Reducing brassinosteroid signalling enhances grain yield in semi-dwarf wheat.

Modern green revolution varieties of wheat (Triticum aestivum L.) confer semi-dwarf and lodging-resistant plant architecture owing to the Reduced height-B1b (Rht-B1b) and Rht-D1b alleles1. However, both Rht-B1b and Rht-D1b are gain-of-function mutant alleles encoding gibberellin signalling repressors that stably repress plant growth and negatively affect nitrogen-use efficiency and grain filling2-5. Therefore, the green revolution varieties of wheat harbouring Rht-B1b or Rht-D1b usually produce smaller grain and require higher nitrogen fertilizer inputs to maintain their grain yields. Here we describe a strategy to design semi-dwarf wheat varieties without the need for Rht-B1b or Rht-D1b alleles. We discovered that absence of Rht-B1 and ZnF-B (encoding a RING-type E3 ligase) through a natural deletion of a haploblock of about 500 kilobases shaped semi-dwarf plants with more compact plant architecture and substantially improved grain yield (up to 15.2%) in field trials. Further genetic analysis confirmed that the deletion of ZnF-B induced the semi-dwarf trait in the absence of the Rht-B1b and Rht-D1b alleles through attenuating brassinosteroid (BR) perception. ZnF acts as a BR signalling activator to facilitate proteasomal destruction of the BR signalling repressor BRI1 kinase inhibitor 1 (TaBKI1), and loss of ZnF stabilizes TaBKI1 to block BR signalling transduction. Our findings not only identified a pivotal BR signalling modulator but also provided a creative strategy to design high-yield semi-dwarf wheat varieties by manipulating the BR signal pathway to sustain wheat production.

Brassinosteroid signals cooperate with katanin-mediated microtubule severing to control stamen filament elongation.

Proper stamen filament elongation is essential for pollination and plant reproduction. Plant hormones are extensively involved in every stage of stamen development; however, the cellular mechanisms by which phytohormone signals couple with microtubule dynamics to control filament elongation remain unclear. Here, we screened a series of Arabidopsis thaliana mutants showing different microtubule defects and revealed that only those unable to sever microtubules, lue1 and ktn80.1234, displayed differential floral organ elongation with less elongated stamen filaments. Prompted by short stamen filaments and severe decrease in KTN1 and KTN80s expression in qui-2 lacking five BZR1-family transcription factors (BFTFs), we investigated the crosstalk between microtubule severing and brassinosteroid (BR) signaling. The BFTFs transcriptionally activate katanin-encoding genes, and the microtubule-severing frequency was severely reduced in qui-2. Taken together, our findings reveal how BRs can regulate cytoskeletal dynamics to coordinate the proper development of reproductive organs.

Salicylic acid attenuates brassinosteroid signaling via protein de-S-acylation.

Brassinosteroids (BRs) are important plant hormones involved in many aspects of development. Here, we show that BRASSINOSTEROID SIGNALING KINASEs (BSKs), key components of the BR pathway, are precisely controlled via de-S-acylation mediated by the defense hormone salicylic acid (SA). Most Arabidopsis BSK members are substrates of S-acylation, a reversible protein lipidation that is essential for their membrane localization and physiological function. We establish that SA interferes with the plasma membrane localization and function of BSKs by decreasing their S-acylation levels, identifying ABAPT11 (ALPHA/BETA HYDROLASE DOMAIN-CONTAINING PROTEIN 17-LIKE ACYL PROTEIN THIOESTERASE 11) as an enzyme whose expression is quickly induced by SA. ABAPT11 de-S-acylates most BSK family members, thus integrating BR and SA signaling for the control of plant development. In summary, we show that BSK-mediated BR signaling is regulated by SA-induced protein de-S-acylation, which improves our understanding of the function of protein modifications in plant hormone cross talk.

Brassinosteroid-dependent phosphorylation of PHOSPHATE STARVATION RESPONSE2 reduces its DNA-binding ability in rice.

