Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells
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We previously showed that eight laccase genes (Lac 1–Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147–155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO4 in the reaction mixtures increased in the range from 1 to 100 μM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.
KeywordsBright yellow-2 (BY-2) Dilignols Heterologous expression Lignin synthesis Multicopper oxidase
We are grateful to Dr. Yuko Ohashi of the National Institute of Agrobiological Sciences for providing a binary vector pBE2113-GUS, Dr. Tsuyoshi Kaneta of Ehime University for technical help in transformation of tobacco BY-2 cells, and Professor Masahiro Inouhe of Ehime University for use of equipment. This work was supported in part by a project grant from Ehime University (Rudimentary Research Support to Y.S.).
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