RESUMEN
Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.
Asunto(s)
Alcaloides , Aporfinas , Aristolochia , Sistema Enzimático del Citocromo P-450 , Filogenia , Proteínas de Plantas , Aporfinas/metabolismo , Aristolochia/enzimología , Aristolochia/metabolismo , Aristolochia/genética , Aristolochia/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Alcaloides/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/enzimología , Raíces de Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Flores/enzimología , Flores/genética , Flores/metabolismo , Tallos de la Planta/metabolismo , Tallos de la Planta/enzimología , Tallos de la Planta/genéticaRESUMEN
Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.
Asunto(s)
Aristolochia/metabolismo , Proteínas de Dominio MADS/metabolismo , Aristolochia/genética , Aristolochia/crecimiento & desarrollo , Evolución Biológica , Flores/crecimiento & desarrollo , Proteínas de Dominio MADS/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Aristolochic acid released from decaying Aristolochia clematitis weed is contaminating soil and food crops in Eastern Europe and is one of the major causes to Balkan endemic nephropathy. Measures should be taken to prevent people from being exposed to these highly potent phytotoxins. Research needs to develop remediation methods.
Asunto(s)
Aristolochia/química , Ácidos Aristolóquicos/toxicidad , Nefropatía de los Balcanes/etiología , Contaminantes del Suelo/química , Aristolochia/crecimiento & desarrollo , Aristolochia/metabolismo , Productos Agrícolas/efectos de los fármacos , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/metabolismo , Humanos , Contaminantes del Suelo/toxicidad , Contaminantes del Agua/química , Contaminantes del Agua/toxicidadRESUMEN
Aristolochia fimbriata (Aristolochiaceae) is a member of an early diverging lineage of flowering plants and a promising candidate for evo-devo studies. Aristolochia flowers exhibit a unique floral synorganization that consists of a monosymmetric and petaloid calyx formed by three congenitally fused sepals, and a gynostemium formed by the congenital fusion between stamens and the stigmatic region of the carpels. This floral ground plan atypical in the magnoliids can be used to evaluate the role of floral organ identity MADS-box genes during early flower evolution. In this study, we present in situ hybridization experiments for the homologs of the canonical C-, D-, and E-class genes. Spatiotemporal expression of the C-class gene AfimAG is restricted to stamens, ovary, and ovules, suggesting a conserved stamen and carpel identity function, consistent with that reported in core-eudicots and monocots. The D-class gene AfimSTK is detected in the anthers, the stigmas, the ovary, the ovules, the fruit, and the seeds, suggesting conserved roles in ovule and seed identity and unique roles in stamens, ovary, and fruit development. In addition, AfimSTK expression patterns in areas of organ abscission and dehiscence zones suggest putative roles linked to senescence processes. We found that both E-class genes are expressed in the anthers and the ovary; however, AfimSEP2 exhibits higher expression compared to AfimSEP1. These findings provide a comprehensive picture of the ancestral expression patterns of the canonical MADS-box floral organ identity genes and the foundations for further comparative analyses in other magnoliids.
Asunto(s)
Aristolochia/metabolismo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Dominio MADS/metabolismo , Proteínas de Plantas/metabolismo , Aristolochia/anatomía & histología , Aristolochia/genética , Flores/anatomía & histología , Flores/genética , Duplicación de Gen , Genoma de Planta , Proteínas de Dominio MADS/genética , Filogenia , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
This study aimed to examine the transfer of nanoparticles within a terrestrial food chain. Oviposited eggs of the swallowtail butterfly (Atrophaneura alcinous) were hatched on the leaves of the host plant (Aristolochia debilis), and the root stock and root hairs were submerged in a suspension of 10 µg/ml titanium dioxide nanoparticles (TiO2-NPs) in a 100 ml bottle. The presence of TiO2-NPs in the veins of the leaves was confirmed by X-ray analytical microscopy (X-ray AM). The hatched 1st instar larvae fed on the leaves to moult into 2nd instar larvae. Small agglomerates of TiO2-NPs less than 150 nm in diameter were identified in the vascular tissue of the exposed plant, the midgut and the excreta of the larvae by transmission electron microscopy. The image of Ti elemental mapping by X-ray AM was analysed with the quantitative spatial information mapping (QSIM) technique. The results demonstrated that TiO2-NPs were transferred from the plant to the larvae and they were disseminated throughout the environment via larval excreta.