Brassinosteroids (BRs) are widely used as plant growth regulators in modern agriculture. Understanding how BRs regulate nutrient signaling is crucial for reducing fertilizer usage. Here we elucidate that the central BR signaling inhibitor GSK3/SHAGGY-LIKE KINASE2 (GSK2) interacts directly with and phosphorylates PHOSPHATE STARVATION RESPONSE2 (OsPHR2), the key regulator of phosphate (Pi) signaling, to suppress its transcription factor activity in rice (Oryza sativa). We identify a critical phosphorylation site at serine residue S269 of OsPHR2 and demonstrate that phosphorylation by GSK2 or phosphor-mimic mutation of S269 substantially impairs the DNA-binding activity of OsPHR2, and thus diminishes expression of OsPHR2-induced genes and reduces Pi levels. Like BRs, Pi starvation noticeably induces GSK2 instability. We further show that this site-specific phosphorylation event is conserved in Arabidopsis (Arabidopsis thaliana), but varies among the PHR-family members, being present only in most land plants. These results unveil a distinctive post-transcriptional regulatory mechanism in Pi signaling by which BRs promote Pi acquisition, with a potential contribution to the environmental adaptability of plants during their evolution.

Arabidopsis protein S-acyl transferases positively mediate BR signaling through S-acylation of BSK1.

Protein S-acyl transferases (PATs) catalyze S-acylation, a reversible post-translational modification critical for membrane association, trafficking, and stability of substrate proteins. Many plant proteins are potentially S-acylated but few have corresponding PATs identified. By using genomic editing, confocal imaging, pharmacological, genetic, and biochemical assays, we demonstrate that three Arabidopsis class C PATs positively regulate BR signaling through S-acylation of BRASSINOSTEROID-SIGNALING KINASE1 (BSK1). PAT19, PAT20, and PAT22 associate with the plasma membrane (PM) and the trans-Golgi network/early endosome (TGN/EE). Functional loss of all three genes results in a plethora of defects, indicative of reduced BR signaling and rescued by enhanced BR signaling. PAT19, PAT20, and PAT22 interact with BSK1 and are critical for the S-acylation of BSK1, and for BR signaling. The PM abundance of BSK1 was reduced by functional loss of PAT19, PAT20, and PAT22 whereas abolished by its S-acylation-deficient point mutations, suggesting a key role of S-acylation in its PM targeting. Finally, an active BR analog induces vacuolar trafficking and degradation of PAT19, PAT20, or PAT22, suggesting that the S-acylation of BSK1 by the three PATs serves as a negative feedback module in BR signaling.

Transcription factor BES1 interacts with HSFA1 to promote heat stress resistance of plants.

Heat stress is a major environmental stress type that can limit plant growth and development. To survive sudden temperature increases, plants utilize the heat shock response, an ancient signaling pathway. Initial results had suggested a role for brassinosteroids (BRs) in this response. Brassinosteroids are growth-promoting steroid hormones whose activity is mediated by transcription factors of the BES1/BZR1 subfamily. Here, we provide evidence that BES1 can contribute to heat stress signaling. In response to heat, BES1 is activated even in the absence of BRs and directly binds to heat shock elements (HSEs), known binding sites of heat shock transcription factors (HSFs). HSFs of the HSFA1 type can interact with BES1 and facilitate its activity in HSE binding. These findings lead us to propose an extended model of the heat stress response in plants, in which the recruitment of BES1 is a means of heat stress signaling cross-talk with a central growth regulatory pathway.

Salicylic acid-activated BIN2 phosphorylation of TGA3 promotes Arabidopsis PR gene expression and disease resistance.

