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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.
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