Gambogic acid and juglone inhibit RNase P through distinct mechanisms.

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 µM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.

Cesium tolerance is enhanced by a chemical which binds to BETA-GLUCOSIDASE 23 in Arabidopsis thaliana.

Cesium (Cs) is found at low levels in nature but does not confer any known benefit to plants. Cs and K compete in cells due to the chemical similarity of Cs to potassium (K), and can induce K deficiency in cells. In previous studies, we identified chemicals that increase Cs tolerance in plants. Among them, a small chemical compound (C17H19F3N2O2), named CsToAcE1, was confirmed to enhance Cs tolerance while increasing Cs accumulation in plants. Treatment of plants with CsToAcE1 resulted in greater Cs and K accumulation and also alleviated Cs-induced growth retardation in Arabidopsis. In the present study, potential target proteins of CsToAcE1 were isolated from Arabidopsis to determine the mechanism by which CsToAcE1 alleviates Cs stress, while enhancing Cs accumulation. Our analysis identified one of the interacting target proteins of CsToAcE1 to be BETA-GLUCOSIDASE 23 (AtßGLU23). Interestingly, Arabidopsis atßglu23 mutants exhibited enhanced tolerance to Cs stress but did not respond to the application of CsToAcE1. Notably, application of CsToAcE1 resulted in a reduction of Cs-induced AtßGLU23 expression in wild-type plants, while this was not observed in a high affinity transporter mutant, athak5. Our data indicate that AtßGLU23 regulates plant response to Cs stress and that CsToAcE1 enhances Cs tolerance by repressing AtßGLU23. In addition, AtHAK5 also appears to be involved in this response.

Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte.

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria.

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

Overexpression of AtBBD1, Arabidopsis Bifunctional Nuclease, Confers Drought Tolerance by Enhancing the Expression of Regulatory Genes in ABA-Mediated Drought Stress Signaling.

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.

A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase.

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.

Chemical Biology in Auxin Research.

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.

Identification of novel compounds that inhibit SnRK2 kinase activity by high-throughput screening.

Abscisic acid (ABA) is a major phytohormone that regulates abiotic stress responses and development. SNF1-rerated protein kinase 2 (SnRK2) is a key regulator of ABA signaling. To isolate compounds which directly affect SnRK2 activity, we optimized a fluorescence-based system for high-throughput screening (HTS) of SnRK2 kinase regulators. Using this system, we screened a chemical library consisting of 16,000 compounds and identified ten compounds (INH1-10) as potential SnRK2 inhibitors. Further characterization of these compounds by in vitro phosphorylation assays confirmed that three of the ten compounds were SnRK2-specific kinase inhibitors. In contrast, seven of ten compounds inhibited ABA-responsive gene expression in Arabidopsis cells. From these results, INH1 was identified as a SnRK2-specific inhibitor in vitro and in vivo. We propose that INH1 could be a lead compound of chemical tools for studying ABA responses in various plant species.

The Arabidopsis phosphatase PP2C49 negatively regulates salt tolerance through inhibition of AtHKT1;1.

Type 2C protein phosphatases (PP2Cs) are the largest protein phosphatase family. PP2Cs dephosphorylate substrates for signaling in Arabidopsis, but the functions of most PP2Cs remain unknown. Here, we characterized PP2C49 (AT3G62260, a Group G PP2C), which regulates Na+ distribution under salt stress and is localized to the cytoplasm and nucleus. PP2C49 was highly expressed in root vascular tissues and its disruption enhanced plant tolerance to salt stress. Compared with wild type, the pp2c49 mutant contained more Na+ in roots but less Na+ in shoots and xylem sap, suggesting that PP2C49 regulates shoot Na+ extrusion. Reciprocal grafting revealed a root-based mechanism underlying the salt tolerance of pp2c49. Systemic Na+ distribution largely depends on AtHKT1;1 and loss of function of AtHKT1;1 in the pp2c49 background overrode the salt tolerance of pp2c49, resulting in salt sensitivity. Furthermore, compared with plants overexpressing PP2C49 in the wild-type background, plants overexpressing PP2C49 in the athtk1;1 mutant background were sensitive to salt, like the athtk1;1 mutants. Moreover, protein-protein interaction and two-voltage clamping assays demonstrated that PP2C49 physically interacts with AtHKT1;1 and inhibits the Na+ permeability of AtHKT1;1. This study reveals that PP2C49 negatively regulates AtHKT1;1 activity and thus determines systemic Na+ allocation during salt stress.

Auxin-Regulated Reversible Inhibition of TMK1 Signaling by MAKR2 Modulates the Dynamics of Root Gravitropism.

Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1-3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1-4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7-10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.

NADPH Oxidase-derived ROS promote mitochondrial alkalization under salt stress in Arabidopsis root cells.