The plant defense hormone, salicylic acid (SA), plays essential roles in immunity and systemic acquired resistance. Salicylic acid induced by the pathogen is perceived by the receptor nonexpressor of pathogenesis-related genes 1 (NPR1), which is recruited by TGA transcription factors to induce the expression of pathogenesis-related (PR) genes. However, the mechanism by which post-translational modifications affect TGA's transcriptional activity by salicylic acid signaling/pathogen infection is not well-established. Here, we report that the loss-of-function mutant of brassinosteroid insensitive2 (BIN2) and its homologs, bin2-3 bil1 bil2, causes impaired pathogen resistance and insensitivity to SA-induced PR gene expression, whereas the gain-of-function mutant, bin2-1, exhibited enhanced SA signaling and immunity against the pathogen. Our results demonstrate that salicylic acid activates BIN2 kinase, which in turn phosphorylates TGA3 at Ser33 to enhance TGA3 DNA binding ability and NPR1-TGA3 complex formation, leading to the activation of PR gene expression. These findings implicate BIN2 as a new component of salicylic acid signaling, functioning as a key node in balancing brassinosteroid-mediated plant growth and SA-induced immunity.

Green means go: Green light promotes hypocotyl elongation via brassinosteroid signaling.

Although many studies have elucidated the mechanisms by which different wavelengths of light (blue, red, far-red, or ultraviolet-B [UV-B]) regulate plant development, whether and how green light regulates plant development remains largely unknown. Previous studies reported that green light participates in regulating growth and development in land plants, but these studies have reported conflicting results, likely due to technical problems. For example, commercial green light-emitting diode light sources emit a little blue or red light. Here, using a pure green light source, we determined that unlike blue, red, far-red, or UV-B light, which inhibits hypocotyl elongation, green light promotes hypocotyl elongation in Arabidopsis thaliana and several other plants during the first 2-3 d after planting. Phytochromes, cryptochromes, and other known photoreceptors do not mediate green-light-promoted hypocotyl elongation, but the brassinosteroid (BR) signaling pathway is involved in this process. Green light promotes the DNA binding activity of BRI1-EMS-SUPPRESSOR 1 (BES1), a master transcription factor of the BR pathway, thus regulating gene transcription to promote hypocotyl elongation. Our results indicate that pure green light promotes elongation via BR signaling and acts as a shade signal to enable plants to adapt their development to a green-light-dominant environment under a canopy.

Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis.

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.

Signaling by the EPFL-ERECTA family coordinates female germline specification through the BZR1 family in Arabidopsis.

In most flowering plants, the female germline is initiated in the subepidermal L2 layer of ovule primordia forming a single megaspore mother cell (MMC). How signaling from the L1 (epidermal) layer could contribute to the gene regulatory network (GRN) restricting MMC formation to a single cell is unclear. We show that EPIDERMAL PATTERNING FACTOR-like (EPFL) peptide ligands are expressed in the L1 layer, together with their ERECTA family (ERf) receptor kinases, to control female germline specification in Arabidopsis thaliana. EPFL-ERf dependent signaling restricts multiple subepidermal cells from acquiring MMC-like cell identity by activating the expression of the major brassinosteroid (BR) receptor kinase BRASSINOSTEROID INSENSITIVE 1 and the BR-responsive transcription factor BRASSINOZOLE RESISTANT 1 (BZR1). Additionally, BZR1 coordinates female germline specification by directly activating the expression of a nucleolar GTP-binding protein, NUCLEOSTEMIN-LIKE 1 (NSN1), which is expressed in early-stage ovules excluding the MMC. Mutants defective in this GRN form multiple MMCs resulting in a strong reduction of seed set. In conclusion, we uncovered a ligand/receptor-like kinase-mediated signaling pathway acting upstream and coordinating BR signaling via NSN1 to restrict MMC differentiation to a single subepidermal cell.

The osmotic stress-activated receptor-like kinase DPY1 mediates SnRK2 kinase activation and drought tolerance in Setaria.

