RESUMEN
Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-l-methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor-treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N- and C-terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.
Asunto(s)
Benzofuranos/metabolismo , Compuestos de Bifenilo/metabolismo , Proteína O-Metiltransferasa/metabolismo , Secuencia de Aminoácidos , Malus , Datos de Secuencia Molecular , Proteína O-Metiltransferasa/química , Proteína O-Metiltransferasa/genética , Pyrus , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Malus/enzimología , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Sorbus/enzimología , Secuencia de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/química , Células Cultivadas , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , ADN Complementario/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Cinética , Malus/genética , Malus/microbiología , Microsomas/metabolismo , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/química , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sesquiterpenos/química , Sorbus/genética , Fracciones Subcelulares/enzimología , Especificidad por Sustrato , Nicotiana/metabolismo , FitoalexinasRESUMEN
Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²âº and K⺠at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.
Asunto(s)
Benzoatos/metabolismo , Cinamatos/metabolismo , Coenzima A Ligasas/metabolismo , Hypericum/citología , Hypericum/enzimología , Sesquiterpenos/metabolismo , Xantonas/metabolismo , Secuencia de Aminoácidos , Benzoatos/química , Cationes , Células Cultivadas , Cromatografía Líquida de Alta Presión , Clonación Molecular , Coenzima A Ligasas/química , Coenzima A Ligasas/genética , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Hypericum/genética , Cinética , Datos de Secuencia Molecular , Fenilanina Amoníaco-Liasa/genética , Fenilanina Amoníaco-Liasa/metabolismo , Filogenia , Transporte de Proteínas , Alineación de Secuencia , Sesquiterpenos/química , Espectrometría de Masa por Ionización de Electrospray , Fracciones Subcelulares/enzimología , Especificidad por Sustrato , Xantonas/química , FitoalexinasRESUMEN
Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.
Asunto(s)
Genes de Plantas , Malus/genética , Complejos Multienzimáticos/genética , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Inmunohistoquímica , Malus/enzimología , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/genéticaRESUMEN
Sorbus aucuparia cell cultures accumulate biphenyl and dibenzofuran phytoalexins in response to elicitor treatment. These polyketide derivatives arise from the starter substrate benzoyl-CoA, the biosynthesis of which is largely unresolved. Two CoA ligases involved are cinnamate:CoA ligase and benzoate:CoA ligase, which were assumed to be related in S. aucuparia to the ubiquitous 4-coumarate:CoA ligase (4CL). cDNAs encoding three distinct 4CLs from elicitor-treated S. aucuparia cell cultures were isolated using RT-PCR and RACE techniques and functionally expressed in Escherichia coli as His(6)-tagged proteins (Sa4CL2 and Sa4CL3) or GST-fusion protein (Sa4CL1). All three isoenzymes preferred 4-coumaric acid over cinnamic acid in spectrophotometric assays and failed to utilize benzoic acid in radioisotopic assays. After elicitor treatment of S. aucuparia cell cultures, the transcript levels of all three Sa4CLs increased but were significantly lower than the maximum expression rates of the phenylalanine ammonia-lyase (PAL) and biphenyl synthase 1 (BIS1) genes. The substrate specificities and the expression profiles indicate that the three 4CL isoenzymes are not involved in benzoyl-CoA biosynthesis in S. aucuparia cell cultures. Sa4CL3 and PAL transcripts also accumulated in response to light treatment. Phylogenetically, Sa4CL1 and Sa4CL2 belong to the class I cluster and Sa4CL3 groups in the class II cluster. Sa4CL3 contains a 49-amino acid N-terminal extension, which includes a chloroplast sorting signal.
Asunto(s)
Coenzima A Ligasas/química , Sorbus/enzimología , Secuencia de Aminoácidos , Ácido Benzoico/química , Ácidos Cafeicos/química , Células Cultivadas , Quitosano/química , Cinamatos/química , Clonación Molecular , Coenzima A Ligasas/genética , Ácidos Cumáricos/química , Perfilación de la Expresión Génica , Isoenzimas/química , Luz , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Fenilanina Amoníaco-Liasa , Filogenia , Propionatos , Sorbus/química , Sorbus/genética , Especificidad por SustratoRESUMEN
Cell cultures of Sorbus aucuparia respond to the addition of chitosan with the accumulation of the biphenyl phytoalexin aucuparin. The carbon skeleton of this inducible defense compound is formed by biphenyl synthase (BIS) from benzoyl-CoA and three molecules of malonyl-CoA. The formation of benzoyl-CoA proceeds via benzaldehyde as an intermediate. Benzaldehyde dehydrogenase (BD), which converts benzaldehyde into benzoic acid, was detected in cell-free extracts from S. aucuparia cell cultures. BD and BIS were induced by chitosan treatment. The preferred substrate for BD was benzaldehyde (K(m)=49 microM). Cinnamaldehyde and various hydroxybenzaldehydes were relatively poor substrates. BD activity was strictly dependent on the presence of NAD(+) as a cofactor (K(m)=67 microM).