RESUMEN
Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an â¼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from â¼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.
Asunto(s)
Ácido Abscísico/antagonistas & inhibidores , Quinolinas/síntesis química , Triazoles/síntesis química , Ácido Abscísico/agonistas , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Benzamidas/síntesis química , Benzamidas/química , Proteínas Portadoras/metabolismo , Química Clic/métodos , Ciclohexanos/síntesis química , Ciclohexanos/química , Expresión Génica , Germinación , Modelos Moleculares , Reguladores del Crecimiento de las Plantas/metabolismo , Quinolinas/farmacología , Semillas/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico , Triazoles/farmacologíaRESUMEN
The plant hormone abscisic acid (ABA) plays a critical role in various environmental stress responses and has long been expected to be used in agriculture. However, the practical use of ABA has been limited, mainly because of its photoinstability and rapid biodegradation. We previously developed photostable ABA agonists, BP2A and Me 1',4'-trans-diol BP2A, in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. This finding validated our structure-based approach in designing photostable agonists and provided a basis for developing a more potent or long-lasting ABA agonist. In this study, we synthesized novel BP2A analogs in which the cyclohexenone ring was modified to avoid catabolism by the ABA metabolic enzyme, ABA 8'-hydroxylase. All synthesized analogs showed higher photostability than BP2A under sunlight. In an Arabidopsis seed germination assay, (+)-compounds 5 and 6 with a tetralone ring displayed significantly stronger ABA agonist activity than (+)-BP2A. In contrast, in the in vitro phosphatase assays, both compounds showed comparable or weaker ABA receptor (PYL1) agonistic activity than (+)-BP2A, suggesting that the stronger ABA-like activity of (+)-5 and (+)-6 may arise from their metabolic stability in vivo. This study provides data relevant to designing photostable and persistent ABA agonists.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/farmacología , Ácido Abscísico/química , Reguladores del Crecimiento de las Plantas , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
The molecular mechanisms controlling underwater elongation are based extensively on studies on internode elongation in the monocot rice (Oryza sativa) and petiole elongation in Rumex rosette species. Here, we characterize underwater growth in the dicot Nasturtium officinale (watercress), a wild species of the Brassicaceae family, in which submergence enhances stem elongation and suppresses petiole growth. We used a genome-wide transcriptome analysis to identify the molecular mechanisms underlying the observed antithetical growth responses. Though submergence caused a substantial reconfiguration of the petiole and stem transcriptome, only little qualitative differences were observed between both tissues. A core submergence response included hormonal regulation and metabolic readjustment for energy conservation, whereas tissue-specific responses were associated with defense, photosynthesis, and cell wall polysaccharides. Transcriptomic and physiological characterization suggested that the established ethylene, abscisic acid (ABA), and GA growth regulatory module for underwater elongation could not fully explain underwater growth in watercress. Petiole growth suppression is likely attributed to a cell cycle arrest. Underwater stem elongation is driven by an early decline in ABA and is not primarily mediated by ethylene or GA. An enhanced stem elongation observed in the night period was not linked to hypoxia and suggests an involvement of circadian regulation.
Asunto(s)
Nasturtium , Oryza , Rumex , Ácido Abscísico , Giberelinas , Oryza/genética , AguaRESUMEN
The physiological functions of the plant hormone abscisic acid (ABA) are triggered by interactions between PYR/PYL/RCAR receptors (PYLs) and group-A protein phosphatases 2C (PP2Cs). PYL agonists/antagonists capable of inducing/disrupting these interactions would be valuable in investigating the regulatory mechanisms of ABA signaling. Previously, we developed (+)-PAO4, a high-affinity PYL antagonist, by conformationally restricting the S-hexyl chain of our first reported PYL antagonist, 3'-hexylsulfanyl-ABA. Although (+)-PAO4 shows a greater binding affinity for Arabidopsis PYL5 compared with 3'-hexylsulfanyl-ABA, it is not able to completely block the ABA responses both in vitro and in vivo. Therefore, we designed novel conformationally restricted PYL antagonists in which the O-butyl chain of (+)-PAO4 was replaced with a pentyl (PAC4), a pentyne (PAT3) or a pentadiyne (PATT1) chain. (+)-PAT3 and (+)-PATT1 suppressed the ABA-induced inhibition of Arabidopsis seed germination more strongly than (+)-PAO4, but contrary to expectations, the affinity of each compound for PYL5 was almost the same as that of (+)-PAO4. Subsequent biochemical analyses revealed that unlike (+)-PAO4, (+)-PAT3 and (+)-PATT1 completely abolished ABA-induced PYL-PP2C interactions without partial agonistic activities. The superior PYL antagonist functions of (+)-PAT3 and (+)-PATT1 over (+)-PAO4 may explain their potent antagonistic activities against exogenous ABA in vivo. Furthermore, (+)-PAT3 and (+)-PATT1 also suppressed ABA responses in rice, indicating that both compounds are useful chemical tools for ABA-signaling studies, not only in dicots but also in monocots.
