Plant Hormone Receptors Research Articles

GMMA Can Stabilize Proteins Across Different Functional Constraints

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2–6 °C in thermal inactivation temperature and no decrease in functionality.

Repurposing plant hormone receptors as chemically-inducible genetic switches for dynamic regulation in yeast

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.

The cell surface is the place to be for brassinosteroid perception and responses.

Adjusting the microenvironment around the cell surface is critical to responding to external cues or endogenous signals and to maintaining cell activities. In plant cells, the plasma membrane is covered by the cell wall and scaffolded with cytoskeletal networks, which altogether compose the cell surface. It has long been known that these structures mutually interact, but the mechanisms that integrate the whole system are still obscure. Here we spotlight the brassinosteroid (BR) plant hormone receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) since it represents an outstanding model for understanding cell surface signalling and regulation. We summarize how BRI1 activity and dynamics are controlled by plasma membrane components and their associated factors to fine-tune signalling. The downstream signals, in turn, manipulate cell surface structures by transcriptional and post-translational mechanisms. Moreover, the changes in these architectures impact BR signalling, resulting in a feedback loop formation. This Review discusses how BRI1 and BR signalling function as central hubs to integrate cell surface regulation.

Resolving binding pathways and solvation thermodynamics of plant hormone receptors

Plant hormones are small molecules that regulate plant growth, development, and responses to biotic and abiotic stresses. They are specifically recognized by the binding site of their receptors. In this work, we resolved the binding pathways for eight classes of phytohormones (auxin, jasmonate, gibberellin, strigolactone, brassinosteroid, cytokinin, salicylic acid, and abscisic acid) to their canonical receptors using extensive molecular dynamics simulations. Furthermore, we investigated the role of water displacement and reorganization at the binding site of the plant receptors through inhomogeneous solvation theory. Our findings predict that displacement of water molecules by phytohormones contributes to free energy of binding via entropy gain and is associated with significant free energy barriers for most systems analyzed. Also, our results indicate that displacement of unfavorable water molecules in the binding site can be exploited in rational agrochemical design. Overall, this study uncovers the mechanism of ligand binding and the role of water molecules in plant hormone perception, which creates new avenues for agrochemical design to target plant growth and development.

High-Performance Cannabinoid Sensor Empowered by Plant Hormone Receptors and Antifouling Magnetic Nanorods.

The misuse of cannabinoids and their synthetic variants poses significant threats to public health, necessitating the development of advanced techniques for detection of these compounds in biological or environmental samples. Existing methods face challenges like lengthy sample pretreatment and laborious antifouling steps. Herein, we present a novel sensing platform using magnetic nanorods coated with zwitterionic polymers for the simple, rapid, and sensitive detection of cannabinoids in biofluids. Our technique utilizes the engineered derivatives of the plant hormone receptor Pyrabactin Resistance 1 (PYR1) as drug recognition elements and employs the chemical-induced dimerization (CID) mechanism for signal development. Additionally, the magnetic nanorods facilitate efficient target capture and reduce the assay duration. Moreover, the zwitterionic polymer coating exhibits excellent antifouling capability, preserving excellent sensor performance in complex biofluids. Our sensors detect cannabinoids in undiluted biofluids like serum, saliva, and urine with a low limit of detection (0.002 pM in saliva and few pM in urine and serum) and dynamic ranges spanning up to 9 orders of magnitude. Moreover, the PYR1 derivatives demonstrate high specificity even in the presence of multiple interfering compounds. This work opens new opportunities for sensor development, showcasing the excellent performance of antifouling magnetic nanorods that can be compatible with different recognition units, including receptors and antibodies, for detecting a variety of targets.

Plant Regulation Functions of Novel Phthalimide Compounds Based on AtPYL2.

