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1.
Proc Natl Acad Sci U S A ; 120(40): e2221286120, 2023 10 03.
Article in English | MEDLINE | ID: mdl-37756337

ABSTRACT

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.


Subject(s)
Arabidopsis Proteins , Arabidopsis , F-Box Proteins , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Receptors, Cell Surface/metabolism , Indoleacetic Acids/metabolism , F-Box Proteins/metabolism , Gene Expression Regulation, Plant
2.
Pest Manag Sci ; 79(4): 1305-1315, 2023 Apr.
Article in English | MEDLINE | ID: mdl-36458868

ABSTRACT

BACKGROUND: Auxin herbicides have been used for selective weed control for 75 years and they continue to be amongst the most widely used weed control agents globally. The auxin herbicides fall into five chemical classes, with two herbicides not classified, and in all cases it is anticipated that recognition in the plant starts with binding to the Transport Inhibitor Response 1 (TIR1) family of auxin receptors. There is evidence that some classes of auxins act selectively with certain clades of receptors, although a comprehensive structure-activity relationship has not been available. RESULTS: Using purified receptor proteins to measure binding efficacy we have conducted quantitative structure activity relationship (qSAR) assays using representative members of the three receptor clades in Arabidopsis, TIR1, AFB2 and AFB5. Complementary qSAR data for biological efficacy at the whole-plant level using root growth inhibition and foliar phytotoxicity assays have also been analyzed for each family of auxin herbicides, including for the afb5-1 receptor mutant line. CONCLUSIONS: Comparisons of all these assays highlight differences in receptor selectivity and some systematic differences between results for binding in vitro and activity in vivo. The results could provide insights into weed spectrum differences between the different classes of auxin herbicides, as well as the potential resistance and cross-resistance implications for this herbicide class. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Herbicides , Herbicides/pharmacology , Indoleacetic Acids/pharmacology , Arabidopsis Proteins/metabolism , Receptors, Cell Surface/metabolism
3.
Plant Sci ; 284: 161-176, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31084869

ABSTRACT

Although the stringent response has been known for more than half a century and has been well studied in bacteria, only the research of the past 19 years revealed that the homologous mechanism is conserved in plants. The plant RelA/SpoT Homolog (RSH) genes have been identified and characterized in a limited number of plant species, whereas products of their catalytic activities, (p)ppGpp (alarmones), have been shown to accumulate mainly in chloroplasts. Here, we identified full-length sequences of the Ipomoea nil RSH genes (InRSH1, InRSH2 and InCRSH), determined their copy number in the I. nil genome as well as the structural conservancy between InRSHs and their Arabidopsis and rice orthologs. We showed that InRSHs are differentially expressed in I. nil organ tissues and that only InRSH2 is upregulated in response to salt, osmotic and drought stress. Our results of the E. coli relA/spoT mutant complementation test suggest that InRSH1 is likely a (p)ppGpp hydrolase, InCRSH - synthetase and InRSH2 shows both activities. Finally, we referred our results to the recently published I. nil genomic and proteomic data and uncovered the complexity of the I. nil RSH family as well as potential ways of the InRSH transcriptional regulation.


Subject(s)
Ipomoea nil/genetics , Plant Proteins/genetics , Transcription Factor RelA/genetics , Gene Expression Regulation, Plant/genetics , Genes, Plant/genetics , Phylogeny , Sequence Alignment , Sequence Analysis, DNA , Stress, Physiological
4.
ACS Chem Biol ; 13(9): 2585-2594, 2018 09 21.
Article in English | MEDLINE | ID: mdl-30138566

ABSTRACT

Indole-3-acetic acid (auxin) is considered one of the cardinal hormones in plant growth and development. It regulates a wide range of processes throughout the plant. Synthetic auxins exploit the auxin-signaling pathway and are valuable as herbicidal agrochemicals. Currently, despite a diversity of chemical scaffolds all synthetic auxins have a carboxylic acid as the active core group. By applying bio-isosteric replacement we discovered that indole-3-tetrazole was active by surface plasmon resonance spectrometry, showing that the tetrazole could initiate assembly of the Transport Inhibitor Resistant 1 (TIR1) auxin coreceptor complex. We then tested the tetrazole's efficacy in a range of whole plant physiological assays and in protoplast reporter assays, which all confirmed auxin activity, albeit rather weak. We then tested indole-3-tetrazole against the AFB5 homologue of TIR1, finding that binding was selective against TIR1, absent with AFB5. The kinetics of binding to TIR1 are contrasted to those for the herbicide picloram, which shows the opposite receptor preference, as it binds to AFB5 with far greater affinity than to TIR1. The basis of the preference of indole-3-tetrazole for TIR1 was revealed to be a single residue substitution using molecular docking, and assays using tir1 and afb5 mutant lines confirmed selectivity in vivo. Given the potential that a TIR1-selective auxin might have for unmasking receptor-specific actions, we followed a rational design, lead optimization campaign, and a set of chlorinated indole-3-tetrazoles was synthesized. Improved affinity for TIR1 and the preference for binding to TIR1 was maintained for 4- and 6-chloroindole-3-tetrazoles, coupled with improved efficacy in vivo. This work expands the range of auxin chemistry for the design of receptor-selective synthetic auxins.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , F-Box Proteins/metabolism , Herbicides/metabolism , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Receptors, Cell Surface/metabolism , Tetrazoles/metabolism , Arabidopsis/growth & development , Halogenation , Herbicides/chemical synthesis , Herbicides/chemistry , Indoleacetic Acids/chemical synthesis , Indoleacetic Acids/chemistry , Molecular Docking Simulation , Plant Growth Regulators/chemical synthesis , Plant Growth Regulators/chemistry , Protein Binding , Tetrazoles/chemical synthesis , Tetrazoles/chemistry
5.
Plant Physiol ; 177(4): 1704-1716, 2018 08.
Article in English | MEDLINE | ID: mdl-29934297

