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.

Revisiting the role and mechanism of ELF3 in circadian clock modulation.

The gene encoding EARLY FLOWERING3 (ELF3) is necessary for photoperiodic flowering and the normal regulation of circadian rhythms. It provides important information at the cellular level to uncover the biological mechanisms that improve plant growth and development. ELF3 interactions with transcription factors such as BROTHER OF LUX ARRHYTHMO (BOA), LIGHT-REGULATED WD1 (LWD1), PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), PHYTOCHROME-INTERACTING FACTOR 7 (PIF7), and LUX ARRHYTHMO (LUX) suggest a role in evening complex (EC) independent pathways, demanding further investigation to elucidate the EC-dependent versus EC-independent mechanisms. The ELF3 regulation of flowering time about photoperiod and temperature variations can also optimize crop cultivation across diverse latitudes. In this review paper, we summarize how ELF3's role in the circadian clock and light-responsive flowering control in crops offers substantial potential for scientific advancement and practical applications in biotechnology and agriculture. Despite its essential role in crop adaptation, very little is known in many important crops. Consequently, comprehensive and targeted research is essential for extrapolating ELF3-related insights from Arabidopsis to other crops, utilizing both computational and experimental methodologies. This research should prioritize investigations into ELF3's protein-protein interactions, post-translational modifications, and genomic targets to elucidate its contribution to accurate circadian clock regulation.

Phytochrome-interacting factors play shared and distinct roles in regulating shade avoidance responses in Populus trees.

Plants adjust their growth and development in response to changing light caused by canopy shade. The molecular mechanisms underlying shade avoidance responses have been widely studied in Arabidopsis and annual crop species, yet the shade avoidance signalling in woody perennial trees remains poorly understood. Here, we first showed that PtophyB1/2 photoreceptors serve conserved roles in attenuating the shade avoidance syndrome (SAS) in poplars. Next, we conducted a systematic identification and characterization of eight PtoPIF genes in Populus tomentosa. Knocking out different PtoPIFs led to attenuated shade responses to varying extents, whereas overexpression of PtoPIFs, particularly PtoPIF3.1 and PtoPIF3.2, led to constitutive SAS phenotypes under normal light and enhanced SAS responses under simulated shade. Notably, our results revealed that distinct from Arabidopsis PIF4 and PIF5, which are major regulators of SAS, the Populus homologues PtoPIF4.1 and PtoPIF4.2 seem to play a minor role in controlling shade responses. Moreover, we showed that PtoPIF3.1/3.2 could directly activate the expression of the auxin biosynthetic gene PtoYUC8 in response to shade, suggesting a conserved PIF-YUC-auxin pathway in modulating SAS in tree. Overall, our study provides insights into shared and divergent functions of PtoPIF members in regulating various aspects of the SAS in Populus.

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.

Functions of Phytochrome Interacting Factors (PIFs) in Adapting Plants to Biotic and Abiotic Stresses.

Plants possess the remarkable ability to sense detrimental environmental stimuli and launch sophisticated signal cascades that culminate in tailored responses to facilitate their survival, and transcription factors (TFs) are closely involved in these processes. Phytochrome interacting factors (PIFs) are among these TFs and belong to the basic helix-loop-helix family. PIFs are initially identified and have now been well established as core regulators of phytochrome-associated pathways in response to the light signal in plants. However, a growing body of evidence has unraveled that PIFs also play a crucial role in adapting plants to various biological and environmental pressures. In this review, we summarize and highlight that PIFs function as a signal hub that integrates multiple environmental cues, including abiotic (i.e., drought, temperature, and salinity) and biotic stresses to optimize plant growth and development. PIFs not only function as transcription factors to reprogram the expression of related genes, but also interact with various factors to adapt plants to harsh environments. This review will contribute to understanding the multifaceted functions of PIFs in response to different stress conditions, which will shed light on efforts to further dissect the novel functions of PIFs, especially in adaption to detrimental environments for a better survival of plants.

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

Genome-wide identification and comprehensive analysis of the phytochrome-interacting factor (PIF) gene family in wheat.