Novel agents contain the structure of phthalimide, which has antibacterial, insecticidal, and herbicidal activities. Recently, studies reported that these compounds can bind to plant hormone receptors and play important regulatory roles. In this study, the functions of agents were studied with in vitro and in vivo assays. The abscisic acid (ABA) receptor pyrabactin resistant-like 2 (PYL2) protein in Arabidopsis thaliana was expressed, purified, and crystallized; the analysis results of the crystal structure showed three AtPYL2 subunits in each asymmetric unit. The affinity of compounds Z1-Z11 to the AtPYL2 protein was tested by microscale thermophoresis (MST) and then verified by isothermal titration calorimetry (ITC). Furthermore, the binding pockets were found using molecular docking to verify the target relationships. Relevant in vivo assays for seed germination and a root growth assay were conducted, with the plant samples being treated with target compounds. The results show that the compounds Z3, Z5, and Z10 target AtPYL2 and that the dissociation constants for binding by MST were 3.59, 3.54, and 3.97 μmol/L, respectively, among them, and the molecular docking results showed that compounds Z3, Z5, and Z10 formed hydrophobic interactions with amino acid residues through hydrogen or halogen bonding. This highlights their potential as an ABA receptor protein agonist. On the other hand, in vivo, compounds Z3, Z5, and Z10 had different inhibitory effects on seed germination, with compound Z5 inhibiting the root growth of A. thaliana and compound Z10 affecting root growth. In conclusion, these compounds could regulate plant growth and could be further developed as new plant-regulating agents.

Difference in the ligand affinity among redundant plant hormone receptors of rice OsCOI1a/1b/2-OsJAZs.

(3R, 7S)-jasmonoyl-L-isoleucine (JA-Ile) is a lipid-derived plant hormone that regulates plant responses, including biotic/abiotic stress adaptation. In the plant cells, JA-Ile is perceived by COI1-JAZ co-receptor by causing protein-protein interaction between COI1 and JAZ proteins to trigger gene expressions. In this study, we focused on Oryza sativa, a model monocot and an important crop, with 45 possible OsCOI-OsJAZ co-receptor pairs composed of three OsCOI homologs (OsCOI1a, OsCOI1b, and OsCOI2) and 15 OsJAZ homologs. We performed fluorescein anisotropy and pull-down assays to examine the affinity between JA-Ile and OsCOI1a/1b/2-OsJAZ1-15 co-receptor pairs. The results revealed a remarkable difference in the modes of ligand perception by OsCOI1a/1b and OsCOI2. Recently, the unique function of OsCOI2 in some of the JA-responses were revealed. Our current results will lead to the possible development of OsCOI2-selective synthetic ligand.

Staying vigilant: NLR monitors virus invasion.

Phytohormones play important roles in plant immunity. Recently, Chen et al. discovered that the tomato spotted wilt virus attacks the plant hormone receptor to promote infection. Plants evolved an immune receptor to mimic the attacked hormone receptors to recognize the virus, thereby activating a robust immune response.

Information potential of an ubiquitous phytochemical cue.

This article is a Commentary on Li et al. (2023), 238: 2099–2112.

Subtype-selective agonists of plant hormone co-receptor COI1-JAZs identified from the stereoisomers of coronatine

Severe genetic redundancy is particularly clear in gene families encoding plant hormone receptors, each subtype sharing redundant and specific functions. Genetic redundancy of receptor family members represents a major challenge for the functional dissection of each receptor subtype. A paradigmatic example is the perception of the hormone (+)-7-iso-jasmonoyl-L-isoleucine, perceived by several COI1-JAZ complexes; the specific role of each receptor subtype still remains elusive. Subtype-selective agonists of the receptor are valuable tools for analyzing the responses regulated by individual receptor subtypes. We constructed a stereoisomer library consisting of all stereochemical isomers of coronatine (COR), a mimic of the plant hormone (+)-7-iso-jasmonoyl-L-isoleucine, to identify subtype-selective agonists for COI1-JAZ co-receptors in Arabidopsis thaliana and Solanum lycopersicum. An agonist selective for the Arabidopsis COI1-JAZ9 co-receptor efficiently revealed that JAZ9 is not involved in most of the gene downregulation caused by COR, and the degradation of JAZ9-induced defense without inhibiting growth.

