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1.
Plant Physiol ; 2024 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-38833284

RESUMEN

P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.

6.
Plant J ; 112(3): 664-676, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36069460

RESUMEN

Vacuolar H+ -ATPase (V-ATPase) has diverse functions related to plant development and growth. It creates the turgor pressure that drives cell growth by generating the energy needed for the active transport of solutes across the tonoplast. V-ATPase is a large protein complex made up of multiheteromeric subunits, some of which have unknown functions. In this study, a forward genetics-based strategy was employed to identify the vab3 mutant, which displayed resistance to isoxaben, a cellulose synthase inhibitor that could induce excessive transverse cell expansion. Map-based cloning and genetic complementary assays demonstrated that V-ATPase B subunit 3 (VAB3) is associated with the observed insensitivity of the mutant to isoxaben. Analysis of the vab3 mutant revealed defective ionic homeostasis and hypersensitivity to salt stress. Treatment with a V-ATPase inhibitor exacerbated ionic tolerance and cell elongation defects in the vab3 mutant. Notably, exogenous low-dose Ca2+ or Na+ could partially restore isoxaben resistance of the vab3 mutant, suggesting a relationship between VAB3-regulated cell growth and ion homeostasis. Taken together, the results of this study suggest that the V-ATPase subunit VAB3 is required for cell growth and ion homeostasis in Arabidopsis.


Asunto(s)
Arabidopsis , ATPasas de Translocación de Protón Vacuolares , Arabidopsis/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Benzamidas/farmacología , Benzamidas/metabolismo , Homeostasis
9.
Int J Mol Sci ; 23(8)2022 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-35457090

RESUMEN

Hydrogen sulfide (H2S) serves as an important gaseous signaling molecule that is involved in intra- and intercellular signal transduction in plant-environment interactions. In plants, H2S is formed in sulfate/cysteine reduction pathways. The activation of endogenous H2S and its exogenous application has been found to be highly effective in ameliorating a wide variety of stress conditions in plants. The H2S interferes with the cellular redox regulatory network and prevents the degradation of proteins from oxidative stress via post-translational modifications (PTMs). H2S-mediated persulfidation allows the rapid response of proteins in signaling networks to environmental stimuli. In addition, regulatory crosstalk of H2S with other gaseous signals and plant growth regulators enable the activation of multiple signaling cascades that drive cellular adaptation. In this review, we summarize and discuss the current understanding of the molecular mechanisms of H2S-induced cellular adjustments and the interactions between H2S and various signaling pathways in plants, emphasizing the recent progress in our understanding of the effects of H2S on the PTMs of proteins. We also discuss future directions that would advance our understanding of H2S interactions to ultimately mitigate the impacts of environmental stresses in the plants.


Asunto(s)
Sulfuro de Hidrógeno , Reguladores del Crecimiento de las Plantas , Gases/metabolismo , Sulfuro de Hidrógeno/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/metabolismo , Transducción de Señal , Estrés Fisiológico
10.
Plant Physiol ; 189(3): 1190-1191, 2022 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-35417022
11.
Plant Physiol ; 189(2): 450-451, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35266546
13.
Nat Commun ; 11(1): 6191, 2020 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-33273474

RESUMEN

In plants, light-dependent activation of de novo fatty acid synthesis (FAS) is partially mediated by acetyl-CoA carboxylase (ACCase), the first committed step for this pathway. However, it is not fully understood how plants control light-dependent FAS regulation to meet the cellular demand for acyl chains. We report here the identification of a gene family encoding for three small plastidial proteins of the envelope membrane that interact with the α-carboxyltransferase (α-CT) subunit of ACCase and participate in an original mechanism restraining FAS in the light. Light enhances the interaction between carboxyltransferase interactors (CTIs) and α-CT, which in turn attenuates carbon flux into FAS. Knockouts for CTI exhibit higher rates of FAS and marked increase in absolute triacylglycerol levels in leaves, more than 4-fold higher than in wild-type plants. Furthermore, WRINKLED1, a master transcriptional regulator of FAS, positively regulates CTI1 expression by direct binding to its promoter. This study reveals that in addition to light-dependent activation, "envelope docking" of ACCase permits fine-tuning of fatty acid supply during the plant life cycle.


Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Arabidopsis/metabolismo , Ácidos Grasos/biosíntesis , Membranas Intracelulares/metabolismo , Acetatos/metabolismo , Acetil-CoA Carboxilasa/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Radioisótopos de Carbono , Regulación del Desarrollo de la Expresión Génica , Luz , Simulación del Acoplamiento Molecular , Plastidios/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Protoplastos/metabolismo
14.
J Biol Chem ; 295(29): 9901-9916, 2020 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-32467229

RESUMEN

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.


Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas de Cloroplastos/metabolismo , Fotosíntesis/fisiología , Acetil-CoA Carboxilasa/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Concentración de Iones de Hidrógeno
15.
Plant Mol Biol ; 98(3): 275-287, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30311174

RESUMEN

KEY MESSAGE: A new synthetic auxin AAL1 with new structure was identified. Different from known auxins, it has weak effects. By AAL1, we found specific amino acids could restore the effects of auxin with similar structure. Auxin, one of the most important phytohormones, plays crucial roles in plant growth, development and environmental response. Although many critical regulators have been identified in auxin signaling pathway, some factors, especially those with weak fine-tuning roles, are still yet to be discovered. Through chemical genetic screenings, we identified a small molecule, Auxin Activity Like 1 (AAL1), which can effectively inhibit dark-grown Arabidopsis thaliana seedlings. Genetic screening identified AAL1 resistant mutants are also hyposensitive to indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). AAL1 resistant mutants such as shy2-3c and ecr1-2 are well characterized as mutants in auxin signaling pathway. Genetic studies showed that AAL1 functions through auxin receptor Transport Inhibitor Response1 (TIR1) and its functions depend on auxin influx and efflux carriers. Compared with known auxins, AAL1 exhibits relatively weak effects on plant growth, with 20 µM and 50 µM IC50 (half growth inhibition chemical concentration) in root and hypocotyl growth respectively. Interestingly, we found the inhibitory effects of AAL1 and IAA could be partially restored by tyrosine and tryptophan respectively, suggesting some amino acids can also affect auxin signaling pathway in a moderate manner. Taken together, our results demonstrate that AAL1 acts through auxin signaling pathway, and AAL1, as a weak auxin activity analog, provides us a tool to study weak genetic interactions in auxin pathway.


Asunto(s)
Arabidopsis/metabolismo , Hidrocarburos Aromáticos/metabolismo , Ácidos Indolacéticos/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Hidrocarburos Aromáticos/farmacología , Luz , Redes y Vías Metabólicas , Estructura Molecular , Mutación , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Plantones , Transducción de Señal
16.
Methods Mol Biol ; 1795: 143-148, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29846925

RESUMEN

Active molecules uncovered through chemical genetics studies have provided unique molecular genetic tools with which to study specific life processes. Different strategies have been developed to study the modes of action of these small molecules, especially for the target identification, including affinity chromatography (for target identification) and genetic/genomic methods. In this chapter we describe the protocols for a conventional forward genetics screening against seed germination inhibitors to study their working mechanism in model plant Arabidopsis. Such methods have been applied to study small molecules germostatin and triplin, the copper ion-binding small molecule.


Asunto(s)
Compuestos de Anilina/farmacología , Pruebas Genéticas , Germinación/efectos de los fármacos , Germinación/genética , Semillas/crecimiento & desarrollo , Semillas/genética , Sulfuros/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , ADN Bacteriano/genética , Descubrimiento de Drogas , Resistencia a Medicamentos/genética , Marcadores Genéticos , Mutación , Bibliotecas de Moléculas Pequeñas
17.
PLoS Genet ; 13(4): e1006703, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28388654

RESUMEN

Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in Arabidopsis thaliana. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown Arabidopsis seedlings through ethylene signaling pathway. ran1-1 and ran1-2 are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of ran1-1 and ran1-2. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in Arabidopsis, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Catión/genética , Cobre/metabolismo , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas Transportadoras de Cobre , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , N-Metiltransferasa de Histona-Lisina , Humanos , Transporte Iónico/genética , Desarrollo de la Planta , Plantas Modificadas Genéticamente , Proteínas de Unión al ARN , Plantones/genética , Transducción de Señal , Tiourea/análogos & derivados , Factores de Transcripción/metabolismo , Proteína de Unión al GTP ran
18.
Plant Physiol ; 173(4): 2356-2369, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28193765

