Jasmonic Acid Inhibits Auxin-Induced Lateral Rooting Independently of the CORONATINE INSENSITIVE1 Receptor.

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (-)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (-)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (-)-JA and (+)-coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation.

Chemical Activation of EDS1/PAD4 Signaling Leading to Pathogen Resistance in Arabidopsis.

In a chemical screen we identified thaxtomin A (TXA), a phytotoxin from plant pathogenic Streptomyces scabies, as a selective and potent activator of FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) expression in Arabidopsis (Arabidopsis thaliana). TXA induction of FMO1 was unrelated to the production of reactive oxygen species (ROS), plant cell death or its known inhibition of cellulose synthesis. TXA-stimulated FMO1 expression was strictly dependent on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) but independent of salicylic acid (SA) synthesis via ISOCHORISMATE SYNTHASE1 (ICS1). TXA induced the expression of several EDS1/PAD4-regulated genes, including EDS1, PAD4, SENESCENCE ASSOCIATED GENE101 (SAG101), ICS1, AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) and PATHOGENESIS-RELATED PROTEIN1 (PR1), and accumulation of SA. Notably, enhanced ALD1 expression did not result in accumulation of the product pipecolic acid (PIP), which promotes FMO1 expression during biologically induced systemic acquired resistance. TXA treatment preferentially stimulated expression of PAD4 compared with EDS1, which was mirrored by PAD4 protein accumulation, suggesting that TXA leads to increased PAD4 availability to form EDS1-PAD4 signaling complexes. Also, TXA treatment of Arabidopsis plants led to enhanced disease resistance to bacterial and oomycete infection, which was dependent on EDS1 and PAD4, as well as on FMO1 and ICS1. Collectively, the data identify TXA as a potentially useful chemical tool to conditionally activate and interrogate EDS1- and PAD4-controlled pathways in plant immunity.

Identification of the Natural Product Rotihibin†A as a TOR Kinase Signaling Inhibitor by Unbiased Transcriptional Profiling.

Bioactive natural products are important starting points for developing chemical tools for biological research. For elucidating their bioactivity profile, biological systems with concise complexity such as cell culture systems are frequently used, whereas unbiased investigations in more complex multicellular systems are only rarely explored. Here, we demonstrate with the natural product Rotihibin A and the plant research model system Arabidopsis thaliana that unbiased transcriptional profiling enables a rapid, label-free, and compound economic evaluation of a natural product's bioactivity profile in a complex multicellular organism. To this end, we established a chemical synthesis of Rotihibin A as well as that of structural analogues, followed by transcriptional profiling-guided identification and validation of Rotihibin A as a TOR signaling inhibitor (TOR=target of rapamycin). These findings illustrate that a combined approach of transcriptional profiling and natural product research may represent a technically simple approach to streamline the development of chemical tools from natural products even for biologically complex multicellular biological systems.

Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

A chemical inhibitor of jasmonate signaling targets JAR1 in Arabidopsis thaliana.

Jasmonates are lipid-derived plant hormones that regulate plant defenses and numerous developmental processes. Although the biosynthesis and molecular function of the most active form of the hormone, (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), have been unraveled, it remains poorly understood how the diversity of bioactive jasmonates regulates such a multitude of plant responses. Bioactive analogs have been used as chemical tools to interrogate the diverse and dynamic processes of jasmonate action. By contrast, small molecules impairing jasmonate functions are currently unknown. Here, we report on jarin-1 as what is to our knowledge the first small-molecule inhibitor of jasmonate responses that was identified in a chemical screen using Arabidopsis thaliana. Jarin-1 impairs the activity of JA-Ile synthetase, thereby preventing the synthesis of the active hormone, JA-Ile, whereas closely related enzymes are not affected. Thus, jarin-1 may serve as a useful chemical tool in search for missing regulatory components and further dissection of the complex jasmonate signaling networks.

Comparative analysis of MAMP-induced calcium influx in Arabidopsis seedlings and protoplasts.

Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells.

Repression of sucrose/ultraviolet B light-induced flavonoid accumulation in microbe-associated molecular pattern-triggered immunity in Arabidopsis.

Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch.

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Chemical and genetic exploration of jasmonate biosynthesis and signaling paths.

Jasmonates are lipid-derived compounds that act as signals in plant stress responses and developmental processes. Enzymes participating in biosynthesis of jasmonic acid (JA) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants have helped to define the pathway for synthesis of jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA, and to identify the F-box protein COI1 as central regulatory unit. Details on the molecular mechanism of JA signaling were recently unraveled by the discovery of JAZ proteins that together with the adaptor protein NINJA and the general co-repressor TOPLESS form a transcriptional repressor complex. The current model of JA perception and signaling implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ proteins for degradation by the 26S proteasome pathway, thereby allowing MYC2 and other transcription factors to activate gene expression. Chemical strategies, as integral part of jasmonate research, have helped the establishment of structure-activity relationships and the discovery of (+)-7-iso-JA-L-Ile as the major bioactive form of the hormone. The transient nature of its accumulation highlights the need to understand catabolism and inactivation of JA-Ile and recent studies indicate that oxidation of JA-Ile by cytochrome P450 monooxygenase is the major mechanism for turning JA signaling off. Plants contain numerous JA metabolites, which may have pronounced and differential bioactivity. A major challenge in the field of plant lipid signaling is to identify the cognate receptors and modes of action of these bioactive jasmonates/oxylipins.

12-hydroxyjasmonic acid glucoside is a COI1-JAZ-independent activator of leaf-closing movement in Samanea saman.

Jasmonates are ubiquitously occurring plant growth regulators with high structural diversity that mediate numerous developmental processes and stress responses. We have recently identified 12-O-ß-D-glucopyranosyljasmonic acid as the bioactive metabolite, leaf-closing factor (LCF), which induced nyctinastic leaf closure of Samanea saman. We demonstrate that leaf closure of isolated Samanea pinnae is induced upon stereospecific recognition of (-)-LCF, but not by its enantiomer, (+)-ent-LCF, and that the nonglucosylated derivative, (-)-12-hydroxyjasmonic acid also displays weak activity. Similarly, rapid and cell type-specific shrinkage of extensor motor cell protoplasts was selectively initiated upon treatment with (-)-LCF, whereas flexor motor cell protoplasts did not respond. In these bioassays related to leaf movement, all other jasmonates tested were inactive, including jasmonic acid (JA) and the potent derivates JA-isoleucine and coronatine. By contrast, (-)-LCF and (-)-12-hydroxyjasmonic acid were completely inactive with respect to activation of typical JA responses, such as induction of JA-responsive genes LOX2 and OPCL1 in Arabidopsis (Arabidopsis thaliana) or accumulation of plant volatile organic compounds in S. saman and lima bean (Phaseolus lunatus), generally considered to be mediated by JA-isoleucine in a COI1-dependent fashion. Furthermore, application of selective inhibitors indicated that leaf movement in S. saman is mediated by rapid potassium fluxes initiated by opening of potassium-permeable channels. Collectively, our data point to the existence of at least two separate JA signaling pathways in S. saman and that 12-O-ß-D-glucopyranosyljasmonic acid exerts its leaf-closing activity through a mechanism independent of the COI1-JAZ module.

Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele.