Co-regulatory effects of hormone and mRNA-miRNA module on flower bud formation of Camellia oleifera.

Few flower buds in a high-yield year are the main factors restricting the yield of Camellia oleifera in the next year. However, there are no relevant reports on the regulation mechanism of flower bud formation. In this study, hormones, mRNAs, and miRNAs were tested during flower bud formation in MY3 ("Min Yu 3," with stable yield in different years) and QY2 ("Qian Yu 2," with less flower bud formation in a high-yield year) cultivars. The results showed that except for IAA, the hormone contents of GA3, ABA, tZ, JA, and SA in the buds were higher than those in the fruit, and the contents of all hormones in the buds were higher than those in the adjacent tissues. This excluded the effect of hormones produced from the fruit on flower bud formation. The difference in hormones showed that 21-30 April was the critical period for flower bud formation in C. oleifera; the JA content in MY3 was higher than that in QY2, but a lower concentration of GA3 contributed to the formation of the C. oleifera flower bud. JA and GA3 might have different effects on flower bud formation. Comprehensive analysis of the RNA-seq data showed that differentially expressed genes were notably enriched in hormone signal transduction and the circadian system. Flower bud formation in MY3 was induced through the plant hormone receptor TIR1 (transport inhibitor response 1) of the IAA signaling pathway, the miR535-GID1c module of the GA signaling pathway, and the miR395-JAZ module of the JA signaling pathway. In addition, the expression of core clock components GI (GIGANTEA) and CO (CONSTANS) in MY3 increased 2.3-fold and 1.8-fold over that in QY2, respectively, indicating that the circadian system also played a role in promoting flower bud formation in MY3. Finally, the hormone signaling pathway and circadian system transmitted flowering signals to the floral meristem characteristic genes LFY (LEAFY) and AP1 (APETALA 1) via FT (FLOWERING LOCUS T) and SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CO 1) to regulate flower bud formation. These data will provide the basis for understanding the mechanism of flower bud alternate formation and formulating high yield regulation measures for C. oleifera.

Functional reconstitution and structural characterization of the plant hormone receptor ETR1 in lipid nanodiscs.

The plant hormone receptor ETR1 regulates many highly relevant agronomic processes. Today, significant functional and structural questions remain unanswered regarding its multi-pass transmembrane sensor domain able to bind and respond to the gaseous plant hormone ethylene at femtomolar concentrations. A significant reason for this is the lack of structural data on full-length ETR1 in a lipid environment. Herein, we present the functional reconstitution of recombinant full-length ETR1 purified and solubilized from a bacterial host into lipid nanodiscs, allowing the study of the purified plant receptor for the first time in a detergent-free membrane-like environment.

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds

Optimization of hydration sites in plant hormone receptors for agrochemical design

Phytohormones are endogenous small molecules derived from common metabolic pathways that regulate key processes in plants, such as defense, development, and growth. Plant hormones are specifically recognized by the binding pocket in their receptors. Phytohormone receptors' agonists and antagonists have the potential to become useful agrochemicals. For this reason, it is necessary to characterize the factors determining the binding affinity and selectivity of these ligands to their receptors. In this work, we employed extensive molecular dynamics simulations coupled with Markov state models to study the binding of eight major classes of plant hormones (auxin, jasmonate, gibberellin, strigolactone, brassinosteroid, cytokinin, salicylic acid, and abscisic acid) to their receptors.