Plant genomes encode many receptor-like kinases (RLKs) that localize to the cell surface and perceive a wide variety of environmental cues to initiate downstream signaling cascades. Whether these RLKs participate in dehydration stress signaling in plants is largely unknown. DROOPY LEAF1 (DPY1), a leucine-rich repeat (LRR)-RLK, was recently shown to regulate plant architecture by orchestrating early brassinosteroid signaling in foxtail millet (Setaria italica). Here, we show that DPY1 is essential for the acclimation of foxtail millet to drought stress. DPY1 can be phosphorylated and activated in response to osmotic stress and is required for more than half of osmotic stress-induced global phosphorylation events, including the phosphorylation of sucrose nonfermenting kinase 2s (SnRK2s), the central kinases involved in osmotic stress. DPY1 acts upstream of STRESS-ACTIVATED PROTEIN KINASE 6 (SAPK6, a subclass I SnRK2) and is required for full SAPK6 activation, thereby allowing regulation of downstream genes to mount a response against drought stress. These signaling events are largely independent of DPY1-mediated brassinosteroid signaling. The DPY1-SAPK6 module is specific to seed plants and is absent in ancestral nonseed plants. Our findings reveal a dehydration stress-activated RLK that plays an indispensable role in osmotic stress signaling and mediates SnRK2 activation at the cell surface.

An endoplasmic reticulum-associated degradation-related E2-E3 enzyme pair controls grain size and weight through the brassinosteroid signaling pathway in rice.

Grain size is an important agronomic trait, but our knowledge about grain size determination in crops is still limited. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a special ubiquitin proteasome system that is involved in degrading misfolded or incompletely folded proteins in the ER. Here, we report that SMALL GRAIN 3 (SMG3) and DECREASED GRAIN SIZE 1 (DGS1), an ERAD-related E2-E3 enzyme pair, regulate grain size and weight through the brassinosteroid (BR) signaling pathway in rice (Oryza sativa). SMG3 encodes a homolog of Arabidopsis (Arabidopsis thaliana) UBIQUITIN CONJUGATING ENZYME 32, which is a conserved ERAD-associated E2 ubiquitin conjugating enzyme. SMG3 interacts with another grain size regulator, DGS1. Loss of function of SMG3 or DGS1 results in small grains, while overexpression of SMG3 or DGS1 leads to long grains. Further analyses showed that DGS1 is an active E3 ubiquitin ligase and colocates with SMG3 in the ER. SMG3 and DGS1 are involved in BR signaling. DGS1 ubiquitinates the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and affects its accumulation. Genetic analysis suggests that SMG3, DGS1, and BRI1 act together to regulate grain size and weight. In summary, our findings identify an ERAD-related E2-E3 pair that regulates grain size and weight, which gives insight into the function of ERAD in grain size control and BR signaling.

Brassinosteroids promote etiolated apical structures in darkness by amplifying the ethylene response via the EBF-EIN3/PIF3 circuit.

Germinated plants grow in darkness until they emerge above the soil. To help the seedling penetrate the soil, most dicot seedlings develop an etiolated apical structure consisting of an apical hook and folded, unexpanded cotyledons atop a rapidly elongating hypocotyl. Brassinosteroids (BRs) are necessary for etiolated apical development, but their precise role and mechanisms remain unclear. Arabidopsis thaliana SMALL AUXIN UP RNA17 (SAUR17) is an apical-organ-specific regulator that promotes production of an apical hook and closed cotyledons. In darkness, ethylene and BRs stimulate SAUR17 expression by transcription factor complexes containing PHYTOCHROME-INTERACTING FACTORs (PIFs), ETHYLENE INSENSITIVE 3 (EIN3), and its homolog EIN3-LIKE 1 (EIL1), and BRASSINAZOLE RESISTANT1 (BZR1). BZR1 requires EIN3 and PIFs for enhanced DNA-binding and transcriptional activation of the SAUR17 promoter; while EIN3, PIF3, and PIF4 stability depends on BR signaling. BZR1 transcriptionally downregulates EIN3-BINDING F-BOX 1 and 2 (EBF1 and EBF2), which encode ubiquitin ligases mediating EIN3 and PIF3 protein degradation. By modulating the EBF-EIN3/PIF protein-stability circuit, BRs induce EIN3 and PIF3 accumulation, which underlies BR-responsive expression of SAUR17 and HOOKLESS1 and ultimately apical hook development. We suggest that in the etiolated development of apical structures, BRs primarily modulate plant sensitivity to darkness and ethylene.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

The transcriptional hub SHORT INTERNODES1 integrates hormone signals to orchestrate rice growth and development.

