The structure of Arabidopsis phytochrome A reveals topological and functional diversification among the plant photoreceptor isoforms.

Plants employ a divergent cohort of phytochrome (Phy) photoreceptors to govern many aspects of morphogenesis through reversible photointerconversion between inactive Pr and active Pfr conformers. The two most influential are PhyA whose retention of Pfr enables sensation of dim light, while the relative instability of Pfr for PhyB makes it better suited for detecting full sun and temperature. To better understand these contrasts, we solved, by cryo-electron microscopy, the three-dimensional structure of full-length PhyA as Pr. Like PhyB, PhyA dimerizes through head-to-head assembly of its C-terminal histidine kinase-related domains (HKRDs), while the remainder assembles as a head-to-tail light-responsive platform. Whereas the platform and HKRDs associate asymmetrically in PhyB dimers, these lopsided connections are absent in PhyA. Analysis of truncation and site-directed mutants revealed that this decoupling and altered platform assembly have functional consequences for Pfr stability of PhyA and highlights how plant Phy structural diversification has extended light and temperature perception.

Mechanisms of UV-B light-induced photoreceptor UVR8 nuclear localization dynamics.

Light regulates the subcellular localization of plant photoreceptors, a key step in light signaling. Ultraviolet-B radiation (UV-B) induces the plant photoreceptor UV RESISTANCE LOCUS 8 (UVR8) nuclear accumulation, where it regulates photomorphogenesis. However, the molecular mechanism for the UV-B-regulated UVR8 nuclear localization dynamics is unknown. With fluorescence recovery after photobleaching (FRAP), cell fractionation followed by immunoblotting and co-immunoprecipitation (Co-IP) assays we tested the function of UVR8-interacting proteins including CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 in the regulation of UVR8 nuclear dynamics in Arabidopsis thaliana. We showed that UV-B-induced rapid UVR8 nuclear translocation is independent of COP1, which previously was shown to be required for UV-B-induced UVR8 nuclear accumulation. Instead, we provide evidence that the UV-B-induced UVR8 homodimer-to-monomer photo-switch and the concurrent size reduction of UVR8 enables its monomer nuclear translocation, most likely via free diffusion. Nuclear COP1 interacts with UV-B-activated UVR8 monomer, thereby promoting UVR8 nuclear retention. Conversely, RUP1and RUP2, whose expressions are induced by UV-B, inhibit UVR8 nuclear retention via attenuating the UVR8-COP1 interaction, allowing UVR8 to exit the nucleus. Collectively, our data suggest that UV-B-induced monomerization of UVR8 promotes its nuclear translocation via free diffusion. In the nucleus, COP1 binding promotes UVR8 monomer nuclear retention, which is counterbalanced by the major negative regulators RUP1 and RUP2.

Plant photoreceptors and their signalling components in chloroplastic anterograde and retrograde communication.

The red phytochrome and blue cryptochrome plant photoreceptors play essential roles in promoting genome-wide changes in nuclear and chloroplastic gene expression for photomorphogenesis, plastid development, and greening. While their importance in anterograde signalling has been long recognized, the molecular mechanisms involved remain under active investigation. More recently, the intertwining of the light signalling cascades with the retrograde signals for the optimization of chloroplast functions has been acknowledged. Advances in the field support the participation of phytochromes, cryptochromes, and key light-modulated transcription factors, including HY5 and the PIFs, in the regulation of chloroplastic biochemical pathways that produce retrograde signals, including the tetrapyrroles and the chloroplastic MEP-isoprenoids. Interestingly, in a feedback loop, the photoreceptors and their signalling components are targets themselves of these retrograde signals, aimed at optimizing photomorphogenesis to the status of the chloroplasts, with GUN proteins functioning at the convergence points. High light and shade are also conditions where the photoreceptors tune growth responses to chloroplast functions. Interestingly, photoreceptors and retrograde signals also converge in the modulation of dual-localized proteins (chloroplastic/nuclear) including WHIRLY and HEMERA/pTAC12, whose functions are required for the optimization of photosynthetic activities in changing environments and are proposed to act themselves as retrograde signals.

Physiological Analysis of Phototropic Responses to Blue and Red Light in Arabidopsis.

