Dual regulation of stomatal development by brassinosteroid in Arabidopsis hypocotyls.

Stomata are epidermal pores that are essential for water evaporation and gas exchange in plants. Stomatal development is orchestrated by intrinsic developmental programs, hormonal controls, and environmental cues. The steroid hormone brassinosteroid (BR) inhibits stomatal lineage progression by regulating BIN2 and BSL proteins in leaves.

Transcription factors BZR1 and PAP1 cooperate to promote anthocyanin biosynthesis in Arabidopsis shoots.

Anthocyanins play critical roles in protecting plant tissues against diverse stresses. The complicated regulatory networks induced by various environmental factors modulate the homeostatic level of anthocyanins. Here, we show that anthocyanin accumulation is induced by brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana) shoots and shed light on the underlying regulatory mechanism.

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.

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.

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.

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.

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.

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.

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.

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 -derived organic hydroperoxide resistance regulatory (OhrR) 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 OhrR via S-thiolation of OhrR on anti-FLAG antibody-coated beads, which led to a strong increase of fluorescence intensity in the supernatant after pulldown.

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.

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 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.

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.

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.

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).

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.

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.

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.

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.

Functional insights of plant GSK3-like kinases: multi-taskers in diverse cellular signal transduction pathways.

The physiological importance of GSK3-like kinases in plants emerged when the functional role of plant GSK3-like kinases represented by BIN2 was first elucidated in the brassinosteroid (BR)-regulated signal transduction pathway. While early studies focused more on understanding how GSK3-like kinases regulate BR signaling, recent studies have implicated many novel substrates of GSK3-like kinases that are involved in a variety of cellular processes as well as BR signaling. Plant GSK3-like kinases play diverse roles in physiological and developmental processes such as cell growth, root and stomatal cell development, flower development, xylem differentiation, light response, and stress responses.

Rapid detection of protein phosphatase activity using Zn(II)-coordinated gold nanosensors based on His-tagged phosphopeptides.

We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution.

Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum.

An in vitro enzyme assay using radioisotope-labeled (3) H-castasterone ((3) H-CS) or (32) P-ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography-mass spectrometry (GC-MS) analysis using non-isotope-labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C-23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay.