Characterization of Thermoresponsive Photobody Dynamics.

Photobodies (PBs) are subnuclear membraneless organelles that self-assemble via the condensation of the plant photoreceptor and thermosensor phytochrome B (phyB). Changes in the light and temperature environment directly modulate PB formation and maintenance by altering the number and size of PBs. In thermomorphogenesis, increases in the ambient temperature incrementally reduce the number of PBs, suggesting that individual PBs possess distinct thermostabilities. Here, we describe a detailed protocol for characterizing cell type-specific PB dynamics induced by warm temperatures in Arabidopsis.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

OptoREACT: Optogenetic Receptor Activation on Nonengineered Human T Cells.

Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

The crosstalk of far-red energy and signaling defines the regulation of photosynthesis, growth, and flowering in tomatoes.

This study investigated the effect of light intensity and signaling on the regulation of far-red (FR)-induced alteration in photosynthesis. The low (LL: 440 µmol m-2 s-1) and high (HL: 1135 µmol m-2 s-1) intensity of white light with or without FR (LLFR: 545 µmol m-2 s-1 including 115 µmol m-2 s-1; HLFR: 1254 µmol m-2 s-1 + 140 µmol m-2 s-1) was applied on the tomato cultivar (Solanum Lycopersicon cv. Moneymaker) and mutants of phytochrome A (phyA) and phytochrome B (phyB1, and phyB2). Both light intensity and FR affected plant morphological traits, leaf biomass, and flowering time. Irrespective of genotype, flowering was delayed by LLFR and accelerated by HLFR compared to the corresponding light intensity without FR. In LLFR, a reduced energy flux through the electron transfer chain along with a reduced energy dissipation per reaction center improved the maximum quantum yield of PSII, irrespective of genotype. HLFR increased net photosynthesis and gas exchange properties in a genotype-dependent manner. FR-dependent regulation of hormones was affected by light signaling. It appeared that PHYB affected the levels of abscisic acid and salicylic acid while PHYA took part in the regulation of CK in FR-exposed plants. Overall, light intensity and signaling of FR influenced plants' photosynthesis and growth by altering electron transport, gas exchange, and changes in the level of endogenous hormones.

Expression, purification and crystallization of the photosensory module of phytochrome B (phyB) from Sorghum bicolor.

Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1-Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1-655, 114-655 and 114-458, respectively), each C-terminally tagged with His6, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Šresolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Šresolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.

COP1 controls light-dependent chromatin remodeling.

Light is a crucial environmental factor that impacts various aspects of plant development. Phytochromes, as light sensors, regulate myriads of downstream genes to mediate developmental reprogramming in response to changes in environmental conditions. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is an E3 ligase for a number of substrates in light signaling, acting as a central repressor of photomorphogenesis. The interplay between phytochrome B (phyB) and COP1 forms an antagonistic regulatory module that triggers extensive gene expression reprogramming when exposed to light. Here, we uncover a role of COP1 in light-dependent chromatin remodeling through the regulation of VIL1 (VIN3-LIKE 1)/VERNALIZATION 5, a Polycomb protein. VIL1 directly interacts with phyB and regulates photomorphogenesis through the formation of repressive chromatin loops at downstream growth-promoting genes in response to light. Furthermore, we reveal that COP1 governs light-dependent formation of chromatin loop and limiting a repressive histone modification to fine-tune expressions of growth-promoting genes during photomorphogenesis through VIL1.

Phytochrome B inhibits the activity of phytochrome-interacting factor 7 involving phase separation.

Shade-intolerant plants sense changes in the light environment and trigger shade-avoidance syndrome in the presence of neighboring vegetation. Phytochrome-interacting factor 7 (PIF7) is an essential regulator that integrates shade signals into plant transcriptional networks. While the regulation of PIF7 under shade conditions has been well studied, the mechanism that represses PIF7 activity under white light remains ambiguous. Here, we report that PIF7 forms nuclear puncta containing phase-separated liquid-like condensates. Phytochrome B (phyB) then binds to dephosphorylated PIF7 and promotes its condensed phase of PIF7 under white light. The phyB-PIF7 condensate subsequently inhibits the DNA-binding activity of PIF7. However, shade inactivation of phyB causes the dissociation of phyB-PIF7 condensates and allows unbound PIF7 to promote the transcription of shade-induced genes. This reversible transcriptional condensation via phase separation provides sessile organisms with the flexibility of gene control to adapt to their surrounding environment.

Engineering of an Optogenetic T Cell Receptor Compatible with Fluorescence-Based Readouts.

