Kenichi Hitomi

Early abscisic acid signal transduction mechanisms: newly discovered components and newly emerging questions

The plant hormone abscisic acid (ABA) regulates many key processes in plants, including seed germination and development and abiotic stress tolerance, particularly drought resistance. Understanding early events in ABA signal transduction has been a major goal of plant research. The recent identification of the PYRABACTIN (4-bromo-N-[pyridin-2-yl methyl]naphthalene-1-sulfonamide) RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family of ABA receptors and their biochemical mode of action represents a major breakthrough in the field. The solving of PYR/RCAR structures provides a context for resolving mechanisms mediating ABA control of protein-protein interactions for downstream signaling. Recent studies show that a pathway based on PYR/RCAR ABA receptors, PROTEIN PHOSPHATASE 2Cs (PP2Cs), and SNF1-RELATED PROTEIN KINASE 2s (SnRK2s) forms the primary basis of an early ABA signaling module. This pathway interfaces with ion channels, transcription factors, and other targets, thus providing a mechanistic connection between the phytohormone and ABA-induced responses. This emerging PYR/RCAR-PP2C-SnRK2 model of ABA signal transduction is reviewed here, and provides an opportunity for testing novel hypotheses concerning ABA signaling. We address newly emerging questions, including the potential roles of different PYR/RCAR isoforms, and the significance of ABA-induced versus constitutive PYR/RCAR-PP2C interactions. We also consider how the PYR/RCAR-PP2C-SnRK2 pathway interfaces with ABA-dependent gene expression, ion channel regulation, and control of small molecule signaling. These exciting developments provide researchers with a framework through which early ABA signaling can be understood, and allow novel questions about the hormone response pathway and possible applications in stress resistance engineering of plants to be addressed.

Structural mechanism of abscisic acid binding and signaling by dimeric PYR1.

ABA Receptor Up Close Plants face a variety of environmental stresses, including drought, salinity, and cold. In the face of such stresses, the plant hormone abscisic acid (ABA) triggers adaptive physiological responses. Nishimura et al. (p. 1373, published online 22 October; see the Perspective by Sussman and Phillips) have now analyzed the crystal structure of one member of the ABA receptor family, PYR1 (pyrabactin resistance 1). The ABA molecule binds within an internal pocket of PYR1, where it probably induces a conformational change. The plant hormone responsible for drought tolerance signals by inducing conformational changes in its dimeric protein receptor. The phytohormone abscisic acid (ABA) acts in seed dormancy, plant development, drought tolerance, and adaptive responses to environmental stresses. Structural mechanisms mediating ABA receptor recognition and signaling remain unknown but are essential for understanding and manipulating abiotic stress resistance. Here, we report structures of pyrabactin resistance 1 (PYR1), a prototypical PYR/PYR1-like (PYL)/regulatory component of ABA receptor (RCAR) protein that functions in early ABA signaling. The crystallographic structure reveals an α/β helix–grip fold and homodimeric assembly, verified in vivo by coimmunoprecipitation. ABA binding within a large internal cavity switches structural motifs distinguishing ABA-free “open-lid” from ABA-bound “closed-lid” conformations. Small-angle x-ray scattering suggests that ABA signals by converting PYR1 to a more compact, symmetric closed-lid dimer. Site-directed PYR1 mutants designed to disrupt hormone binding lose ABA-triggered interactions with type 2C protein phosphatase partners in planta.

Identification of a New Cryptochrome Class: Structure, Function, and Evolution

Cryptochrome flavoproteins, which share sequence homology with light-dependent DNA repair photolyases, function as photoreceptors in plants and circadian clock components in animals. Here, we coupled sequencing of an Arabidopsis cryptochrome gene with phylogenetic, structural, and functional analyses to identify a new cryptochrome class (cryptochrome DASH) in bacteria and plants, suggesting that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. The cryptochrome crystallographic structure, reported here for Synechocystis cryptochrome DASH, reveals commonalities with photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features. Whole genome transcriptional profiling together with experimental confirmation of DNA binding indicated that Synechocystis cryptochrome DASH functions as a transcriptional repressor.

