RESUMEN
The salicinoids are anti-herbivore phenolic glycosides unique to the Salicaceae (Populus and Salix). They consist of a salicyl alcohol glucoside core, which is usually further acylated with benzoic, cinnamic or phenolic acids. While salicinoid structures are well known, their biosynthesis remains enigmatic. Recently, two enzymes from poplar, salicyl alcohol benzoyl transferase and benzyl alcohol benzoyl transferase, were shown to catalyze the production of salicyl benzoate, a predicted potential intermediate in salicinoid biosynthesis. Here, we used transcriptomics and co-expression analysis with these two genes to identify two UDP-glucose-dependent glycosyltransferases (UGT71L1 and UGT78M1) as candidate enzymes in this pathway. Both recombinant enzymes accepted only salicyl benzoate, salicylaldehyde and 2-hydroxycinnamic acid as glucose acceptors. Knocking out the UGT71L1 gene by CRISPR/Cas9 in poplar hairy root cultures led to the complete loss of salicortin, tremulacin and tremuloidin, and a partial reduction of salicin content. This demonstrated that UGT71L1 is required for synthesis of the major salicinoids, and suggested that an additional route can lead to salicin. CRISPR/Cas9 knockouts for UGT78M1 were not successful, and its in vivo role thus remains to be determined. Although it has a similar substrate preference and predicted structure as UGT71L1, it appears not to contribute to the synthesis of salicortin, tremulacin and tremuloidin, at least in roots. The demonstration of UGT71L1 as an enzyme of salicinoid biosynthesis will open up new avenues for the elucidation of this pathway.
Asunto(s)
Glicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Populus/enzimología , Alcoholes Bencílicos , Glucósidos/biosíntesis , Glicosiltransferasas/química , Glicosiltransferasas/genética , Redes y Vías Metabólicas , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Populus/genética , Populus/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long-distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate-limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4-coumaroyl-shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4-coumaroyl-esters and -amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4-coumaroyl-shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4-coumaroyl-shikimate poorly in vitro, but were able to use alternative substrates, in particular 4-coumaroyl-anthranilate. Thus, caffeoyl-shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non-seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4-coumaroyl-anthranilate, while 4-coumaroyl-shikimate was converted to lower extents. Despite having in vitro activity with 4-coumaroyl-shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss-of-function phenotype, suggesting distinct properties also in vivo.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Evolución Molecular , Lignina/biosíntesis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/metabolismo , Briófitas/metabolismo , Bryopsida/metabolismo , Sistema Enzimático del Citocromo P-450/clasificación , Magnoliopsida/metabolismo , Filogenia , Proteínas de Plantas/clasificación , Populus , Pteris/metabolismo , Selaginellaceae/metabolismo , Ácido ShikímicoRESUMEN
Occurrence of stomata on both leaf surfaces (amphistomaty) promotes higher stomatal conductance and photosynthesis while simultaneously increasing exposure to potential disease agents in black cottonwood (Populus trichocarpa). A genome-wide association study (GWAS) with 2.2M single nucleotide polymorphisms generated through whole-genome sequencing found 280 loci associated with variation in adaxial stomatal traits, implicating genes regulating stomatal development and behavior. Strikingly, numerous loci regulating plant growth and response to biotic and abiotic stresses were also identified. The most significant locus was a poplar homologue of SPEECHLESS (PtSPCH1). Individuals possessing PtSPCH1 alleles associated with greater adaxial stomatal density originated primarily from environments with shorter growing seasons (e.g. northern latitudes, high elevations) or with less precipitation. PtSPCH1 was expressed in developing leaves but not developing stem xylem. In developing leaves, RNA sequencing showed patterns of coordinated expression between PtSPCH1 and other GWAS-identified genes. The breadth of our GWAS results suggests that the evolution of amphistomaty is part of a larger, complex response in plants. Suites of genes underpin this response, retrieved through genetic association to adaxial stomata, and show coordinated expression during development. We propose that the occurrence of amphistomaty in P. trichocarpa involves PtSPCH1 and reflects selection for supporting rapid growth over investment in immunity.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Plantas/metabolismo , Estomas de Plantas/fisiología , Populus/fisiología , Alelos , Clima , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estudio de Asociación del Genoma Completo , Genotipo , Geografía , Fenotipo , Desarrollo de la Planta , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Estomas de Plantas/genética , Polimorfismo de Nucleótido Simple/genética , Populus/genética , Populus/crecimiento & desarrollo , Populus/inmunología , Carácter Cuantitativo Heredable , Especificidad de la EspecieRESUMEN
The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non-seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate-specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes.
