Expression and functional analyses of a putative phenylcoumaran benzylic ether reductase in Arabidopsis thaliana

Abstract

A candidate gene for phenylcoumaran benzylic ether reductase in Arabidopsis thaliana encodes a peptide with predicted functional activity and plays a crucial role in secondary metabolism. Phenylcoumaran benzylic ether reductase (PCBER) is thought to be an enzyme crucial in the biosynthesis of 8-5'-linked neolignans. Genes of the enzyme have been isolated and characterized in several plant species. In this study, we cloned cDNA and the 5'-untranslated region of one PCBER candidate gene (At4g39230, designated AtPCBER1) from Arabidopsis thaliana. At the amino acid level, AtPCBER1 shows high sequence identity (64-71 %) with PCBERs identified from other plant species. Expression analyses of AtPCBER1 by reverse transcriptase-polymerase chain reaction and histochemical analysis of transgenic plants harboring the 5'-untranslated region of AtPCBER1 linked with gus coding sequence indicate that expression is induced by wounding and is expressed in most tissues, including flower, stem, leaf, and root. Catalytic analysis of recombinant AtPCBER1 with neolignan and lignans in the presence of NADPH suggests that the protein can reduce not only the 8-5'-linked neolignan, dehydrodiconiferyl alcohol, but also 8-8' linked lignans, pinoresinol, and lariciresinol, with lower activities. To investigate further, we performed metabolomic analyses of transgenic plants in which the target gene was up- or down-regulated. Our results indicate no significant effects of AtPCBER1 gene regulation on plant growth and development; however, levels of some secondary metabolites, including lignans, flavonoids, and glucosinolates, differ between wild-type and transgenic plants. Taken together, our findings indicate that AtPCBER1 encodes a polypeptide with PCBER activity and has a critical role in the biosynthesis of secondary metabolites in A. thaliana.