Plant UVR8 Photoreceptor Senses UV-B by Tryptophan-Mediated Disruption of Cross-Dimer Salt Bridges

Donuts Dissociate In Arabidopsis, the UVR8 protein responds to ultraviolet-B (UV-B) light by dissociating into monomers, which are then available to interact with downstream factors that enact the plants response to light. Christie et al. (p. 1492, published online 9 February; see the cover and see the Perspective by Gardner and Correa) have now determined the crystal structure of UVR8. Without ultraviolet-B light, UVR8 dimerizes, with two donut-shaped monomers joined by a network of salt bridges. Close-packing of a pyramid of tryptophan residues permits exciton coupling that is key to UV-B perception. Electron transfer after UV-B perception could dissociate the salt bridges that hold the dimer together and release monomeric UVR8 to initiate light-induced signaling. A tryptophan pyramid allows a dimeric protein to perceive ultraviolet light without an additional chromophore. The recently identified plant photoreceptor UVR8 (UV RESISTANCE LOCUS 8) triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light through an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine retunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.

Binding and Catalytic Properties of Xenopus (6-4) Photolyase

Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6–4]T in double-stranded DNA with high affinity (K D = 10−9) and to T[6–4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (K D = ∼2 × 10−8). Majority of the T[6–4]T-(6-4) photolyase complex dissociates very slowly (k off = 2.9 × 10−5s−1). Its absolute action spectrum without a second chromophore in the 350–600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (φ) of repair is approximately 0.11. The fully reduced form (E-FADH−) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis,syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.

FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis

Abscisic acid (ABA) is a plant hormone that regulates plant growth and development and mediates abiotic stress responses. Direct cellular monitoring of dynamic ABA concentration changes in response to environmental cues is essential for understanding ABA action. We have developed ABAleons: ABA-specific optogenetic reporters that instantaneously convert the phytohormone-triggered interaction of ABA receptors with PP2C-type phosphatases to send a fluorescence resonance energy transfer (FRET) signal in response to ABA. We report the design, engineering and use of ABAleons with ABA affinities in the range of 100–600 nM to map ABA concentration changes in plant tissues with spatial and temporal resolution. High ABAleon expression can partially repress Arabidopsis ABA responses. ABAleons report ABA concentration differences in distinct cell types, ABA concentration increases in response to low humidity and NaCl in guard cells and to NaCl and osmotic stress in roots and ABA transport from the hypocotyl to the shoot and root. DOI: http://dx.doi.org/10.7554/eLife.01739.001

Direct observation of a photoinduced radical pair in a cryptochrome blue-light photoreceptor.

Proteins from the photolyase/cryptochrome family share their three-dimensional fold, sequence homology, and the redox-active flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse activities.[1] In response to blue or UV-A light, they function physiologically in DNA repair, entrainment of the circadian clock, or other processes.[1-3] Members of the photolyase/cryptochrome family have been identified in various organisms ranging from bacteria to plants, animals and humans.[1] Within this family, a phylogenetic cluster of genes originally identified from Arabidopsis and Synechocystis encode cryptochrome-like proteins, which are distinct from previously characterized “classic” plant (represented by Arabidopsis HY4) or animal (represented by Drosophila and Homo sapiens) cryptochromes, yet more closely resemble the latter.[4] Remarkably, the cryptochromes from this new cluster (Cry-DASH) have now been found through all kingdoms of life.[5] While multiple biological functions have been discussed, the availability of stable, recombinantly expressed, Cry-DASH proteins from diverse species provides the means of deciphering cryptochrome protein chemistry. Results from recent experiments point to the direction that Cry-DASH could work as a transcriptional regulator,[4, 5] as well as a DNA repair enzyme for single-stranded DNA.[6] Other experimental results suggest the participation of Cry-DASH in circadian input pathways.[7, 8]