RESUMEN

Abscisic acid (ABA), the most important stress-induced phytohormone, regulates seed dormancy, germination, plant senescence, and the abiotic stress response. ABA signaling is repressed by group A type 2C protein phosphatases (PP2Cs), and then ABA binds to its receptor of the ACTIN RESISTANCE1 (PYR1), PYR1-LIKE (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family, which, in turn, inhibits PP2Cs and activates downstream ABA signaling. The agonist/antagonist of ABA receptors have the potential to reveal the ABA signaling machinery and to become lead compounds for agrochemicals; however, until now, no broad-spectrum antagonists of ABA receptors blocking all PYR/PYL-PP2C interactions have been identified. Here, using chemical genetics screenings, we identified ABA ANTAGONIST1 (AA1), the first broad-spectrum antagonist of ABA receptors in Arabidopsis (Arabidopsis thaliana). Physiological analyses revealed that AA1 is sufficiently active to block ABA signaling. AA1 interfered with all the PYR/PYL-HAB1 interactions, and the diminished PYR/PYL-HAB1 interactions, in turn, restored the activity of HAB1. AA1 binds to all 13 members. Molecular dockings, the non-AA1-bound PYL2 variant, and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2. Using AA1, we tested the genetic relationships of ABA receptors with other core components of ABA signaling, demonstrating that AA1 is a powerful tool with which to sidestep this genetic redundancy of PYR/PYLs. In addition, the application of AA1 delays leaf senescence. Thus, our study developed an efficient broad-spectrum antagonist of ABA receptors and demonstrated that plant senescence can be chemically controlled through AA1, with a simple and easy-to-synthesize structure, allowing its availability and utility as a chemical probe synthesized in large quantities, indicating its potential application in agriculture.


Asunto(s)
Ácido Abscísico/metabolismo , Receptores de Superficie Celular/metabolismo , Ácido Abscísico/farmacología , Agroquímicos/química , Agroquímicos/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Unión Proteica/efectos de los fármacos , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/genética , Plantones/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/farmacología , Técnicas del Sistema de Dos Híbridos
19.
Plant Signal Behav ; 11(4): e1144000, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26918467

RESUMEN

Seed dormancy and germination are the most important adaptive traits of seed plants, which control the germination in a proper space and time. Internal genetic factors together with environmental cues govern seed dormancy and germination. Abscisic acid (ABA), a key phytohormone induces seed dormancy and inhibits seed germination through its molecular genetic signaling network responding the seed inherent physiological and environmental factors. Recently, auxin has been shown to be another phytohormone that induces seed dormancy. We have recently shown that germonstatin (GS), a small synthetic molecule identified by high through-put chemical genetic screenings, inhibits seed germination through up-regulating auxin signaling and inducing auxin biosynthesis. GERMOSTATIN RESISTANCE LOCUS 1 (GSR1) encodes a plant homeodomain (PHD) finger protein and is responsible for GS seed germination inhibition. Its knockdown mutant gsr1 displays decreased dormancy. In this report, we show that GS is not an ABA analog and provided 2 other GS-resistant mutants related to the chemical's function in seed germination inhibition other than gsr1, suggesting that GS may have pleiotropic effects through targeting different pathway governing seed germination.


Asunto(s)
Compuestos de Anilina/farmacología , Arabidopsis/crecimiento & desarrollo , Pleiotropía Genética/efectos de los fármacos , Germinación/efectos de los fármacos , Semillas/crecimiento & desarrollo , Sulfuros/farmacología , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Semillas/efectos de los fármacos
20.
Plant J ; 85(1): 3-15, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26611158

RESUMEN

Seed dormancy and germination are important physiological processes during the life cycle of a seed plant. Recently, auxin has been characterized as a positive regulator that functions during seed dormancy and as a negative regulator during germination. Through chemical genetic screenings, we have identified a small molecule, germostatin (GS), which effectively inhibits seed germination in Arabidopsis. GSR1 (germostatin resistance locus 1) encodes a tandem plant homeodomain (PHD) finger protein, identified by screening GS-resistant mutants. Certain PHD fingers of GSR1 are capable of binding unmethylated H3K4, which has been reported as an epigenetic mark of gene transcriptional repression. Biochemical studies show that GSR1 physically interacts with the transcriptional repressor ARF16 and attenuates the intensity of interaction of IAA17/ARF16 by directly interacting with IAA17 to release ARF16. Further results show that axr3-1, arf10 arf16 are hyposensitive to GS, and gsr1 not only resists auxin-mediated inhibition of seed germination but also displays decreased dormancy. We therefore propose that GSR1 may form a co-repressor with ARF16 to regulate seed germination. Besides promoting auxin biosynthesis via upregulating expression of YUCCA1, GS also enhances auxin responses by inducing degradation of DΙΙ-VENUS and upregulating expression of DR5-GFP. In summary, we identified GSR1 as a member of the auxin-mediated seed germination genetic network, and GS, a small non-auxin molecule that specifically acts on auxin-mediated seed germination.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Homeodominio/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes Reporteros , Germinación , Proteínas de Homeodominio/genética , Mutación , Latencia en las Plantas , Semillas/genética , Semillas/fisiología , Transducción de Señal , Factores de Transcripción/genética , Transcripción Genética
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