expressed in baculovirus infected insect cells and generated GI protein that was more stable than that expressed in E. coli, but was still insoluble, possibly due to aggregation. Attempts were made to solubilise GI protein, and its localisation in insect cells was visualised using fluorescence microscopy. Analysis of the regulation of GI mRNA and protein levels in Arabidopsis mutant and wild-type plants revealed the involvement of several different photoreceptors. Under long day conditions, ZEITLUPE (ZTL) was the only photoreceptor found to be essential for GI mRNA and protein cycling. Studies in individual colours of light and darkness demonstrated that PHYTOCHROME A, ZTL, and CRYPTOCHROME 1 (CRY1) and/or CRY2 are responsible for the regulation of GI mRNA cycling. The same photoreceptors are involved in post-transcriptional regulation of GI, with CRY1 and/or CRY2 seemingly causing de-stabilisation of GI in the dark. Detailed analysis of GI mRNA regulation demonstrated that GI transcript is more stable in the morning than in the afternoon.