Optogenetics offers a set of tools for the precise manipulation of signaling pathways. Here we exploit optogenetics to experimentally change the kinetics of protein-protein interactions on demand. We had developed a system in which the interaction of a modified T cell receptor (TCR) with an engineered ligand can be controlled by light. The ligand was the plant photoreceptor phytochrome B (PhyB) and the TCR included a TCRß chain fused to GFP and a mutated PhyB-interacting factor (PIFS), resulting in the GFP-PIFS-TCR. We failed to engineer a nonfluorescent PIFS-fused TCR, since PIFS did not bind to PhyB when omitting GFP. Here we tested nine different versions of PIFS-fused TCRs. We found that the SNAP-PIFS-TCR was expressed well on the surface, bound to PhyB, and subsequently elicited activation signals. This receptor could be combined with a GFP reporter system in which the expression of GFP is driven by the transcription factor NF-AT.

FERONIA, the kinase that phosphorylates PhyB.

The phosphorylation status of phyB changes dynamically in response to environmental conditions and critically governs the corresponding plant's responses. However, the kinase(s) that phosphorylates phyB is/are still unknown. Liu et al. have not only identified the kinase that phosphorylates phyB but also revealed its biological implications during salt stress.

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.

EARLY FLOWERING 3 interactions with PHYTOCHROME B and PHOTOPERIOD1 are critical for the photoperiodic regulation of wheat heading time.

The photoperiodic response is critical for plants to adjust their reproductive phase to the most favorable season. Wheat heads earlier under long days (LD) than under short days (SD) and this difference is mainly regulated by the PHOTOPERIOD1 (PPD1) gene. Tetraploid wheat plants carrying the Ppd-A1a allele with a large deletion in the promoter head earlier under SD than plants carrying the wildtype Ppd-A1b allele with an intact promoter. Phytochromes PHYB and PHYC are necessary for the light activation of PPD1, and mutations in either of these genes result in the downregulation of PPD1 and very late heading time. We show here that both effects are reverted when the phyB mutant is combined with loss-of-function mutations in EARLY FLOWERING 3 (ELF3), a component of the Evening Complex (EC) in the circadian clock. We also show that the wheat ELF3 protein interacts with PHYB and PHYC, is rapidly modified by light, and binds to the PPD1 promoter in planta (likely as part of the EC). Deletion of the ELF3 binding region in the Ppd-A1a promoter results in PPD1 upregulation at dawn, similar to PPD1 alleles with intact promoters in the elf3 mutant background. The upregulation of PPD1 is correlated with the upregulation of the florigen gene FLOWERING LOCUS T1 (FT1) and early heading time. Loss-of-function mutations in PPD1 result in the downregulation of FT1 and delayed heading, even when combined with the elf3 mutation. Taken together, these results indicate that ELF3 operates downstream of PHYB as a direct transcriptional repressor of PPD1, and that this repression is relaxed both by light and by the deletion of the ELF3 binding region in the Ppd-A1a promoter. In summary, the regulation of the light mediated activation of PPD1 by ELF3 is critical for the photoperiodic regulation of wheat heading time.

Two Distinct Molecular Types of Phytochrome A in Plants: Evidence of Existence and Implications for Functioning.

Phytochrome (phy) system in plants comprising a small number of phytochromes with phyA and phyB as major ones is responsible for acquiring light information in the red-far-red region of the solar spectrum. It provides optimal strategy for plant development under changing light conditions throughout all its life cycle beginning from seed germination and seedling establishment to fruiting and plant senescence. The phyA was shown to participate in the regulation of this cycle which is especially evident at its early stages. It mediates three modes of reactions-the very low and low fluence responses (VLFR and LFR) and the high irradiance responses (HIR). The phyA is the sole light receptor in the far-red spectral region responsible for plant's survival under a dense plant canopy where light is enriched with the far-red component. Its appearance is believed to be one of the main factors of plants' successful evolution. So far, it is widely accepted that one molecular phyA species is responsible for its complex functional manifestations. In this review, the evidence of the existence of two distinct phyA types-major, light-labile and soluble phyA' and minor, relatively light-stable and amphiphilic phyA″-is presented as what may account for the diverse modes of phyA action.

SALT OVERLY SENSITIVE2 stabilizes phytochrome-interacting factors PIF4 and PIF5 to promote Arabidopsis shade avoidance.