RESUMEN
Lignin, one of the most abundant biopolymers on Earth, derives from the plant phenolic metabolism. It appeared upon terrestrialization and is thought critical for plant colonization of land. Early diverging land plants do not form lignin, but already have elements of its biosynthetic machinery. Here we delete in a moss the P450 oxygenase that defines the entry point in angiosperm lignin metabolism, and find that its pre-lignin pathway is essential for development. This pathway does not involve biochemical regulation via shikimate coupling, but instead is coupled with ascorbate catabolism, and controls the synthesis of the moss cuticle, which prevents desiccation and organ fusion. These cuticles share common features with lignin, cutin and suberin, and may represent the extant representative of a common ancestor. Our results demonstrate a critical role for the ancestral phenolic metabolism in moss erect growth and cuticle permeability, consistent with importance in plant adaptation to terrestrial conditions.
Asunto(s)
Bryopsida/metabolismo , Lignina/metabolismo , Fenoles/metabolismo , Ácido Ascórbico/metabolismo , Evolución Biológica , Bryopsida/genética , Sistema Enzimático del Citocromo P-450/genética , Desecación , Embryophyta , Técnicas de Inactivación de Genes , Magnoliopsida/metabolismo , Lípidos de la Membrana , Componentes Aéreos de las Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
KEY MESSAGE: Douglas-fir transcriptomics. Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is economically important with extensive breeding programs and seed trade. However, the molecular genetics of its seed development are largely unknown. We developed a transcriptome resource covering key developmental stages of megagametophytes over time: prefertilization, fertilization, embryogenesis, and early, unfertilized abortion. RNA sequencing reads were assembled de novo into 105,505 predicted high-confidence transcripts derived from 34,521 predicted genes. Expression levels were estimated based on alignment of the original reads to the reference. Megagametophytes express a distinct set of genes compared to those of vegetative tissues. Transcripts related to signaling, protein turnover, and RNA biogenesis have lower expression values in vegetative tissues, whereas cell wall remodeling, solute transport, and seed storage protein transcripts have higher expression values in megagametophytes. Seed storage protein transcripts become very abundant in both pollinated and unpollinated megagametophytes over time, even in aborting ovules. However, the absence of protein storage bodies in unfertilized megagametophytes suggests extensive posttranscriptional mechanisms that either inhibit storage protein translation or their aggregation into protein bodies. This novel transcriptome resource provides a foundation for further important insights into conifer seed development.
