RESUMO
The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.
Assuntos
Adenilil Ciclases , Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Ácidos Indolacéticos , Receptores de Superfície Celular , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Mutação , Gravitropismo , Raízes de Plantas/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Sistemas do Segundo MensageiroRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Receptores de Superfície Celular/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.
Assuntos
Homeostase , Ácidos Indolacéticos , Arabidopsis , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismoRESUMO
The natural auxin indole-3-acetic acid (IAA) is a key regulator of many aspects of plant growth and development. Synthetic auxin herbicides such as 2,4-D mimic the effects of IAA by inducing strong auxinic-signaling responses in plants. To determine the mechanism of 2,4-D resistance in a Sisymbrium orientale (Indian hedge mustard) weed population, we performed a transcriptome analysis of 2,4-D-resistant (R) and -susceptible (S) genotypes that revealed an in-frame 27-nucleotide deletion removing nine amino acids in the degron tail (DT) of the auxin coreceptor Aux/IAA2 (SoIAA2). The deletion allele cosegregated with 2,4-D resistance in recombinant inbred lines. Further, this deletion was also detected in several 2,4-D-resistant field populations of this species. Arabidopsis transgenic lines expressing the SoIAA2 mutant allele were resistant to 2,4-D and dicamba. The IAA2-DT deletion reduced binding to TIR1 in vitro with both natural and synthetic auxins, causing reduced association and increased dissociation rates. This mechanism of synthetic auxin herbicide resistance assigns an in planta function to the DT region of this Aux/IAA coreceptor for its role in synthetic auxin binding kinetics and reveals a potential biotechnological approach to produce synthetic auxin-resistant crops using gene-editing.
Assuntos
Ácido 2,4-Diclorofenoxiacético , Brassicaceae/genética , Resistência a Herbicidas/genética , Inseticidas , Proteínas de Plantas/genética , Receptores de Superfície Celular/genética , Deleção de Sequência , Brassicaceae/metabolismo , Dicamba , Simulação de Acoplamento Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , RNA de Plantas/genética , Receptores de Superfície Celular/metabolismo , Análise de Sequência de RNA/métodosRESUMO
Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.
Assuntos
Ácidos Indolacéticos , Reguladores de Crescimento de Plantas , Ácidos Indolacéticos/farmacologia , Benzoatos , Oxigenases de Função Mista/genética , Cinamatos/farmacologia , Regulação da Expressão Gênica de PlantasRESUMO
Upon exposure to light, etiolated Arabidopsis seedlings form adventitious roots (AR) along the hypocotyl. While processes underlying lateral root formation are studied intensively, comparatively little is known about the molecular processes involved in the initiation of hypocotyl AR. AR and LR formation were studied using a small molecule named Hypocotyl Specific Adventitious Root INducer (HYSPARIN) that strongly induces AR but not LR formation. HYSPARIN does not trigger rapid DR5-reporter activation, DII-Venus degradation or Ca2+ signalling. Transcriptome analysis, auxin signalling reporter lines and mutants show that HYSPARIN AR induction involves nuclear TIR1/AFB and plasma membrane TMK auxin signalling, as well as multiple downstream LR development genes (SHY2/IAA3, PUCHI, MAKR4 and GATA23). Comparison of the AR and LR induction transcriptome identified SAURs, AGC kinases and OFP transcription factors as specifically upregulated by HYSPARIN. Members of the SAUR19 subfamily, OFP4 and AGC2 suppress HYS-induced AR formation. While SAUR19 and OFP subfamily members also mildly modulate LR formation, AGC2 regulates only AR induction. Analysis of HYSPARIN-induced AR formation uncovers an evolutionary conservation of auxin signalling controlling LR and AR induction in Arabidopsis seedlings and identifies SAUR19, OFP4 and AGC2 kinase as novel regulators of AR formation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Proteínas de Arabidopsis/metabolismo , Plântula , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/metabolismoRESUMO
Polar auxin transport is a quintessential feature of higher plant physiology and it has been known for many years that some of the primary drivers of polar auxin transport are the PIN-formed (PIN) auxin efflux proteins. Formative research established many key biochemical features of the transport system and discovered inhibitors such as 1-naphthylphthalamic acid (NPA), but the mechanism of action of PINs has remained elusive. This changed in 2022 with the publication of high-resolution structures of the membrane-spanning domains of three PIN proteins. The atomic structures and associated activity assays reveal that PINs use an elevator mechanism to transport auxin anions out of the cell. NPA was shown to be a competitive inhibitor that traps PINs in their inward-open conformation. The secrets of the hydrophilic cytoplasmic loop of PIN proteins remain to be discovered.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Transporte Biológico , Ácidos Indolacéticos/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Glycopolymers are potent candidates for biomedical applications by exploiting multivalent carbohydrate-lectin interactions. Owing to their specific recognition capabilities, glycosylated polymers can be utilized for targeted drug delivery to certain cell types bearing the corresponding lectin receptors. A fundamental challenge in glycopolymer research, however, is the specificity of recognition to receptors binding to the same sugar unit (e.g., mannose). Variation of polymer backbone chirality has emerged as an effective method to distinguish between lectins on a molecular level. Herein, we present a facile route toward producing glycopolymers with a defined tacticity based on a step-growth polymerization technique using click chemistry. A set of polymers have been fabricated and further functionalized with mannose moieties to enable lectin binding to receptors relevant to the immune system (mannose-binding lectin, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin, and dendritic and thymic epithelial cell-205). Surface plasmon resonance spectrometry was employed to determine the kinetic parameters of the step-growth glycopolymers. The results highlight the importance of structural complexity in advancing glycopolymer synthesis, yet multivalency remains a main driving force in lectin recognition.