Asunto(s)
Aristolochia/metabolismo , Mariposas Diurnas/fisiología , Larva/fisiología , Hojas de la Planta/metabolismo , Titanio/química , Animales , Aristolochia/parasitología , Transporte Biológico , Microanálisis por Sonda Electrónica , Heces/química , Femenino , Cadena Alimentaria , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Oviposición/fisiología , Hojas de la Planta/parasitología , Raíces de Plantas/metabolismo , Titanio/metabolismo , Cigoto/fisiologíaRESUMEN
Malaria is a life-threatening disease caused by parasites transmitted to people and animals through the bites of infected mosquitoes. We biosynthesized silver nanoparticles (AgNP) using Aristolochia indica extract as reducing and stabilizing agent. AgNP were characterized by UV-vis spectroscopy, FTIR, SEM, EDX and XRD. In laboratory, LC50 of A. indica extract against Anopheles stephensi ranged from 262.66 (larvae I) to 565.02 ppm (pupae). LC50 of AgNP against A. stephensi ranged from 3.94 (larvae I) to 15.65 ppm (pupae). In the field, the application of A. indica extract and AgNP (10 × LC50) leads to 100% larval reduction after 72 h. In laboratory, 24-h predation efficiency of Diplonychus indicus against A. stephensi larvae was 33% (larvae II) and 57% (larvae III). In AgNP-contaminated environment (1 ppm), it was 45.5% (larvae II) and 71.75% (larvae III). Overall, A. indica-synthesized AgNP may be considered as newer and safer control tools against Anopheles vectors.
Asunto(s)
Anopheles/efectos de los fármacos , Aristolochia/metabolismo , Insecticidas/farmacología , Nanopartículas del Metal/química , Plata/química , Animales , Tecnología Química Verde , Insecticidas/química , Insecticidas/metabolismo , Larva/efectos de los fármacos , Hojas de la Planta/metabolismoRESUMEN
Flower monosymmetry contributes to specialized interactions between plants and their insect pollinators. In the magnoliids, flower monosymmetry is exhibited only in the Aristolochiaceae (Piperales). Aristolochia flowers develop a calyx-derived monosymmetric perianth that enhances pollination success by a flytrap mechanism. Aristolochia arborea forms additionally a special perianth outgrowth that mimics a mushroom to attract flies, the mushroom mimicry structure (MMS). In core eudicots, members of the CYC2 clade of TCP transcription factors are key regulators of corolla monosymmetry establishment. The CYC2 clade arose via core eudicot-specific duplications from ancestral CYC/TB1 genes. CYC/TB1 genes are also thought to affect monosymmetry formation in early diverging eudicot and monocot species. Here, we demonstrate that CYC/TB1 genes, named CYC-like genes (CYCL) are present in basal angiosperms and magnoliids. Expression analyses in A. arborea indicate that CYCL genes participate in perianth and MMS differentiation processes and do not support a CYCL gene function in initial flower monosymmetry formation. Heterologous CYCL and CYC2 gene overexpression studies in Arabidopsis show that Aristolochia CYCL proteins only perform a CYC2-like function when the CYCL TCP domain is replaced by a CYC2 domain. Comparative TCP domain analyses revealed that an LxxLL motif, known to mediate protein-protein interactions, evolved in the second helix of the TCP domain in the CYC2 lineage and contributes to CYC2-related functions. Our data imply that divergent evolution of the CYC/TB1 lineages caused significant changes in their coding regions, which together with cis-regulatory changes established the key CYC2 function in regulating eudicot flower monosymmetry.
