Transcriptional regulation of lignin biosynthesis by tobacco LIM protein in transgenic woody plant

Lignin is a complex phenolic plant polymer that is essential for mechanical support, defense, and water transport in higher plants. The AC-rich motif, Pal-box is an important cis-acting element for gene expression involved in phenylpropanoid biosynthesis. A cDNA clone (Ntlim1) encoding a Pal-box binding protein was isolated by Southwestern screening. The deduced amino acid sequence is highly similar to the members of the LIM protein family that contain zinc finger motif. Moreover, the Ntlim1 had a specific DNA binding ability and transiently activated the transcription of beta-glu-curonidase reporter gene driven by the Pal-box sequence in tobacco protoplasts. The results of transient expression assays with tobacco cultured cells showed that fusion proteins between GFP and Ntlim1 can enter nuclei. The transgenic tobacco with antisense Ntlim1 showed low level of transcripts from some key phenylpropanoid pathway genes such as, phenylalanine ammonia-lyase (PAL), hydroxycinnamate CoA ligase (4CL) and cinnamyl alcohol dehydrogenase (CAD). A 27% reduction of lignin content was observed in the transgenic tobacco with antisense Ntlim1. We succeeded the production of transgenic woody plants (Eucalyptus camaldulensis) with low lignin content by introduction of the antisense Ntlim1.