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Molecular genetic and structural studies have revealed the mechanisms of fundamental components of key auxin regulatory pathways consisting of auxin biosynthesis, transport, and signaling. Chemical biology methods applied in auxin research have been greatly expanded through the understanding of auxin regulatory pathways. Many small-molecule modulators of auxin metabolism, transport, and signaling have been generated on the basis of the outcomes of genetic and structural studies on auxin regulatory pathways. These chemical modulators are now widely used as essential tools for dissecting auxin biology in diverse plants. This review covers the structures, primary targets, modes of action, and applications of chemical tools in auxin biosynthesis, transport, and signaling., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1 -overexpression lines ( AtBBD1 -OX), single knockout atbbd1 , and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1 -OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1 -OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1 -OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.

Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing a pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between a leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor's island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of the PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel "druggable" binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.

Glutamate receptor-like channels (GLRs) play important roles in numerous plant physiological processes. GLRs are homologous to ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in vertebrates. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.58- and 1.75-Å resolution, respectively. Our structures show a fold similar to that of iGluRs, but with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The mutation R133A strongly increases the constitutive activity of the channel, suggesting that the LBD mutated at the residue critical for agonist binding produces a more stable closed clamshell conformation. Furthermore, our structures explain the promiscuity of GLR activation by different amino acids, confirm evolutionary conservation of structure between GLRs and iGluRs, and predict common molecular principles of their gating mechanisms driven by bilobed clamshell-like LBDs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The binding of strigolactones to their receptor, the α/β hydrolase DWARF14 (D14), leads to the modulation of transcriptional activity by destabilization of specific transcriptional corepressors via proteasomal degradation. Subsequently, strigolactones also promote D14 degradation by the same pathway. Here we describe an innovative quantitative bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to the firefly luciferase, developed to identify new strigolactone analogs capable to activate the strigolactone signaling.

Abscisic acid (ABA) although complicated and expensive to produce, plays an important role in signalling responsible for regulation of developmental manifestations such as seed maturation and surviving through stress conditions. Hence, development of cost effective molecules with minimal side effects that mimic the functions of ABA is the need of the hour. In this agreement, we screened a series of 27 in-house synthesized 3-methyleneisoindolin-1-one molecules over three ABA receptors (PYR1, PYL1, and PYL3). The commercial ABA agonist Pyrabactin was taken as a standard ligand in this study. The top three molecules for each receptor were selected and further evaluated to estimate the dynamical contribution and complex stability via Molecular Mechanics-Poisson Boltzmann surface area calculations. Two molecules (Mol26 and Mol25) showed higher binding free energy and stable complex conformation for PYL3 in comparison to Pyrabactin. This study revealed the structural basis of the binding mechanism of 3-methyleneisoindolin-1-one molecules with ABA receptors. Mol26 and Mol25 were identified for the development of specific PYL3 agonists with a vast potential in agriculture to accentuate the ABA like action in plants., (Copyright © 2020. Published by Elsevier Masson SAS.)

In plants, biosynthesized ABA undergoes two important physiological processes of signal transduction and metabolism simultaneously. In this study, we described a class of ABA receptor agonist/antagonist switching probes APA n , which can regulate the agonistic activity or antagonistic activity according to the length of a 6'-alkoxyl chain. From APA1 to APA6, with the extension of the alkoxyl chain, it showed a gradually increased receptor-binding potential and decreased HAB1 inhibition activity. Theoretical analysis based on molecular docking and molecular dynamics simulation revealed that some factors outside the ligand-binding pocket in receptors could also affect the binding of the ligand to the receptor, for example, the van der Waals interaction between the alkyl chain in APA n and the 3'-tunnel of ABA receptors made it bind more tightly than iso -PhABA. This enhanced binding made it an antagonist rather than a weakened agonist.

Abscisic acid (ABA) is a ubiquitous phytohormone, plays important roles in several physiological processes, including stress adaptation, flowering, seed germination, fruit ripening, and leaf senescence etc. ABA binds with START domain proteins called Pyrabactin Resistance1 (PYR1)/PYR1-like (PYL)/Regulatory Components of ABA Receptors (RCARs) and controls the activity of PP2C phosphatase proteins and in turn the ABA-dependent signaling pathway. Fourteen ABA receptors have been identified in the model plant Arabidopsis thaliana and have shown to be involved in various biological functions. Under field conditions, exogenous application of ABA produces inadequate physiological response due to its rapid conversion into the biologically inactive metabolites. ABA shows selective binding preferences to PYL receptor subtypes and hence produces pleiotropic physiological and phenotypic effects which limit the usage of ABA in agriculture. An agrochemical meant for ameliorating the undesirable physiological effect of the plant should ideally have positive biological attributes without affecting the normal growth, development, and yield. Therefore, to overcome the limitations of ABA for its usage in various agricultural applications, several types of ABA-mimicking agents have been developed. Many compounds have been identified as having significant ABA-agonist/antagonist activity and can be employed to reverse the excessive/moderate ABA action. The present review highlights the potential usage of ABA signaling modulators for managing agronomic and postharvest traits. Besides, designing, development and versatile usage of ABA-mimicking compounds displaying ABA agonists and antagonist activities are discussed in detail., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca 2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for l-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology., Competing Interests: The authors declare no competing interest.

Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The plant hormone abscisic acid (ABA) regulates the development of various plant organs including seeds, roots, and fruits, and significantly contributes to abiotic stress responses, especially to drought. Since recent climate changes are adversely affecting crop cultivation, enhancement of plant stress tolerance by regulation of ABA signaling would be an important strategy. In the plant genome, ABA receptors are encoded by multiple genes constituting three subfamilies; however, functional differences among them remain unclear. To enhance desired effects of ABA, the biological functions of the receptor family warrant clarification. This study aimed to determine the functional differences among ABA receptors in plants. We screened small-molecule ligands binding to specific receptors, using a chemical array. In vitro evaluation of hit compounds using 11 Arabidopsis ABA receptors revealed that (+)-3'-alkyl ABAs served as agonists for different receptors depending on the length of their 3'-alkyl chains. Combinatorial in vitro and physiological effects of these compounds on the stomata, seeds, and seedlings indicated that, along with subfamily III, receptors of subfamily II are important to induce strong drought responses. Among (+)-3'-alkyl ABAs assessed herein, (+)-3'-butyl ABA induced a transcriptional response and stomatal closure but only slightly inhibited seed germination and growth, suggesting that it enhances drought tolerance. In silico docking simulation and site-directed mutagenesis revealed the amino acid residues contributing to the selective agonist activity of the (+)-3'-alkyl ABAs. These results provide novel insights into the structure and biological effects of 3'-derivatives of ABA and a basis for agrochemical development.

HTL/KAI2, a member of the α/β-fold hydrolase superfamily, is known to be a receptor-like protein of lactone compounds and that triggers seed germination of Arabidopsis. However, the endogenous ligand and physiological roles of HTL/KAI2 have remained unclear. To understand the mechanism underlying seed germination involved in HTL/KAI2 signaling, it is necessary to identify the endogenous ligand of HTL/KAI2. To date, even a biosynthetic mutant of the ligand has not yet been isolated. Because exogenous agonistic chemicals can only be purchased in small amounts at high prices, the limited supply of those chemicals has hampered any large-scale experiments, such as mutant screening. Therefore, easily synthesized and scalable artificial agonist would remove the limitation of the chemical supply and contribute to the identification of the endogenous ligand of HTL/KAI2 and/or the biosynthetic mutants. In this study, we demonstrated that designed chemicals with a phenoxyfuranone scaffold potently stimulated seed germination via HTL/KAI2 in Arabidopsis. As a result of screening of these chemicals, we selected a representative compound with convincing selectivity. Here in, we provide a new promising synthetic agonist of HTL/KAI2., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown 1,2 . Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis 3-7 . In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium 8-10 . The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.

Pyrabactin resistance 1 (PYR1) and related abscisic acid (ABA) receptors are new targets for manipulating plant drought tolerance. Here, we identify and use PYR1 hypersensitive mutants to define ligand binding hotspots and show that these can guide improvements in agonist potency. One hotspot residue defined, A160, is part of a pocket that is occupied by ABA's C6 methyl or by the toluyl methyl of the synthetic agonist quinabactin (QB). A series of QB analogues substituted at the toluyl position were synthesized and provide up to 10-fold gain in activity in vitro. Furthermore, we demonstrate that hypersensitive receptors can be used to improve the sensitivity of a previously described mammalian cell ABA-regulated transcriptional circuit by three orders of magnitude. Collectively, our data show that the systematic mapping of hypersensitivity sites in a ligand-binding pocket can help guide ligand optimization and tune the sensitivity of engineered receptors.