Asunto(s)
Pseudotsuga/genética , Semillas/genética , Transcriptoma , Fertilización , Anotación de Secuencia Molecular , Óvulo Vegetal/embriología , Óvulo Vegetal/genética , Óvulo Vegetal/crecimiento & desarrollo , Proteínas de Plantas/genética , Pseudotsuga/embriología , Pseudotsuga/crecimiento & desarrollo , Semillas/embriología , Semillas/crecimiento & desarrollo , Análisis de Secuencia de ARNRESUMEN
Forest trees generally show high levels of local adaptation and efforts focusing on understanding adaptation to climate will be crucial for species survival and management. Here, we address fundamental questions regarding the molecular basis of adaptation in undomesticated forest tree populations to past climatic environments by employing an integrative quantitative genetics and landscape genomics approach. Using this comprehensive approach, we studied the molecular basis of climate adaptation in 433 Populus trichocarpa (black cottonwood) genotypes originating across western North America. Variation in 74 field-assessed traits (growth, ecophysiology, phenology, leaf stomata, wood, and disease resistance) was investigated for signatures of selection (comparing QST-FST) using clustering of individuals by climate of origin (temperature and precipitation). 29,354 SNPs were investigated employing three different outlier detection methods and marker-inferred relatedness was estimated to obtain the narrow-sense estimate of population differentiation in wild populations. In addition, we compared our results with previously assessed selection of candidate SNPs using the 25 topographical units (drainages) across the P. trichocarpa sampling range as population groupings. Narrow-sense QST for 53% of distinct field traits was significantly divergent from expectations of neutrality (indicating adaptive trait variation); 2,855 SNPs showed signals of diversifying selection and of these, 118 SNPs (within 81 genes) were associated with adaptive traits (based on significant QST). Many SNPs were putatively pleiotropic for functionally uncorrelated adaptive traits, such as autumn phenology, height, and disease resistance. Evolutionary quantitative genomics in P. trichocarpa provides an enhanced understanding regarding the molecular basis of climate-driven selection in forest trees and we highlight that important loci underlying adaptive trait variation also show relationship to climate of origin. We consider our approach the most comprehensive, as it uncovers the molecular mechanisms of adaptation using multiple methods and tests. We also provide a detailed outline of the required analyses for studying adaptation to the environment in a population genomics context to better understand the species' potential adaptive capacity to future climatic scenarios.
Asunto(s)
Evolución Biológica , Genómica , Populus/genética , Colombia Británica , Clima , Genoma de Planta , Genotipo , Geografía , Oregon , Polimorfismo de Nucleótido Simple/genética , Análisis de Componente Principal , Carácter Cuantitativo Heredable , Selección GenéticaRESUMEN
Contrasting with the wealth of information available on the multiple roles of jasmonates in plant development and defense, knowledge about the functions and the biosynthesis of hydroxylated oxylipins remains scarce. By expressing the caleosin RESPONSIVE TO DESSICATION20 (RD20) in Saccharomyces cerevisiae, we show that the recombinant protein possesses an unusual peroxygenase activity with restricted specificity toward hydroperoxides of unsaturated fatty acid. Accordingly, Arabidopsis (Arabidopsis thaliana) plants overexpressing RD20 accumulate the product 13-hydroxy-9,11,15-octadecatrienoic acid, a linolenate-derived hydroxide. These plants exhibit elevated levels of reactive oxygen species (ROS) associated with early gibberellin-dependent flowering and abscisic acid hypersensitivity at seed germination. These phenotypes are dependent on the presence of active RD20, since they are abolished in the rd20 null mutant and in lines overexpressing RD20, in which peroxygenase was inactivated by a point mutation of a catalytic histidine residue. RD20 also confers tolerance against stress induced by Paraquat, Rose Bengal, heavy metal, and the synthetic auxins 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid. Under oxidative stress, 13-hydroxy-9,11,15-octadecatrienoic acid still accumulates in RD20-overexpressing lines, but this lipid oxidation is associated with reduced ROS levels, minor cell death, and delayed floral transition. A model is discussed where the interplay between fatty acid hydroxides generated by RD20 and ROS is counteracted by ethylene during development in unstressed environments.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Unión al Calcio/metabolismo , Ácidos Grasos Insaturados/metabolismo , Desarrollo de la Planta , Especies Reactivas de Oxígeno/metabolismo , Ácido Abscísico/metabolismo , Etilenos/metabolismo , Flores/fisiología , Germinación , Giberelinas/metabolismo , Estrés Oxidativo , Latencia en las Plantas , Especificidad por SustratoRESUMEN