Sun-loving plants trigger the shade avoidance syndrome (SAS) to compete against their neighbors for sunlight. Phytochromes are plant red (R) and far-red (FR) light photoreceptors that play a major role in perceiving the shading signals and triggering SAS. Shade induces a reduction in the level of active phytochrome B (phyB), thus increasing the abundance of PHYTOCHROME-INTERACTING FACTORS (PIFs), a group of growth-promoting transcription factors. However, whether other factors are involved in modulating PIF activity in the shade remains largely obscure. Here, we show that SALT OVERLY SENSITIVE2 (SOS2), a protein kinase essential for salt tolerance, positively regulates SAS in Arabidopsis thaliana. SOS2 directly phosphorylates PIF4 and PIF5 at a serine residue close to their conserved motif for binding to active phyB. This phosphorylation thus decreases their interaction with phyB and posttranslationally promotes PIF4 and PIF5 protein accumulation. Notably, the role of SOS2 in regulating PIF4 and PIF5 protein abundance and SAS is more prominent under salt stress. Moreover, phyA and phyB physically interact with SOS2 and promote SOS2 kinase activity in the light. Collectively, our study uncovers an unexpected role of salt-activated SOS2 in promoting SAS by modulating the phyB-PIF module, providing insight into the coordinated response of plants to salt stress and shade.

The Arabidopsis endosperm is a temperature-sensing tissue that implements seed thermoinhibition through phyB.

Seed thermoinhibition, the repression of germination under high temperatures, prevents seedling establishment under potentially fatal conditions. Thermoinhibition is relevant for phenology and agriculture, particularly in a warming globe. The temperature sensing mechanisms and signaling pathways sustaining thermoinhibition are unknown. Here we show that thermoinhibition in Arabidopsis thaliana is not autonomously controlled by the embryo but is rather implemented by the endosperm. High temperature is sensed through endospermic phyB by accelerating its reversion from the active signaling Pfr form into the inactive Pr form, as previously described in seedlings. This leads to thermoinhibition mediated by PIFs, mainly PIF1, PIF3 and PIF5. Endospermic PIF3 represses the expression of the endospermic ABA catabolic gene CYP707A1 and promotes endospermic ABA accumulation and release towards the embryo to block its growth. Furthermore, endospermic ABA represses embryonic PIF3 accumulation that would otherwise promote embryonic growth. Hence, under high temperatures PIF3 exerts opposite growth responses in the endosperm and embryo.

FAR-RED INSENSITIVE 219 and phytochrome B corepress shade avoidance via modulating nuclear speckle formation.

Plants can sense the shade from neighboring plants by detecting a reduction of the red:far-red light (R:FR) ratio. Phytochrome B (phyB) is the primary photoreceptor that perceives shade light and regulates jasmonic acid (JA) signaling. However, the molecular mechanisms underlying phyB and JA signaling integration in shade responses remain largely unknown. Here, we show the interaction of phyB and FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) in a functional demand manner in Arabidopsis (Arabidopsis thaliana) seedling development. Genetic evidence and interaction studies indicated that phyB and FIN219 synergistically and negatively regulate shade-induced hypocotyl elongation. Moreover, phyB interacted with various isoforms of FIN219 under high and low R:FR light. Methyl jasmonate (MeJA) treatment, FIN219 mutation, and PHYBOE digalactosyldiacylglycerol synthase1-1 (dgd1-1) plants, which show increased levels of JA, altered the patterns of phyB-associated nuclear speckles under the same conditions. Surprisingly, PHYBOE dgd1-1 showed a shorter hypocotyl phenotype than its parental mutants under shade conditions. Microarray assays using PHYBOE and PHYBOE fin219-2 indicated that PHYB overexpression substantially affects defense response-related genes under shade light and coregulates expression of auxin-responsive genes with FIN219. Thus, our findings reveal that phyB substantially crosstalks with JA signaling through FIN219 to modulate seedling development under shade light.

Phytochrome B photobodies are comprised of phytochrome B and its primary and secondary interacting proteins.

Phytochrome B (phyB) is a plant photoreceptor that forms a membraneless organelle called a photobody. However, its constituents are not fully known. Here, we isolated phyB photobodies from Arabidopsis leaves using fluorescence-activated particle sorting and analyzed their components. We found that a photobody comprises ~1,500 phyB dimers along with other proteins that could be classified into two groups: The first includes proteins that directly interact with phyB and localize to the photobody when expressed in protoplasts, while the second includes proteins that interact with the first group proteins and require co-expression of a first-group protein to localize to the photobody. As an example of the second group, TOPLESS interacts with PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and localizes to the photobody when co-expressed with PCH1. Together, our results support that phyB photobodies include not only phyB and its primary interacting proteins but also its secondary interacting proteins.

The Arabidopsis Phytoglobin 2 mediates phytochrome B (phyB) light signaling responses during somatic embryogenesis.

