Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene induces significant changes in phenotype, soluble phenolic pools and lignin in transgenic tobacco

Abstract

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.

Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco

“…This is due to the increased accumulation of cinnamaldehydes in the lignin monomers. In contrast, down-regulation of L. leucocephala CCR gene resulted in an orange-brown colouration of xylem in transgenic tobacco, which was accounted by the presence of ferulic and sinapic acids (Prashant et al 2011). Moreover, among transgenic lines, the coloration is either homogenous in the stem or limited to defined zones.…”

“…Antisense RNA technology was used earlier to specifically reduce the expression of many plant genes (Van Blockland et al 1993;Watson and Grierson 1993), including genes involved in lignin biosynthetic pathway (Boudet and Grima-Pettenati 1996;Campbell and Sederoff 1996;Prashant et al 2011). …”

“…For instance, down-regulation of cinnamate-4-hydroxylase (Sewalt et al 1997) and 4-coumarate-CoA ligase in tobacco (Kajita et al 1997) or in poplar (Hu et al 1998) resulted in noticeable reduction of lignin content. In tobacco where cinnamoyl CoA reductase was down-regulated, 1.5 to 2-fold decrease in lignin content was recorded in the most severely depressed line, which displayed altered growth and development together with collapsed vessel elements Prashant et al 2011). While these studies result in lignin reduction, the accompanying changes in plant structure certainly reduce their applicability.…”

Isolation of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase gene promoters from Leucaena leucocephala, a leguminous tree species, and characterization of tissue-specific activity in transgenic tobacco

“…We recently reported that down-regulation of L. leucocephala CCR gene under the constitutive promoter CaMV 35S significantly reduces lignin content, and also affects growth and development of the transgenic tobacco plants (Prashant et al 2011). To overcome such risks and challenges associated with down-regulating lignin content in the whole plant, it would be preferable to express lignin biosynthetic pathway genes under vascular tissue-specific promoters.…”

Anatomical Changes During Transition from Juvenile to Adult Wood in Branch and Main Trunk Xylem of Subabul (Leucaena leucocephala[Lam.] de Wit)

“…L. leucocephala is an exotic in India and is cultivated in various parts of the country as a source of forage as well as paper pulp. Although the paper industry mainly uses bamboos, Eucalyptus, Acasia, Populus, Casuarina, Picea, and Pinus species as a source for the pulp production worldwide, Leucaena species are exclusively used in India because of their easy acclimatization and high rate of biomass production (Prashant et al, 2011). In India, about 25% of raw material comes from this genus.…”