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.

A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation.

The Xishuangbanna (XIS) cucumber (Cucumis sativus var. xishuangbannanesis) is a semiwild variety that has many distinct agronomic traits. Here, long reads generated by Nanopore sequencing technology helped assembling a high-quality genome (contig N50 = 8.7 Mb) of landrace XIS49. A total of 10,036 structural/sequence variations (SVs) were identified when comparing with Chinese Long (CL), and known SVs controlling spines, tubercles, and carpel number were confirmed in XIS49 genome. Two QTLs of hypocotyl elongation under low light, SH3.1 and SH6.1, were fine-mapped using introgression lines (donor parent, XIS49; recurrent parent, CL). SH3.1 encodes a red-light receptor Phytochrome B (PhyB, CsaV3_3G015190). A ∼4 kb region with large deletion and highly divergent regions (HDRs) were identified in the promoter of the PhyB gene in XIS49. Loss of function of this PhyB caused a super-long hypocotyl phenotype. SH6.1 encodes a CCCH-type zinc finger protein FRIGIDA-ESSENTIAL LIKE (FEL, CsaV3_6G050300). FEL negatively regulated hypocotyl elongation but it was transcriptionally suppressed by long terminal repeats retrotransposon insertion in CL cucumber. Mechanistically, FEL physically binds to the promoter of CONSTITUTIVE PHOTOMORPHOGENIC 1a (COP1a), regulating the expression of COP1a and the downstream hypocotyl elongation. These above results demonstrate the genetic mechanism of cucumber hypocotyl elongation under low light.

What is going on inside of phytochrome B photobodies?

Plants exhibit an enormous phenotypic plasticity to adjust to changing environmental conditions. For this purpose, they have evolved mechanisms to detect and measure biotic and abiotic factors in their surroundings. Phytochrome B exhibits a dual function, since it serves as a photoreceptor for red and far-red light as well as a thermosensor. In 1999, it was first reported that phytochromes not only translocate into the nucleus but also form subnuclear foci upon irradiation by red light. It took more than 10 years until these phytochrome speckles received their name; these foci were coined photobodies to describe unique phytochrome-containing subnuclear domains that are regulated by light. Since their initial discovery, there has been much speculation about the significance and function of photobodies. Their presumed roles range from pure experimental artifacts to waste deposits or signaling hubs. In this review, we summarize the newest findings about the meaning of phyB photobodies for light and temperature signaling. Recent studies have established that phyB photobodies are formed by liquid-liquid phase separation via multivalent interactions and that they provide diverse functions as biochemical hotspots to regulate gene expression on multiple levels.

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.

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.

Modulation of starch synthesis in Arabidopsis via phytochrome B-mediated light signal transduction.

Starch is a major storage carbohydrate in plants and is critical in crop yield and quality. Starch synthesis is intricately regulated by internal metabolic processes and external environmental cues; however, the precise molecular mechanisms governing this process remain largely unknown. In this study, we revealed that high red to far-red (high R:FR) light significantly induces the synthesis of leaf starch and the expression of synthesis-related genes, whereas low R:FR light suppress these processes. Arabidopsis phytochrome B (phyB), the primary R and FR photoreceptor, was identified as a critical positive regulator in this process. Downstream of phyB, basic leucine zipper transcription factor ELONGATED HYPOCOTYL5 (HY5) was found to enhance starch synthesis, whereas the basic helix-loop-helix transcription factors PHYTOCHROME INTERACTING FACTORs (PIF3, PIF4, and PIF5) inhibit starch synthesis in Arabidopsis leaves. Notably, HY5 and PIFs directly compete for binding to a shared G-box cis-element in the promoter region of genes encoding starch synthases GBSS, SS3, and SS4, which leads to antagonistic regulation of their expression and, consequently, starch synthesis. Our findings highlight the vital role of phyB in enhancing starch synthesis by stabilizing HY5 and facilitating PIFs degradation under high R:FR light conditions. Conversely, under low R:FR light, PIFs predominantly inhibit starch synthesis. This study provides insight into the physiological and molecular functions of phyB and its downstream transcription factors HY5 and PIFs in starch synthesis regulation, shedding light on the regulatory mechanism by which plants synchronize dynamic light signals with metabolic cues to module starch synthesis.

Interplay among photoreceptors determines the strategy of coping with excess light in tomato.

This study investigates photoreceptor's role in the adaption of photosynthetic apparatus to high light (HL) intensity by examining the response of tomato wild type (WT) (Solanum lycopersicum L. cv. Moneymaker) and tomato mutants (phyA, phyB1, phyB2, cry1) plants to HL. Our results showed a photoreceptor-dependent effect of HL on the maximum quantum yield of photosystem II (Fv/Fm) with phyB1 exhibiting a decrease, while phyB2 exhibiting an increase in Fv/Fm. HL resulted in an increase in the efficient quantum yield of photosystem II (ΦPSII) and a decrease in the non-photochemical quantum yields (ΦNPQ and ΦN0) solely in phyA. Under HL, phyA showed a significant decrease in the energy-dependent quenching component of NPQ (qE), while phyB2 mutants showed an increase in the state transition (qT) component. Furthermore, ΔΔFv/Fm revealed that PHYB1 compensates for the deficit of PHYA in phyA mutants. PHYA signaling likely emerges as the dominant effector of PHYB1 and PHYB2 signaling within the HL-induced signaling network. In addition, PHYB1 compensates for the role of CRY1 in regulating Fv/Fm in cry1 mutants. Overall, the results of this research provide valuable insights into the unique role of each photoreceptor and their interplay in balancing photon energy and photoprotection under HL condition.

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.

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.

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.

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.

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.

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.

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.

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.