The shikimate pathway leads to the biosynthesis of aromatic amino acids essential for protein biosynthesis and the production of a wide array of plant secondary metabolites. Among them, quinate is an astringent feeding deterrent that can be formed in a single step reaction from 3-dehydroquinate catalyzed by quinate dehydrogenase (QDH). 3-Dehydroquinate is also the substrate for shikimate biosynthesis through the sequential actions of dehydroquinate dehydratase (DQD) and shikimate dehydrogenase (SDH) contained in a single protein in plants. The reaction mechanism of QDH resembles that of SDH. The poplar genome encodes five DQD/SDH-like genes (Poptr1 to Poptr5), which have diverged into two distinct groups based on sequence analysis and protein structure prediction. In vitro biochemical assays proved that Poptr1 and -5 are true DQD/SDHs, whereas Poptr2 and -3 instead have QDH activity with only residual DQD/SDH activity. Poplar DQD/SDHs have distinct expression profiles suggesting separate roles in protein and lignin biosynthesis. Also, the QDH genes are differentially expressed. In summary, quinate (secondary metabolism) and shikimate (primary metabolism) metabolic activities are encoded by distinct members of the same gene family, each having different physiological functions.
Asunto(s)
Populus/metabolismo , Ácido Quínico/metabolismo , Ácido Shikímico/metabolismo , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Perfilación de la Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Populus/genética , Homología de Secuencia de AminoácidoRESUMEN
In order to uncover the genetic basis of phenotypic trait variation, we used 448 unrelated wild accessions of black cottonwood (Populus trichocarpa) from much of its range in western North America. Extensive data from large-scale trait phenotyping (with spatial and temporal replications within a common garden) and genotyping (with a 34 K Populus single nucleotide polymorphism (SNP) array) of all accessions were used for gene discovery in a genome-wide association study (GWAS). We performed GWAS with 40 biomass, ecophysiology and phenology traits and 29,355 filtered SNPs representing 3518 genes. The association analyses were carried out using a Unified Mixed Model accounting for population structure effects among accessions. We uncovered 410 significant SNPs using a Bonferroni-corrected threshold (P<1.7×10(-6)). Markers were found across 19 chromosomes, explained 1-13% of trait variation, and implicated 275 unique genes in trait associations. Phenology had the largest number of associated genes (240 genes), followed by biomass (53 genes) and ecophysiology traits (25 genes). The GWAS results propose numerous loci for further investigation. Many traits had significant associations with multiple genes, underscoring their genetic complexity. Genes were also identified with multiple trait associations within and/or across trait categories. In some cases, traits were genetically correlated while in others they were not.
Asunto(s)
Genética de Población , Polimorfismo de Nucleótido Simple , Populus/genética , Biomasa , Ecología , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , América del Norte , Fenotipo , Populus/fisiología , Carácter Cuantitativo HeredableRESUMEN
BACKGROUND: Nitrogen uptake, reallocation within the plant, and between subcellular compartments involves ammonium, nitrate and peptide transporters. Ammonium transporters are separated into two distinct families (AMT1 and AMT2), each comprised of five members on average in angiosperms. Nitrate transporters also form two discrete families (NRT1 and NRT2), with angiosperms having four NRT2s, on average. NRT1s share an evolutionary history with peptide transporters (PTRs). The NRT1/PTR family in land plants usually has more than 50 members and contains also members with distinct activities, such as glucosinolate and abscisic acid transport. RESULTS: Phylogenetic reconstructions of each family across 20 land plant species with available genome sequences were supplemented with subcellular localization and transmembrane topology predictions. This revealed that both AMT families diverged prior to the separation of bryophytes and vascular plants forming two distinct clans, designated as supergroups, each. Ten supergroups were identified for the NRT1/PTR family. It is apparent that nitrate and peptide transport within the NRT1/PTR family is polyphyletic, that is, nitrate and/or peptide transport likely evolved multiple times within land plants. The NRT2 family separated into two distinct clans early in vascular plant evolution. Subsequent duplications occurring prior to the eudicot/monocot separation led to the existence of two AMT1, six AMT2, 31 NRT1/PTR, and two NRT2 clans, designated as groups. CONCLUSION: Phylogenetic separation of groups suggests functional divergence within the angiosperms for each family. Distinct groups within the NRT1/PTR family appear to separate peptide and nitrate transport activities as well as other activities contained within the family, for example nitrite transport. Conversely, distinct activities, such as abscisic acid and glucosinolate transport, appear to have recently evolved from nitrate transporters.
