Biochemical and genetic approaches to modulate phototropin photoreceptor sensitivity

The necessity of light for plants to sustain their autotrophic lifestyle has made the optimization of growth to maximize light capture a crucial strategy for survival in light limiting environments. Increases in light capture can be achieved through alterations in plant architecture, such as modifications to leaf position and stem length. Responses to the light environment are mediated by a network of photoreceptor proteins, which sense specific wavelengths of light and respond to light excitation by initiating signaling. Higher plants respond to red and far-red light through the phytochrome family, blue light through cryptochromes, the zeitlupe family, and phototropins, and UV-B light through the UV RESISTANCE LOCUS 8 photoreceptor. Of these photoreceptor proteins, the phototropins (phots) are perhaps the most closely tied to photosynthetic efficiency. Higher plant phots, phot1 and phot2, mediate leaf expansion to maximize the surface area available for light capture as well as control movement and positioning responses, such as petiole inclination, movement towards more favorable light conditions through phototropism, and, at a cellular level, chloroplast movement. Furthering the role of phots in optimizing responses upstream of photosynthesis, phot1 and phot2 also control stomatal opening in response to blue light, allowing the uptake of carbon dioxide into the leaf for fixation into sugars. In general, these responses are redundantly coordinated by both phot1 and phot2, with phot1 acting as the primary sensor due to its greater sensitivity. Because of the profound effect phots have on photosynthetic competence, the studies presented here examine phot1 with the goal of understanding the physiological role of phot1 sensitivity in plants and explore the possibility that enhancing phot1 sensitivity could increase plant growth. Phots consist of two N-terminal light sensing LOV (Light, Oxygen or Voltage) domains, LOV1 and LOV2, coupled to a serine/threonine kinase domain at the C-terminus. Each of the LOV domains bind a flavin mononucleotide (FMN) chromophore that allows these domains to perceive blue light. In darkness, FMN is non-covalently bound within each of the LOV domains, which repress the activity of the kinase domain. When FMN is excited by blue light, a covalent bond is formed between a conserved cysteine residue present within each LOV domain and FMN. LOV2 specifically is coupled to the kinase domain through two alpha helices, Ja and A’a, which become disordered following the formation of the covalent photoadduct. The unfolding of these alpha helices relieves repression of the kinase domain, initiating signaling. The onset of phot1 signaling is characterized by phot1 autophosphorylation and the dephosphorylation of the phot1 signaling partner NONPHOTOTROPIC HYPOCOTYL 3 (NPH3). Over time, the covalent photoadduct decays and phot1 returns to its inactive dark state, completing the photocycle. The chemistry of the phot1 photocycle in vitro is understood in detail, but its downstream signaling following activation remains relatively elusive, with only a handful of signaling partners and phosphorylation substrates identified. For the sensitivity of phot1 to be thoroughly explored, how the phot1 photocycle affects plant growth as well as how phot1 activity is modulated by signaling partners needed to be addressed. Therefore, a biochemical approach was used to introduce mutations within LOV2 to slow its dark reversion to prolong signaling and investigate how this modulates phot1 sensitivity in vitro and in planta, and, secondly, a genetic strategy was employed to uncover whether any signaling processes can modulate phot1 sensitivity in plants. Compared to other photoreceptors that receive blue light through LOV domains, dark reversion of phot1 following a light stimulus is relatively fast, with the lit state lasting only approximately 15 minutes, while other LOV domains remain activated for many hours. To generate slow photocycle mutants of phot1, previous characterizations of slow photocycling LOV domains were exploited to engineer the phot1 photocycle to have a slower dark reversion by introducing mutations into LOV2. To study the photocycle in vitro, the phot1 light-sensing module consisting of the LOV1 and LOV2 domains (LOV1+LOV2) was heterologously expressed and purified from E. coli and the photocycle was measured spectrophotometrically. Using this approach, 13 LOV2 variants were generated and examined to identify slow photocycle mutants. Three mutations in LOV2, N476L, V478I, and L558I, were found to slow the LOV1+LOV2 photocycle in vitro. Following identification, these mutations were introduced into full-length phot1 expressed heterologously in insect cells to verify the autophosphorylation activity of each mutant. Following the characterization of the candidate slow photocycle mutants in vitro, each phot1 photocycle mutant was examined in planta in a phot1phot2 double mutant background to see whether possession of a slow photocycle increased phot1 sensitivity. Of the three candidate mutations, V478I and L558I were verified as possessing a slow dark reversion through the phosphorylation status of NPH3. NPH3 is dephosphorylated in a phot1- dependent manner following light treatment; it was found that in the presence of wild-type phot1, the phosphorylated form of NPH3 is recovered around one hour following a return to darkness after phot1 stimulation by blue light. By contrast, the dephosphorylated state of NPH3 was sustained in phot1-V478I and -L558I for a substantially longer period of time, consistent with a slow phot1 photocycle and prolonged phot1 activation in these mutants. Surprisingly, it was found that these mutants were less sensitive than wild-type phot1 for phototropism in response to low intensity light treatments. Furthermore, biomass accumulation was not increased in the phot1-L558I mutant under growth conditions consisting of very low light. While the photocycle mutants did not exhibit increased sensitivity or growth in response to continuous light treatments, evidence from collaborators indicated that phot1-L558I is more efficient than wild-type phot1 for the chloroplast accumulation response following brief pulses of blue light. While the role of the phot1 photocycle under continuous irradiation remained unclear, this enhanced chloroplast accumulation response implies that the phot1 photocycle is important for its sensitivity to brief irradiations. Unlike phot1, further work with phot2 later indicated that introducing a slow photocycle mutation to phot2 LOV2 can significantly increase growth in a phot1phot2 mutant background under continuous low light. To investigate other factors that may affect phot1 sensitivity, a genetic screen was undertaken in an attempt to identify suppressors of phot1 activity. The LOV2Kinase (L2K) transgenic line, which expresses a truncated version of phot1 in a phot1phot2 double mutant background, was previously found to be unable to respond to low-intensity blue light, though it can mediate phot1 responses when the light intensity is increased. Because L2K possesses this conditional phenotype, random mutations were introduced into the genome of L2Kexpressing plants and a screen was established to identify mutants that were able to respond to low-intensity light with the hypothesis that those mutations could lie within suppressors of phot1 activity, allowing L2K to signal under circumstances where it ordinarily could not. Using this approach, three independent candidate suppressor mutants were identified that had increased sensitivity for the petiole positioning response under low light. One suppressor mutant was identified as a novel allele of the phytochrome B red light receptor, the second is likely to be a mutant of the transcription factor SQUAMOSA PROMOTOR BINDING LIKE 14, and the identity of the third candidate suppressor is still not known, though it overexpressed the L2K protein. These candidate suppressors may represent novel modulators of phot1 activity and possible mechanisms for how these candidate suppressors may act on phot1 activity are discussed