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Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of phot1-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1 D806N -GFP but did enhance its dimerization, suggesting that phot1 dimerization is independent of phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis revealed that the interaction between phot1-GFP and a marker of sterol-rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1 D806N -GFP and AtRem1.3-mCherry, indicating that BL facilitates the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as organizing platforms essential for the proper function of activated phot1 at the PM., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

Tryptophan residues at the dimer interface of the plant photoreceptor UVR8 promote monomerisation after UV-B absorption via a so far unknown mechanism. Using FTIR spectroscopy we assign light-induced structural transitions of UVR8 mainly to amino acid side chains without major transformations of the secondary structure of the physiologically relevant C-terminal extension. Additionally, we assign the monomerisation associated increase and red shift of the UVR8 tryptophan emission to a photoinduced rearrangement of tryptophan side chains and a relocation of the aspartic acid residues D96 and D107, respectively. By illumination dependent emission spectroscopy we furthermore determined the quantum yield of photoinduced monomerisation to 20 ± 8%.

Light is a key environmental factor that drives many aspects of plant growth and development. Phototropism, the reorientation of growth toward or away from light, represents one of these important adaptive processes. Modern studies of phototropism began with experiments conducted by Charles Darwin demonstrating that light perception at the shoot apex of grass coleoptiles induces differential elongation in the lower epidermal cells. This led to the discovery of the plant growth hormone auxin and the Cholodny-Went hypothesis attributing differential tropic bending to lateral auxin relocalization. In the past two decades, molecular-genetic analyses in the model flowering plant Arabidopsis thaliana has identified the principal photoreceptors for phototropism and their mechanism of activation. In addition, several protein families of auxin transporters have been identified. Despite extensive efforts, however, it still remains unclear as to how photoreceptor activation regulates lateral auxin transport to establish phototropic growth. This review aims to summarize major developments from over the last century and how these advances shape our current understanding of higher plant phototropism. Recent progress in phototropism research and the way in which this research is shedding new light on old concepts, including the Cholodny-Went hypothesis, is also highlighted.

Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.

Phototropin is the designation originally assigned to a recently characterized chromoprotein that serves as a photoreceptor for phototropism. Phototropin is a light-activated autophosphorylating serine/threonine kinase that binds two flavin mononucleotide (FMN) molecules that function as blue light-absorbing chromophores. Each FMN molecule is bound in a rigid binding pocket within specialized PAS (PER-ARNT-SIM superfamily) domains, known as LOV (light, oxygen, or voltage) domains. This article reviews the detailed photobiological and biochemical characterization of the light-activated phosphorylation reaction of phototropin and follows the sequence of events leading to the cloning, sequencing, and characterization of the gene and the subsequent biochemical characterization of its encoded protein. It then considers recent biochemical and photochemical evidence that light activation of phototropin involves the formation of a cysteinyl adduct at the C(4a) position of the FMN chromophores. Adduct formation causes a major conformational change in the chromophores and a possible conformational change in the protein moiety as well. The review concludes with a brief discussion of the evidence for a second phototropin-like protein in Arabidopsis and rice. Possible roles for this photoreceptor are discussed.