Asunto(s)
Proteínas de Transporte de Anión/clasificación , Proteínas de Transporte de Catión/clasificación , Embryophyta/clasificación , Evolución Molecular , Proteínas de Transporte de Membrana/clasificación , Proteínas de Plantas/clasificación , Compuestos de Amonio/metabolismo , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Embryophyta/genética , Embryophyta/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Familia de Multigenes , Transportadores de Nitrato , Nitratos/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
In plants, genes may sustain extensive pleiotropic functional properties by individually affecting multiple, distinct traits. We discuss results from three genome-wide association studies of approximately 400 natural poplar (Populus trichocarpa) accessions phenotyped for 60 ecological/biomass, wood quality, and rust fungus resistance traits. Single-nucleotide polymorphisms (SNPs) in the poplar ortholog of the class III homeodomain-leucine zipper transcription factor gene REVOLUTA (PtREV) were significantly associated with three specific traits. Based on SNP associations with fungal resistance, leaf drop, and cellulose content, the PtREV gene contains three potential regulatory sites within noncoding regions at the gene's 3' end, where alternative splicing and messenger RNA processing actively occur. The polymorphisms in this region associated with leaf abscission and cellulose content are suggested to represent more recent variants, whereas the SNP associated with leaf rust resistance may be more ancient, consistent with REV's primary role in auxin signaling and its functional evolution in supporting fundamental processes of vascular plant development.
Asunto(s)
Pleiotropía Genética , Técnicas Genéticas , Proteínas de Plantas/genética , Populus/genética , Desequilibrio de Ligamiento/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Flores/metabolismo , Monoterpenos/metabolismo , Monoterpenos Acíclicos , Proteínas de Arabidopsis/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Flores/genética , Regulación de la Expresión Génica de las Plantas , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Mutación , Plantas Modificadas Genéticamente , Saccharomyces cerevisiae/genética , Nicotiana/genéticaRESUMEN
Metabolic plasticity, which largely relies on the creation of new genes, is an essential feature of plant adaptation and speciation and has led to the evolution of large gene families. A typical example is provided by the diversification of the cytochrome P450 enzymes in plants. We describe here a retroposition, neofunctionalization, and duplication sequence that, via selective and local amino acid replacement, led to the evolution of a novel phenolic pathway in Brassicaceae. This pathway involves a cascade of six successive hydroxylations by two partially redundant cytochromes P450, leading to the formation of N1,N5-di(hydroxyferuloyl)-N10-sinapoylspermidine, a major pollen constituent and so-far-overlooked player in phenylpropanoid metabolism. This example shows how positive Darwinian selection can favor structured clusters of nonsynonymous substitutions that are needed for the transition of enzymes to new functions.