Phytochrome-interacting factors (PIFs) are essential transcription factors for plant growth, development, and stress responses. Although PIF genes have been extensively studied in many plant species, they have not been thoroughly investigated in wheat. Here, we identified 18 PIF genes in cultivated hexaploid wheat (Triticum aestivum L). Phylogenetic analysis, exon-intron structures, and motif compositions revealed the presence of four distinct groups of TaPIFs. Genome-wide collinearity analysis of PIF genes revealed the evolutionary history of PIFs in wheat, Oryza sativa, and Brachypodium distachyon. Cis-regulatory element analysis suggested that TaPIF genes indicated participated in plant development and stress responses. Subcellular localization assays indicated that TaPIF2-1B and TaPIF4-5B were transcriptionally active. Both were found to be localized to the nucleus. Gene expression analyses demonstrated that TaPIFs were primarily expressed in the leaves and were induced by various biotic and abiotic stresses and phytohormone treatments. This study provides new insights into PIF-mediated stress responses and lays a strong foundation for future investigation of PIF genes in wheat.

Rice PIFs: Critical regulators in rice development and stress response.

Phytochrome-interacting factors (PIFs) belong to a subfamily of the basic helix-loop-helix (bHLH) family of transcription factors, which serve as a "hub" for development and growth of plants. They have the capability to regulate the expression of many downstream genes, integrate multiple signaling pathways, and act as a signaling center within the cell. In rice (Oryza sativa), the PIF family genes, known as OsPILs, play a crucial part in many different aspects. OsPILs play a crucial role in regulating various aspects of photomorphogenesis, skotomorphogenesis, plant growth, and development in rice. These vital processes include chlorophyll synthesis, plant gravitropism, plant height, flowering, and response to abiotic stress factors such as low temperature, drought, and high salt. Additionally, OsPILs are involved in controlling several important agronomic traits in rice. Some OsPILs members coordinate with each other to function. This review summarizes and prospects the latest research progress on the biological functions of OsPILs transcription factors and provides a reference for further exploring the functions and mechanism of OsPILs.

Near-infrared light and PIF4 promote plant antiviral defense by enhancing RNA interference.

The molecular mechanism underlying phototherapy and light treatment, which utilize various wavelength spectra of light, including near-infrared (NIR), to cure human and plant diseases, is obscure. Here we revealed that NIR light confers antiviral immunity by positively regulating PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-activated RNA interference (RNAi) in plants. PIF4, a central transcription factor involved in light signaling, accumulates to high levels under NIR light in plants. PIF4 directly induces the transcription of two essential components of RNAi, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and ARGONAUTE 1 (AGO1), which play important roles in resistance to both DNA and RNA viruses. Moreover, the pathogenic determinant ßC1 protein, which is evolutionarily conserved and encoded by betasatellites, interacts with PIF4 and inhibits its positive regulation of RNAi by disrupting PIF4 dimerization. These findings shed light on the molecular mechanism of PIF4-mediated plant defense and provide a new perspective for the exploration of NIR antiviral treatment.

On the Role of a Conserved Tryptophan in the Chromophore Pocket of Cyanobacteriochrome.

The cyanobacteriochrome Slr1393 can be photoconverted between a red (Pr) and green absorbing form (Pg). The recently determined crystal structures of both states suggest a major movement of Trp496 from a stacking interaction with ring D of the phycocyanobilin (PCB) chromophore in Pr to a position outside the chromophore pocket in Pg. Here, we investigated the role of this amino acid during photoconversion in solution using engineered protein variants in which Trp496 was substituted by natural and non-natural amino acids. These variants and the native protein were studied by various spectroscopic techniques (UV-vis absorption, fluorescence, IR, NIR and UV resonance Raman) complemented by theoretical approaches. Trp496 is shown to affect the electronic transition of PCB and to be essential for the thermal equilibrium between Pr and an intermediate state O600. However, Trp496 is not required to stabilize the tilted orientation of ring D in Pr, and does not play a role in the secondary structure changes of Slr1393 during the Pr/Pg transition. The present results confirm the re-orientation of Trp496 upon Pr â†’ Pg conversion, but do not provide evidence of a major change in the microenvironment of this residue. Structural models indicate the penetration of water molecules into the chromophore pocket in both Pr and Pg states and thus water-Trp contacts, which can readily account for the subtle spectral changes between Pr and Pg. Thus, we conclude that reorientation of Trp496 during the Pr-to-Pg photoconversion in solution is not associated with a major change in the dielectric environment in the two states.

Engineering Bacteriophytochrome-coupled Photoactivated Adenylyl Cyclases for Enhanced Optogenetic cAMP Modulation.

Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.