Assuntos
Lectinas , Manose , Lectinas/química , Manose/química , Carboidratos , Ressonância de Plasmônio de Superfície , Polímeros/químicaRESUMO
BACKGROUND: Whether increased BMI is associated with an increased risk of venous thromboembolism (VTE) is controversial. Despite this, BMI > 40 kg/m 2 remains a common cutoff for lower limb arthroplasty eligibility. Current United Kingdom national guidelines list obesity as a risk factor for VTE, but these are based on evidence that has largely failed to differentiate between potentially minor (distal deep vein thrombosis [DVT]), and more harmful (pulmonary embolism [PE] and proximal DVT) diagnoses. Determining the association between BMI and the risk of clinically important VTE is needed to improve the utility of national risk stratification tools. QUESTIONS/PURPOSES: (1) In patients undergoing lower limb arthroplasty, is BMI 40 kg/m 2 or higher (morbid obesity) associated with an increased risk of PE or proximal DVT within 90 days of surgery, compared with patients with BMI less than 40 kg/m 2 ? (2) What proportion of investigations ordered for PE and proximal DVT were positive in patients with morbid obesity who underwent lower limb arthroplasty compared with those with BMI less than 40 kg/m 2 ? METHODS: Data were collected retrospectively from the Northern Ireland Electronic Care Record, a national database recording patient demographics, diagnoses, encounters, and clinical correspondence. Between January 2016 and December 2020, 10,217 primary joint arthroplasties were performed. Of those, 21% (2184 joints) were excluded; 2183 were in patients with multiple arthroplasties and one had no recorded BMI. All 8033 remaining joints were eligible for inclusion, 52% of which (4184) were THAs, 44% (3494) were TKAs, and 4% (355) were unicompartmental knee arthroplasties; all patients had 90 days of follow-up. The Wells score was used to guide the investigations. Indications for CT pulmonary angiography for suspected PE included pleuritic chest pain, reduced oxygen saturations, dyspnea, or hemoptysis. Indications for ultrasound scans for suspected proximal DVT included leg swelling, pain, warmth, or erythema. Distal DVTs were recorded as negative scans because we do not treat them with modified anticoagulation. The division of categories was set at BMI 40 kg/m 2 , a common clinical cutoff used in surgical eligibility algorithms. Patients were grouped according to WHO BMI categories to assess for the following confounding variables: sex, age, American Society of Anesthesiologists grade, joint replaced, VTE prophylaxis, grade of operative surgeon, and implant cement status. RESULTS: We found no increase in the odds of PE or proximal DVT in any WHO BMI category. When comparing patients with BMI less than 40 kg/m 2 with those with a BMI of 40 kg/m 2 or higher, there was no difference in the odds of PE (0.8% [58 of 7506] versus 0.8% [four of 527]; OR 1.0 [95% CI 0.4 to 2.8]; p > 0.99) or proximal DVT (0.4% [33 of 7506] versus 0.2% [one of 527]; OR 2.3 [95% CI 0.3 to 17.0]; p = 0.72). Of those who received diagnostic imaging, 21% (59 of 276) of CT pulmonary angiograms and 4% (34 of 718) of ultrasounds were positive for patients with BMI less than 40 kg/m 2 compared with 14% (four of 29; OR 1.6 [95% CI 0.6 to 4.5]; p = 0.47) and 2% (one of 57; OR 2.7 [95% CI 0.4 to 18.6]; p = 0.51) for patients with BMI 40 kg/m 2 or higher. There was no difference in the percentage of CT pulmonary angiograms ordered (4% [276 of 7506] versus 5% [29 of 527]; OR 0.7 [95% CI 0.5 to 1.0]; p = 0.07) or ultrasounds ordered (10% [718 of 7506] versus 11% [57 of 527]; OR 0.9 [95% CI 0.7 to 1.2]; p = 0.49) for BMI less than 40 kg/m 2 and BMI 40 kg/m 2 or higher. CONCLUSION: Increased BMI should not preclude individuals from lower limb arthroplasty based on suspected risk of clinically important VTE. National VTE risk stratification tools should be based on evidence assessing clinically relevant VTE (specifically, proximal DVT, PE, or death of thromboembolism) only. LEVEL OF EVIDENCE: Level III, therapeutic study.
