Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling.

SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.

In Planta Labeling Using a Clickable ER-Disrupting Probe Suggests a Role for Oleosins in Arabidopsis Seedling ER Integrity.

Several small-molecule perturbagens of the plant endomembrane system are known, but few selectively disrupt endoplasmic reticulum (ER) structure and function. We conducted a microscopy-based screen for small-molecule disruptors of ER structure and discovered eroonazole, a 1,2-4-triazole that induces extensive ER vesiculation in Arabidopsis seedlings. To identify eroonazole targets, we synthesized a clickable photoaffinity derivative and used it for whole-seedling labeling experiments. These reveal that the probe labels multiple oleosins, plant membrane proteins that stabilize ER-derived lipid droplets. Oleosin labeling is absent in an oleosin1234 quadruple mutant and reduced using an inactive analog. Cellular analyses of the ER in the quadruple mutant demonstrate that oleosins are required for normal ER structure during seed germination and suggest that perturbation of oleosin function by eroonazole underlies its effects on seedling ER structure.

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.

Dynamic control of plant water use using designed ABA receptor agonists.

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.

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors.

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

Small Molecule Probes of ABA Biosynthesis and Signaling.

The phytohormone ABA mediates many physiological and developmental responses, and its key role in plant water relations has fueled efforts to improve crop water productivity by manipulating ABA responses. ABA's core signaling components are encoded by large gene families, which has hampered functional studies using classical genetic approaches due to redundancy. Chemical approaches can complement genetic approaches and have the advantage of delivering both biological probes and potential agrochemical leads; these benefits have spawned the discovery and design of new chemical modulators of ABA signaling and biosynthesis, which have contributed to the identification of ABA receptors and helped to define PYR1 and related subfamily III receptors as key cellular targets for chemically manipulating water productivity. In this review, we provide an overview of small molecules that have helped dissect both ABA signaling and metabolic pathways. We further discuss how the insights gleaned using ABA probe molecules might be translated to improvements in crop water productivity and future opportunities for development of small molecules that affect ABA metabolism and signaling.

Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology.

The treatment of a biological system with small molecules to specifically perturb cellular functions is commonly referred to as chemical biology. Small molecules are used commercially as drugs, herbicides, and fungicides in different systems, but in recent years they are increasingly exploited as tools for basic research. For instance, chemical genetics involves the discovery of small-molecule effectors of various cellular functions through screens of compound libraries. Whereas the drug discovery field has largely been driven by target-based screening approaches followed by drug optimization, chemical genetics in plant systems tends to be fueled by more general phenotype-based screens, opening the possibility to identify a wide range of small molecules that are not necessarily directly linked to the process of interest. Here, we provide an overview of the current progress in chemical genetics in plants, with a focus on the discoveries regarding small molecules identified in screens designed with a basic biology perspective. We reflect on the possibilities that lie ahead and discuss some of the potential pitfalls that might be encountered upon adopting a given chemical genetics approach.

Danger-associated peptide signaling in Arabidopsis requires clathrin.

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H(+)-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

The brassinosteroid chemical toolbox.

Chemical biology approaches have been instrumental in understanding the mode of action of brassinosteroids, a group of plant steroid hormones essential for plant development and growth. The small molecules used for such approaches include inhibitors of biosynthetic enzymes and signaling components. Additionally, recent structural data on the brassinosteroid receptor complex together with its ligand brassinolide, the most active brassinosteroid, and knowledge on its different analogs have given us a better view on the recognition of the hormone and signaling initiation. Moreover, a fluorescently labeled brassinosteroid enabled the visualization of the receptor-ligand pair in the cell. Given the insights obtained, small molecules will continue to provide new opportunities for probing brassinosteroid biosynthesis and for unraveling the dynamic and highly interconnected signaling.

Target identification strategies in plant chemical biology.

The current needs to understand gene function in plant biology increasingly require more dynamic and conditional approaches opposed to classic genetic strategies. Gene redundancy and lethality can substantially complicate research, which might be solved by applying a chemical genetics approach. Now understood as the study of small molecules and their effect on biological systems with subsequent target identification, chemical genetics is a fast developing field with a strong history in pharmaceutical research and drug discovery. In plant biology however, chemical genetics is still largely in the starting blocks, with most studies relying on forward genetics and phenotypic analysis for target identification, whereas studies including direct target identification are limited. Here, we provide an overview of recent advances in chemical genetics in plant biology with a focus on target identification. Furthermore, we discuss different strategies for direct target identification and the possibilities and challenges for plant biology.

Mitochondrial perturbation negatively affects auxin signaling.

Mitochondria are crucial players in the signaling and metabolic homeostasis of the plant cell. The molecular components that orchestrate the underlying processes, however, are largely unknown. Using a chemical biology approach, we exploited the responsiveness of Arabidopsis UDP-glucosyltransferase-encoding UGT74E2 towards mitochondrial perturbation in order to look for novel mechanisms regulating mitochondria-to-nucleus communication. The most potent inducers of UGT74E2 shared a (2-furyl)acrylate (FAA) substructure that negatively affected mitochondrial function and was identified before as an auxin transcriptional inhibitor. Based on these premises, we demonstrated that perturbed mitochondria negatively affect the auxin signaling machinery. Moreover, chemical perturbation of polar auxin transport and auxin biosynthesis was sufficient to induce mitochondrial retrograde markers and their transcript abundance was constitutively elevated in the absence of the auxin transcriptional activators ARF7 and ARF19.

The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis.

Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, brassinosteroid insensitive1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.

Small molecules for dissecting endomembrane trafficking: a cross-systems view.

Endomembrane trafficking has a key role for ensuring homeostasis, growth and development, hormonal signaling, and adaptation of eukaryotes to the constantly changing environmental conditions. The complex organization of the endomembrane system implies the need for searching novel tools to specifically probe the regulatory components and dissect the tightly interconnected vesicle transport pathways. Here, we review the large-scale chemical genetic screens, which led to the identification of small molecules with an impact on various parts of the vesicle trafficking network. We discuss the similarities and differences in the organization of the endomembrane systems in yeasts, mammals, and plants based on studies of small molecules and their effects on trafficking hubs, routes, and conserved protein targets.