Glenn R. Hicks

The phytohormone jasmonoyl-L-isoleucine (JA-Ile) regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein Coronatine Insensitive 1 (COI1) and a Jasmonate (JA) ZIM-domain (JAZ) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signaling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI–JAZ interaction and degradation of JAZ1 and JAZ9 in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation, and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana) COI1–JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1–JAZ also occurs in the liverwort Marchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signaling, J4 works as an antagonist of the closely related auxin signaling pathway, preventing Transport Inhibitor Response1/Aux–indole-3-acetic acid interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression, and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways. One-sentence summary: A chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and nonvascular plants.

The phytohormone JA-Ile regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein COI1 (Coronatine Insensitive 1) and a JAZ (Jasmonate ZIM-domain) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signalling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI-JAZ interaction and degradation of JAZ1 and JAZ9 in vivo . One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis ( Arabidopsis thaliana) COI1-JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1- JAZ also occurs in the liverwort Marchantia polymorpha , suggesting the mode of action is conserved in land plants. Besides JA signalling, J4 works as an antagonist of the closely-related auxin signalling pathway, preventing TIR1/Aux-IAA interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways. One sentence summary: A chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and non-vascular plants.

The cellulose synthase inhibitor Endosidin20 is an excellent tool for manipulating cellulose synthesis in order to produce cellulose products with desired prope

Cellulose is synthesized by rosette structured cellulose synthase (CESA) complexes (CSCs), each of which is composed of multiple units of CESAs in three different isoforms. CSCs rely on vesicle trafficking for delivery to the plasma membrane where they catalyze cellulose synthesis. Although the rosette structured CSCs were observed decades ago, it remains unclear what amino acids in plant CESA that directly participate in cellulose catalytic synthesis. It is also not clear how the catalytic activity of CSCs influences their efficient transport at the subcellular level. Here we report characterization of the small molecule Endosidin20 (ES20) and present evidence that it represents a new CESA inhibitor. We show data from chemical genetic analyses, biochemical assays, structural modeling, and molecular docking to support our conclusion that ES20 targets the catalytic site of Arabidopsis CESA6. Further, chemical genetic analysis reveals important amino acids that potentially form the catalytic site of plant CESA6. Using high spatiotemporal resolution live-cell imaging, we found that inhibition of CSC catalytic activity by inhibitor treatment, or by creating missense mutation at amino acids in the predicted catalytic site, causes reduced efficiency in CSC transport to the plasma membrane. Our results show that the catalytic activity of plant CSCs is integrated with subcellular trafficking dynamics. One sentence summary: Endosidin20 targets cellulose synthase at the catalytic site to inhibit cellulose synthesis and the inhibition of catalytic activity reduces cellulose synthase complex delivery to the plasma membrane.

Interactions between plant cells and the environment rely on modulation of protein receptors, transporters, channels, and lipids at the plasma membran

Significance: In plants, the retromer complex (VPS35, VPS29, VPS26) has been implicated in retrograde traffic from the endosomes back to the trans -Golgi network. Nevertheless, vps35 mutants exhibit impaired anterograde traffic toward the vacuole (synthetic, endocytic, and autophagic). This paradox remains an open question. We show that the small molecule endosidin17 (ES17) targets VPS35, which disrupts its association with RABG3f. This, in turn, impairs retromer complex membrane anchoring, producing vps35 mutant-like phenotypes. Using ES17, we found that VPS35–RABG3f binding precedes the release of RABG3f–GTP to interact with the vacuolar homotypic fusion and vacuole protein sorting (HOPS) complex, which is required for fusion of late compartments with the tonoplast. Thus, VPS35–RABG3f interaction constitutes a checkpoint before proceeding with membrane fusion.

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Saccharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development.

The endomembrane trafficking network is highly complex and dynamic, with both conventional and so-called unconventional routes which are in essence recently discovered pathways that are poorly understood in plants. One approach to dissecting endomembrane pathways that we have pioneered is the use of chemical biology. Classical genetic manipulations often deal with indirect pleiotropic phenotypes resulting from the perturbation of key players of the trafficking routes. Many of these difficulties can be circumvented using small molecules to modify or disrupt the function or localization of key proteins regulating these pathways. In this review, we summarize how small molecules have been used as probes to define these pathways, and how they could be used to increase current knowledge of unconventional protein secretion pathways.