ABSTRACT

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.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cyclopentanes/metabolism , Indoleacetic Acids/metabolism , Oxylipins/metabolism , Plant Roots/growth & development , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cyclopentanes/pharmacology , Gene Expression Regulation, Plant/drug effects , Indenes/pharmacology , Oxylipins/pharmacology , Plant Growth Regulators/metabolism , Plant Roots/genetics , Plants, Genetically Modified , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Seedlings/drug effects , Seedlings/growth & development , Seedlings/metabolism , Signal Transduction
6.
J Exp Bot ; 69(2): 265-275, 2018 01 04.
Article in English | MEDLINE | ID: mdl-28992122

ABSTRACT

Herbicides are an important asset in ensuring food security, especially when faced with an ever-increasing demand on food production to feed the global population. The current selection of herbicides is increasingly encountering resistance in agricultural weeds they once targeted effectively. It is imperative that new compounds or more effective modes of action are discovered in order to overcome this resistance. This cheminformatics review looks at current herbicides and evaluates their physiochemical properties on a class-by-class basis. We focus in particular on the synthetic auxin herbicides, Herbicide Resistance Action Committee class O, analyzing these against herbicides more generally and for class-specific features such as mobility in plant vasculature. We summarise the physiochemical properties of all 24 compounds used commercially as auxins and relate these results to ongoing approaches to novel auxin discovery. We introduce an interactive, open source cheminformatics tool known as DataWarrior for herbicide discovery, complete with records for over 300 herbicidal compounds. We hope this tool helps researchers as part of a rational approach to not only auxin discovery but agrochemical discovery in general.


Subject(s)
Herbicides , Indoleacetic Acids , Herbicides/chemistry , Indoleacetic Acids/chemistry , Informatics
7.
Planta ; 246(5): 817-842, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28948393

ABSTRACT

MAIN CONCLUSION: Plant RSH proteins are able to synthetize and/or hydrolyze unusual nucleotides called (p)ppGpp or alarmones. These molecules regulate nuclear and chloroplast transcription, chloroplast translation and plant development and stress response. Homologs of bacterial RelA/SpoT proteins, designated RSH, and products of their activity, (p)ppGpp-guanosine tetra-and pentaphosphates, have been found in algae and higher plants. (p)ppGpp were first identified in bacteria as the effectors of the stringent response, a mechanism that orchestrates pleiotropic adaptations to nutritional deprivation and various stress conditions. (p)ppGpp accumulation in bacteria decreases transcription-with exception to genes that help to withstand or overcome current stressful situations, which are upregulated-and translation as well as DNA replication and eventually reduces metabolism and growth but promotes adaptive responses. In plants, RSH are nuclei-encoded and function in chloroplasts, where alarmones are produced and decrease transcription, translation, hormone, lipid and metabolites accumulation and affect photosynthetic efficiency and eventually plant growth and development. During senescence, alarmones coordinate nutrient remobilization and relocation from vegetative tissues into seeds. Despite the high conservancy of RSH protein domains among bacteria and plants as well as the bacterial origin of plant chloroplasts, in plants, unlike in bacteria, (p)ppGpp promote chloroplast DNA replication and division. Next, (p)ppGpp may also perform their functions in cytoplasm, where they would promote plant growth inhibition. Furthermore, (p)ppGpp accumulation also affects nuclear gene expression, i.a., decreases the level of Arabidopsis defense gene transcripts, and promotes plants susceptibility towards Turnip mosaic virus. In this review, we summarize recent findings that show the importance of RSH and (p)ppGpp in plant growth and development, and open an area of research aiming to understand the function of plant RSH in response to stress.


Subject(s)
Guanosine Pentaphosphate/metabolism , Ligases/metabolism , Plant Development , Plants/enzymology , Adaptation, Physiological , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis/growth & development , Chloroplasts/metabolism , Ligases/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plants/genetics , Stress, Physiological
8.
Methods Mol Biol ; 1497: 159-191, 2017.
Article in English | MEDLINE | ID: mdl-27864766

ABSTRACT

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.