Asunto(s)
Flores/crecimiento & desarrollo , Magnoliopsida/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Aristolochia/metabolismo , Flores/metabolismo , Magnoliopsida/crecimiento & desarrollo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Insect herbivores contend with various plant traits that are presumed to function as feeding deterrents. Paradoxically, some specialist insect herbivores might benefit from some of these plant traits, for example by sequestering plant chemical defenses that herbivores then use as their own defense against natural enemies. Larvae of the butterfly species Battus philenor (L.) (Papilionidae) sequester toxic alkaloids (aristolochic acids) from their Aristolochia host plants, rendering larvae and adults unpalatable to a broad range of predators. We studied the importance of two putative defensive traits in Aristolochia erecta: leaf toughness and aristolochic acid content, and we examined the effect of intra- and interplant chemical variation on the chemical phenotype of B. philenor larvae. It has been proposed that genetic variation for sequestration ability is "invisible to natural selection" because intra- and interindividual variation in host-plant chemistry will largely eliminate a role for herbivore genetic variation in determining an herbivore's chemical phenotype. We found substantial intra- and interplant variation in leaf toughness and in the aristolochic acid chemistry in A. erecta. Based on field observations and laboratory experiments, we showed that first-instar larvae preferentially fed on less tough, younger leaves and avoided tougher, older leaves, and we found no evidence that aristolochic acid content influenced first-instar larval foraging. We found that the majority of variation in the amount of aristolochic acid sequestered by larvae was explained by larval family, not by host-plant aristolochic acid content. Heritable variation for sequestration is the predominant determinant of larval, and likely adult, chemical phenotype. This study shows that for these highly specialized herbivores that sequester chemical defenses, traits that offer mechanical resistance, such as leaf toughness, might be more important determinants of early-instar larval foraging behavior and development compared to plant chemical defenses.
Asunto(s)
Aristolochia/fisiología , Ácidos Aristolóquicos/metabolismo , Mariposas Diurnas/fisiología , Herbivoria , Larva/fisiología , Animales , Aristolochia/química , Aristolochia/metabolismo , Ácidos Aristolóquicos/análisis , Mariposas Diurnas/genética , Mariposas Diurnas/metabolismo , Variación Genética , Hojas de la Planta/química , Hojas de la Planta/fisiología , Selección GenéticaRESUMEN
Volatile and semi-volatile secondary metabolites, as well as aristolochic acids (AA), present in leaves, stems, and flowers of Aristolochia ringens were determined by gas chromatography (GC)-mass spectrometry (MS) and high-performance liquid chromatography (HPLC) methods, respectively. Metabolite isolation was performed using different extraction techniques: microwave-assisted hydrodistillation (MWHD), supercritical fluid extraction, and headspace solid-phase microextraction (HS-SPME). The chemical composition of the extracts and oils was established by GC-MS. The determinations of AAI and AAII were conducted by methanolic extraction of different plant parts followed by HPLC analysis. Essential oil yields from leaves and stems were 0.008 +/- 0.0022% and 0.047 +/- 0.0026%, respectively. Aristolochia ringens flowers did not yield essential oil under MWHD. Sesquiterpene hydrocarbons (66%) were the main compounds in the essential oil isolated from leaves whereas monoterpene hydrocarbons (73%) predominated in the stems essential oil. Yields of extracts isolated by SFE from leaves, stems, and flowers were 4 +/- 1.8%, 1.2 +/- 0.25%, and 4 +/- 1.8%, respectively. In vivo HS-SPME of flowers isolated compounds with known unpleasant smells such as volatile aldehydes and short-chain carboxylic acids. HPLC analysis detected the presence of AAII in the flowers of Aristolochia ringens at a concentration of 610 +/- 47 mg/kg of dried flower.
Asunto(s)
Aristolochia/química , Aristolochia/metabolismo , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/metabolismo , Cromatografía Líquida de Alta Presión , Flores/química , Flores/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Tallos de la Planta/química , Tallos de la Planta/metabolismo , VolatilizaciónRESUMEN
A native female-specific chemoreceptive protein of a swallowtail butterfly [oviposition stimulant binding protein (OSBP)] was shown to specifically bind to aristolochic acid, a main stimulant for oviposition from its host plant. Oviposition stimulants are recognized by chemoreceptive organs of insects. OSBP isolated previously from the chemoreceptive organs was assumed to bind to an oviposition stimulant. Using a highly sensitive fluorescent micro-binding assay, we clarified OSBP bound to aristolochic acid. Three-dimensional molecular modeling revealed the structure of the OSBP-aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant as a ligand in insects.
