RESUMO
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.
Assuntos
Coenzima A Ligases/química , Sorbus/enzimologia , Sequência de Aminoácidos , Ácido Benzoico/química , Ácidos Cafeicos/química , Células Cultivadas , Quitosana/química , Cinamatos/química , Clonagem Molecular , Coenzima A Ligases/genética , Ácidos Cumáricos/química , Perfilação da Expressão Gênica , Isoenzimas/química , Luz , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Fenilalanina Amônia-Liase , Filogenia , Propionatos , Sorbus/química , Sorbus/genética , Especificidade por SubstratoRESUMO
The Rosaceous subtribe Pyrinae (formerly subfamily Maloideae) is well-known for its economically important fruit trees, such as apple and pear, and also includes Sorbus aucuparia. Elicitor-treated S. aucuparia cell cultures are used to study the biosynthesis of the Pyrinae-specific phytoalexins, biphenyls and dibenzofurans. Three biphenyls (aucuparin, noraucuparin, 2'-hydroxyaucuparin) and a dibenzofuran (eriobofuran) were isolated and structure elucidated using GC-MS and NMR. A second dibenzofuran of low abundance was tentatively assigned as noreriobofuran. Treatment of S. aucuparia cell cultures with yeast extract induced the formation of aucuparin as the major phytoalexin. In contrast, addition of preparations from the fire blight bacterium, Erwinia amylovora, and the scab-causing fungus, Venturia inaequalis, resulted in accumulation of eriobofuran as the major inducible constituent. Methyl jasmonate was a poor elicitor. The observations are suggestive of a biogenic relationship between biphenyls and dibenzofurans. Elicitor-treated S. aucuparia cell cultures provide an interesting in vitro system for studying biphenyl and dibenzofuran metabolism in the economically valuable Pyrinae.
