Small molecule RHP1 promotes root hair tip growth by acting upstream of the RHD6-RSL4-dependent transcriptional pathway and ROP signaling in plants.

Root hairs are single-cell projections in the root epidermis. The presence of root hairs greatly expands the root surface, which facilitates soil anchorage and the absorption of water and nutrients. Root hairs are also the ideal system to study the mechanism of polar growth. Previous research has identified many important factors that control different stages of root hair development. Using a chemical genetics screen, in this study we report the identification of a steroid molecule, RHP1, which promotes root hair growth at nanomolar concentrations without obvious change of other developmental processes. We further demonstrate that RHP1 specifically affects tip growth with no significant influence on cell fate or planar polarity. We also show that RHP1 promotes root hair tip growth via acting upstream of the RHD6-RSL4-dependent transcriptional pathway and ROP GTPase-guided local signaling. Finally, we demonstrate that RHP1 exhibits a wide range of effects on different plant species in both monocots and dicots. This study of RHP1 will not only help to dissect the mechanism of root hair tip growth, but also provide a new tool to modify root hair growth in different plant species.

A small molecule antagonizes jasmonic acid perception and auxin responses in vascular and nonvascular plants.

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.

Endosidin20 Targets the Cellulose Synthase Catalytic Domain to Inhibit Cellulose Biosynthesis.

Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.

Interaction between VPS35 and RABG3f is necessary as a checkpoint to control fusion of late compartments with the vacuole.

Vacuoles are essential organelles in plants, playing crucial roles, such as cellular material degradation, ion and metabolite storage, and turgor maintenance. Vacuoles receive material via the endocytic, secretory, and autophagic pathways. Membrane fusion is the last step during which prevacuolar compartments (PVCs) and autophagosomes fuse with the vacuole membrane (tonoplast) to deliver cargoes. Protein components of the canonical intracellular fusion machinery that are conserved across organisms, including Arabidopsis thaliana, include complexes, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), that catalyze membrane fusion, and homotypic fusion and vacuole protein sorting (HOPS), that serve as adaptors which tether cargo vesicles to target membranes for fusion under the regulation of RAB-GTPases. The mechanisms regulating the recruitment and assembly of tethering complexes are not well-understood, especially the role of RABs in this dynamic regulation. Here, we report the identification of the small synthetic molecule Endosidin17 (ES17), which interferes with synthetic, endocytic, and autophagic traffic by impairing the fusion of late endosome compartments with the tonoplast. Multiple independent target identification techniques revealed that ES17 targets the VPS35 subunit of the retromer tethering complex, preventing its normal interaction with the Arabidopsis RAB7 homolog RABG3f. ES17 interference with VPS35-RABG3f interaction prevents the retromer complex to endosome anchoring, resulting in retention of RABG3f. Using multiple approaches, we show that VPS35-RABG3f-GTP interaction is necessary to trigger downstream events like HOPS complex assembly and fusion of late compartments with the tonoplast. Overall, our results support a role for the interaction of RABG3f-VPS35 as a checkpoint in the control of traffic toward the vacuole.

Merging roads: chemical tools and cell biology to study unconventional protein secretion.

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.

Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase.

Ethylene is an important phytohormone that promotes the ripening of fruits and senescence of flowers thereby reducing their shelf lives. Specific ethylene biosynthesis inhibitors would help to decrease postharvest loss. Here, we identify pyrazinamide (PZA), a clinical drug used to treat tuberculosis, as an inhibitor of ethylene biosynthesis in Arabidopsis thaliana, using a chemical genetics approach. PZA is converted to pyrazinecarboxylic acid (POA) in plant cells, suppressing the activity of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), the enzyme catalysing the final step of ethylene formation. The crystal structures of Arabidopsis ACO2 in complex with POA or 2-Picolinic Acid (2-PA), a POA-related compound, reveal that POA/2-PA bind at the active site of ACO, preventing the enzyme from interacting with its natural substrates. Our work suggests that PZA and its derivatives may be promising regulators of plant metabolism, in particular ethylene biosynthesis.

Firmly Planted, Always Moving.

I was a budding pianist immersed in music in Leningrad, in the Soviet Union (now Saint Petersburg, Russia), when I started over, giving up sheet music for the study of ciliates. In a second starting-over story, I emigrated to the United States, where I switched to studying carbohydrate-binding plant lectin proteins, dissecting plant vesicular trafficking, and isolating novel glycosyltransferases responsible for making cell wall polysaccharides. I track my journey as a plant biologist from student to principal investigator to founding director of the Center for Plant Cell Biology and then director of the Institute for Integrative Genome Biology at the University of California, Riverside. I discuss implementing a new vision as the first and (so far) only female editor in chief of Plant Physiology, as well as how my laboratory helped develop chemical genomics tools to study the functions of essential plant proteins. Always wanting to give back what I received, I discuss my present efforts to develop female scientist leadership in Chinese universities and a constant theme throughout my life: a love of art and travel.

Different Endomembrane Trafficking Pathways Establish Apical and Basal Polarities.

The endomembrane system is an interconnected network required to establish signal transduction, cell polarity, and cell shape in response to developmental or environmental stimuli. In the model plant Arabidopsis thaliana, there are numerous markers to visualize polarly localized plasma membrane proteins utilizing endomembrane trafficking. Previous studies have shown that the large ARF-GEF GNOM plays a key role in the establishment of basal (rootward) polarity, whereas the apically (shootward) polarized membrane proteins undergo sorting via different routes. However, the mechanism that maintains apical polarity is largely unknown. Here, we used a chemical genomic approach and identified the compound endosidin 16 (ES16), which perturbed apically localized plasma membrane proteins without affecting basal polarity. We demonstrated that ES16 is an inhibitor for recycling of apical, lateral, and nonpolar plasma membrane proteins as well as biosynthetic secretion, leaving the basal proteins as the only exceptions not subject to ES16 inhibition. Further evidence from pharmaceutical and genetic data revealed that ES16 effects are mediated through the regulation of small GTPase RabA proteins and that RabA GTPases work in concert with the BIG clade ARF-GEF to modulate the nonbasal trafficking. Our results reveal that ES16 defines a distinct pathway for endomembrane sorting routes and is essential for the establishment of cell polarity.

Drug Affinity Responsive Target Stability (DARTS) to Resolve Protein-Small Molecule Interaction in Arabidopsis.

Target identification remains a challenging step in plant chemical genomics approaches. Drug affinity responsive target stability (DARTS) represents a straightforward technique to identify small molecules' protein targets and assist in the characterization of interactions between small molecules and putative targets identified by other methods. When a small molecule interacts with a protein, it has the potential to stabilize the protein's structure, resulting in a reduced susceptibility to protease action. During the DARTS procedure, protein extracts are treated with proteolytic enzymes, and only proteins that bind to the small molecule are protected from proteolysis. DARTS represents a protocol independent of the molecule's mechanism of action or chemical structure. Another advantage of DARTS is that it does not require additional modifications or tagging of the small molecule. The protocols outlined in this article describe in detail the DARTS technique applied to plant proteins and propose several detection procedures according to protein abundance. © 2017 by John Wiley & Sons, Inc.

Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains.

The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae.

BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana.

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF-defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)-Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.