Asunto(s)
Ácidos Aristolóquicos/metabolismo , Mariposas Diurnas/fisiología , Proteínas de Insectos/metabolismo , Oviposición , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Animales , Aristolochia/metabolismo , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacología , Bioensayo , Mariposas Diurnas/metabolismo , Femenino , Fluorescencia , Colorantes Fluorescentes/química , Proteínas de Insectos/química , Microscopía Fluorescente , Modelos Moleculares , Oviposición/efectos de los fármacos , Conformación ProteicaRESUMEN
Biflavones, a chalcone-flavone, and a tetraflavonoid with a new carbon skeleton were isolated from the leaves of Aristolochia ridicula. Their structures were determined by chemical derivatizations and spectrometric analyses.
Asunto(s)
Aristolochia/química , Flavonoides/química , Flavonoides/metabolismo , Aristolochia/metabolismo , Estructura Molecular , Hojas de la Planta/químicaRESUMEN
Two new aristolactams, aristolactam E (1) and aristolactam-AIIIa-6-O-beta-D-glucoside (2), three novel benzoyl benzyltetrahydroisoquinoline ether N-oxide alkaloids, aristoquinoline A (3), aristoquinoline B (4), and aristoquinoline C (5), and a new biphenyl ether, aristogin F (6), together with 62 known compounds have been isolated from the root and stem of Aristolochia elegans Mast. The structures of the new natural products were established on the basis of spectral evidence. Some of the isolated compounds were examined for their antioxidative and antityrosinase activities. Occurrence of the isoquinolones, biphenyl ethers, and benzoyl benzyltetrahydroisoquinoline ether alkaloids in the same plant indicated the definite possibility of these metabolites as biotransformation intermediates of bisbenzyltetrahydroisoquinoline alkaloids. This can be useful to solve the catabolic process of bisbenzyltetrahydroisoquinoline alkaloids.
Asunto(s)
Alcaloides/química , Aristolochia/química , Alcaloides/aislamiento & purificación , Alcaloides/metabolismo , Aristolochia/metabolismo , Compuestos de Bifenilo/química , Dicroismo Circular , Éteres/química , Glucósidos/química , Lactamas/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Extractos Vegetales/química , Raíces de Plantas/química , Tallos de la Planta/química , Quinolinas/químicaRESUMEN
Plant small heat-stress proteins (sHSPs) have been shown to be expressed not only after exposure to elevated temperatures, but also at particular developmental stages such as embryogenesis, microsporogenesis, and fruit maturation. This paper presents new data on the occurrence of sHSPs in vegetative tissues, their tissue-specific distribution, and cellular localization. We have found sHSPs in 1-year-old twigs of Acer platanoides L. and Sambucus nigra L. and in the liana Aristolochia macrophylla Lamk. exclusively in the winter months. In tendrils of Aristolochia, sHSPs were localized in vascular cambium cells. After budding, in spring, these proteins were no longer present. Furthermore, accumulation of sHSPs was demonstrated in tubers and bulbs of Allium cepa L., Amaryllis ( Hippeastrum hybridum hort.), Crocus albiflorus L., Hyacinthus orientalis L., Narcissus pseudonarcissus L., Tulipa gesneriana L., and Solanum tuberosum L. (potato). In potato tubers and bulb scales of Narcissus the stress proteins were localized in the central vacuoles of storage parenchyma cells. In order to obtain more information on a possible functional correlation between storage proteins and sHSPs, the accumulation of both types of protein in tobacco seeds during seed ripening and germination was monitored. The expression of sHSPs and globulins started simultaneously at about the 17th day after anthesis. During seed germination the sHSPs disappeared in parallel with the storage proteins. Furthermore, in embryos of transgenic tobacco plants, which do not contain any protein bodies or storage proteins, no sHSPs were found. Thus, the occurrence of sHSPs in perennial plant storage organs seems to be associated with the presence of storage proteins.