Asunto(s)
Brassicaceae/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Evolución Molecular , Polen/crecimiento & desarrollo , Espermidina/análogos & derivados , Arabidopsis/genética , Arabidopsis/metabolismo , Secuencia de Bases , Brassica napus/genética , Brassica napus/crecimiento & desarrollo , Brassica napus/metabolismo , Brassicaceae/genética , Brassicaceae/crecimiento & desarrollo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Duplicación de Gen , Hidroxilación , Redes y Vías Metabólicas , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/metabolismo , Interferencia de ARN , Retroelementos , Selección Genética , Espermidina/metabolismoRESUMEN
An approach based on an in silico analysis predicted that CYP77A4, a cytochrome P450 that so far has no identified function, might be a fatty acid-metabolizing enzyme. CYP77A4 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for cytochrome P450 expression. Lauric acid (C(12:0)) was converted into a mixture of hydroxylauric acids when incubated with microsomes from yeast expressing CYP77A4. A variety of physiological C(18) fatty acids were tested as potential substrates. Oleic acid (cis-Delta(9)C(18:1)) was converted into a mixture of omega-4- to omega-7-hydroxyoleic acids (75%) and 9,10-epoxystearic acid (25%). Linoleic acid (cis,cis-Delta(9),Delta(12)C(18:2)) was exclusively converted into 12,13-epoxyoctadeca-9-enoic acid, which was then converted into diepoxide after epoxidation of the Delta(9) unsaturation. Chiral analysis showed that 9,10-epoxystearic acid was a mixture of 9S/10R and 9R/10S in the ratio 33 : 77, whereas 12,13-epoxyoctadeca-9-enoic acid presented a strong enantiomeric excess in favor of 12S/13R, which represented 90% of the epoxide. Neither stearic acid (C(18:0)) nor linolelaidic acid (trans,trans-Delta(9),Delta(12)C(18:2)) was metabolized, showing that CYP77A4 requires a double bond, in the cis configuration, to metabolize C(18) fatty acids. CYP77A4 was also able to catalyze the in vitro formation of the three mono-epoxides of alpha-linolenic acid (cis,cis,cis-Delta(9),Delta(12),Delta(15)C(18:3)), previously described as antifungal compounds. Epoxides generated by CYP77A4 are further metabolized to the corresponding diols by epoxide hydrolases located in microsomal and cytosolic subcellular fractions from Arabidopsis thaliana. The concerted action of CYP77A4 with epoxide hydrolases and hydroxylases allows the production of compounds involved in plant-pathogen interactions, suggesting a possible role for CYP77A4 in plant defense.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Biocatálisis , Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Epoxi/metabolismo , Ácidos Grasos/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografía Líquida de Alta Presión , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Citosol/metabolismo , Ácidos Grasos/química , Expresión Génica , Hidrólisis , Microsomas/metabolismo , Estructura MolecularRESUMEN
BACKGROUND: Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s) is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism. RESULTS: We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here. CONCLUSION: The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/genética , Sistema Enzimático del Citocromo P-450/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes/genética , Arabidopsis/efectos de los fármacos , Ciclopentanos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Vectores Genéticos , Internet , Mutación/genética , Especificidad de Órganos/efectos de los fármacos , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/biosíntesis , Reguladores del Crecimiento de las Plantas/farmacología , Plastidios/enzimología , Plastidios/genética , Reproducibilidad de los Resultados , Triterpenos/metabolismoRESUMEN
BACKGROUND: Plants are exposed to attack from a large variety of herbivores. Feeding insects can induce substantial changes of the host plant transcriptome. Arabidopsis thaliana has been established as a relevant system for the discovery of genes associated with response to herbivory, including genes for specialized (i.e. secondary) metabolism as well as genes involved in plant-insect defence signalling. RESULTS: Using a 70-mer oligonucleotide microarray covering 26,090 gene-specific elements, we monitored changes of the Arabidopsis leaf transcriptome in response to feeding by diamond back moth (DBM; Plutella xylostella) larvae. Analysis of samples from a time course of one hour to 24 hours following onset of DBM feeding revealed almost three thousand (2,881) array elements (including 2,671 genes with AGI annotations) that were differentially expressed (>2-fold; p[t-test] < 0.05) of which 1,686 also changed more than twofold in expression between at least two time points of the time course with p(ANOVA) < 0.05. While the majority of these transcripts were up-regulated within 8 h upon onset of insect feeding relative to untreated controls, cluster analysis identified several distinct temporal patterns of transcriptome changes. Many of the DBM-induced genes fall into ontology groups annotated as stress response, secondary metabolism and signalling. Among DBM-induced genes associated with plant signal molecules or phytohormones, genes associated with octadecanoid signalling were clearly overrepresented. We identified a substantial number of differentially expressed genes associated with signal transduction in response to DBM feeding, and we compared there expression profiles with those of previously reported transcriptome responses induced by other insect herbivores, specifically Pieris rapae, Frankliniella occidentalis, Bemisia tabaci,Myzus persicae, and Brevicoryne brassicae. CONCLUSION: Arabidopsis responds to feeding DBM larvae with a drastic reprogramming of the transcriptome, which has considerable overlap with the response induced by other insect herbivores. Based on a meta-analysis of microarray data we identified groups of transcription factors that are either affected by multiple forms of biotic or abiotic stress including DBM feeding or, alternatively, were responsive to DBM herbivory but not to most other forms of stress.