Mandipropamid as a chemical inducer of proximity for in vivo applications

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

Plant hormone functions and interactions in biological systems

Plant hormone functions and interactions in biological systems

The karrikin signaling regulator SMAX1 controls Lotus japonicus root and root hair development by suppressing ethylene biosynthesis

An evolutionarily ancient plant hormone receptor complex comprising the α/β-fold hydrolase receptor KARRIKIN INSENSITIVE 2 (KAI2) and the F-box protein MORE AXILLARY GROWTH 2 (MAX2) mediates a range of developmental responses to smoke-derived butenolides called karrikins (KARs) and to yet elusive endogenous KAI2 ligands (KLs). Degradation of SUPPRESSOR OF MAX2 1 (SMAX1) after ligand perception is considered to be a key step in KAR/KL signaling. However, molecular events which regulate plant development downstream of SMAX1 removal have not been identified. Here we show that Lotus japonicus SMAX1 is specifically degraded in the presence of KAI2 and MAX2 and plays an important role in regulating root and root hair development. smax1 mutants display very short primary roots and elongated root hairs. Their root transcriptome reveals elevated ethylene responses and expression of ACC Synthase 7 (ACS7), which encodes a rate-limiting enzyme in ethylene biosynthesis. smax1 mutants release increased amounts of ethylene and their root phenotype is rescued by treatment with ethylene biosynthesis and signaling inhibitors. KAR treatment induces ACS7 expression in a KAI2-dependent manner and root developmental responses to KAR treatment depend on ethylene signaling. Furthermore, in Arabidopsis, KAR-induced root hair elongation depends on ACS7 Thus, we reveal a connection between KAR/KL and ethylene signaling in which the KAR/KL signaling module (KAI2-MAX2-SMAX1) regulates the biosynthesis of ethylene to fine-tune root and root hair development, which are important for seedling establishment at the beginning of the plant life cycle.

Transcriptome analysis reveals that Populus tomentosa hybrid poplar 741 responds to blue light treatment by regulating growth-related genes and their metabolic pathways

In this study, Populus tomentosa hybrid poplar 741 seedlings were used for transcriptome analysis; growth characteristics under blue-light treatment were examined at different durations (1, 2, and 5 d), with white-light treatment used as the control, to reveal the response mechanism under blue light. The results showed height growth of poplar 741 were higher under blue light than under control at 1, 2, and 5 d, and increased by 380%, 216%, and 173%, respectively. The chlorophyll and carotenoid contents increased under blue light, indicating blue light was beneficial to photosynthetic pigments biosynthesis. The net photosynthetic rate (Pn), stomatal conductance, and Fv/Fm values were higher under blue light, with Pn, which was significantly increased by 20.47% at 2 d and 83.68% at 5 d, indicating blue light improved photosynthesis. In total, 505, 755, and 680 differentially expressed genes (DEGs) were detected at 1, 2, and 5 d, respectively. Twenty-six DEGs related to chlorophyll, photosynthesis, plant hormone, and blue light receptors were identified, and most of these were upregulated, indicating poplar 741 coped with blue light by regulating the expression of DEGs in porphyrin and chlorophyll metabolism, terpenoid backbone biosynthesis, diterpenoid biosynthesis, and circadian rhythm-plant pathways. This study revealed the physiological responses of poplar 741 to blue light and identified the potential pathways and candidate genes, which will provide information to explore the growth regulatory mechanism of poplar 741 under different light qualities.

Genome-wide phylogenetic and structural analysis reveals the molecular evolution of the ABA receptor gene family.

The plant hormone abscisic acid (ABA) plays a crucial role during the plant life cycle as well as in adaptive responses to environmental stresses. The core regulatory components of ABA signaling in plants are the pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor family (PYLs), which comprise the largest plant hormone receptor family known. They act as negative regulators of members of the protein phosphatase type 2C family. Due to the biological importance of PYLs, many researchers have focused on their genetic redundancy and consequent functional divergence. However, little is understood of their evolution and its impact on the generation of regulatory diversity. In this study, we identify positive selection and functional divergence in PYLs through phylogenetic reconstruction, gene structure and expression pattern analysis, positive selection analysis, functional divergence analysis, and structure comparison. We found the correlation of desensitization of PYLs under specific modifications in the molecular recognition domain with functional diversification. Hence, an interesting antagonistic co-evolutionary mechanism is proposed for the functional diversification of ABA receptor family proteins. We believe a compensatory evolutionary pathway may have occurred.