The DELLA protein family and gibberellin signal transduction : : 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

Gibberellins (GA) are plant hormones that promote important aspects of growth such as seed germination, leaf expansion, trichome initiation, transition to flowering, stem elongation, flower and fruit development. Genetic and molecular data indicate that growth and development are a default state and that the DELLA proteins are key repressors of GAmediated growth and development. Mutant analysis indicates that GA does not directly promote growth; rather it overcomes the repression of the DELLA proteins by causing them to be degraded. The N-terminal domain of the DELLA proteins is involved in the perception of the GA signal and the C-terminal domain mediates the repression of GA responses. A GA-bound receptor recognises the DELLA proteins and interacts with an F-box E3 ligase, the DELLA proteins are then poly-ubiquitinated and degraded through the ubiquitin-26S proteasome system. However, the DELLA proteins are also post-translationally modified which affects their activity. It is believed the DELLAs are modified both with O-linked N-Acetyl glucosamine for stability and phosphate groups to mark them for F-box recognition. However, the precise nature, and role of these modifications is yet to be shown. DELLA-repressive action is mediated by interaction with other proteins and not through direct DNA binding. Few DELLA-interacting proteins are known. Apple and Kiwifruit DELLA repressor, GID1 GA receptor and SLY1/ GID2 F-box orthologues were identified in their respective sequence databases. Relative amount and location of the orthologous transcript sequences was examined through qPCR and reporter gene experiments. Apple qPCR experiments indicated relatively high levels of DELLA transcripts in developmentally arrested tissues. Kiwifruit experiments present a more complicated picture, with high relative levels of DELLAs in the actively expanding tissues, however, concomitant with this were high relative levels of the GID1 and GID2 transcripts. Each transcript was found in every tissue studied and indicated complex developmental transcriptional control. Both direct and indirect immunoprecipitation experiments utilising a novel tag were performed in GA-deficient plant backgrounds in order to isolate DELLA proteins and their interacting proteins likely targeted by DELLA repressive function. Proteins from these experiments were identified from their peptides in mass spectrometry analysis and database query. Several transcription factors, kinases, proteins involved in RNA processing and protein components involved in hormonal signalling pathways other than GA were present. DELLA repression complex formation was also investigated with two-dimensional electrophoresis and western blotting, and indicated a dominant repressive complex at approximately 160 kDa, with additional multiple larger complexes of up to 600 kDa.