Participation of miR165a in the Phytochrome Signal Transduction in Maize (Zea mays L.) Leaves under Changing Light Conditions.

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles.

A cytosol-tethered YHB variant of phytochrome B retains photomorphogenic signaling activity.

The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks therein. This necessitates changes in protein-protein interactions of phyB in the cytosol, about which little is presently known. Via introduction of a nucleus-excluding G767R mutation into the dominant, constitutively active phyBY276H (YHB) allele, we explore the functional consequences of expressing a cytosol-localized YHBG767R variant in transgenic Arabidopsis seedlings. We show that YHBG767R elicits selective constitutive photomorphogenic phenotypes in dark-grown phyABCDE null mutants, wild type and other phy-deficient genotypes. These responses include light-independent apical hook opening, cotyledon unfolding, seed germination and agravitropic hypocotyl growth with minimal suppression of hypocotyl elongation. Such phenotypes correlate with reduced PIF3 levels, which implicates cytosolic targeting of PIF3 turnover or PIF3 translational inhibition by YHBG767R. However, as expected for a cytoplasm-tethered phyB, YHBG767R elicits reduced light-mediated signaling activity compared with similarly expressed wild-type phyB in phyABCDE mutant backgrounds. YHBG767R also interferes with wild-type phyB light signaling, presumably by formation of cytosol-retained and/or otherwise inactivated heterodimers. Our results suggest that cytosolic interactions with PIFs play an important role in phyB signaling even under physiological conditions.

RcOST1L phosphorylates RcPIF4 for proteasomal degradation to promote flowering in rose.

Flowering is a vital agronomic trait that determines the economic value of most ornamental plants. The flowering time of rose (Rosa spp.) is photoperiod insensitive and is thought to be tightly controlled by light intensity, although the detailed molecular mechanism remains unclear. Here, we showed that rose plants flower later under low-light (LL) intensity than under high-light (HL) intensity, which is mainly related to the stability of PHYTOCHROME-INTERACTING FACTORs (RcPIFs) mediated by OPEN STOMATA 1-Like (RcOST1L) under different light intensity regimes. We determined that HL conditions trigger the rapid phosphorylation of RcPIFs before their degradation. A yeast two-hybrid screen identified the kinase RcOST1L as interacting with RcPIF4. Moreover, RcOST1L positively regulated rose flowering and directly phosphorylated RcPIF4 on serine 198 to promote its degradation under HL conditions. Additionally, phytochrome B (RcphyB) enhanced RcOST1L-mediated phosphorylation of RcPIF4 via interacting with the active phyB-binding motif. RcphyB was activated upon HL and recruited RcOST1L to facilitate its nuclear accumulation, in turn leading to decreased stability of RcPIF4 and flowering acceleration. Our findings illustrate how RcPIF abundance safeguards proper rose flowering under different light intensities, thus uncovering the essential role of RcOST1L in the RcphyB-RcPIF4 module in flowering.

Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis.

Hypocotyl elongation in Arabidopsis is widely utilized as a readout for phytochrome B (phyB) signaling and thermomorphogenesis. Hypocotyl elongation is gated by the circadian clock and, therefore, it occurs at distinct times depending on day length or seasonal cues. In short-day conditions, hypocotyl elongation occurs mainly at the end of nighttime when phyB reverts to the inactive form. In contrast, in long-day conditions, hypocotyl elongation occurs during the daytime when phyB is in the photoactivated form. Warm temperatures can induce hypocotyl growth in both long-day and short-day conditions. However, the corresponding daytime and nighttime temperature responses reflect distinct underpinning mechanisms. Here, we describe assays for dissecting the mechanisms between daytime and nighttime thermoresponsive hypocotyl elongation.

Measurement of Phytochrome B Thermal Reversion Rates In Vivo.

Thermal reversion of phytochromes is the light-independent but strongly temperature-dependent relaxation of the light-activated Pfr form of phytochromes back into the inactive Pr ground state. The thermal reversion rates of different phytochromes vary considerably. For phytochrome B (phyB), thermal reversion represents a critical parameter affecting phyB activity as it reduces the active phyB Pfr pool, accelerated by increasing temperatures. Phytochromes are dimers existing in three different states: Pfr-Pfr homodimer, Pfr-Pr heterodimer, and Pr-Pr homodimer. Consequently, thermal reversion occurs in two steps, with Pfr-Pfr to Pfr-Pr reversion being much slower than reversion from Pfr-Pr to Pr-Pr. To measure thermal reversion in vivo, the relative proportion of Pfr in relation to the total amount of phytochrome (Ptot) must be determined in living samples. This is accomplished by in vivo spectroscopy utilizing dual wavelength ratiospectrophotometers, optimized for assaying phytochromes in highly scattering plant material. The method is depending on the photoreversibility of phytochromes displaying light-induced absorbance changes in response to actinic irradiation. In this chapter, we describe the experimental design and explain step-by-step the calculations necessary to determine the thermal reversion rates of phyB in vivo, taking into account phytochrome dimerization.

Phase Separation of Phytochrome B in HEK293T Cells.

In this method, we employed HEK293T cells to express the plant photoreceptor phytochrome B (phyB). Through the application of various treatments such as phycocyanobilin (PCB) supplementation, red light exposure, and temperature adjustments, the phyB proteins exhibited liquid-liquid phase separation, leading to the formation of biomolecular condensates. Here, we present a comprehensive description of the protein expression, cell treatment, and imaging capture procedures. This detailed guide provides step-by-step instructions on how to induce phase separation of phyB proteins in HEK293T cells. By utilizing this approach, researchers can investigate the physicochemical characteristics and dynamic formation process of phyB photobodies with precision.

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.

