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.

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.

Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases.

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development and immunity in plants, but the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLAGELLIN SENSING 2 (FLS2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We surprisingly found that the loss of the BSU1 family phosphatases activates effector-triggered immunity and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine 251. Mutation of serine 251 reduces BSU1's ability to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress effector-triggered immunity and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine 764 upon brassinosteroid signalling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signalling specificities for diverse plant receptor kinases.

Brassinosteroids enhance salicylic acid-mediated immune responses by inhibiting BIN2 phosphorylation of clade I TGA transcription factors in Arabidopsis.

Salicylic acid (SA) plays an important role in plant immune response, including resistance to pathogens and systemic acquired resistance. Two major components, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES (NPRs) and TGACG motif-binding transcription factors (TGAs), are known to mediate SA signaling, which might also be orchestrated by other hormonal and environmental changes. Nevertheless, the molecular and functional interactions between SA signaling components and other cellular signaling pathways remain poorly understood. Here we showed that the steroid plant hormone brassinosteroid (BR) promotes SA responses by inactivating BR-INSENSITIVE 2 (BIN2), which inhibits the redox-sensitive clade I TGAs in Arabidopsis. We found that both BR and the BIN2 inhibitor bikinin synergistically increase SA-mediated physiological responses, such as resistance to Pst DC3000. Our genetic and biochemical analyses indicated that BIN2 functionally interacts with TGA1 and TGA4, but not with other TGAs. We further demonstrated that BIN2 phosphorylates Ser-202 of TGA4, resulting in the suppression of the redox-dependent interaction between TGA4 and NPR1 as well as destabilization of TGA4. Consistently, transgenic Arabidopsis overexpressing TGA4-YFP with a S202A mutation displayed enhanced SA responses compared to the wild-type TGA4-YFP plants. Taken together, these results suggest a novel crosstalk mechanism by which BR signaling coordinates the SA responses mediated by redox-sensitive clade I TGAs.

Inhibition of 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE expression by brassinosteroid reduces carotenoid accumulation in Arabidopsis.

Unlike the indispensable function of the steroid hormone brassinosteroid (BR) in regulating plant growth and development, the metabolism of secondary metabolites regulated by BR is not well known. Here we show that BR reduces carotenoid accumulation in Arabidopsis seedlings. BR-deficient or BR-insensitive mutants accumulated higher content of carotenoids than wild-type plants, whereas BR treatment reduced carotenoid content. We demonstrated that BR transcriptionally suppresses 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) expression involved in carotenogenesis via plastoquinone production. We found that the expression of HPPD displays an oscillation pattern that is expressed more strongly in dark than in light conditions. Moreover, BR appeared to inhibit HPPD expression more strongly in darkness than in light, leading to suppression of a diurnal oscillation of HPPD expression. BR-responsive transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) directly bound to the promoter of HPPD, and HPPD suppression by BR was increased in the bzr1-1D gain-of-function mutation. Interestingly, dark-induced HPPD expression did not cause carotenoid accumulation, due to down-regulation of other carotenoid biosynthetic genes in the dark. Our results suggest that BR regulates different physiological responses in dark and light through inhibition of HPPD expression.

Chaperone-like protein DAY plays critical roles in photomorphogenesis.

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

Gold nanoparticle-assisted SELEX as a visual monitoring platform for the development of small molecule-binding DNA aptasensors.

To resolve time-consuming and imperceptible monitoring problems in the traditional systematic evolution of ligands by exponential enrichment (SELEX), we report gold nanoparticle-assisted SELEX (GNP-SELEX) as a visual, proofreading, and self-monitoring platform and its application to small molecule-binding single-stranded DNA (ssDNA) aptasensors. Through the colorimetric changes between rounds, GNP-SELEX enabled the rapid determination of target-specific aptamer library enrichment with neither target modification nor extra monitoring process. We identified ssDNA aptamers with high selectivity and binding affinity by targeting two small molecules (brassinolide; BL and bisphenol A; BPA) as a model. The rational design of selected aptamers by 3D molecular simulation increased their ability to detect BL or BPA in real samples as bioreceptors. These results suggest that GNP-SELEX is useful as a self-monitoring platform to discover ssDNA aptamers as well as to develop aptasensors for diverse targets in a rapid and simple way.