The plasma membrane NADPH Oxidase-derived ROS as signaling molecules play crucial roles in salt stress response. As the motor organelle of cells, mitochondria are also important for salt tolerance. However, the possible interaction between NADPH Oxidase-derived ROS and mitochondria is not well studied. Here, a transgenic Arabidopsis expressing mitochondrial matrix-targeted pH-sensitive indicator cpYFP was used to monitor the pH dynamics in root cells under salt stress. A significant alkalization in mitochondria was observed when the root was exposed to NaCl or KCl, but not osmotic stress such as isotonic mannitol. Interestingly, when pretreated with the NADPH Oxidase inhibitor DPI, the mitochondrial alkalization in root cells was largely abolished. Genetic evidence further showed that salt-induced mitochondrial alkalization was significantly reduced in the loss of function mutant atrbohF . Pretreatment with endocytosis-related inhibitor PAO or TyrA23, which inhibited the ROS accumulation under salt treatment, almost abolished this effect. Furthermore, [Ca2+]cyt increase might also play important roles by affecting ROS generation to mediate salt-induced mitochondrial alkalization as indicated by treatment with plasma membrane Ca2+ channel inhibitor LaCl3 and mitochondrial Ca2+ uniporter inhibitor Ruthenium Red. Together, these results suggest that the plasma membrane NADPH Oxidase-derived ROS promote the mitochondrial alkalization under salt treatment, providing a possible link between different cellular compartments under salt stress.

Close Temporal Relationship between Oscillating Cytosolic K+ and Growth in Root Hairs of Arabidopsis.

Root hair elongation relies on polarized cell expansion at the growing tip. As a major osmotically active ion, potassium is expected to be continuously assimilated to maintain cell turgor during hair tip growth. However, due to the lack of practicable detection methods, the dynamics and physiological role of K+ in hair growth are still unclear. In this report, we apply the small-molecule fluorescent K+ sensor NK3 in Arabidopsis root hairs for the first time. By employing NK3, oscillating cytoplasmic K+ dynamics can be resolved at the tip of growing root hairs, similar to the growth oscillation pattern. Cross-correlation analysis indicates that K+ oscillation leads the growth oscillations by approximately 1.5 s. Artificially increasing cytoplasmic K+ level showed no significant influence on hair growth rate, but led to the formation of swelling structures at the tip, an increase of cytosolic Ca2+ level and microfilament depolymerization, implying the involvement of antagonistic regulatory factors (e.g., Ca2+ signaling) in the causality between cytoplasmic K+ and hair growth. These results suggest that, in each round of oscillating root hair elongation, the oscillatory cell expansion accelerates on the heels of cytosolic K+ increment, and decelerates with the activation of antagonistic regulators, thus forming a negative feedback loop which ensures the normal growth of root hairs.

Cell death resulted from loss of fumarylacetoacetate hydrolase in Arabidopsis is related to phytohormone jasmonate but not salicylic acid.

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step in Tyr degradation pathway essential to animals but not well understood in plants. Previously, we found that mutation of SSCD1 encoding Arabidopsis FAH causes cell death under short day, which uncovered an important role of Tyr degradation pathway in plants. Since phytohormones salicylic acid (SA) and jasmonate (JA) are involved in programmed cell death, in this study, we investigated whether sscd1 cell death is related to SA and JA, and found that (1) it is accompanied by up-regulation of JA- and SA-inducible genes as well as accumulation of JA but not SA; (2) it is repressed by breakdown of JA signaling but not SA signaling; (3) the up-regulation of reactive oxygen species marker genes in sscd1 is repressed by breakdown of JA signaling; (4) treatment of wild-type Arabidopsis with succinylacetone, an abnormal metabolite caused by loss of FAH, induces expression of JA-inducible genes whereas treatment with JA induces expression of some Tyr degradation genes with dependence of JA signaling. These results demonstrated that cell death resulted from loss of FAH in Arabidopsis is related to JA but not SA, and suggested that JA signaling positively regulates sscd1 cell death by up-regulating Tyr degradation.

APAn, a Class of ABA Receptor Agonism/Antagonism Switching Probes.

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 APAn, 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 APAn 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.

Recurrent requirement for the m6A-ECT2/ECT3/ECT4 axis in the control of cell proliferation during plant organogenesis.

mRNA methylation at the N6-position of adenosine (m6A) enables multiple layers of post-transcriptional gene control, often via RNA-binding proteins that use a YT521-B homology (YTH) domain for specific m6A recognition. In Arabidopsis, normal leaf morphogenesis and rate of leaf formation require m6A and the YTH-domain proteins ECT2, ECT3 and ECT4. In this study, we show that ect2/ect3 and ect2/ect3/ect4 mutants also exhibit slow root and stem growth, slow flower formation, defective directionality of root growth, and aberrant flower and fruit morphology. In all cases, the m6A-binding site of ECT proteins is required for in vivo function. We also demonstrate that both m6A methyltransferase mutants and ect2/ect3/ect4 exhibit aberrant floral phyllotaxis. Consistent with the delayed organogenesis phenotypes, we observe particularly high expression of ECT2, ECT3 and ECT4 in rapidly dividing cells of organ primordia. Accordingly, ect2/ect3/ect4 mutants exhibit decreased rates of cell division in leaf and vascular primordia. Thus, the m6A-ECT2/ECT3/ECT4 axis is employed as a recurrent module to stimulate plant organogenesis, at least in part by enabling rapid cellular proliferation.