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

Homologous flavoproteins from the photolyase (PHR)/cryptochrome (CRY) family use the FAD cofactor in PHRs to catalyze DNA repair and in CRYs to tune the circadian clock and control development. To help address how PHR/CRY members achieve these diverse functions, we determined the crystallographic structure of Arabidopsis thaliana (6-4) PHR (UVR3), which is strikingly (>65%) similar in sequence to human circadian clock CRYs. The structure reveals a substrate-binding cavity specific for the UV-induced DNA lesion, (6-4) photoproduct, and cofactor binding sites different from those of bacterial PHRs and consistent with distinct mechanisms for activities and regulation. Mutational analyses were combined with this prototypic structure for the (6-4) PHR/clock CRY cluster to identify structural and functional motifs: phosphate-binding and Pro-Lys-Leu protrusion motifs constricting access to the substrate-binding cavity above FAD, sulfur loop near the external end of the Trp electron-transfer pathway, and previously undefined C-terminal helix. Our results provide a detailed, unified framework for investigations of (6-4) PHRs and the mammalian CRYs. Conservation of key residues and motifs controlling FAD access and activities suggests that regulation of FAD redox properties and radical stability is essential not only for (6-4) photoproduct DNA repair, but also for circadian clock-regulating CRY functions. The structural and functional results reported here elucidate archetypal relationships within this flavoprotein family and suggest how PHRs and CRYs use local residue and cofactor tuning, rather than larger structural modifications, to achieve their diverse functions encompassing DNA repair, plant growth and development, and circadian clock regulation.

Structural Tuning of the Fluorescent Protein iLOV for Improved Photostability

Background: iLOV is a fluorescent flavoprotein engineered from the plant blue light receptor phototropin. Results: Structures reveal altered protein-chromophore interactions within the flavin-binding cavity of iLOV when compared with its progenitors. Directed evolution further anchored the chromophore to increase iLOV photostability by an order of magnitude. Conclusion: Improving iLOV photostability by constraining its fluorophore establishes a framework for fine-tuning fluorescence. Significance: Enhanced photostability increases iLOV utility as an oxygen-independent fluorescent reporter. Fluorescent proteins derived from light, oxygen, or voltage (LOV) domains offer advantages over green fluorescent protein (GFP) from their small size and efficacy under anaerobic conditions. The flavoprotein improved LOV (iLOV) was engineered from the blue light receptor phototropin as a reporter of viral infection. To inform the molecular basis for the improved, photoreversible, fluorescent properties of iLOV, we employed directed evolution and determined five LOV crystallographic structures. Comparative structural analyses between iLOV and its progenitors reveal mutation-induced constraints in the environment of the flavin mononucleotide (FMN) chromophore; in iLOV, the methyl group of Thr-394 “crowds” the FMN isoalloxazine ring, Leu-470 triggers side chain “flipping” of Leu-472, and the terminal FMN phosphate shows increased anchoring. We further engineered iLOV variants that are readily detectable in bacterial and mammalian cells due to order-of-magnitude photostability increases. Structure determination of a resulting representative photostable iLOV (phiLOV) variant reveals additional constraints on the chromophore. Aromatic residues Tyr-401 and Phe-485 in phiLOV sandwich the FMN isoalloxazine ring from both sides, whereas Ser-390 anchors the side chain of FMN-interacting Gln-489 Our combined structural and mutational results reveal that constraining the FMN fluorophore yields improved photochemical properties for iLOV and its new photostable derivative. These findings provide a framework for structural fine-tuning of LOV scaffold proteins to maximize their potential as oxygen-independent fluorescent reporters.

Tetrameric architecture of the circadian clock protein KaiB. A novel interface for intermolecular interactions and its impact on the circadian rhythm.

Cyanobacteria are among the simplest organisms that show daily rhythmicity. Their circadian rhythms consist of the localization, interaction, and accumulation of various proteins, including KaiA, KaiB, KaiC, and SasA. We have determined the 1.9-Å resolution crystallographic structure of the cyanobacterial KaiB clock protein from Synechocystis sp. PCC6803. This homotetrameric structure reveals a novel KaiB interface for protein-protein interaction; the protruding hydrophobic helix-turn-helix motif of one subunit fits into a groove between two β-strands of the adjacent subunit. A cyanobacterial mutant, in which the Asp-Lys salt bridge mediating this tetramer-forming interaction is disrupted by mutation of Asp to Gly, exhibits severely impaired rhythmicity (a short free-running period; ∼19 h). The KaiB tetramer forms an open square, with positively charged residues around the perimeter. KaiB is localized on the phospholipid-rich membrane and translocates to the cytosol to interact with the other Kai components, KaiA and KaiC. KaiB antagonizes the action of KaiA on KaiC, and shares a sequence-homologous domain with the SasA kinase. Based on our structure, we discuss functional roles for KaiB in the circadian clock.