RESUMO
cDNA and genomic clones of cinnamoyl CoA reductase measuring 1011 and 2992 bp were isolated from a leguminous pulpwood tree Leucaena leucocephala, named as LlCCR. The cDNA exhibited 80-85% homology both at the nucleotide and amino acid levels with other known sequences. The genomic sequence contained five exons and four introns. Sense and antisense constructs of LlCCR were introduced in tobacco plants to up and down-regulate this key enzyme of lignification. The primary transformants showed a good correlation between CCR transcript levels and its activity. Most of the CCR down-regulated lines displayed stunted growth and development, wrinkled leaves and delayed senescence. These lines accumulated unusual phenolics like ferulic and sinapic acids in cell wall. Histochemical staining suggested reduction in aldehyde units and increased syringyl over guaiacyl (S/G) ratio of lignin. Anatomical studies showed thin walled, elongated xylem fibres, collapsed vessels with drastic reduction of secondary xylem. The transmission electron microscopic studies revealed modification of ultrastructure and topochemical distribution of wall polysaccharides and lignin in the xylem fibres. CCR down-regulated lines showed increased thickness of secondary wall layers and poor lignification of S2 and S3 wall layers. The severely down-regulated line AS17 exhibited 24.7% reduction of Klason lignin with an increase of 15% holocellulose content. Contrarily, the CCR up-regulated lines exhibited robust growth, development and significant increase in lignin content. The altered lignin profiles observed in transgenic tobacco lines support a role for CCR down-regulation in improving wood properties of L. leucocephala exclusively used in the pulp and paper industry of India.
Assuntos
Aldeído Oxirredutases/metabolismo , Fabaceae/enzimologia , Lignina/análise , Fenóis/análise , Proteínas de Plantas/metabolismo , Aldeído Oxirredutases/genética , Celulose/análise , Celulose/metabolismo , DNA Complementar/genética , DNA de Plantas/genética , Regulação para Baixo , Fabaceae/genética , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Microscopia Eletrônica de Transmissão , Fenóis/metabolismo , Fenótipo , Proteínas de Plantas/genética , Caules de Planta/anatomia & histologia , Caules de Planta/ultraestrutura , Plantas Geneticamente Modificadas/genética , Nicotiana/química , Nicotiana/genética , Transformação GenéticaRESUMO
Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.