Subject(s)
Arabidopsis Proteins/metabolism , F-Box Proteins/metabolism , Indoleacetic Acids/metabolism , Plant Proteins/metabolism , Receptors, Cell Surface/metabolism , Arabidopsis/metabolism , Biological Assay/methods , Gene Expression Regulation, Plant/physiology , Kinetics , Ligands , Signal Transduction/physiology
9.
Plant Cell ; 27(11): 3038-64, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26566919

ABSTRACT

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.


Subject(s)
Arabidopsis/immunology , Immunosuppression Therapy , Pathogen-Associated Molecular Pattern Molecules/metabolism , Plant Immunity/genetics , Plant Leaves/immunology , Pseudomonas syringae/physiology , Transcription, Genetic , Arabidopsis/genetics , Arabidopsis/microbiology , Base Sequence , Chromatin/metabolism , Gene Expression Profiling , Gene Expression Regulation, Plant , Gene Ontology , Gene Regulatory Networks , Genes, Plant , Molecular Sequence Data , Nucleotide Motifs/genetics , Plant Diseases/genetics , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/microbiology , Promoter Regions, Genetic/genetics , Pseudomonas syringae/growth & development , Transcription Factors/metabolism
10.
Planta ; 239(2): 437-54, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24213153

ABSTRACT

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca²âº-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca²âº storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma-style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.


Subject(s)
Calcium/metabolism , Calreticulin/genetics , Gene Expression Regulation, Plant , Petunia/genetics , Amino Acid Sequence , Base Sequence , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calreticulin/metabolism , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Plant/chemistry , DNA, Plant/genetics , Flowers/cytology , Flowers/genetics , Flowers/physiology , Gene Expression , Homeostasis , Molecular Sequence Data , Petunia/cytology , Petunia/physiology , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Pollen/cytology , Pollen/genetics , Pollen/physiology , Pollination , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, DNA
11.
Postepy Biochem ; 52(1): 87-93, 2006.
Article in Polish | MEDLINE | ID: mdl-16869306

ABSTRACT

The stringent response is a key regulatory reaction exhibited by bacteria in response to amino acid deprivation or carbon, nitrogen and phosphate limitation. In E. coli, the products of the relA and spoT genes regulate accumulation of the effector molecules of the stringent response--guanosine tetra- and pentaphosphates (ppGpp and pppGpp respectively). These unusual hyperphosphorylated nucleotides bind directly to the bacterial RNA polymerase and change transcriptional activity of some genes--transcription repression of genes associated with the translational apparatus and the simultaneous upregulation of genes encoding metabolic enzymes, especially those involved in amino acid biosynthesis, protein hydrolysis and coding for the sigma S factor. Sigma S factor binds to the core of the bacterial RNA polymerase and induce stationary-phase-specific and stress-response-specific genes expression.


Subject(s)
Adaptation, Physiological/genetics , Bacterial Physiological Phenomena , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/genetics , Amino Acids/biosynthesis , Amino Acids/deficiency , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Guanosine Pentaphosphate/genetics , Guanosine Pentaphosphate/metabolism , Guanosine Tetraphosphate/genetics , Guanosine Tetraphosphate/metabolism , Models, Genetic , Protein Biosynthesis/drug effects , Sigma Factor/metabolism , Transcription, Genetic
12.
Postepy Biochem ; 52(1): 94-100, 2006.
Article in Polish | MEDLINE | ID: mdl-16869307

ABSTRACT

All living organisms possess adaptive responses to environmental stresses that are essential to ensuring cell survival. One of them is the stringent response, initially discovered forty years ago in the gram-negative model organism E. coli. Recently plant homologues to the bacterial relA/spoT genes were identified (RSH genes--RelA/SpoT Homologues). Also the products of rsh proteins activity--(p)ppGpp were identified in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to some biotic and abiotic stresses. Elevation of ppGpp levels was elicited also by treatment with plant hormones. What is more--in vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp. It is supposed that plant stringent response is a conserve stress-response pathway possibly operating via regulation of chloroplast gene expression and, thus, the regulation of plastid metabolism.


Subject(s)
Adaptation, Physiological/genetics , Chloroplasts/metabolism , Guanosine Pentaphosphate/metabolism , Plant Growth Regulators/genetics , Plant Physiological Phenomena , Cell Fractionation , DNA, Plant/metabolism , Escherichia coli/genetics , Guanosine Pentaphosphate/biosynthesis , Guanosine Tetraphosphate/biosynthesis , Guanosine Tetraphosphate/metabolism , Molecular Sequence Data , Phylogeny , Plant Growth Regulators/metabolism , Plant Proteins/metabolism , RNA/biosynthesis , Sequence Homology, Amino Acid
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