Plants have evolved sophisticated mechanisms to coordinate their growth and stress responses via integrating various phytohormone signaling pathways. However, the precise molecular mechanisms orchestrating integration of the phytohormone signaling pathways remain largely obscure. In this study, we found that the rice (Oryza sativa) short internodes1 (shi1) mutant exhibits typical auxin-deficient root development and gravitropic response, brassinosteroid (BR)-deficient plant architecture and grain size as well as enhanced abscisic acid (ABA)-mediated drought tolerance. Additionally, we found that the shi1 mutant is also hyposensitive to auxin and BR treatment but hypersensitive to ABA. Further, we showed that OsSHI1 promotes the biosynthesis of auxin and BR by activating the expression of OsYUCCAs and D11, meanwhile dampens ABA signaling by inducing the expression of OsNAC2, which encodes a repressor of ABA signaling. Furthermore, we demonstrated that 3 classes of transcription factors, AUXIN RESPONSE FACTOR 19 (OsARF19), LEAF AND TILLER ANGLE INCREASED CONTROLLER (LIC), and OsZIP26 and OsZIP86, directly bind to the promoter of OsSHI1 and regulate its expression in response to auxin, BR, and ABA, respectively. Collectively, our results unravel an OsSHI1-centered transcriptional regulatory hub that orchestrates the integration and self-feedback regulation of multiple phytohormone signaling pathways to coordinate plant growth and stress adaptation.

Hormonal interactions in the regulation of plant development.

Plants exhibit a unique developmental flexibility to ever-changing environmental conditions. To achieve their profound adaptability, plants are able to maintain permanent stem cell populations and form new organs during the entire plant life cycle. Signaling substances, called plant hormones, such as auxin, cytokinin, abscisic acid, brassinosteroid, ethylene, gibberellin, jasmonic acid, and strigolactone, govern and coordinate these developmental processes. Physiological and genetic studies have dissected the molecular components of signal perception and transduction of the individual hormonal pathways. However, over recent years it has become evident that hormones do not act only in a linear pathway. Hormonal pathways are interconnected by a complex network of interactions and feedback circuits that determines the final outcome of the individual hormone actions. This raises questions about the molecular mechanisms underlying hormonal cross talk and about how these hormonal networks are established, maintained, and modulated throughout plant development.

SUMO/deSUMOylation of the BRI1 brassinosteroid receptor modulates plant growth responses to temperature.

Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. Here we identify SUMOylation as a new modification targeting BRI1 to regulate its activity. BRI1 is SUMOylated in planta on two lysine residues, and the levels of BRI1 SUMO conjugates are controlled by the Desi3a SUMO protease. Loss of Desi3a leads to hypersensitivity to BRs, indicating that Desi3a acts as a negative regulator of BR signaling. Besides, we demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and to enhanced BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response.

HD-ZIP III-dependent local promotion of brassinosteroid synthesis suppresses vascular cell division in Arabidopsis root apical meristem.