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

DNA damage observed during plant immune responses is reported to be an intrinsic component of plant immunity. However, other immune responses may suppress DNA damage to maintain host genome integrity. Here, we show that immunity-related DNA damage can be abrogated by preventing cell death triggered by Nucleotide-binding, Leucine-rich-repeat immune Receptors (NLRs). SNI1 (suppressor of npr1-1, inducible 1), a subunit of the structural maintenance of chromosome (SMC) 5/6 complex, was reported to be a negative regulator of systemic acquired resistance (SAR) and to be necessary for controlling DNA damage. We find that cell death and DNA damage in sni1 loss-of-function mutants are prevented by mutations in the NLR signaling component EDS1. Similar to sni1, elevated DNA damage is seen in other autoimmune mutants with cell death lesions, including camta3, pub13 and vad1, but not in dnd1, an autoimmune mutant with no visible cell death. We find that as in sni1, DNA damage in camta3 is EDS1-dependent, but that it is also NLR-dependent. Using the NLR RPM1 as a model, we also show that extensive DNA damage is observed when an NLR is directly triggered by effectors. We also find that the expression of DNA damage repair (DDR) genes in mutants with cell death lesions is down regulated, suggesting that degraded DNA that accumulates during cell death is a result of cellular dismantling and is not sensed as damaged DNA that calls for repair. Our observations also indicate that SNI1 is not directly involved in SAR or DNA damage accumulation.

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.

Drought stress is a major threat to crop production, but effective methods to mitigate the adverse effects of drought are not available. Here, we report that adding fluorine atoms in the benzyl ring of the abscisic acid (ABA) receptor agonist AM1 optimizes its binding to ABA receptors by increasing the number of hydrogen bonds between the compound and the surrounding amino acid residues in the receptor ligand-binding pocket. The new chemicals, known as AMFs, have long-lasting effects in promoting stomatal closure and inducing the expression of stress-responsive genes. Application of AMFs or transgenic overexpression of the receptor PYL2 in Arabidopsis and soybean plants confers increased drought resistance. The greatest increase in drought resistance is achieved when AMFs are applied to the PYL2-overexpression transgenic plants. Our results demonstrate that the combining of potent chemicals with transgenic overexpression of an ABA receptor is very effective in helping plants combat drought stress.

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure-activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The phytohormone abscisic acid (ABA) plays a crucial role in mediating plant growth and development by recruiting genetically redundant ABA receptors. To overcome its oxidation inactivation, we developed a novel ABA analog named 2',3'-benzo-iso-ABA (iso-PhABA) and studied its function and structural characterization with A. thaliana ABA receptors. The (+)-iso-PhABA form showed much higher ABA-like activities than (+)-ABA including inhibitory effects on the seed germination of lettuce and A. thaliana, wheat embryo germination and rice seedling elongation. The PP2C (protein phosphatases 2C) activity assay showed that (+)-iso-PhABA acted as a potent and selective ABA receptor agonist, which is preferred to PYL10. In some cases, (-)-iso-PhABA showed moderate to high activity for the PYL protein inhibiting PP2C activity, suggesting different mechanisms of action of iso-PhABA and ABA. The complex crystal structure of iso-PhABA with PYL10 was determined and elucidated successfully, revealing that (+)-iso-PhABA was better coordinated in the same binding pocket compared to (+)-ABA. Moreover, the detailed interaction network of iso-PhABA/PYL10 was disclosed and involves hydrogen bonds and multiple hydrophobic interactions that provide a robust framework for the design of novel ABA receptor agonists/antagonists.