Microbial Redox Regulator-Enabled Pulldown for Rapid Analysis of Plasma Low-Molecular-Weight Biothiols.

Although low-molecular-weight (LMW) biothiols function as a disease indicator in plasma, rapidly and effectively analyzing them remains challenging in the extracellular oxidative environment due to technical difficulties. Here, we report a newly designed, affinity pulldown platform using a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA for rapid and cost-effective analyses of plasma LMW biothiols. In the presence of organic hydroperoxide, LMW biothiols triggered the rapid dissociation of FAM-labeled dsDNA from FLAG-tagged OhrRBS via S-thiolation of OhrRBS on anti-FLAG antibody-coated beads, which led to a strong increase of fluorescence intensity in the supernatant after pulldown. This method was easily extended by using a reducing agent to detect free and total LMW biothiols simultaneously in mouse plasma. Unlike free plasma LMW biothiols, total plasma LMW biothiols were more elevated in ΔLDLR mice than those in normal mice. Owing to the rapid dissociation of OhrR/dsDNA complexes in response to LMW biothiols, this pulldown platform is immediately suitable for monitoring rapid redox changes in plasma LMW biothiols as well as studying oxidative stress and diseases in blood.

OsBZR1 turnover mediated by OsSK22-regulated U-box E3 ligase OsPUB24 in rice BR response.

Oryza sativa BRASSINAZOLE RESISTANT 1 (OsBZR1) is the closest rice homolog of the Arabidopsis BZR1 and bri1-EMS-SUPPRESSOR 1 (BES1)/BZR2 transcription factors. OsBZR1 plays a central role in the rice brassinosteroid signaling pathway. Despite its functional importance, the control mechanism by which the cellular stability of OsBZR1 is regulated has not yet been fully elucidated. Here, we report that a rice U-box E3 ubiquitin (Ub) ligase OsPUB24 acts as a negative regulator in the BR signaling pathway via the 26S proteasome-dependent degradation of OsBZR1. The ospub24 T-DNA knock-out mutant and Ubi:RNAi-OsPUB24 knock-down rice plants displayed enhanced seedling growth, increased lamina joint bending, and hypersensitivity to brassinolide (BL). The expressions of the BR biosynthetic genes suppressed by BR in a negative feedback loop were lower in the mutant progeny than in the wild-type rice plants, which indicated increased BR responses in the mutant line. OsPUB24 ubiquitinated OsBZR1, resulting in the proteasomal degradation of OsBZR1. In addition, the stability of OsPUB24 was downregulated by BL and bikinin, an inhibitor of Oryza sativa Shaggy/GSK3-like kinase 22 (OsSK22). OsSK22, the homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein kinase, phosphorylated OsPUB24 and elevated the cellular stability of OsPUB24. Our findings suggest that OsPUB24 participates in OsBZR1 turnover, and that the regulatory networks of OsPUB24, OsSK22 and OsBZR1 are crucial for fine-tuning the BR response in rice.

Plant U-Box40 Mediates Degradation of the Brassinosteroid-Responsive Transcription Factor BZR1 in Arabidopsis Roots.

Brassinosteroid (BR) regulates a wide range of physiological responses through the activation of BRASSINAZOLE RESISTANT1 (BZR1), whose activity is tightly controlled by its phosphorylation status and degradation. Although BZR1 appears to be degraded in distinct ways in response to different hormonal or environmental cues, little is known about how BR signaling regulates its degradation. Here we show that the BR-regulated U-box protein PUB40 mediates the proteasomal degradation of BZR1 in a root-specific manner in Arabidopsis (Arabidopsis thaliana). BZR1 levels were strongly reduced by plant U-box40 (PUB40) overexpression, whereas the pub39 pub40 pub41 mutant accumulated much more BZR1 than wild type in roots. The bzr1-1D gain-of-function mutation reduced the interaction with PUB40, which suppressed PUB40-mediated BZR1 degradation in roots. The cell layer-specific expression of PUB40 in roots helps induce selective BZR1 accumulation in the epidermal layer. Both BR treatment and loss-of-function of PUB40 expanded BZR1 accumulation to most cell layers. In addition, BZR1 accumulation increased the resistance of pub39 pub40 pub41 to low inorganic phosphate availability, as observed in bzr1-1D BRASSINOSTEROID-INSENSITIVE2-induced phosphorylation of PUB40, which mainly occurs in roots, gives rise to BZR1 degradation through enhanced binding of PUB40 to BZR1 and PUB40's stability. Our results suggest a molecular mechanism of root-specific BZR1 degradation regulated by BR signaling.