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Mariposas Nocturnas , Animales , Arabidopsis/fisiología , Perfilación de la Expresión Génica , Larva , Análisis de Secuencia por Matrices de Oligonucleótidos , Reguladores del Crecimiento de las Plantas/genética , Hojas de la Planta/genética , Transducción de SeñalRESUMEN
4-coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. Transgenic Arabidopsis plants containing the At4CL1 or At4CL2 promoter fused to the beta-glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation of At4CL and other phenylpropanoid-specific genes, we generated ethyl methyl sulfate mutagenized populations of At4CL1::GUS and At4CL2::GUS transgenic lines and screened approximately 16,000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs enhanced the GUS-silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in the At4CL1::GUS and At4CL2::GUS silenced lines. While silencing in the seedlings of the At4CL1::GUS lines was root specific in seedlings, it affected all organs in the At4CL2::GUS lines. Also, At4CL1::GUS transgene silencing was confined to the transgene but At4CL2::GUS silencing extended to the endogenous At4CL2 gene. Organ-specific silencing of the At4CL1::GUS transgene cannot be explained by current models in the literature.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Epigénesis Genética/fisiología , Silenciador del Gen/fisiología , Plantas Modificadas Genéticamente/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiologíaRESUMEN
Lignin is an important biopolymer that is deposited in secondary cell walls of plant cells (e.g., tracheary elements) and in response to stresses such as wounding. Biosynthesis of lignin monomers occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters, subsequently reduced to the corresponding monolignols (hydroxycinnamoyl alcohols). 4CL is encoded by a family of four genes in Arabidopsis thaliana (At4CL1-At4CL4), which are developmentally regulated and co-expressed with other phenylpropanoid genes. We investigated in detail the wound-induced expression of At4CL1-At4CL4, and found that At4CL1 and At4CL2 mRNA accumulation follows biphasic kinetics over a period of 72 h, while At4CL4 expression is rapidly activated for a period of at least 12 h before declining. In order to localize cis-regulatory elements involved in the developmental and wound-induced regulation of the At4CL gene family members, At4CL promoter-beta-glucuronidase (GUS) reporter gene fusions were constructed and transferred into Arabidopsis plants. Analysis of these plants revealed that the promoter fragments direct discrete and distinct patterns of expression, some of which did not recapitulate expected patterns of wound-induced expression. The locations of regulatory elements associated with the At4CL2 gene were investigated in detail using a series of transgenic Arabidopsis plants containing promoter fragments and parts of the transcribed region of the gene fused to GUS. Positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene were located both in the promoter and transcribed regions of the At4CL2 gene.
Asunto(s)
Arabidopsis/metabolismo , Coenzima A Ligasas/metabolismo , Arabidopsis/genética , Coenzima A Ligasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Intrones , Familia de Multigenes , Regiones Promotoras GenéticasRESUMEN
Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.