Investigation of phototropin blue light receptor function and signalling in arabidopsis

The global success of plants depends largely on their ability to perceive and respond to light, mainly in two regions of the electromagnetic spectrum. Phytochromes are light sensors for the red and far-red wavelengths of light while cryptochromes, phototropins and members of the ZTL/ADO family respond to blue and UV-A wavelengths of light. Phototropins are UV-A/blue-light receptor kinases found ubiquitously in plants from the unicellular green alga Chalmydomonas reinhardtii through bryophytes and pteridophytes up to angiosperms. The model plant Arabidopsis possesses two phototropins (phot1 and phot2) and is the subject of the work presented in this thesis. The general structure of the phototropin protein comprises a photosensory region at the N-terminal that contains two LOV (light, oxygen and voltage sensing) domains and a C-terminal kinase domain belonging to the large AGC family of protein kinases. The LOV domains form a covalent adduct with the chromophore flavin mononucleotide (FMN) in response to illumination with blue light which in turn leads to structural changes throughout the protein resulting in autophosphorylation of the N-terminal region by the kinase domain. Phototropins function redundantly to mediate a number of physiological responses in planta which serve to promote plant fitness and maximise photosynthetic potential. Phototropism, chloroplast accumulation, blue light-induced stomatal opening, leaf expansion and leaf movements can be induced through the activation of both phot1 and phot2 in response to different intensities of light, with phot1 being more light sensitive than phot2. In addition to the functionally redundant responses, phot1 alone is responsible for destabilisation of certain mRNA transcripts and the rapid inhibition of hypocotyl elongation when etiolated seedlings are transferred to blue light, while phot2 is solely responsible for the high light induced chloroplast avoidance response. While much is known about the mechanisms of light perception by the phototropins at the molecular level, and the responses mediated by them have been well described, little is known about their methods of signalling to induce these physiological responses upon photoactivation by blue light. Therefore, the aims of this study were to identify novel phot-interacting proteins and to investigate the modes of phot1 signalling by structure/function analyses in order to better understand the way phototropins elicit signal transduction to downstream components in order to bring about the responses described above. Initially, a yeast two-hybrid screen was carried out to try to identify immediate interacting partners for phot1. The results of the yeast two-hybrid screen are described in Chapter 3. One hundred and thirty yeast colonies containing putative phot1-interacting proteins were identified from the screen and preliminary characterisation of six of these proteins are described in this chapter. Two of the proteins investigated are members of the ADP-ribosylation family which is involved in the regulation of membrane trafficking. The ARF proteins identified show a blue-light-sensitive interaction with phot1 and also interact with phot2. These proteins are of interest given the subcellular movement of phototropins from the plasma membrane after exposure to blue light. The C-terminal kinase domain of phot1 was found to interact with p-glycoprotein 19 (PGP19), a protein involved in polar auxin transport. The interaction between these proteins is interesting because of the role auxin plays in phot1-mediated responses such as phototropism and leaf expansion, and preliminary characterisation of the interaction in vitro is shown in Chapter 3. The implications of a direct link between phototropins and the proteins involved in auxin transport are discussed. A further two proteins identified from the screen are members of the NPH3/RPT2-Like (NRL) family. RPT2 has already been identified as a phot1-interacting protein and identification of this protein increased confidence in the efficacy of the screen to identify genuine interacting proteins. A novel member of the NRL family, designated NPH3-L, was also identified from the screen. Chapter 4 describes the tissue specific and subcellular localisation of NPH3-L and contains results of preliminary investigations into the function of NPH3-L in planta. 14-3-3λ was identified from the screen using full-length phot1 as bait. A 14-3-3 protein has been shown previously to bind to autophosphorylated phototropin in Vicia faba (Kinoshita et al., 2003). Chapter 5 details the localisation of 14-3-3λ at tissue and subcellular levels and shows that 14-3-3λ binding to plant-derived phot1-GFP is both light dependent and induced by receptor autophosphorylation. Creation of GFP-14-3-3λ overexpressing lines in wild-type and phot1-5phot2-1 backgrounds allowed investigation into the roles that light and phototropins play in regulating the subcellular localisation of 14-3-3λ. It is shown that light-induced movement of 14-3-3λ at the plasma membrane is dependent on the presence of endogenous phototropins. Physiological implications of this interaction are discussed. Finally, in order to determine the modes of phototropin signalling, structure/function studies were carried out by expressing different regions of phot1 in a variety of Arabidopsis backgrounds. The results of the structure/function studies are described in Chapter 6. Known phot1-mediated responses were investigated in the transgenic plants to determine the effects of individual domains of phot1. Particular attention was paid to the role of receptor autophosphorylation in phot1-mediated responses to light. A transgenic line overexpressing the LOV2-kinase region of phot1 demonstrates that phot1 autophosphorylation is not the primary signalling event involved in phot1-mediated responses to light and shows that the truncated version of phot1 is sufficient to complement most phot1-mediated responses. This also shows that the LOV1 domain is dispensable and suggests phot1 may signal through phosphorylation of substrates. Comparisons are drawn between phot1 kinase overexpressing lines and inactive phot1 kinase overexpressing lines. Preliminary observations of a transgenic line overexpressing phot1 in a wild-type background indicate that overexpression of phot1 may alter polar auxin transport. Together these studies provide new insights into possible mechanisms of phot1 signalling and the function of major domains of phot1.