Arabidopsis ACC Oxidase 1 Coordinated by Multiple Signals Mediates Ethylene Biosynthesis and Is Involved in Root Development.

Ethylene regulates numerous aspects of plant growth and development. Multiple external and internal factors coordinate ethylene production in plant tissues. Transcriptional and post-translational regulations of ACC synthases (ACSs), which are key enzymes mediating a rate-limiting step in ethylene biosynthesis have been well characterized. However, the regulation and physiological roles of ACC oxidases (ACOs) that catalyze the final step of ethylene biosynthesis are largely unknown in Arabidopsis. Here, we show that Arabidopsis ACO1 exhibits a tissue-specific expression pattern that is regulated by multiple signals, and plays roles in the lateral root development in Arabidopsis. Histochemical analysis of the ACO1 promoter indicated that ACO1 expression was largely modulated by light and plant hormones in a tissue-specific manner. We demonstrated that point mutations in two E-box motifs on the ACO1 promoter reduce the light-regulated expression patterns of ACO1. The aco1-1 mutant showed reduced ethylene production in root tips compared to wild-type. In addition, aco1-1 displayed altered lateral root formation. Our results suggest that Arabidopsis ACO1 integrates various signals into the ethylene biosynthesis that is required for ACO1's intrinsic roles in root physiology.

OST1 Activation by the Brassinosteroid-Regulated Kinase CDG1-LIKE1 in Stomatal Closure.

Crosstalk between signaling pathways is an important feature of complex regulatory networks. How signal crosstalk circuits are tailored to suit different needs of various cell types remains a mystery in biology. Brassinosteroid (BR) and abscisic acid (ABA) antagonistically regulate many aspects of plant growth and development through direct interactions between components of the two signaling pathways. Here, we show that BR and ABA synergistically regulate stomatal closure through crosstalk between the BR-activated kinase CDG1-LIKE1 (CDL1) and the OPEN STOMATA1 (OST1) of the ABA signaling pathway in Arabidopsis thaliana We demonstrate that the cdl1 mutant displayed reduced sensitivity to ABA in a stomatal closure assay, similar to the ost1 mutant. CDL1 and the BR receptor BR-INSENSITIVE1, but not other downstream components of the BR signaling pathway, were required for BR regulation of stomatal movement. Genetic and biochemical experiments demonstrated that CDL1 activates OST1 by phosphorylating it on residue Ser-7. BR increased phosphorylation of OST1, and the BR-induced OST1 activation was abolished in cdl1 mutants. Moreover, we found that ABA activates CDL1 in an OST1-dependent manner. Taken together, our findings illustrate a cell-type-specific BR signaling branch through which BR acts synergistically with ABA in regulating stomatal closure.

Differential expression of primary pair-rule genes during bidirectional regeneration in Perionyx excavatus.

The earthworm Perionyx excavatus is a species highly capable of bidirectional regeneration. Pair-rule genes are thought to have an ancestral function in arthropod segmentation. However, orthologs in annelids (i.e. Capitella teleta) do not exhibit segmental expression in the ectoderm or mesoderm. Their role in regeneration is currently unclear. Here, we report the expression profile of primary pair-rule genes (Pex-EvxA, Pex-EvxB, Pex-RuntA, Pex-RuntB, Pex-Hes1A, Pex-Hes1B, Pex-Hes4A, and Pex-Hey) found in P. excavatus using semi-quantitative reverse transcription-polymerase chain reaction. Our results indicated these genes showed variable expression during bidirectional regeneration. Six of these genes might play diverse and potentially critical roles in head and/or tail regeneration.

Function and molecular regulation of DWARF1 as a C-24 reductase in brassinosteroid biosynthesis in Arabidopsis.