Asunto(s)
Alquinos/farmacología , Proteínas de Arabidopsis/antagonistas & inhibidores , Diseño de Fármacos , Receptores de Superficie Celular/antagonistas & inhibidores , Alquinos/síntesis química , Alquinos/química , Arabidopsis/química , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Germinación/efectos de los fármacos , Conformación Molecular , Receptores de Superficie Celular/metabolismo , Semillas/efectos de los fármacos , Semillas/metabolismoRESUMEN
The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.
Asunto(s)
Ácido Abscísico/análogos & derivados , Proteínas de Arabidopsis/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Reguladores del Crecimiento de las Plantas , Ácido Abscísico/síntesis química , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Germinación/efectos de los fármacos , Lactuca/efectos de los fármacos , Lactuca/metabolismo , Modelos Moleculares , Fosfoproteínas Fosfatasas/metabolismo , Reguladores del Crecimiento de las Plantas/síntesis química , Reguladores del Crecimiento de las Plantas/farmacología , Fenómenos Fisiológicos de las Plantas , Unión Proteica , Raphanus/efectos de los fármacos , Raphanus/metabolismo , Semillas/efectos de los fármacos , Semillas/metabolismo , Relación Estructura-ActividadRESUMEN
Abscisic acid (ABA), a plant hormone, is involved in many plant development processes and environmental stress responses that are regulated by a Pyrabactin Resistant 1 (PYR)/Pyrabactin Resistant-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) receptor protein-mediated signal transduction pathway. In Arabidopsis thaliana, PYL proteins constitute a 14-member family comprising two distinct subclasses: dimeric receptors (PYR1 and PYL1-PYL3) and monomeric receptors (PYL4-PYL13). The individual contributions of PYL subclasses/subtypes with specific physiological actions are still poorly understood; consequently, the development of PYL subclass/subtype-selective agonists should be useful to reveal the different functions of these receptors. In this study, we focused on the ABA analogs 6-nor-ABA and 7'-nor-ABA, which were expected to function as monomeric receptor-selective agonists on the basis of crystal structures of PYL-ABA complexes and sequence alignments of PYL subtypes. In a protein phosphatase 2C (PP2C) assay, the agonist activities of both analogs were lower than those of ABA toward all tested PYL proteins, regardless of subclass/subtype. Nevertheless, we found that 6-nor-ABA acts as a selective agonist at the physiological level: it induced stomatal closure but did not inhibit seed germination and root growth. On the basis of observed inhibitory activity against PP2C among different PYL subtypes, this biological effect of 6-nor-ABA may be attributed to the activity of that agonist on PYL5 and/or PYL6.