RESUMO
BACKGROUND: Cementless total knee arthroplasty (TKA) is the subject of renewed interest. Previous concerns about survivorship have been addressed and there is an appeal in terms of biological fixation and surgical efficiency. However, even surgeon advocates have concerns about the risk of marked subsidence when using this technology in older patients at risk for osteoporosis. METHODS: This was a retrospective analysis of 1,000 consecutive fully cementless mobile bearing TKAs performed at a single institution on women over 75 years of age who had postoperative and 1-year x-rays. The primary outcome was the incidence of subsidence. RESULTS: There were three asymptomatic cases with definite subsidence and change in alignment. In a fourth symptomatic case, the femoral component subsided into varus and the tibia into valgus, thus maintaining alignment which facilitated nonoperative treatment in a 92-year-old. Overall, at 1 year, there were two- liner revisions for infection without recurrence. Five patients had further surgery, of which three were washouts and two were for periprosthetic fractures sustained postoperatively within 1 year. Seven patients had further anesthesia, of which five were manipulations and two were nonrecurrent closed reductions for spinouts. CONCLUSION: Cementless TKA did not have a high risk of subsidence in this at-risk population. In the hands of experienced surgeons, these procedures can be used safely irrespective of bone quality.
Assuntos
Artroplastia do Joelho , Prótese do Joelho , Humanos , Feminino , Idoso , Idoso de 80 Anos ou mais , Artroplastia do Joelho/efeitos adversos , Estudos Retrospectivos , Prótese do Joelho/efeitos adversos , Tíbia/cirurgia , Radiografia , Reoperação , Resultado do TratamentoRESUMO
In a world that will rely increasingly on efficient plant growth for sufficient food, it is important to learn about natural mechanisms of phytohormone action. In this work, the introduction of a fluorophore to an auxin molecule represents a sensitive and non-invasive method to directly visualise auxin localisation with high spatiotemporal resolution. The state-of-the-art multidisciplinary approaches of genetic and chemical biology analysis together with live cell imaging, liquid chromatography-mass spectrometry (LC-MS) and surface plasmon resonance (SPR) methods were employed for the characterisation of auxin-related biological activity, distribution and stability of the presented compounds in Arabidopsis thaliana. Despite partial metabolisation in vivo, these fluorescent auxins display an uneven and dynamic distribution leading to the formation of fluorescence maxima in tissues known to concentrate natural auxin, such as the concave side of the apical hook. Importantly, their distribution is altered in response to different exogenous stimuli in both roots and shoots. Moreover, we characterised the subcellular localisation of the fluorescent auxin analogues as being present in the endoplasmic reticulum and endosomes. Our work provides powerful tools to visualise auxin distribution within different plant tissues at cellular or subcellular levels and in response to internal and environmental stimuli during plant development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Reguladores de Crescimento de Plantas , Raízes de Plantas/metabolismoRESUMO
The phenylpropanoid pathway serves a central role in plant metabolism, providing numerous compounds involved in diverse physiological processes. Most carbon entering the pathway is incorporated into lignin. Although several phenylpropanoid pathway mutants show seedling growth arrest, the role for lignin in seedling growth and development is unexplored. We use complementary pharmacological and genetic approaches to block CINNAMATE-4-HYDROXYLASE (C4H) functionality in Arabidopsis seedlings and a set of molecular and biochemical techniques to investigate the underlying phenotypes. Blocking C4H resulted in reduced lateral rooting and increased adventitious rooting apically in the hypocotyl. These phenotypes coincided with an inhibition in AUX transport. The upstream accumulation in cis-cinnamic acid was found to be likely to cause polar AUX transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem-mediated AUX transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, AUX homeostasis. Our results show that the accumulation of bioactive intermediates and depletion in lignin jointly cause the aberrant phenotypes upon blocking C4H, and demonstrate that proper deposition of lignin is essential for the establishment of AUX distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development.
