Long Nguyen

Chemical Inhibition of a Subset of Arabidopsis thaliana GSK3-like Kinases Activates Brassinosteroid Signaling

Glycogen synthase kinase 3 (GSK3) is a key regulator in signaling pathways in both animals and plants. Three Arabidopsis thaliana GSK3s are shown to be related to brassinosteroid (BR) signaling. In a phenotype-based compound screen we identified bikinin, a small molecule that activates BR signaling downstream of the BR receptor. Bikinin directly binds the GSK3 BIN2 and acts as an ATP competitor. Furthermore, bikinin inhibits the activity of six other Arabidopsis GSK3s. Genome-wide transcript analyses demonstrate that simultaneous inhibition of seven GSK3s is sufficient to activate BR responses. Our data suggest that GSK3 inhibition is the sole activation mode of BR signaling and argues against GSK3-independent BR responses in Arabidopsis. The opportunity to generate multiple and conditional knockouts in key regulators in the BR signaling pathway by bikinin represents a useful tool to further unravel regulatory mechanisms.

A role for the root cap in root branching revealed by the non-auxin probe naxillin

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.

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.

Mitochondrial Defects Confer Tolerance against Cellulose Deficiency

Mitochondrial mutations confer resistance towards cellulose inhibitors, indicating communication between mitochondria and the cell wall to cope with stresses that affect cell wall integrity. Because the plant cell wall provides the first line of defense against biotic and abiotic assaults, its functional integrity needs to be maintained under stress conditions. Through a phenotype-based compound screening approach, we identified a novel cellulose synthase inhibitor, designated C17. C17 administration depletes cellulose synthase complexes from the plasma membrane in Arabidopsis thaliana, resulting in anisotropic cell elongation and a weak cell wall. Surprisingly, in addition to mutations in CELLULOSE SYNTHASE1 (CESA1) and CESA3, a forward genetic screen identified two independent defective genes encoding pentatricopeptide repeat (PPR)-like proteins (CELL WALL MAINTAINER1 [CWM1] and CWM2) as conferring tolerance to C17. Functional analysis revealed that mutations in these PPR proteins resulted in defective cytochrome c maturation and activation of mitochondrial retrograde signaling, as evidenced by the induction of an alternative oxidase. These mitochondrial perturbations increased tolerance to cell wall damage induced by cellulose deficiency. Likewise, administration of antimycin A, an inhibitor of mitochondrial complex III, resulted in tolerance toward C17. The C17 tolerance of cwm2 was partially lost upon depletion of the mitochondrial retrograde regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction with the cell wall. In view of mitochondria being a major target of a variety of stresses, our data indicate that plant cells might modulate mitochondrial activity to maintain a functional cell wall when subjected to stresses.

Chemical genetics uncovers novel inhibitors of lignification, including p-iodobenzoic acid targeting CINNAMATE-4-HYDROXYLASE

Chemical screening for novel inhibitors of lignification uncovered a compound processed in the plant into two such inhibitors, one of which, p-iodobenzoic acid, inhibits CINNAMATE-4-HYDROXYLASE activity. Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization.

TR-DB: An open-access database of compounds affecting the ethylene-induced triple response in Arabidopsis

Small molecules which act as hormone agonists or antagonists represent useful tools in fundamental research and are widely applied in agriculture to control hormone effects. High-throughput screening of large chemical compound libraries has yielded new findings in plant biology, with possible future applications in agriculture and horticulture. To further understand ethylene biosynthesis/signaling and its crosstalk with other hormones, we screened a 12,000 compound chemical library based on an ethylene-related bioassay of dark-grown Arabidopsis thaliana (L.) Heynh. seedlings. From the initial screening, 1313 (∼11%) biologically active small molecules altering the phenotype triggered by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), were identified. Selection and sorting in classes were based on the angle of curvature of the apical hook, the length and width of the hypocotyl and the root. A MySQL-database was constructed (https://chaos.ugent.be/WE15/) including basic chemical information on the compounds, images illustrating the phenotypes, phenotype descriptions and classification. The research perspectives for different classes of hit compounds will be evaluated, and some general screening tips for customized high-throughput screening and pitfalls will be discussed.

Small-Molecule Dissection of Brassinosteroid Signaling

The growth-promoting hormones, the brassinosteroids (BRs), are perceived at the plant cell surface by receptor kinases that transduce the signal to the nucleus by an intracellular cascade of phosphorylation-mediated protein-protein interactions. BR signaling is also regulated by the plant endocytic machinery because the increased endosomal localization of the BR receptor enhances the BR responses. Chemical genetics is a powerful approach to identify new components in redundant signaling networks and to characterize highly dynamic processes, such as endocytosis. Here, we describe a screen in Arabidopsis thaliana seedlings for small molecules that affect hypocotyl elongation under continuous light conditions, indicative for an effect on BR responses. The compounds identified in this screen were used to dissect endomembrane trafficking of the BR receptor, BR INSENSITIVE1, a process that is essential for BR signal transduction.

Small-molecule screens to study lateral root development.

Development of the root system is essential for proper plant growth and development. Extension of the root system is achieved by the continuous establishment of new meristems in existing parental root tissues, which leads to the development of lateral roots. This process of lateral root organogenesis consists of different developmental stages, which are all controlled by the plant hormone auxin. In this chapter, we describe a screening method in Arabidopsis thaliana to identify small synthetic molecules that interfere with the process of lateral root development during specific developmental stages.

Nonselective chemical inhibition of Sec7 domain-containing ARF GEFs in Arabidopsis

A chemical genetic approach uncovered the small molecule Secdin, which impairs multiple endomembrane trafficking routes by targeting the Arabidopsis 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.

Multi-Parametric Screening in Arabidopsis thaliana Seedlings

Phenotypic screening and subsequent target identification approaches are very valuable to identify chemical probes that can be used to explore the connection between phenotypes and biological pathways. However, assessing a phenotypic effect in plants in a high-throughput fashion is a challenging task and often requires expensive readout devices. In this chapter, we describe a cost-effective multi-parametric screening procedure that is compatible with liquid-handling systems and that enables the assessment of phenotypes in Arabidopsis thaliana seedlings in an automated way.