Heat Shock Factor A1s are required for phytochrome-interacting factor 4-mediated thermomorphogenesis in Arabidopsis.

Thermomorphogenesis and the heat shock (HS) response are distinct thermal responses in plants that are regulated by PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and HEAT SHOCK FACTOR A1s (HSFA1s), respectively. Little is known about whether these responses are interconnected and whether they are activated by similar mechanisms. An analysis of transcriptome dynamics in response to warm temperature (28°C) treatment revealed that 30 min of exposure activated the expression of a subset of HSFA1 target genes in Arabidopsis thaliana. Meanwhile, a loss-of-function HSFA1 quadruple mutant (hsfa1-cq) was insensitive to warm temperature-induced hypocotyl growth. In hsfa1-cq plants grown at 28°C, the protein and transcript levels of PIF4 were greatly reduced, and the circadian rhythm of many thermomorphogenesis-related genes (including PIF4) was disturbed. Additionally, the nuclear localization of HSFA1s and the binding of HSFA1d to the PIF4 promoter increased following warm temperature exposure, whereas PIF4 overexpression in hsfa1-cq partially rescued the altered warm temperature-induced hypocotyl growth of the mutant. Taken together, these results suggest that HSFA1s are required for PIF4 accumulation at a warm temperature, and they establish a central role for HSFA1s in regulating both thermomorphogenesis and HS responses in Arabidopsis.

Inhibition of demethylase by IOX1 modulates chromatin accessibility to enhance NSCLC radiation sensitivity through attenuated PIF1.

Chromatin accessibility is a critical determinant of gene transcriptional expression and regulated by histones modification. However, the potential for manipulating chromatin accessibility to regulate radiation sensitivity remains unclear. Our findings demonstrated that the histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), could enhance the radiosensitivity of non-small cell lung cancer (NSCLC) in vitro and in vivo. Mechanistically, IOX1 treatment reduced chromatin accessibility in the promoter region of DNA damage repair genes, leading to decreased DNA repair efficiency and elevated DNA damage induced by γ irradiation. Notably, IOX1 treatment significantly reduced both chromatin accessibility and the transcription of phytochrome interacting factor 1 (PIF1), a key player in telomere maintenance. Inhibition of PIF1 delayed radiation-induced DNA and telomeric DNA damage repair, as well as increased radiosensitivity of NSCLC in vitro and in vivo. Further study indicated that the above process was regulated by a reduction of transcription factor myc-associated zinc finger protein (MAZ) binding to the distal intergenic region of the PIF1. Taken together, IOX1-mediated demethylase inactivation reduced chromatin accessibility, leading to elevated telomere damage which is partly due to PIF1 inhibition, thereby enhancing NSCLC radiosensitivity.

Genome-wide identification and molecular expression profile analysis of FHY3/FAR1 gene family in walnut (Juglans sigillata L.) development.

BACKGROUND: Juglans sigillata L. (walnut) has a high economic value for nuts and wood and has been widely grown and eaten around the world. Light plays an important role in regulating the development of the walnut embryo and promoting nucleolus enlargement, which is one of the factors affecting the yield and quality of walnut. However, little is known about the effect of light on the growth and quality of walnuts. Studies have shown that far red prolonged hypocotyl 3 (FHY3) and far red damaged response (FAR1) play important roles in plant growth, light response, and resistance. Therefore, FHY3/FAR1 genes were identified in walnuts on a genome-wide basis during their growth and development to reveal the potential regulation mechanisms involved in walnut kernel growth and development. RESULTS: In the present study, a total of 61 FHY3/FAR1 gene family members in walnuts have been identified, ranging in length from 117 aa to 895 aa. These gene family members have FHY3 or FAR1 conserved domains, which are unevenly distributed on the 15 chromosomes (Chr) of the walnut (except for the Chr16). All 61 FHY3/FAR1 genes were divided into five subclasses (I, II, III, IV, and V) by phylogenetic tree analysis. The results indicated that FHY3/FAR1 genes in the same subclasses with similar structures might be involved in regulating the growth and development of walnut. The gene expression profiles were analyzed in different walnut kernel varieties (Q, T, and F). The result showed that some FHY3/FAR1 genes might be involved in the regulation of walnut kernel ripening and seed coat color formation. Seven genes (OF07056-RA, OF09665-RA, OF24282-RA, OF26012-RA, OF28029-RA, OF28030-RA, and OF08124-RA) were predicted to be associated with flavonoid biosynthetic gene regulation cis-acting elements in promoter sequences. RT-PCR was used to verify the expression levels of candidate genes during the development and color change of walnut kernels. In addition, light responsiveness and MeJA responsiveness are important promoter regulatory elements in the FHY3/FAR1 gene family, which are potentially involved in the light response, growth, and development of walnut plants. CONCLUSION: The results of this study provide a valuable reference for supplementing the genomic sequencing results of walnut, and pave the way for further research on the FHY3/FAR1 gene function of walnut.