Recognition of microbe-associated molecular patterns (MAMPs), conserved structures typical of a microbial class, triggers immune responses in eukaryotes. This is accompanied by a diverse set of physiological responses that are thought to enhance defense activity in plants. However, the extent and mechanisms by which MAMP-induced events contribute to host immunity are poorly understood. Here we reveal Arabidopsis priority in sweet life4 (psl4) and psl5 mutants that are insensitive to the bacterial elongation factor (EF)-Tu epitope elf18 but responsive to flagellin epitope flg22. PSL4 and PSL5, respectively, identify beta- and alpha-subunits of endoplasmic reticulum-resident glucosidase II, which is essential for stable accumulation and quality control of the elf18 receptor EFR but not the flg22 receptor FLS2. We notice that EFR signaling is partially and differentially impaired without a significant decrease of the receptor steady-state levels in 2 weakly dysfunctional gIIalpha alleles, designated psl5-1 and rsw3. Remarkably, rsw3 plants exhibit marked supersusceptibility against a virulent bacterial phytopathogen despite nearly intact coactivation of MAPKs, reactive oxygen species, ethylene biosynthesis, and callose deposition in response to elf18, demonstrating that these signaling outputs alone are insufficient to mount effective immunity. However, rsw3 plants fail to maintain high transcript levels of defense-promoting WRKY, PR1, and PR2 genes at late time points (4 to 24 h) after elf18 elicitation. This points to an unexpected separation between initial and sustained activation of EFR-mediated signaling in the absence of proper glucosidase II-mediated endoplasmic reticulum quality control. Our findings strongly suggest the importance of sustained MAMP receptor signaling as a key step in the establishment of robust immunity.

Accumulation of isochorismate-derived 2,3-dihydroxybenzoic 3-O-beta-D-xyloside in arabidopsis resistance to pathogens and ageing of leaves.

An intricate network of hormone signals regulates plant development and responses to biotic and abiotic stress. Salicylic acid (SA), derived from the shikimate/isochorismate pathway, is a key hormone in resistance to biotrophic pathogens. Several SA derivatives and associated modifying enzymes have been identified and implicated in the storage and channeling of benzoic acid intermediates or as bioactive molecules. However, the range and modes of action of SA-related metabolites remain elusive. In Arabidopsis, Enhanced Disease Susceptibility 1 (EDS1) promotes SA-dependent and SA-independent responses in resistance against pathogens. Here, we used metabolite profiling of Arabidopsis wild type and eds1 mutant leaf extracts to identify molecules, other than SA, whose accumulation requires EDS1 signaling. Nuclear magnetic resonance and mass spectrometry of isolated and purified compounds revealed 2,3-dihydroxybenzoic acid (2,3-DHBA) as an isochorismate-derived secondary metabolite whose accumulation depends on EDS1 in resistance responses and during ageing of plants. 2,3-DHBA exists predominantly as a xylose-conjugated form (2-hydroxy-3-beta-O-D-xylopyranosyloxy benzoic acid) that is structurally distinct from known SA-glucose conjugates. Analysis of DHBA accumulation profiles in various Arabidopsis mutants suggests an enzymatic route to 2,3-DHBA synthesis that is under the control of EDS1. We propose that components of the EDS1 pathway direct the generation or stabilization of 2,3-DHBA, which as a potentially bioactive molecule is sequestered as a xylose conjugate.

A chemical screen for suppressors of the avrRpm1-RPM1-dependent hypersensitive cell death response in Arabidopsis thaliana.

Arabidopsis thaliana RPM1 encodes an intracellular immune sensor that conditions disease resistance to Pseudomonas syringae expressing the type III effector protein AvrRpm1. Conditional expression of this type III effector in a transgenic line carrying avrRpm1 under the control of a steroid-inducible promoter results in RPM1-dependent cell death that resembles the cell death response of the incompatible RPM1-avrRpm1 plant-bacterium interaction. This line was previously used in a genetic screen, which revealed two genes that likely function in the folding of pre-activation RPM1. We established a chemical screen for small molecules that suppress steroid-inducible and RPM1-avrRpm1-dependent cell death in Arabidopsis seedlings. Screening of a library comprising 6,800 compounds of natural origin identified two trichothecene-type mycotoxins, 4,15-diacetoxyscirpenol (DAS) and neosolaniol (NEO), which are synthesized by Fusarium and other fungal species. However, protein blot analysis revealed that DAS and NEO inhibit AvrRpm1 synthesis rather than suppress RPM1-mediated responses. This inhibition of translational activity likely explains the survival of the seedlings under screening conditions. Likewise, flg22-induced defense responses are also impaired at the translational, but not the transcriptional, level by DAS or NEO. Unexpectedly, both compounds not only prevented AvrRpm1 synthesis, but rather caused an apparent hyper-accumulation of RPM1 and HSP70. The hyper-accumulation phenotype is likely unrelated to the ribotoxic function of DAS and NEO and could be due to an inhibitory activity on the proteolytic machinery of Arabidopsis or elicitor-like activities of type A trichothecenes.