Spatiotemporal control of cell division in the meristem is vital for plant growth. In the stele of the root apical meristem (RAM), procambial cells divide periclinally to increase the number of vascular cell files. Class III homeodomain leucine zipper (HD-ZIP III) proteins are key transcriptional regulators of RAM development and suppress the periclinal division of vascular cells in the stele; however, the mechanism underlying the regulation of vascular cell division by HD-ZIP III transcription factors (TFs) remains largely unknown. Here, we performed transcriptome analysis to identify downstream genes of HD-ZIP III and found that HD-ZIP III TFs positively regulate brassinosteroid biosynthesis-related genes, such as CONSTITUTIVE PHOTOMORPHOGENIC DWARF (CPD), in vascular cells. Introduction of pREVOLUTA::CPD in a quadruple loss-of-function mutant of HD-ZIP III genes partly rescued the phenotype in terms of the vascular defect in the RAM. Treatment of a quadruple loss-of-function mutant, a gain-of-function mutant of HD-ZIP III, and the wild type with brassinosteroid and a brassinosteroid synthesis inhibitor also indicated that HD-ZIP III TFs act together to suppress vascular cell division by increasing brassinosteroid levels. Furthermore, brassinosteroid application suppressed the cytokinin response in vascular cells. Together, our findings suggest that the suppression of vascular cell division by HD-ZIP III TFs is caused, at least in part, by the increase in brassinosteroid levels through the transcriptional activation of brassinosteroid biosynthesis genes in the vascular cells of the RAM. This elevated brassinosteroid level suppresses cytokinin response in vascular cells, inhibiting vascular cell division in the RAM.

Comparative analysis of BZR1/BES1 family transcription factors in Arabidopsis.

Brassinazole Resistant 1 (BZR1) and bri1 EMS Suppressor 1 (BES1) are key transcription factors that mediate brassinosteroid (BR)-responsive gene expression in Arabidopsis. The BZR1/BES1 family is composed of BZR1, BES1, and four BES1/BZR1 homologs (BEH1-BEH4). However, little is known about whether BEHs are regulated by BR signaling in the same way as BZR1 and BES1. We comparatively analyzed the functional characteristics of six BZR1/BES1 family members and their regulatory mechanisms in BR signaling using genetic and biochemical analyses. We also compared their subcellular localizations regulated by the phosphorylation status, interaction with GSK3-like kinases, and heterodimeric combination. We found that all BZR1/BES1 family members restored the phenotypic defects of bri1-5 by their overexpression. Unexpectedly, BEH2-overexpressing plants showed the most distinct phenotype with enhanced BR responses. RNA-Seq analysis indicated that overexpression of both BZR1 and BEH2 regulates BR-responsive gene expression, but BEH2 has a much greater proportion of BR-independent gene expression than BZR1. Unlike BZR1 and BES1, the BR-regulated subcellular translocation of the four BEHs was not tightly correlated with their phosphorylation status. Notably, BEH1 and BEH2 are predominantly localized in the nucleus, which induces the nuclear accumulation of other BZR1/BES1 family proteins through heterodimerization. Altogether, our comparative analyses suggest that BEH1 and BEH2 play an important role in the functional interaction between BZR1/BES1 family transcription factors.

BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth.

The BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor family plays an essential role in plant brassinosteroid (BR) signaling, but the signaling mechanism through which BZR1 and its homologs cooperate with certain coactivators to facilitate transcription of target genes remains incompletely understood. In this study, we used an efficient protein interaction screening system to identify blue-light inhibitor of cryptochromes 1 (BIC1) as a new BZR1-interacting protein in Arabidopsis thaliana. We show that BIC1 positively regulates BR signaling and acts as a transcriptional coactivator for BZR1-dependent activation of BR-responsive genes. Simultaneously, BIC1 interacts with the transcription factor PIF4 to synergistically and interdependently activate expression of downstream genes including PIF4 itself, and to promote plant growth. Chromatin immunoprecipitation assays demonstrate that BIC1 and BZR1/PIF4 interdependently associate with the promoters of common target genes. In addition, we show that the interaction between BIC1 and BZR1 is evolutionally conserved in the model monocot plant Triticum aestivum (bread wheat). Together, our results reveal mechanistic details of BR signaling mediated by a transcriptional activation module BIC1/BZR1/PIF4 and thus provide new insights into the molecular mechanisms underlying the integration of BR and light signaling in plants.