Phytochromes transmit photoperiod information via the evening complex in Brachypodium.

BACKGROUND: Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. RESULTS: We find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night. CONCLUSIONS: We propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.

Role of Phytochromes in Red Light-Regulated Alternative Splicing in Arabidopsis thaliana: Impactful but Not Indispensable.

Light is both the main source of energy and a key environmental signal for plants. It regulates not only gene expression but also the tightly related processes of splicing and alternative splicing (AS). Two main pathways have been proposed to link light sensing with the splicing machinery. One occurs through a photosynthesis-related signal, and the other is mediated by photosensory proteins, such as red light-sensing phytochromes. Here, we evaluated the relative contribution of each of these pathways by performing a transcriptome-wide analysis of light regulation of AS in plants that do not express any functional phytochrome (phyQ). We found that an acute 2-h red-light pulse in the middle of the night induces changes in the splicing patterns of 483 genes in wild-type plants. Approximately 30% of these genes also showed strong light regulation of splicing patterns in phyQ mutant plants, revealing that phytochromes are important but not essential for the regulation of AS by R light. We then performed a meta-analysis of related transcriptomic datasets and found that different light regulatory pathways can have overlapping targets in terms of AS regulation. All the evidence suggests that AS is regulated simultaneously by various light signaling pathways, and the relative contribution of each pathway is highly dependent on the plant developmental stage.

The PIF1/PIF3-MED25-HDA19 transcriptional repression complex regulates phytochrome signaling in Arabidopsis.

Light signals are perceived by photoreceptors, triggering the contrasting developmental transition in dark-germinated seedlings. Phytochrome-interacting factors (PIFs) are key regulators of this transition. Despite their prominent functions in transcriptional activation, little is known about PIFs' roles in transcriptional repression. Here, we provide evidence that histone acetylation is involved in regulating phytochrome-PIFs signaling in Arabidopsis. The histone deacetylase HDA19 interacts and forms a complex with PIF1 and PIF3 and the Mediator subunit MED25. The med25/hda19 double mutant mimics and enhances the phenotype of pif1/pif3 in both light and darkness. HDA19 and MED25 are recruited by PIF1/PIF3 to the target loci to reduce histone acetylation and chromatin accessibility, providing a mechanism for PIF1/PIF3-mediated transcriptional repression. Furthermore, MED25 forms liquid-like condensates, which can compartmentalize PIF1/PIF3 and HDA19 in vitro and in vivo, and the number of MED25 puncta increases in darkness. Collectively, our study establishes a mechanism wherein PIF1/PIF3 interact with HDA19 and MED25 to mediate transcriptional repression in the phytochrome signaling pathway and suggests that condensate formation with Mediator may explain the distinct and specific transcriptional activity of PIF proteins.

Conserved tyrosine in phytochromes controls the photodynamics through steric demand and hydrogen bonding capabilities.

Using ultrafast spectroscopy and site-specific mutagenesis, we demonstrate the central role of a conserved tyrosine within the chromophore binding pocket in the forward (Pr â†’ Pfr) photoconversion of phytochromes. Taking GAF1 of the knotless phytochrome All2699g1 from Nostoc as representative member of phytochromes, it was found that the mutations have no influence on the early (<30 ps) dynamics associated with conformational changes of the chromophore in the excited state. Conversely, they drastically impact the extended protein-controlled excited state decay (>100 ps). Thus, the steric demand, position and H-bonding capabilities of the identified tyrosine control the chromophore photoisomerization while leaving the excited state chromophore dynamics unaffected. In effect, this residue operates as an isomerization-steric-gate that tunes the excited state lifetime and the photoreaction efficiency by modulating the available space of the chromophore and by stabilizing the primary intermediate Lumi-R. Understanding the role of such a conserved structural element sheds light on a key aspect of phytochrome functionality and provides a basis for rational design of optimized photoreceptors for biotechnological applications.