SNARE-protein-mediated disease resistance at the plant cell wall.

Failure of pathogenic fungi to breach the plant cell wall constitutes a major component of immunity of non-host plant species--species outside the pathogen host range--and accounts for a proportion of aborted infection attempts on 'susceptible' host plants (basal resistance). Neither form of penetration resistance is understood at the molecular level. We developed a screen for penetration (pen) mutants of Arabidopsis, which are disabled in non-host penetration resistance against barley powdery mildew, Blumeria graminis f. sp. hordei, and we isolated the PEN1 gene. We also isolated barley ROR2 (ref. 2), which is required for basal penetration resistance against B. g. hordei. The genes encode functionally homologous syntaxins, demonstrating a mechanistic link between non-host resistance and basal penetration resistance in monocotyledons and dicotyledons. We show that resistance in barley requires a SNAP-25 (synaptosome-associated protein, molecular mass 25 kDa) homologue capable of forming a binary SNAP receptor (SNARE) complex with ROR2. Genetic control of vesicle behaviour at penetration sites, and plasma membrane location of PEN1/ROR2, is consistent with a proposed involvement of SNARE-complex-mediated exocytosis and/or homotypic vesicle fusion events in resistance. Functions associated with SNARE-dependent penetration resistance are dispensable for immunity mediated by race-specific resistance (R) genes, highlighting fundamental differences between these two resistance forms.

Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress.

Xanthone production in Hypericum perforatum (HP) suspension cultures in response to elicitation by Agrobacterium tumefaciens co-cultivation has been studied. RNA blot analyses of HP cells co-cultivated with A. tumefaciens have shown a rapid up-regulation of genes encoding important enzymes of the general phenylpropanoid pathway (PAL, phenylalanine ammonia lyase and 4CL, 4-coumarate:CoA ligase) and xanthone biosynthesis (BPS, benzophenone synthase). Analyses of HPLC chromatograms of methanolic extracts of control and elicited cells (HP cells that were co-cultivated for 24h with A. tumefaciens) have revealed a 12-fold increase in total xanthone concentration and also the emergence of many xanthones after elicitation. Methanolic extract of elicited cells exhibited significantly higher antioxidant and antimicrobial competence than the equivalent extract of control HP cells indicating that these properties have been significantly increased in HP cells after elicitation. Four major de novo synthesized xanthones have been identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone. Antioxidant and antimicrobial characterization of these de novo xanthones have revealed that xanthones play dual function in plant cells during biotic stress: (1) as antioxidants to protect the cells from oxidative damage and (2) as phytoalexins to impair the pathogen growth.

Jasmonates meet fatty acids: functional analysis of a new acyl-coenzyme A synthetase family from Arabidopsis thaliana.

