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

Abstract

Both cDNA including 5 0 UTR and 3 0 UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium 9 Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5 0 UTR and 283 bases of 3 0 UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7-32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in upregulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.

Transcriptome Analysis of an Anthracnose-Resistant Tea Plant Cultivar Reveals Genes Associated with Resistance to Colletotrichum camelliae

“…Down-regulating CCR affects the composition of soluble phenolics by increasing various ferulate derivatives in Arabidopsis , poplar, and tobacco [ 20 – 23 ]. A similar pattern was observed in CAD-downregulated tobacco [ 24 ]. Reducing lignin content may trigger signaling pathways that activate stress genes [ 25 ].…”

Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes

“…Numerous studies have reported that CAD is a key enzyme in the biosynthesis of lignin and plays an essential role in plant development associated with plant biomass ( Mitchell et al, 1994 ; Feuillet et al, 1995 ; Halpin et al, 1998 ; Bagniewska-Zadworna et al, 2014 ; Pan et al, 2014 ; Choi et al, 2016 ). Downregulation of CAD expression or its knockout causes a reduction of lignin, which leads to severe plant dwarfism and a decreased biomass ( Sirisha et al, 2012 ; Anderson et al, 2015 ; Ozparpucu et al, 2017 ; Ponniah et al, 2017 ) as well as decreased plant resistance to pathogens ( Bagniewska-Zadworna et al, 2014 ; Preisner et al, 2014 ; Rong et al, 2016 ). However, whether an increase in total lignin via CAD overexpression can affect the biosynthesis of terpenoids, such as artemisinin biosynthesis in A. annua , remains uninvestigated.…”

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

“…Previous characterization of CAD-deficient plants revealed a reddishorange coloration in the lignifying cells (Ralph et al, 1997;Sibout et al, 2005;Sirisha et al, 2012). For wild-type and fah1 plants, disruption of both CADC and CADD were required to observe this phenotype; however, lignified cells in cadc C4H-F5H are pigmented, and this coloration is even more intense in cadd C4H-F5H.…”