Characterisation of the interactions of RGL1 : a negative regulator of gibberellin signalling : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry, Massey University, Palmerston North, New Zealand.

Abstract

The gibberellins are a family of phytohormones that promote many aspects of plant development. Central to the function of gibberellins are the DELLA regulatory proteins. The DELLA proteins actively repress cell differentiation and elongation, but are degraded upon perception of gibberellin, thus relieving repression of gibberellin responses. The GID1-family gibberellin receptors and DELLA-specific F-box proteins are essential for the gibberellin-induced degradation of the DELLA proteins. Importantly, the direct interaction between gibberellin-bound GID1-family gibberellin receptors and the N-terminal domain of DELLA proteins is a prerequisite for proteasomal degradation through recruitment of the F-box proteins. To increase understanding of gibberellin signalling, I have characterised a gibberellin-dependent GID1-DELLA-F-box protein signalling switch in Arabidopsis thaliana. First, I have characterised a suite of anti-DELLA antibodies for detection of four endogenous A. thaliana DELLA proteins, GIBBERELLIC ACID-INSENSITIVE (GAI), REPRESSOR OF GA1-3 (RGA), RGA-LIKE-1 (RGL1), and RGA-LIKE-2 (RGL2). Using these monoclonal antibodies against the conserved motifs of DELLA proteins, I showed that residues Asp/Glu/Leu/Leu within the signature DELLA motif are not essential for interaction of RGL1 with GID1A. Further, in vitro interaction assays allowed modelling a two-step conformational change within the N-terminal domain of RGL1 upon interaction with gibberellin-bound GID1A. Together with interaction assays in yeast two- and three-hybrid systems, these experiments provided three clues to the mechanism of GID1A-RGL1-SLY1 gibberellin signalling switch: i) N- to C- interdomain interactions of RGL1 regulate its accessibility to SLY1; ii) the N-terminal domain of RGL1 undergoes conformational rearrangement upon interaction with gibberellin-GID1A; iii) the conformational changes of the N-terminal domain of RGL1 primes the C-terminal domain for the recruitment of SLY1. I have also isolated two novel RGL1-interacting proteins, the myrosinase THIOGLUCOSIDE GLUCOHYDROLASE-2 (TGG2) and GERMIN-LIKE-PROTEIN-1 (GLP1), through affinity-purification from nuclear extract and mass spectrometry fingerprinting. Neither protein has yet been implicated in gibberellin signalling. Therefore, the identification of these novel components may help resolve several uncharacterised aspects of gibberellin signalling.