Analysis of phototropin membrane localisation and function in Arabidopsis

The ability of plants to respond to environmental cues is crucial for the success of the organism. Light is an important source of energy for the plant as well as providing information for plant growth and development. Plant responses to the electromagnetic spectrum are controlled by a number of different photoreceptors. Phytochromes respond to red and far-red light and are responsible for controlling photomorphogenesis in seedlings (Chen and Chory, 2011). The recently identified UVR8 responds to UV-B light and prevents damage to the plant from these harmful wavelengths (Rizzini et al., 2011). Responses to UVA/blue light are controlled by three different photoreceptors: the cryptochromes that are involved in the circadian clock as well as regulating a number of aspects of photomorphogenesis (Chaves et al., 2011); the Zeitlupe family which control the circadian clock and flowering responses (Demarsy and Fankhauser, 2009) and finally the phototropins which regulate the light-dependent processes that increase photosynthetic efficiency of plants (Christie, 2007, Christie and Murphy, 2013). Within Arabidopsis there are two phototropins, phototropin 1 (phot1) and phototropin 2 (phot2), which share approximately 60% sequence identity at the amino acid level (Christie et al., 2002). The phototropins have also been identified in algae, ferns and mosses as well as higher plants where their mode of action appears to be conserved (Onodera et al., 2005). The phototropins function redundantly to control phototropism, chloroplast movement, leaf flattening and positioning, stomatal opening whilst phot1 alone controls the rapid inhibition of hypocotyl growth upon transfer of dark-grown seedlings to light (Christie, 2007). The phototropins are serine/threonine (Ser/Thr) kinases consisting of two Light, Oxygen and Voltage (LOV) domains in the N terminus and a Ser/Thr kinase at the C terminus (Figure 1). In darkness, the protein associates with the plasma membrane and upon illumination partially internalises to cytosolic strands. Illumination with blue light also results in the the LOV domains forming a covalent linkage with the chromophore, Flavin Mononucleotide (FMN) and the protein undergoes autophosphorylation at a number of serine residues including upstream of the LOV1 domain and between the LOV1 and LOV2 linker region, as well as within the kinase activation loop (Inoue et al., 2008; Sullivan et al., 2008). Whilst the physiological functions of the phototropins from Arabidopsis are well characterised, the function of the membrane association and subsequent internalisation is unknown. Therefore the aims of this project were to identify the mechanism of phot1 association with the membrane and to determine if there are specific regions of membrane interaction within the kinase domain of phot1, since the C-terminus of phot2 including the kinase domain is known to direct localisation to the plasma membrane (Kong et al., 2007). It was therefore of interest to analyse the kinase domain of various phototropins to assess if there are sequences that may direct protein localisation to the plasma membrane. Chapter 3 describes the Lysine Rich Motif (LRM) that was identified and subsequently mutated to assess localisation when transformed in to the phot1-5 phot2-1 double mutant as well as subsequent complementation analysis of the physiological responses controlled by phot1. The analysis suggests that phot1 may interact with the membrane through a lipid interaction that is not mediated by the LRM. The consequences of these results are discussed further in the chapter. Further analysis of the role played by the kinase domain in membrane localisation of phot1 is described in the truncation analysis in Chapter 4. A similar approach to the deletion analysis employed by Kong et al. (2013) was used. A predicted secondary structure was generated to ensure that truncations were performed outside secondary structures that may be important for the protein structure. The insect cell system as well as transient expression in N. benthamaiana provided convenient methods to analyse various truncations of the kinase domain of phot1. The method was also used to analyse the membrane association of phot2. The effect of 1-butanol treatment on Arabidopsis seedlings was also investigated in relation to phot1 associating with the plasma membrane by interaction with lipids such as phosphatdic acid (PA). The work presented in this chapter suggests that there may be more than one region of phot1 that interacts with the membrane and the complications of this are discussed further. In order to investigate the function of internalised phot1 after irradiation, the protein was constitutively targeted to the membrane using a farnesyl tag. A farnesylation sequence targets proteins to the membrane via a lipid modification (Sorek, Bloch and Yalovsky, 2009). The farnesyl-tagged phot1-GFP was transformed into the phot1-5 phot2-1 double mutant and the physiological responses controlled by the phototropin were assessed. The results of targeting phot1-GFP to the membrane are shown in Chapter 6. Constitutive targeting of phot1-GFP to the plasma membrane showed that the soluble protein visualised after blue light illumination is, perhaps surprisingly, dispensable for the physiological responses tested. There may however be a role to play in the fine-tuning of the Arabidopsis response to illumination. Together these studies provide new perceptions in the possible mechanism of membrane attachment of phot1. Finally, investigation of the phototropin from the algae Ostreococcus tauri reveal that, while it can mediate some phot-regulated responses when expressed in the phot1-5 phot2-1 double mutant the protein is not functional in phototropism providing a unique phototropin that could be used to investigate the downstream control of this response. These findings highlight key differences between the mode of action of plant and algal phototropins that have so far gone unrecognised.