Design of potent ABA receptor antagonists based on a conformational restriction approach.

The physiological functions of the plant hormone abscisic acid (ABA) are triggered by interactions between PYR/PYL/RCAR receptors (PYLs) and group-A protein phosphatases 2C (PP2Cs). PYL agonists/antagonists capable of inducing/disrupting these interactions would be valuable in investigating the regulatory mechanisms of ABA signaling. Previously, we developed (+)-PAO4, a high-affinity PYL antagonist, by conformationally restricting the S-hexyl chain of our first reported PYL antagonist, 3'-hexylsulfanyl-ABA. Although (+)-PAO4 shows a greater binding affinity for Arabidopsis PYL5 compared with 3'-hexylsulfanyl-ABA, it is not able to completely block the ABA responses both in vitro and in vivo. Therefore, we designed novel conformationally restricted PYL antagonists in which the O-butyl chain of (+)-PAO4 was replaced with a pentyl (PAC4), a pentyne (PAT3) or a pentadiyne (PATT1) chain. (+)-PAT3 and (+)-PATT1 suppressed the ABA-induced inhibition of Arabidopsis seed germination more strongly than (+)-PAO4, but contrary to expectations, the affinity of each compound for PYL5 was almost the same as that of (+)-PAO4. Subsequent biochemical analyses revealed that unlike (+)-PAO4, (+)-PAT3 and (+)-PATT1 completely abolished ABA-induced PYL-PP2C interactions without partial agonistic activities. The superior PYL antagonist functions of (+)-PAT3 and (+)-PATT1 over (+)-PAO4 may explain their potent antagonistic activities against exogenous ABA in vivo. Furthermore, (+)-PAT3 and (+)-PATT1 also suppressed ABA responses in rice, indicating that both compounds are useful chemical tools for ABA-signaling studies, not only in dicots but also in monocots.

The AtGSTU7 gene influences glutathione-dependent seed germination under ABA and osmotic stress in Arabidopsis.

Glutathione S-transferases (GSTs) play important roles in metabolism and detoxification of plant cells. However, their functions during development are less well understood. Arabidopsis AtGSTU7 (AT2G29420) encodes a Tau class GST. Here we provide the AtGSTU7 was abundantly expressed in seeds and in roots at an early stage of germination. AtGSTU7 expression was repressed by exogenous ABA and promoted by osmotic stress. A null mutant of AtGSTU7 (atgstu7) accumulated higher contents of reduced GSH and decreased amounts of endogenous H2O2 in seedlings. The atgstu7 plants showed decreased osmotic tolerance during seed germination, which was influenced by GSH and ABI3 gene expression. The results suggested that AtGSTU7 involvement in seed germination is mediated by GSH-ROS homeostasis and ABA signaling.

Protein phosphatase 2A promotes stomatal development by stabilizing SPEECHLESS in Arabidopsis.

Stomatal guard cells control gas exchange that allows plant photosynthesis but limits water loss from plants to the environment. In Arabidopsis, stomatal development is mainly controlled by a signaling pathway comprising peptide ligands, membrane receptors, a mitogen-activated protein kinase (MAPK) cascade, and a set of transcription factors. The initiation of the stomatal lineage requires the activity of the bHLH transcription factor SPEECHLESS (SPCH) with its partners. Multiple kinases were found to regulate SPCH protein stability and function through phosphorylation, yet no antagonistic protein phosphatase activities have been identified. Here, we identify the conserved PP2A phosphatases as positive regulators of Arabidopsis stomatal development. We show that mutations in genes encoding PP2A subunits result in lowered stomatal production in Arabidopsis Genetic analyses place the PP2A function upstream of SPCH. Pharmacological treatments support a role for PP2A in promoting SPCH protein stability. We further find that SPCH directly binds to the PP2A-A subunits in vitro. In plants, nonphosphorylatable SPCH proteins are less affected by PP2A activity levels. Thus, our research suggests that PP2A may function to regulate the phosphorylation status of the master transcription factor SPCH in stomatal development.

Molecular Mechanisms of Brassinosteroid-Mediated Responses to Changing Environments in Arabidopsis.

Plant adaptations to changing environments rely on integrating external stimuli into internal responses. Brassinosteroids (BRs), a group of growth-promoting phytohormones, have been reported to act as signal molecules mediating these processes. BRs are perceived by cell surface receptor complex including receptor BRI1 and coreceptor BAK1, which subsequently triggers a signaling cascade that leads to inhibition of BIN2 and activation of BES1/BZR1 transcription factors. BES1/BZR1 can directly regulate the expression of thousands of downstream responsive genes. Recent studies in the model plant Arabidopsis demonstrated that BR biosynthesis and signal transduction, especially the regulatory components BIN2 and BES1/BZR1, are finely tuned by various environmental cues. Here, we summarize these research updates and give a comprehensive review of how BR biosynthesis and signaling are modulated by changing environments and how these changes regulate plant adaptive growth or stress tolerance.