Gibberellin (GA) is a major plant hormone that regulates plant growth and development and is widely used as a plant growth regulator in agricultural production. There is an increasing demand for function-limited GA mimics due to the limitations on the agronomical application of GA to crops, including GA's high cost of producing and its leading to the crops' lodging. AC94377, a substituted phthalimide, is a chemical that mimics the growth-regulating activity of GAs in various plants, despite its structural difference. Although AC94377 is widely studied in many weeds and crops, its mode of action as a GA mimic is largely unknown. In this study, we confirmed that AC94377 displays GA-like activities in Arabidopsis (Arabidopsis thaliana) and demonstrated that AC94377 binds to the Arabidopsis GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor (AtGID1), forms the AtGID1-AC94377-DELLA complex, and induces the degradation of DELLA protein. Our results also indicated that AC94377 is selective for a specific subtype among three AtGID1s and that the selectivity of AC94377 is attributable to a single residue at the entrance to the hydrophobic pocket of GID1. We conclude that AC94377 is a GID1 agonist with selectivity for a specific subtype of GID1, which could be further developed and used as a function-limited regulator of plant growth in both basic study and agriculture., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Agricultural productivity is dictated by water availability and consequently drought is the major source of crop losses worldwide. The phytohormone abscisic acid (ABA) is elevated in response to water deficit and modulates drought tolerance by reducing water consumption and inducing other drought-protective responses. The recent identification of ABA receptors, elucidation of their structures and understanding of the core ABA signaling network has created new opportunities for agrochemical development. An unusually large gene family encodes ABA receptors and, until recently, it was unclear if selective or pan-agonists would be necessary for modulating water use. The recent identification of the selective agonist quinabactin has resolved this issue and defined Pyrabactin Resistance 1 (PYR1) and its close relatives as key targets for water use control. This review provides an overview of the structure and function of ABA receptors, progress in the development of synthetic agonists, and the use of orthogonal receptors to enable agrochemical control in transgenic plants., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The specific cochaperonin, chloroplast chaperonin (Cpn)20, consisting of two tandem GroES-like domains, is present abundantly in plant and algal chloroplasts, in addition to Cpn10, which is similar in size to GroES. How Cpn20 oligomers, containing six or eight 10-kDa domains, cooperate with the heptameric ring of chaperonin at the same time as encountering symmetry mismatch is unclear. In the present study, we characterized the functional cooperation of cochaperonins, including two plastidic Cpn20 homo-oligomers from Arabidopsis (AtCpn20) and Chlamydomonas (CrCPN20), and one algal CrCPNs hetero-oligomer, consisting of three cochaperonins, CrCPN11, CrCPN20 and CrCPN23, with two chaperonins, Escherichia coli GroEL and Chlamydomonas CrCPN60. AtCpn20 and CrCPNs were functional for assisting both chaperonins in folding model substrates ribulose bisphosphate carboxylase oxygenase from Rhodospirillum rubrum (RrRubisco) in vitro and complementing GroES function in E. coli. CrCPN20 cooperated only with CrCPN60 (and not GroEL) to refold RrRubisco in vitro and showed differential complementation with the two chaperonins in E. coli. Cochaperonin concatamers, consisting of six to eight covalently linked 10-kDa domains, were functionally similar to their respective native forms. Our results indicate that symmetrical match between chaperonin and cochaperonin is not an absolute requisite for functional cooperation., (© 2015 FEBS.)

Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules.

Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H(+)-adenosine triphosphatase 2 at Ser(899), mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion.

Abscisic acid (ABA) is the most important hormone for plants to resist drought and other abiotic stresses. ABA binds directly to the PYR/PYL family of ABA receptors, resulting in inhibition of type 2C phosphatases (PP2C) and activation of downstream ABA signaling. It is envisioned that intervention of ABA signaling by small molecules could help plants to overcome abiotic stresses such as drought, cold and soil salinity. However, chemical instability and rapid catabolism by plant enzymes limit the practical application of ABA itself. Here we report the identification of a small molecule ABA mimic (AM1) that acts as a potent activator of multiple members of the family of ABA receptors. In Arabidopsis, AM1 activates a gene network that is highly similar to that induced by ABA. Treatments with AM1 inhibit seed germination, prevent leaf water loss, and promote drought resistance. We solved the crystal structure of AM1 in complex with the PYL2 ABA receptor and the HAB1 PP2C, which revealed that AM1 mediates a gate-latch-lock interacting network, a structural feature that is conserved in the ABA-bound receptor/PP2C complex. Together, these results demonstrate that a single small molecule ABA mimic can activate multiple ABA receptors and protect plants from water loss and drought stress. Moreover, the AM1 complex crystal structure provides a structural basis for designing the next generation of ABA-mimicking small molecules.

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1-AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta.

The regulation of gene expression by the hormone auxin is a crucial mechanism in plant development. We have shown that the Arabidopsis F-box protein TIR1 is a receptor for auxin, and our recent structural work has revealed the molecular mechanism of auxin perception. TIR1 is the substrate receptor of the ubiquitin-ligase complex SCF(TIR1). Auxin binding enhances the interaction between TIR1 and its substrates, the Aux/IAA repressors, thereby promoting the ubiquitination and degradation of Aux/IAAs, altering the expression of hundreds of genes. TIR1 is the prototype of a new class of hormone receptor and the first example of an SCF ubiquitin-ligase modulated by a small molecule. Here, we describe the design, synthesis, and characterization of a series of auxin agonists and antagonists. We show these molecules are specific to TIR1-mediated events in Arabidopsis, and their mode of action in binding to TIR1 is confirmed by x-ray crystallographic analysis. Further, we demonstrate the utility of these probes for the analysis of TIR1-mediated auxin signaling in the moss Physcomitrella patens. Our work not only provides a useful tool for plant chemical biology but also demonstrates an example of a specific small-molecule inhibitor of F-box protein-substrate recruitment. Substrate recognition and subsequent ubiquitination by SCF-type ubiquitin ligases are central to many cellular processes in eukaryotes, and ubiquitin-ligase function is affected in several human diseases. Our work supports the idea that it may be possible to design small-molecule agents to modulate ubiquitin-ligase function therapeutically.