Asunto(s)
Ácido Abscísico/química , Proteínas de Arabidopsis/química , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Modelos Moleculares , Raíces de PlantasRESUMEN
The physiological functions of abscisic acid (ABA) are regulated by a signal transduction pathway involving cytosolic ABA receptors, which include 14 PYR/PYL/RCAR (PYL) proteins in Arabidopsis. The development of a PYL antagonist could be a valuable tool to improve our understanding of the roles of ABA. We previously developed 3'-hexylsulfanyl-ABA (AS6), whose S-hexyl chain blocks protein phosphatase 2C (PP2C) binding by steric hindrance. This finding not only validated our structure-based approach to the design of a PYL antagonist, but also provided a basis for the development of a more potent or subclass/subtype selective PYL antagonist. In the present study, we synthesized a conformationally restricted analog of AS6, namely propenyl-ABA with an O-butyl chain (PAO4), to improve the affinity for PYL proteins by reducing the entropic penalty for binding to the receptors. In seed germination assays, (+)-PAO4 was a slightly stronger antagonist than AS6 in Arabidopsis and a significantly stronger antagonist in lettuce. Analysis of the thermodynamic parameters associated with the formation of the Arabidopsis PYL-(+)-PAO4 complex revealed that (+)-PAO4 binds more strongly to PYL5 than AS6 owing to an entropic advantage. In PP2C assays, this enhancement effect was observed only for the monomeric PYL subclass containing PYL5, suggesting that (+)-PAO4 is more effective than AS6 in physiological events involving monomeric PYL proteins as ABA receptors.
Asunto(s)
Ácido Abscísico/química , Ácido Abscísico/fisiología , Proteínas de Arabidopsis/antagonistas & inhibidores , Diseño de Fármacos , Conformación Molecular , ArabidopsisRESUMEN
A novel chemical tool compound that is an antagonist of brassinolide (BL, 1)-induced rice lamina joint inclination was developed. Although 2-O-, 3-O-, 22-O-, or 23-O-methylation of BL causes a critical decrease in biological activity,(5) a crystal structure of the extracellular leucine-rich repeat (LRR) domain of BRASSINOSTEROID-INSENSITIVE I (BRI1) bound to BL(3,4) indicates that the loss of activity of the O-methylated BL may result from not only the low affinity to BRI1, but also from blocking the interaction with another BR signaling factor, a partner protein of BRI1 (e.g., BRI1-ASSOCIATED KINASE 1, BAK1). On the basis of this hypothesis we synthesized the BL 2,3-acetonide 2, the 22,23-acetonide 3, and the 2,3:22,23-diacetonide 4 to assess the possibility of 2-O- and 3-O- or/and 22-O- and 23-O-alkylated BL as an antagonist in BR signaling evoked by exogenously applied BL. The 2,3-acetonide 2 more strongly inhibited the lamina inclination caused by BL relative to the 22,23-acetonide 3, whereas the diacetonide 4 had no effect most likely due to its increased hydrophobicity. This suggested that the 2,3-hydroxyl groups of BL play a more significant role in the interaction with a BRI1 partner protein rather than BRI1 itself in rice lamina joint inclination. Taken together it was demonstrated that BL, the most potent agonist of BRI1, is transformed into an antagonist by functionalization of the 2,3-dihydroxyl groups as the acetonide. This finding opens the door to the potential development of a chemical tool that modulates protein-protein interactions in the BR signaling pathway to dissect the BR-dependent processes.
Asunto(s)
Brasinoesteroides/síntesis química , Brasinoesteroides/farmacología , Oryza/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/antagonistas & inhibidores , Esteroides Heterocíclicos/farmacología , Brasinoesteroides/química , Estructura Molecular , Oryza/crecimiento & desarrollo , Esteroides Heterocíclicos/síntesis química , Esteroides Heterocíclicos/químicaRESUMEN
BACKGROUND: Carotenoids are a major class of plant pigments and fulfill many functions in different organisms that either produce or consume them. Although the color of the stamina of tea (Camellia sinensis) flowers is clearly due to the presence of carotenoids, the carotenoid profile and content remain to be discovered. RESULTS: We investigated the carotenoid profile of tea flowers and determined changes in concentrations over the floral development. The flowers contained oxygenated xanthophylls such as neoxanthin, lutein and zeaxanthin, as well as the hydrocarbons ß-carotene and α-carotene. Flowers of the tea plant contain to vegetables comparable amounts of carotenoids. The content of 9'-cis-epoxycarotenoids, which serve as abscisic acid precursors, as well as changes in concentration of abscisic acid were studied. The concentrations of carotenoids decreased whereas the abscisic acid content increased over the floral development. Exogenously applied S-abscisic acid affected water uptake, flower opening and carotenoid accumulation. CONCLUSION: In summary, this paper reports, for the first time, the carotenoid profile and content of tea flowers. The study revealed that carotenoids in tea flowers are an interesting target in respect of possible applications of tea flower extracts as well as biological functions of abscisic acid during floral development.