Isolation of Phytochrome B Photobodies.

Phytochrome B (phyB), a plant photoreceptor, forms a membraneless organelle known as a photobody. Here, we present a protocol for the isolation of phyB photobodies through fluorescence-activated particle sorting from mature transgenic Arabidopsis leaves expressing phyB-GFP. This protocol involves the isolation of nuclei from frozen ground leaves using sucrose gradient centrifugation, the disruption of nuclear envelopes by sonication, and the subsequent isolation of phyB photobodies through fluorescence-activated particle sorting. We include experimental tips and notes for each step.

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.

Distinguishing individual photobodies using Oligopaints reveals thermo-sensitive and -insensitive phytochrome B condensation at distinct subnuclear locations.

Photobodies (PBs) are membraneless subnuclear organelles that self-assemble via concentration-dependent liquid-liquid phase separation (LLPS) of the plant photoreceptor and thermosensor phytochrome B (PHYB). The current PHYB LLPS model posits that PHYB phase separates randomly in the nucleoplasm regardless of the cellular or nuclear context. Here, we established a robust Oligopaints method in Arabidopsis to determine the positioning of individual PBs. We show surprisingly that even in PHYB overexpression lines - where PHYB condensation would be more likely to occur randomly - PBs positioned at twelve distinct subnuclear locations distinguishable by chromocenter and nucleolus landmarks, suggesting that PHYB condensation occurs nonrandomly at preferred seeding sites. Intriguingly, warm temperatures reduce PB number by inducing the disappearance of specific thermo-sensitive PBs, demonstrating that individual PBs possess different thermosensitivities. These results reveal a nonrandom PB nucleation model, which provides the framework for the biogenesis of spatially distinct individual PBs with diverse environmental sensitivities within a single plant nucleus.

Photobody formation spatially segregates two opposing phytochrome B signaling actions of PIF5 degradation and stabilization.

Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

The PHYB-FOF2-VOZ2 module functions to fine-tune flowering in response to changes in light quality by modulating FLC expression in Arabidopsis.

Proper timing of flowering under different environmental conditions is critical for plant propagation. Light quality is a pivotal environmental cue that plays a critical role in flowering regulation. Plants tend to flower late under light with a high red (R)/far-red (FR) light ratio but early under light with a low R/FR light ratio. However, how plants fine-tune flowering in response to changes in light quality is not well understood. Here, we demonstrate that F-box of Flowering 2 (FOF2), an autonomous pathway-related regulator, physically interacts with VASCULAR PLANT ONE-ZINC FINGER 1 and 2 (VOZ1 and VOZ2), which are direct downstream factors of the R/FR light receptor phytochrome B (PHYB). We show that PHYB physically interacts with FOF2, mediates stabilization of the FOF2 protein under FR light and end-of-day FR light, and enhances FOF2 binding to VOZ2, which leads to degradation of VOZ2 by SCFFOF2 E3 ligase. By contrast, PHYB mediates degradation of FOF2 protein under R light and end-of-day R light. Genetic interaction studies demonstrated that FOF2 functions downstream of PHYB to promote FLC expression and inhibit flowering under both high R/FR light and simulated shade conditions, processes that are partially dependent on VOZ proteins. Taken together, our findings suggest a novel mechanism whereby plants fine-tune flowering time through a PHYB-FOF2-VOZ2 module that modulates FLC expression in response to changes in light quality.

Freezing treatment under light conditions leads to a dramatic enhancement of freezing tolerance in cold-acclimated Arabidopsis.

Overwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short-term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second-phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second-phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C-repeat binding factor and cold-regulated genes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea during the 2PFA treatment. Compared with wild-type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.

Phytochrome B photobody components.

Phytochrome B (phyB) is a red and far-red photoreceptor that promotes light responses. Upon photoactivation, phyB enters the nucleus and forms a molecular condensate called a photobody through liquid-liquid phase separation. Phytochrome B photobody comprises phyB, the main scaffold molecule, and at least 37 client proteins. These clients belong to diverse functional categories enriched with transcription regulators, encompassing both positive and negative light signaling factors, with the functional bias toward the negative factors. The functionally diverse clients suggest that phyB photobody acts either as a trap to capture proteins, including negatively acting transcription regulators, for processes such as sequestration, modification, or degradation or as a hub where proteins are brought into close proximity for interaction in a light-dependent manner.

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.

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.

Applications of fluorescence spectroscopy in the investigation of plant phytochrome invivo.

Fluorometry is an effective research tool in biology and medicine; it is widely used in the study of the photosynthetic pigment apparatus in vivo. This method can be applied to the key plant photoreceptor phytochrome (phy). The fluorescence of phytochrome in plants was recorded for the first time in the group of the author, and a spectrofluorometric technique for its in vivo study was developed. The photophysical and photochemical properties of the pigment were described, and the photoreceptor was shown to be present in plants as two phenomenological types-active (at cryogenic temperatures) and water-soluble (Pr') and inactive and amphiphilic (Pr″). The scheme of the photoreaction explaining their photochemical distinctions was proposed. Phytochrome A was shown to comprise both types (phyA' and phyA″), whereas phytochrome B was only the second type. For phyA', distinct conformers have been detected. phyA' and phyA″ differ by the N-terminus of the molecule, possibly by serine phosphorylation. They mediate, respectively, the very low fluence and high irradiance photoresponses. Light, internal factors (kinase/phosphatase balance, pH), and hormones (jasmonate) were shown to affect the content and functions of the two phyA pools. All this points to the effectiveness of the developed method for invivo investigations of the phytochrome system. The data obtained can be applied in practical terms in agrobiology and light culture, as well as in the use of phytochrome as a new nanotool and a fluorescent probe.

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.

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.