Structure-function analysis of phototropin receptor kinases

The ability of plants to convert energy provided by the sun into a form accessible by heterotrophic organisms ensures that they form a basal part of most ecosystems. However, in addition to being a vital energy source light can also serve as an environmental indicator. In order to maximise light perception, plants have evolved a suite of photosensors with differing sensitivities, which in combination provide detailed information regarding light availability and quality. The major photoreceptor families identified in plants include the phytochromes, which are most sensitive to red and far-red light, and the cryptochromes and phototropins, which are UV-A and blue light receptors. Additional photosensors, including the recently identified ZEITLUPE/ADAGIO family, have roles in modulating the action of these main components. Plants can also respond to UV-B and green light, although the photoreceptors responsible for their detection remain elusive. Phototropins are blue light sensors that are responsible for a range of responses (including phototropism, chloroplast movement and stomatal opening) that combine to increase the photosynthetic efficiency of plants. Initially identified in the model plant Arabidopsis thaliana, phototropins have since been characterised in the unicellular alga Chalmydomonas reinhardtii, pteridophytes and angiosperms. The light sensitivity of phototropins is derived from the action of two highly conserved regions known as LOV domains which subsequently induce activity of an integral serine/threonine kinase domain via movement of a conserved alpha-helix (Jalpha-helix). Although little is known regarding phototropin signal transduction an obvious biochemical consequence of phototropin light stimulation is autophosphorylation. Phototropin autophosphorylation has previously been studied using a baculovirus/insect cell expression system, with the consequences of phototropin mutation on phototropin kinase activity within this heterologous system having comparable effects to those when identically-mutated phototropins were introduced into transgenic Arabidopsis. Whilst the ability of phototropins to act as light-regulated kinases is well established, the mechanism by which this occurs remains unclear. In this study a baculovirus/insect cell expression system is used to further characterise the mode of phototropin autophosphorylation and the functionality of a mutated phototropin 1 which demonstrates increased autophosphorylation activity in this system is assessed in planta using transgenic Arabidopsis. It was initially of interest to further evaluate the mode of phototropin autophosphorylation. For example, it is unclear whether phototropin autophosphorylation occurs via an intramolecular mechanism or whether this process involves cross-phosphorylation between phototropin molecules. In Chapter 3 the mode of phototropin autophosphorylation activity was further examined using the baculovirus/insect cell expression system to assess the effect of protein truncations and specific point mutations on phototropin kinase activity. Such mutational analysis reveals that phototropin 1 is capable of intermolecular phosphorylation in vitro and also suggests that further phototropin autophosphorylation sites exist in addition to those previously mapped. Additionally, the importance of LOV2 and the Jalpha-helix as components of the phototropin receptor activation are confirmed. The implications of such findings for our understanding of phototropin autophosphorylation are discussed. LOV domains bind flavin mononucleotide as a chromophore. The light-induced formation of a covalent adduct between the LOV domain and the associated chromophore is thought to induce conformational changes culminating in phototropin kinase activation. In Chapter 4 the two leading hypotheses proposed to permit signal transmission between the light-sensitive LOV domains and the integral kinase domain are examined. Previous work has shown that photosensitivity within one of the two LOV domains (LOV2) is sufficient for light-regulated kinase activity, suggesting that light-induced kinase activation is primarily induced via LOV2. This allowed the effect of point mutations within LOV2 on the light-regulated kinase activation of the full-length phototropin protein to be assessed using the baculovirus/insect cell expression system. Work presented here suggests that a single point mutation within LOV2 (Gln575 to Leu; Q575L) is sufficient to reduce signal transmission between LOV2 and the phototropin kinase domain whilst not altering the photosensitivity of the LOV2 domain. In contrast, mutation of a highly conserved salt bridge at the surface of LOV2 does not alter LOV2 domain signal transmission. Interestingly, the reduced light-induced kinase activity of the phot1 Q575L mutant could be counteracted by an additional mutation within the Jalpha-helix (Ile608 to Glu; I608E) which has previously been shown to increase the autophosphorylation of mock-treated phot1 in vitro in comparison with a wild-type control. Such findings suggest that conformational changes may occur in sequence to induce phototropin kinase activation. The final component of this work involved transgenic Arabidopsis to examine the in planta functionality of phot1 I608E, which has been shown to have increased autophosphorylation in vitro when expressed using the baculovirus/insect cell system. However, rather than demonstrating phenotypes consistent with a constitutively-active form of phototropin, transgenic Arabidopsis expressing phot1 I608E appeared to have only partial functionality in planta and indeed appeared to inhibit phot-mediated phenotypes when expressed in a wild-type background. Possible explanations of the observed phenotypes are discussed. The studies presented here further advance our knowledge of the light-induced mechanism which results in activation of the phototropin photoreceptor and also provide insight into the potential differing roles of phototropin autophosphorylation in planta.