Asunto(s)
Ácido Abscísico , Camellia sinensis/metabolismo , Carotenoides/metabolismo , Flores/metabolismo , Desarrollo de la Planta , Reguladores del Crecimiento de las Plantas , Agua/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Camellia sinensis/efectos de los fármacos , Camellia sinensis/fisiología , Flores/efectos de los fármacos , Flores/fisiología , Desarrollo de la Planta/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Té , Xantófilas/metabolismo , beta Caroteno/metabolismoRESUMEN
White-rot fungi are the most important group of lignin biodegraders. Phanerochaete sordida YK-624 has higher ligninolytic activity than that of model white-rot fungi. However, the underlying mechanism responsible for lignin degradation by white-rot fungi remains unknown, and the induced compounds isolated from white-rot fungi for lignin degradation have never been studied. In the present study, we tried to screen ligninolytic-inducing compounds produced by P. sordida YK-624. After large-scale incubation of P. sordida YK-624, the culture and mycelium were separated by filtration. After the separation and purification, purified compounds were analyzed by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance. The sterilized unbleached hardwood kraft pulp was used for the initial evaluation of ligninolytic activity. Ergosterol was isolated and identified and it induced the lignin-degrading activity of this fungus. Moreover, we investigated ergosterol metabolites from P. sordida YK-624, and the ergosterol metabolites ergosta-4,7,22-triene-3,6-dione and ergosta-4,6,8(14),22-tetraen-3-one were identified and then chemically synthesized. These compounds significantly improved the lignin-degrading activity of the fungus. This is the first report on the ligninolytic-inducing compounds produced by white-rot fungi.
RESUMEN
After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8'-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18 h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2B, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds.
Asunto(s)
Ácido Abscísico/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Lactonas/farmacología , Orobanchaceae/genética , Proteínas de Plantas/genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario , Perfilación de la Expresión Génica , Germinación , Datos de Secuencia Molecular , Orobanchaceae/efectos de los fármacos , Orobanchaceae/crecimiento & desarrollo , Latencia en las Plantas , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa , Semillas/metabolismo , Análisis de Secuencia de ADN , Triazoles/metabolismoRESUMEN
The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA 8'-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolism. Azole P450 inhibitors bind to the P450 active site by both coordinating to the heme-iron atom via an sp(2) nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from its small molecular size and flexible conformation that allows it to fit into active sites differing in size and shape. To find a selective inhibitor of CYP701A based on this hypothesis, we examined inhibitory activities of three types of UNI analogues, which were conformationally constrained, enlarged in width, and enlarged in length, against recombinant rice CYP701A6 and Arabidopsis CYP707A3. Conformationally restricted analogues, UFAP2 and UFAP2N, inhibited CYP701A6 as strongly as UNI, whereas it inhibited CYP707A3 less than UNI.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Oryza/enzimología , Triazoles/química , Triazoles/farmacología , Sistema Enzimático del Citocromo P-450 , Conformación Molecular , Especificidad por SustratoRESUMEN
We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated logP value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 µM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (±)-Abz-E2B was a considerably weaker inhibitor than (±)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (±)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 µM, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 µM or higher concentration is required, although (±)-Abz-E2B may be useful as an alternative option at a lower concentration.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Oryza/efectos de los fármacos , Triazoles/química , Triazoles/farmacología , Sistema Enzimático del Citocromo P-450 , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Estructura Molecular , Oryza/crecimiento & desarrollo , Proteínas de Plantas , Plantones/efectos de los fármacos , Solubilidad , Especificidad por Sustrato , Agua/químicaRESUMEN
The plant hormone abscisic acid (ABA) plays a central role in adaptive responses to abiotic stresses that adversely affect crop growth and productivity. However, ABA photoinstability limits its use in agriculture. To overcome this drawback, in this study, we developed photostable ABA analogues, the (+)-BP2A compound series (compounds 5-9), in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. All BP2A analogues showed higher stability against UV-B irradiation at 302 nm than ABA, and compounds 6 and 7 barely decomposed even under sunlight. In physiological assays, (+)-BP2A and (+)-compound 7, in which the α,ß-unsaturated carbonyl group of BP2A was reduced, exhibited ABA-like activities, including inhibition of seed germination and induced drought tolerance in Arabidopsis. Biochemical studies revealed that (+)-compound 7, unlike (+)-BP2A, did not activate pyrabactin resistance-like (PYL) receptors in vitro and was converted to (+)-BP2A in plants, suggesting that it functions as a prodrug PYL agonist. Furthermore, (+)-compound 7 inhibited seed germination of tomato, lettuce, and rice. Thus, this compound represents a potential plant growth regulator that induces ABA-type responses in agricultural fields.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Ácido Abscísico , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/metabolismo , Reguladores del Crecimiento de las Plantas/farmacologíaRESUMEN
We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA.