Arabidopsis thaliana contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-CoA synthetase (LACS), 4-coumarate:CoA ligases (4CL), and proteins closely related to 4CLs with unknown activities. The function of these 4CL-like proteins was systematically explored by applying an extensive substrate screen, and it was uncovered that activation of fatty acids is the common feature of all active members of this protein family, thereby defining a new group of fatty acyl-CoA synthetase, which is distinct from the known LACS family. Significantly, four family members also displayed activity towards different biosynthetic precursors of jasmonic acid (JA), including 12-oxo-phytodienoic acid (OPDA), dinor-OPDA, 3-oxo-2(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8), and OPC-6. Detailed analysis of in vitro properties uncovered significant differences in substrate specificity for individual enzymes, but only one protein (At1g20510) showed OPC-8:CoA ligase activity. Its in vivo function was analysed by transcript and jasmonate profiling of Arabidopsis insertion mutants for the gene. OPC-8:CoA ligase expression was activated in response to wounding or infection in the wild type but was undetectable in the mutants, which also exhibited OPC-8 accumulation and reduced levels of JA. In addition, the developmental, tissue- and cell-type specific expression pattern of the gene, and regulatory properties of its promoter were monitored by analysing promoter::GUS reporter lines. Collectively, the results demonstrate that OPC-8:CoA ligase catalyses an essential step in JA biosynthesis by initiating the beta-oxidative chain shortening of the carboxylic acid side chain of its precursors, and, in accordance with this function, the protein is localized in peroxisomes.

High-Throughput Screening of Chemical Compound Libraries for Modulators of Salicylic Acid Signaling by In Situ Monitoring of Glucuronidase-Based Reporter Gene Expression.

Salicylic acid (SA) is a vital phytohormone that is intimately involved in coordination of the complex plant defense response to pathogen attack. Many aspects of SA signaling have been unraveled by classical genetic and biochemical methods using the model plant Arabidopsis thaliana, but many details remain unknown, owing to the inherent limitations of these methods. In recent years, chemical genetics has emerged as an alternative scientific strategy to complement classical genetics by virtue of identifying bioactive chemicals or probes that act selectively on their protein targets causing either activation or inhibition. Such selective tools have the potential to create conditional and reversible chemical mutant phenotypes that may be combined with genetic mutants. Here, we describe a facile chemical screening methodology for intact Arabidopsis seedlings harboring the ß-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-ß-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. We show pilot screens for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis line expressing the SA-inducible PR1p::GUS reporter gene. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.

Members of the abscisic acid co-receptor PP2C protein family mediate salicylic acid-abscisic acid crosstalk.

The interplay between abscisic acid (ABA) and salicylic acid (SA) influences plant responses to various (a)biotic stresses; however, the underlying mechanism for this crosstalk is largely unknown. Here, we report that type 2C protein phosphatases (PP2Cs), some of which are negative regulators of ABA signaling, bind SA. SA binding suppressed the ABA-enhanced interaction between these PP2Cs and various ABA receptors belonging to the PYR/PYL/RCAR protein family. Additionally, SA suppressed ABA-enhanced degradation of PP2Cs and ABA-induced stabilization of SnRK2s. Supporting SA's role as a negative regulator of ABA signaling, exogenous SA suppressed ABA-induced gene expression, whereas the SA-deficient sid2-1 mutant displayed heightened PP2C degradation and hypersensitivity to ABA-induced suppression of seed germination. Together, these results suggest a new molecular mechanism through which SA antagonizes ABA signaling. A better understanding of the crosstalk between these hormones is important for improving the sustainability of agriculture in the face of climate change.

The Root Growth-Regulating Brevicompanine Natural Products Modulate the Plant Circadian Clock.

Plant growth regulating properties of brevicompanines (Brvs), natural products of the fungus Penicillium brevicompactum, have been known for several years, but further investigations into the molecular mechanism of their bioactivity have not been performed. Following chemical synthesis of brevicompanine derivatives, we studied their activity in the model plant Arabidopsis by a combination of plant growth assays, transcriptional profiling, and numerous additional bioassays. These studies demonstrated that brevicompanines cause transcriptional misregulation of core components of the circadian clock, whereas other biological read-outs were not affected. Brevicompanines thus represent promising chemical tools for investigating the regulation of the plant circadian clock. In addition, our study also illustrates the potential of an unbiased -omics-based characterization of bioactive compounds for identifying the often cryptic modes of action of small molecules.

Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana.

The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as "chemical genetics," has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical's bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the ß-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-ß-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.