RESUMEN
As a crucial component of pattern-recognition receptors (PRRs) that recognizing pathogen-associated molecular patterns (PAMPs) and defending against invading pathogens, the Toll-like receptors (TLRs) have been paid extensive attention. While the identification and functional roles of TLRs in innate immunity have been reported in a plenty of organisms, the systematic knowledge of TLRs is still lacking in the red swamp crayfish (Procambarus clarkia). In current study, a total of 7 tlr genes were identified in P. clarkia based on the published transcriptome and genome data. The PcTLRs length varied from 939 to 1517aa and contain typical domains of TLR protein, including transmembrane region, varied LRR and TIR domains. 7 Pctlr genes were distributed in 5 chromosomes and 2 scaffolds. The expression pattern of different Pctlr genes in different tissues (hepatopancreas, gill and muscle) and in response to black may disease (BMD) showed significant difference. In addition, 5 proteins that might interact with PcTLR-2 were predicted, among them the expression pattern of dorsal and relish was consistent with Pctlr-2 in three tissues, while the other genes were not. The PcTLR-2-Dorsal/Relish pathway might play crucial roles in response to BMD infection. The results provided a theoretical foundation for further studies on the molecular mechanisms of TLRs in BMD infection in the red swamp crayfish and provided reference for the research of other crustacean species.
Asunto(s)
Astacoidea , Clarkia , Animales , Astacoidea/genética , Astacoidea/metabolismo , Clarkia/metabolismo , Receptores Toll-Like , Receptores de Reconocimiento de Patrones/genética , Inmunidad Innata/genética , Moléculas de Patrón Molecular Asociado a PatógenosRESUMEN
It has been suggested that gene duplication and polyploidization create opportunities for the evolution of novel characters. However, the connections between the effects of polyploidization and morphological novelties have rarely been examined. In this study, we investigated whether petal pigmentation patterning in an allotetraploid Clarkia gracilis has evolved as a result of polyploidization. Clarkia gracilis is thought to be derived through a recent polyploidization event with two diploid species, C. amoena huntiana and an extinct species that is closely related to C. lassenensis. We reconstructed phylogenetic relationships of the R2R3-MYBs (the regulators of petal pigmentation) from two subspecies of C. gracilis and the two purported progenitors, C. a. huntiana and C. lassenensis. The gene tree reveals that these R2R3-MYB genes have arisen through duplications that occurred before the divergence of the two progenitor species, that is, before polyploidization. After polyploidization and subsequent gene loss, only one of the two orthologous copies inherited from the progenitors was retained in the polyploid, turning it to diploid inheritance. We examined evolutionary changes in these R2R3-MYBs and in their expression, which reveals that the changes affecting patterning (including expression domain contraction, loss-of-function mutation, cis-regulatory mutation) occurred after polyploidization within the C. gracilis lineages. Our results thus suggest that polyploidization itself is not necessary in producing novel petal color patterns. By contrast, duplications of R2R3-MYB genes in the common ancestor of the two progenitors have apparently facilitated diversification of petal pigmentation patterns.
Asunto(s)
Clarkia , Onagraceae , Antocianinas/genética , Clarkia/anatomía & histología , Clarkia/genética , Clarkia/metabolismo , Flores/metabolismo , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas , Onagraceae/metabolismo , Filogenia , Pigmentación/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Salicylic acid methyltransferases (SAMTs) synthesize methyl salicylate (MeSA) using salicylate as the substrate. MeSA synthesized in plants may function as an airborne signal to activate the expression of defense-related genes and could also be a critical mobile signaling molecule that travels from the site of plant infection to establish systemic immunity in the induction of disease resistance. Here the results of QM/MM free energy simulations for the methyl transfer process in Clarkia breweri SAMT (CbSAMT) are reported to determine the origin of the substrate specificity of SAMTs. The free energy barrier for the methyl transfer from S-adenosyl-L-methionine (AdoMet) to 4-hydroxybenzoate in CbSAMT is found to be about 5 kcal/mol higher than that from AdoMet to salicylate, consistent with the experimental observations. It is suggested that the relatively high efficiency for the methylation of salicylate compared to 4-hydroxybenzoate is due, at least in part, to the reason that a part of the stabilization of the transition state (TS) configuration is already reflected in the reactant complex, presumably, through the binding. The results seem to indicate that the creation of the substrate complex (e.g., through mutagenesis and substrate modifications) with its structure closely resembling TS might be fruitful for improving the catalytic efficiency for some enzymes. The results show that the computer simulations may provide important insights into the origin of the substrate specificity for the SABATH family and could be used to help experimental efforts in generating engineered enzymes with altered substrate specificity.
Asunto(s)
Metiltransferasas/química , Proteínas de Plantas/química , Salicilatos/metabolismo , Sitios de Unión , Catálisis , Clarkia/metabolismo , Simulación por Computador , Metilación , Metiltransferasas/metabolismo , Modelos Moleculares , Parabenos/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Unión Proteica , Ingeniería de Proteínas , S-Adenosilmetionina/metabolismo , Ácido Salicílico/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.
Asunto(s)
Clarkia/enzimología , Enzimas/metabolismo , Petunia/enzimología , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Clarkia/genética , Clarkia/metabolismo , Electroforesis en Gel de Poliacrilamida , Enzimas/genética , Eugenol/análogos & derivados , Eugenol/química , Eugenol/metabolismo , Flores/enzimología , Flores/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Petunia/genética , Petunia/metabolismo , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Homología de Secuencia de AminoácidoRESUMEN
The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.