Plants utilize light as sole energy source. To maximize light capture, they are able to detect the light direction and orient themselves toward the light source. This phototropic response is mediated by the plant blue-light photoreceptors phototropin1 and phototropin2 (phot1 and phot2). Although fully differentiated plants also exhibit this response, it can be best observed in etiolated seedlings. Differences in light between the illuminated and shaded site of a seedling stem lead to changes in the auxin distribution, resulting in cell elongation on the shaded site. Since phototropism connects light perception, signaling, and auxin transport, it is of great interest to analyze this response with a fast and simple method. Moreover, pre-exposure to red light enhances the phototropic response via phytochrome A (phyA) and phyB action. Here we describe a method to analyze the phototropic response of Arabidopsis seedlings to blue light and the enhanced response with a red-light pretreatment. With numerous mutants available, its fast germination, and its small size, Arabidopsis is well suited for this analysis. Different genotypes can be simultaneously probed in less than a week.

Optogenetic and Chemical Induction Systems for Regulation of Transgene Expression in Plants: Use in Basic and Applied Research.

Continuous and ubiquitous expression of foreign genes sometimes results in harmful effects on the growth, development and metabolic activities of plants. Tissue-specific promoters help to overcome this disadvantage, but do not allow one to precisely control transgene expression over time. Thus, inducible transgene expression systems have obvious benefits. In plants, transcriptional regulation is usually driven by chemical agents under the control of chemically-inducible promoters. These systems are diverse, but usually contain two elements, the chimeric transcription factor and the reporter gene. The commonly used chemically-induced expression systems are tetracycline-, steroid-, insecticide-, copper-, and ethanol-regulated. Unlike chemical-inducible systems, optogenetic tools enable spatiotemporal, quantitative and reversible control over transgene expression with light, overcoming limitations of chemically-inducible systems. This review updates and summarizes optogenetic and chemical induction methods of transgene expression used in basic plant research and discusses their potential in field applications.

A photoregulatory mechanism of the circadian clock in Arabidopsis.

Cryptochromes (CRYs) are photoreceptors that mediate light regulation of the circadian clock in plants and animals. Here we show that CRYs mediate blue-light regulation of N6-methyladenosine (m6A) modification of more than 10% of messenger RNAs in the Arabidopsis transcriptome, especially those regulated by the circadian clock. CRY2 interacts with three subunits of the METTL3/14-type N6-methyladenosine RNA methyltransferase (m6A writer): MTA, MTB and FIP37. Photo-excited CRY2 undergoes liquid-liquid phase separation (LLPS) to co-condense m6A writer proteins in vivo, without obviously altering the affinity between CRY2 and the writer proteins. mta and cry1cry2 mutants share common defects of a lengthened circadian period, reduced m6A RNA methylation and accelerated degradation of mRNA encoding the core component of the molecular oscillator circadian clock associated 1 (CCA1). These results argue for a photoregulatory mechanism by which light-induced phase separation of CRYs modulates m6A writer activity, mRNA methylation and abundance, and the circadian rhythms in plants.

Cryptochromes are the dominant photoreceptors mediating heliotropic responses of Arabidopsis inflorescences.

Inflorescence movements in response to natural gradients of sunlight are frequently observed in the plant kingdom and are suggested to contribute to reproductive success. Although the physiological and molecular bases of light-mediated tropisms in vegetative organs have been thoroughly investigated, the mechanisms that control inflorescence orientation in response to light gradients under natural conditions are not well understood. In this work, we have used a combination of laboratory and field experiments to investigate light-mediated re-orientation of Arabidopsis thaliana inflorescences. We show that inflorescence phototropism is promoted by photons in the UV and blue spectral range (≤500 nm) and depends on multiple photoreceptor families. Experiments under controlled conditions show that UVR8 is the main photoreceptor mediating the phototropic response to narrowband UV-B radiation, and phototropins and cryptochromes control the response to narrowband blue light. Interestingly, whereas phototropins mediate bending in response to low irradiances of blue, cryptochromes are the principal photoreceptors acting at high irradiances. Moreover, phototropins negatively regulate the action of cryptochromes at high irradiances of blue light. Experiments under natural field conditions demonstrate that cryptochromes are the principal photoreceptors acting in the promotion of the heliotropic response of inflorescences under full sunlight.

Identification of a dual orange/far-red and blue light photoreceptor from an oceanic green picoplankton.

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities.

Grapevine morphological shade acclimation is mediated by light quality whereas hydraulic shade acclimation is mediated by light intensity.

