Cinnamate 4-Hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree
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Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 220.127.116.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.
KeywordsCinnamate 4-Hydroxylase (C4H) Lignin Leucaena leucocephala Phenylpropanoid
The authors thank Council of Scientific and Industrial Research (CSIR, Govt. of India) for funding the project. SK and KP thankfully acknowledge CSIR; and SO thanks Department of Biotechnology (Govt. of India) for giving assistance in the form of Junior and Senior Research Fellowship. We also thank Dr. David Nelson, University of Tennessee for classifying and assigning names to LlC4H proteins.
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