DWARF1 (DWF1) is a sterol C-24 reductase that catalyses the conversion of 24-methylenecholesterol (24-MCHR) to campesterol (CR) in Arabidopsis. A loss-of-function mutant, dwf1, showed similar phenotypic abnormalities to brassinosteroid (BR)-deficient mutants. These abnormalities were reversed in the wild-type phenotype by exogenous application of castasterone (CS) and brassinolide (BL), but not dolichosterone (DS). Accumulation of DS and decreased CS were found in quantitative analysis of endogenous BRs in dwf1. The enzyme solution prepared from dwf1 was unable to convert 6-deoxoDS to 6-deoxoCS and DS to CS, as seen in either wild-type or 35S:DWF1 transgenic plants. This suggests that DWF1 has enzyme activity not only for a sterol C-24 reductase, but also for a BR C-24 reductase that catalyses C-24 reduction of 6-deoxoDS to 6-deoxoCS and of DS to CS in Arabidopsis. Overexpression of DWF1 in a BR-deficient mutant (det2 35S:DWF1) clearly rescued abnormalities found in det2, indicating that DWF1 functions in biosynthesis of active BRs in Arabidopsis. Expression of DWF1 is down-regulated by application of CS and BL and in a BR-dominant mutant, bes1-D. E-boxes in the putative promoter region of DWF1 directly bind to a BR transcription factor, BES1, implying that DWF1 expression is feedback-regulated by BR signaling via BES1. Overall, biosynthesis of 24-methylene BR is an alternative route for generating CS, which is mediated and regulated by DWF1 in Arabidopsis.

Nanocrack-regulated self-humidifying membranes.

The regulation of water content in polymeric membranes is important in a number of applications, such as reverse electrodialysis and proton-exchange fuel-cell membranes. External thermal and water management systems add both mass and size to systems, and so intrinsic mechanisms of retaining water and maintaining ionic transport in such membranes are particularly important for applications where small system size is important. For example, in proton-exchange membrane fuel cells, where water retention in the membrane is crucial for efficient transport of hydrated ions, by operating the cells at higher temperatures without external humidification, the membrane is self-humidified with water generated by electrochemical reactions. Here we report an alternative solution that does not rely on external regulation of water supply or high temperatures. Water content in hydrocarbon polymer membranes is regulated through nanometre-scale cracks ('nanocracks') in a hydrophobic surface coating. These cracks work as nanoscale valves to retard water desorption and to maintain ion conductivity in the membrane on dehumidification. Hydrocarbon fuel-cell membranes with surface nanocrack coatings operated at intermediate temperatures show improved electrochemical performance, and coated reverse-electrodialysis membranes show enhanced ionic selectivity with low bulk resistance.

ARF7 increases the endogenous contents of castasterone through suppression of BAS1 expression in Arabidopsis thaliana.

Homeostasis of brassinosteroids (BRs) maintained by the balance between their biosynthesis and inactivation is important to coordinate the diverse physiological and developmental responses of plants. Although BR signaling regulates the endogenous levels of BRs via negative feedback regulation, it remains largely unknown how the biosynthesis and inactivation of BR are triggered. BAS1 encodes CYP734A1, which inactivates the biologically active BRs via C-26 hydroxylation and is down-regulated by a BR-responsive transcription factor, BZR1. Here it is demonstrated that the expression of the BAS1 gene is regulated by auxin response factors (ARFs) in Arabidopsis thaliana. Two successive E-box motifs on the BAS1 promoter function as BZR1 binding sites and are essential for BR-regulated BAS1 expression. The expression of BAS1 is increased in the arf7 and arf7arf19 mutants. The endogenous level of bioactive BR, castasterone, is greatly decreased in those mutants. ARF7 can bind to the E-box motifs of the BAS1 promoter where BZR1 binds, suggesting that ARF7 and BZR1 mutually compete for the same cis-element of the BAS1 promoter. Additionally, ARF7 directly interacts with BZR1, which inhibits their DNA binding activities and regulation of BAS1 expression. In conclusion, auxin signaling via ARF7 directly modulates the expression of BAS1 by competition with BZR1, thereby increasing the level of castasterone and promoting growth and development in A. thaliana.

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.

Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (µIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the µIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by µIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.