Asunto(s)
Ácido Abscísico , Aminoácidos , Ácido Abscísico/síntesis química , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Aminoácidos/síntesis química , Aminoácidos/química , Aminoácidos/metabolismo , Aminoácidos/farmacología , Proteínas de Arabidopsis/metabolismo , Germinación , Concentración 50 Inhibidora , Estructura Molecular , Oryza/metabolismo , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/metabolismo , Semillas/crecimiento & desarrollo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We developed abscinazole-E1 (Abz-E1), a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A). This inhibitor was designed and synthesized as an enlarged analogue of uniconazole (UNI), a well-known plant growth retardant, which inhibits a gibberellin biosynthetic enzyme (ent-kaurene oxidase, CYP701A) as well as CYP707A. Our results showed that Abz-E1 functions as a potent inhibitor of CYP707A and a poor inhibitor of CYP701A both in vitro and in vivo. Abz-E1 application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Triazoles/química , Arabidopsis/enzimología , Arabidopsis/fisiología , Germinación , Oryza/enzimología , Oryza/fisiología , Proteínas de PlantasRESUMEN
The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8'-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp2 nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Plantas/efectos de los fármacos , Plantas/enzimología , Triazoles/química , Triazoles/farmacología , Arabidopsis/enzimología , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Lactuca/efectos de los fármacos , Lactuca/crecimiento & desarrollo , Modelos Moleculares , Oryza/efectos de los fármacos , Oryza/crecimiento & desarrollo , Desarrollo de la Planta , Reguladores del Crecimiento de las Plantas/síntesis química , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrollo , Triazoles/síntesis químicaRESUMEN
We enlarged the uniconazole (UNI) molecule to find a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, and synthesized various UNI derivatives that were substituted with hydrophilic and hydrophobic groups at the 4-chlorine of the phenyl group of UNI using click chemistry. Considering its potency in ABA 8'-hydroxylase inhibition, its small effect on seedling growth, and its ease of application, UT4, the UNI derivative containing the C4 alkyltriazole, was the best candidate for a highly selective inhibitor of ABA 8'-hydroxylase.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/química , Triazoles/química , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Malus/crecimiento & desarrollo , Oryza/crecimiento & desarrollo , Proteínas de Plantas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrollo , Triazoles/síntesis química , Triazoles/farmacologíaRESUMEN
To develop a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, seed dormancy, and other various physiological events, we designed and synthesized conformationally restricted analogues of uniconazole (UNI), a well-known plant growth retardant, which inhibits a biosynthetic enzyme (ent-kaurene oxidase) of gibberellin as well as ABA 8'-hydroxylase. Although most of these analogues were less effective than UNI in inhibition of ABA 8'-hydroxylase and rice seedling growth, we found that a lactol-bridged analogue with an imidazole is a potent inhibitor of ABA 8'-hydroxylase but not of plant growth. This compound, abscinazole-F1, induced drought tolerance in apple seedlings upon spray treatment with a 10 microM solution.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Reguladores del Crecimiento de las Plantas/metabolismo , Triazoles/química , Triazoles/metabolismo , Ácido Abscísico/metabolismo , Aclimatación , Sistema Enzimático del Citocromo P-450/metabolismo , Sequías , Malus/fisiología , Conformación Molecular , Oryza/crecimiento & desarrollo , Reguladores del Crecimiento de las Plantas/síntesis química , Reguladores del Crecimiento de las Plantas/química , Proteínas de Plantas , Triazoles/síntesis químicaRESUMEN
Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.