Assuntos
Cinamatos , Plântula , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plântula/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismoRESUMO
Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Espermatozoides/metabolismo , Sequência de Bases , Humanos , Limite de Detecção , Masculino , Técnica de Seleção de Aptâmeros/métodosRESUMO
Polar auxin transport plays a pivotal role in plant growth and development. PIN-FORMED (PIN) auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis (Arabidopsis thaliana). PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endocitose , Ácidos Indolacéticos/metabolismo , Fenilacetatos/farmacologia , Arabidopsis/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Gravitropismo/efeitos dos fármacos , Hipocótilo/efeitos dos fármacos , Hipocótilo/crescimento & desenvolvimento , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Transdução de SinaisRESUMO
Star-shaped glycopolymers provide very high binding activities toward lectins. However, a straightforward synthesis method for the preparation of multi-arm glycopolymers in a one-pot approach has been challenging. Herein, we report a rapid synthesis of well-defined multi-arm glycopolymers via Cu(0)-mediated reversible deactivation radical polymerization in aqueous media. d-Mannose acrylamide has been homo- and copolymerized with NIPAM to provide linear arms and then core cross-linked with a bisacrylamide monomer. Thus, the arm length and core size of multi-arm glycopolymers were tuned. Moreover, the stability of multi-arm glycopolymers was investigated, and degradation reactions under acidic or basic conditions were observed. The binding activities of the obtained multi-arm glycopolymers with mannose-specific human lectins, DC-SIGN and MBL, were investigated via surface plasmon resonance spectroscopy. Finally, the encapsulation ability of multi-arm glycopolymers was examined using DHA and Saquinavir below and above the lower critical solution temperature (LCST) of P(NIPAM).
Assuntos
Manose , Polímeros , Humanos , Lectinas de Ligação a Manose , Polimerização , Ressonância de Plasmônio de SuperfícieRESUMO
Lectins are omnipresent carbohydrate binding proteins that are involved in a multitude of biological processes. Unearthing their binding properties is a powerful tool toward the understanding and modification of their functions in biological applications. Herein, we present the synthesis of glycopolymers with a brush architecture via a "grafting from" methodology. The use of a versatile 2-oxazoline inimer was demonstrated to open avenues for a wide range of 2-oxazoline/acrylamide bottle brush polymers utilizing aqueous Cu-mediated reversible deactivation radical polymerization (Cu-RDRP). The polymers in the obtained library were assessed for their thermal properties in aqueous solution and their binding toward the C-type animal lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and mannose-binding lectin (MBL) via surface plasmon resonance spectrometry. The encapsulation properties of a hydrophobic drug-mimicking compound demonstrated the potential use of glyco brush copolymers in biological applications.
Assuntos
Acrilamidas , Moléculas de Adesão Celular , Animais , Embrião de Galinha , Molécula 3 de Adesão Intercelular , Lectinas Tipo C , Oxazóis , Receptores de Superfície CelularRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Ácidos Indolacéticos/metabolismo , Oxilipinas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Indenos/farmacologia , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transdução de SinaisRESUMO
Developmental responses to auxin are regulated by facilitated uptake and efflux, but detailed molecular understanding of the carrier proteins is incomplete. We have used pharmacological tools to explore the chemical space that defines substrate preferences for the auxin uptake carrier AUX1. Total and partial loss-of-function aux1 mutants were assessed against wild-type for dose-dependent resistance to a range of auxins and analogues. We then developed an auxin accumulation assay with associated mathematical modelling to enumerate accurate IC50 values for a small library of auxin analogues. The structure activity relationship data were analysed using molecular field analyses to create a pharmacophoric atlas of AUX1 substrates. The uptake carrier exhibits a very high level of selectivity towards small substrates including the natural indole-3-acetic acid, and the synthetic auxin 2,4-dichlorophenoxyacetic acid. No AUX1 activity was observed for herbicides based on benzoic acid (dicamba), pyridinyloxyacetic acid (triclopyr) or the 6-arylpicolinates (halauxifen), and very low affinity was found for picolinic acid-based auxins (picloram) and quinolinecarboxylic acids (quinclorac). The atlas demonstrates why some widely used auxin herbicides are not, or are very poor substrates. We list molecular descriptors for AUX1 substrates and discuss our findings in terms of herbicide resistance management.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Herbicidas/metabolismo , Ácidos Indolacéticos/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Bioensaio , Indóis/metabolismo , Concentração Inibidora 50 , Modelos Biológicos , Mutação/genética , Raízes de Plantas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Especificidade por Substrato , Nicotiana/citologiaRESUMO
Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.
Assuntos
Cinamatos/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bryopsida/efeitos dos fármacos , Bryopsida/crescimento & desenvolvimento , Cinamatos/química , Cinamatos/farmacologia , Ciclina B/genética , Ciclina B/metabolismo , Regulação da Expressão Gênica de Plantas , Isomerismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Selaginellaceae/efeitos dos fármacos , Selaginellaceae/crescimento & desenvolvimento , Transdução de SinaisRESUMO
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.