MAIN CONCLUSIONS: During the light induction of somatic embryogenesis, phyB-Pfr suppresses Phytoglobin 2, known to elevate nitric oxide (NO). NO depresses Phytochrome Interacting Factor 4 (PIF4) relieving its inhibition on embryogenesis through auxin. An obligatory step of many in vitro embryogenic systems is the somatic-embryogenic transition culminating with the formation of the embryogenic tissue. In Arabidopsis, this transition requires light and is facilitated by high levels of nitric oxide (NO) generated by either suppression of the NO scavenger Phytoglobin 2 (Pgb2), or its removal from the nucleus. Using a previously characterized induction system regulating the cellular localization of Pgb2, we demonstrated the interplay between phytochrome B (phyB) and Pgb2 during the formation of embryogenic tissue. The deactivation of phyB in the dark coincides with the induction of Pgb2 known to reduce the level of NO; consequently, embryogenesis is inhibited. Under light conditions, the active form of phyB depresses the levels of Pgb2 transcripts, thus expecting an increase in cellular NO. Induction of Pgb2 increases Phytochrome Interacting Factor 4 (PIF4) suggesting that high levels of NO repress PIF4. The PIF4 inhibition is sufficient to induce several auxin biosynthetic (CYP79B2, AMI1, and YUCCA 1, 2, and 6) and response (ARF5, 8, and 16) genes, conducive to the formation of the embryonic tissue and production of somatic embryos. Auxin responses mediated by ARF10 and 17 appear to be regulated by Pgb2, possibly through NO, in a PIF4-independent fashion. Overall, this work provides a new and preliminary model integrating Pgb2 (and NO) with phyB in the light regulation of in vitro embryogenesis.

Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction.

Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.

A 5.5-kb LTR-retrotransposon insertion inside phytochrome B gene (CsPHYB) results in long hypocotyl and early flowering in cucumber (Cucumis sativus L.).

KEY MESSAGE: The novel spontaneous long hypocotyl and early flowering (lhef) mutation in cucumber is due to a 5551-bp LTR-retrotransposon insertion in CsPHYB gene encoding PHYTOCHROME B, which plays a major role in regulating photomorphogenic hypocotyl growth and flowering. Hypocotyl length and flowering time are important for establishing high-quality seedlings in modern cucumber production, but little is known for the underlying molecular mechanisms of these two traits. In this study, a spontaneous cucumber long hypocotyl and early flowering mutant was identified and characterized. Based on multiple lines of evidence, we show that cucumber phytochrome B (CsPHYB) is the candidate gene for this mutation, and a 5551-bp LTR-retrotransposon insertion in the first exon of CsPHYB was responsible for the mutant phenotypes. Uniqueness of the mutant allele at CsPHYB was verified in 114 natural cucumber lines. Ectopic expression of the CsPHYB in Arabidopsis phyB mutant rescued the long hypocotyl and early flowering phenotype of phyB-9 mutant. The wild-type CsPHYB protein was localized on the membrane and cytoplasm under white light condition, whereas in the nucleus under red light, it is consistent with its roles as a red-light photoreceptor in Arabidopsis. However, the mutant csphyb protein was localized on the membrane and cytoplasm under both white and red-light conditions. Expression dynamics of CsPHYB and several cell elongation-related genes were positively correlated with hypocotyl elongation; the transcription levels of key positive and negative regulators for flowering time were also consistent with the anthesis dates in the mutant and wild-type plants. Yeast two hybrid and bimolecular fluorescence complementation assays identified physical interactions between CsPHYB and phytochrome interacting factor 3/4 (CsPIF3/4). These findings will provide new insights into the roles of the CsPHYB in cucumber hypocotyl growth and flowering time.

Epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm.

Phytochrome B (phyB) expressed in the epidermis is sufficient to promote red light responses, including the inhibition of hypocotyl elongation and hypocotyl negative gravitropism. Nonetheless, the downstream mechanism of epidermal phyB in promoting light responses had been elusive. Here, we mutagenized the epidermis-specific phyB-expressing line (MLB) using ethyl methanesulfonate (EMS) and characterized a novel mutant allele of RRC1 (rrc1-689), which causes reduced epidermal phyB-mediated red light responses. The rrc1-689 mutation increases the alternative splicing of major clock gene transcripts, including PRR7 and TOC1, disrupting the rhythmic expression of the entire clock and clock-controlled genes. Combined with the result that MLB/prr7 exhibits the same red-hyposensitive phenotypes as MLB/rrc1-689, our data support that the circadian clock is required for the ability of epidermal phyB to promote light responses. We also found that, unlike phyB, RRC1 preferentially acts in the endodermis to maintain the circadian rhythm by suppressing the alternative splicing of core clock genes. Together, our results suggest that epidermal phyB requires RRC1 to promote light responses by activating the circadian rhythm in Arabidopsis thaliana.