Plants acclimate to shade by sensing light signals such as low photosynthetic active radiation (PAR), low blue light (BL) levels and low red-to-far red ratios (R:FR) trough plant photoreceptors cross talk. We previously demonstrated that grapevine is irresponsive to variations in R:FR and that BL-attenuation mediates morphological and architectural responses to shade increasing light interception and absorption efficiencies. However, we wondered if grapevine respond to low R:FR when BL is attenuated at the same time. Our objective was to evaluate if morphological, architectural and hydraulic acclimation to shade is mediated by low R:FR ratios and BL attenuation. To test this, we carried out experiments under natural radiation, manipulating light quality by selective sunlight exclusion and light supplementation. We grew grapevines under low PAR (LP) and four high PAR (HP) treatments: HP, HP plus FR supplementation (HP + FR), HP with BL attenuation (HP-B) and HP with BL attenuation plus FR supplementation (HP-B + FR). We found that plants grown under HP-B and HP-B + FR had similar morphological (stem and petiole length, leaf thickness and area), architectural (laminae' angles) and anatomical (stomatal density) traits than plants grown under LP. However, only LP plants presented lower stomata differentiation, lower δ13C and hence lower water use efficiency. Therefore, even under a BL and R:FR attenuated environment, morphological and architectural responses were modulated by BL but not by variation in R:FR. Meanwhile water relations were affected by PAR intensity but not by changes in light quality. Knowing grapevine responses to light quantity and quality are indispensable to adopt tools or design new cultural management practices that manipulate irradiance in the field intending to improve crop performance.

Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

Phytochrome regulates cellular response plasticity and the basic molecular machinery of leaf development.

Plants are plastic organisms that optimize growth in response to a changing environment. This adaptive capability is regulated by external cues, including light, which provides vital information about the habitat. Phytochrome photoreceptors detect far-red light, indicative of nearby vegetation, and elicit the adaptive shade-avoidance syndrome (SAS), which is critical for plant survival. Plants exhibiting SAS are typically more elongated, with distinctive, small, narrow leaf blades. By applying SAS-inducing end-of-day far-red (EoD FR) treatments at different times during Arabidopsis (Arabidopsis thaliana) leaf 3 development, we have shown that SAS restricts leaf blade size through two distinct cellular strategies. Early SAS induction limits cell division, while later exposure limits cell expansion. This flexible strategy enables phytochromes to maintain control of leaf size through the proliferative and expansion phases of leaf growth. mRNAseq time course data, accessible through a community resource, coupled to a bioinformatics pipeline, identified pathways that underlie these dramatic changes in leaf growth. Phytochrome regulates a suite of major development pathways that control cell division, expansion, and cell fate. Further, phytochromes control cell proliferation through synchronous regulation of the cell cycle, DNA replication, DNA repair, and cytokinesis, and play an important role in sustaining ribosome biogenesis and translation throughout leaf development.

Detection of UVR8 Homodimers and Monomers by Immunoblotting Analysis in Tomato.

The UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor mediating photomorphogenic responses to UV-B. UVR8 exists as homodimer in plants and UV-B induces dissociation of dimeric UVR8 into monomers to initiate responses. The monomer/dimer status of UVR8 is reversible and a dynamic photo-equilibrium is established in plants according to the ambient light conditions. Here we describe a method to detect UVR8 homodimer and monomer by immunoblotting method from tomato (Solanum lycopersicum) plants. The feature of this method is that protein samples are not boiled prior to loading on an SDS-PAGE gel, which allows the detection of UVR8 homodimer and monomers simultaneously with a single antibody.

A constitutively monomeric UVR8 photoreceptor confers enhanced UV-B photomorphogenesis.

The plant ultraviolet-B (UV-B) photoreceptor UVR8 plays an important role in UV-B acclimation and survival. UV-B absorption by homodimeric UVR8 induces its monomerization and interaction with the E3 ubiquitin ligase COP1, leading ultimately to gene expression changes. UVR8 is inactivated through redimerization, facilitated by RUP1 and RUP2. Here, we describe a semidominant, hyperactive allele, namely uvr8-17D, that harbors a glycine-101 to serine mutation. UVR8G101S overexpression led to weak constitutive photomorphogenesis and extreme UV-B responsiveness. UVR8G101S was observed to be predominantly monomeric in vivo and, once activated by UV-B, was not efficiently inactivated. Analysis of a UVR8 crystal structure containing the G101S mutation revealed the distortion of a loop region normally involved in stabilization of the UVR8 homodimer. Plants expressing a UVR8 variant combining G101S with the previously described W285A mutation exhibited robust constitutive photomorphogenesis. This work provides further insight into UVR8 activation and inactivation mechanisms and describes a genetic tool for the manipulation of photomorphogenic responses.

Expression of Tomato UVR8 in Arabidopsis reveals conserved photoreceptor function.

UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor that regulates UV-B photomorphogenesis in plants. UV-B photon perception promotes UVR8 homodimer dissociation into monomer, which is reverted to homodimer post UV-B, forming a complete photocycle. UVR8 monomer interacts with CONSTITUTIVELY PHOTOMORPHOGENEIC 1 (COP1) to initiate UV-B signaling. The function and mechanism of Arabidopsis UVR8 (AtUVR8) are extensively investigated, however, little is known about UVR8 and its signaling mechanisms in other plant species. Tomato is a widely used model plant for horticulture research. In this report we tested whether an ortholog of AtUVR8 in Tomato (SIUVR8) can complement Arabidopsis uvr8 mutant and whether the above-mentioned key signaling mechanisms of UVR8 are conserved. Heterologous expressed SIUVR8 in an Arabidopsis uvr8 null mutant rescued the uvr8 mutant in the tested UV-B responses including hypocotyl elongation, UV-B target gene expression and anthocyanin accumulation, demonstrating that the SIUVR8 is a putative UV-B photoreceptor. Moreover, in response to UV-B, SIUVR8 forms a protein complex with Arabidopsis COP1 in plants, suggesting conserved signaling mechanism. SIUVR8 exhibits similar photocycle as AtUVR8 in plants, which highlights conserved photoreceptor activation and inactivation mechanisms.

Control of Plant Growth and Defense by Photoreceptors: From Mechanisms to Opportunities in Agriculture.

Plants detect and respond to the proximity of competitors using light signals perceived by photoreceptor proteins. A low ratio of red to far-red radiation (R:FR ratio) is a key signal of competition that is sensed by the photoreceptor phytochrome B (phyB). Low R:FR ratios increase the synthesis of growth-related hormones, including auxin and gibberellins, promoting stem elongation and other shade-avoidance responses. Other photoreceptors that help plants to optimize their developmental configuration and resource allocation patterns in the canopy include blue light photoreceptors, such as cryptochromes and phototropins, and UV receptors, such as UVR8. All photoreceptors act by directly or indirectly controlling the activity of two major regulatory nodes for growth and development: the COP1/SPA ubiquitin E3 ligase complex and the PIF transcription factors. phyB is also an important modulator of hormonal pathways that regulate plant defense against herbivores and pathogens, including the jasmonic acid signaling pathway. In this Perspective, we discuss recent advances on the studies of the mechanisms that link photoreceptors with growth and defense. Understanding these mechanisms is important to provide a functional platform for breeding programs aimed at improving plant productivity, stress tolerance, and crop health in species of agronomic interest, and to manipulate the light environments in protected agriculture.

A leap in quantum efficiency through light harvesting in photoreceptor UVR8.

Plants utilize a UV-B (280 to 315 nm) photoreceptor UVR8 (UV RESISTANCE LOCUS 8) to sense environmental UV levels and regulate gene expression to avoid harmful UV effects. Uniquely, UVR8 uses intrinsic tryptophan for UV-B perception with a homodimer structure containing 26 structural tryptophan residues. However, besides 8 tryptophans at the dimer interface to form two critical pyramid perception centers, the other 18 tryptophans' functional role is unknown. Here, using ultrafast fluorescence spectroscopy, computational methods and extensive mutations, we find that all 18 tryptophans form light-harvesting networks and funnel their excitation energy to the pyramid centers to enhance light-perception efficiency. We determine the timescales of all elementary tryptophan-to-tryptophan energy-transfer steps in picoseconds to nanoseconds, in excellent agreement with quantum computational calculations, and finally reveal a significant leap in light-perception quantum efficiency from 35% to 73%. This photoreceptor is the first system discovered so far, to be best of our knowledge, using natural amino-acid tryptophans to form networks for both light harvesting and light perception.

Regulation of PIF4-mediated thermosensory growth.

Ambient temperature has profound impacts on almost every aspect of plant growth and development, including seed germination, stem and petiole elongation, leaf movement, stomata development, flowering, and pathogen defense. Although the signal transduction pathways underlying plant responses to extreme cold and heat temperatures have been well studied, our understanding, at the molecular level, of how plants adjust phenotypic plasticity in response to nonstressful ambient temperature is still rudimentary. This review summarizes studies related to PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), the cardinal regulator of thermoresponsive growth in the model dicotyledonous plant Arabidopsis thaliana, emphasizing recent progress in the light-quality- and photoperiod-dependent regulation of PIF4-mediated thermomorphogenesis.

An Optogenetic Tool for Induced Protein Stabilization Based on the Phaeodactylum tricornutum Aureochrome 1a Light-Oxygen-Voltage Domain.

Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light-oxygen-voltage (LOV) domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254, and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark-state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic adenosine monophosphate production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.