Downregulation of Cinnamoyl-Coenzyme A Reductase in Poplar: Multiple-Level Phenotyping Reveals Effects on Cell Wall Polymer Metabolism and Structure

Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Gent, Belgium.
The Plant Cell (Impact Factor: 9.34). 12/2007; 19(11):3669-91. DOI: 10.1105/tpc.107.054148
Source: PubMed


Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

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Available from: Lapierre Catherine, Sep 30, 2015
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    • "Remarkably, the CCR gene was first cloned in Eucalyptus gunnii (EguCCR) and its identity was confirmed unambiguously by the enzymatic activity of the corresponding recombinant protein (Lacombe et al., 1997). EguCCR cDNA was then used as a probe to clone its orthologs in tobacco (Nicotiana tabacum) (Piquemal et al., 1998), Arabidopsis thaliana (Lauvergeat et al., 2001) and Populus (Lepl e et al., 2007). In line with its key role in controlling lignin content and composition, EguCCR was later shown to co-localize with a quantitative trait locus (QTL) for S : G lignin ratio (Gion et al., 2011). "
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    ABSTRACT: Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification. We performed genome-wide identification of putative members of the lignin gene families, followed by comparative phylogenetic studies focusing on bona fide clades inferred from genes functionally characterized in other species. RNA-seq and microfluid real-time quantitative PCR (RT-qPCR) expression data were used to investigate the developmental and environmental responsive expression patterns of the genes. The phylogenetic analysis revealed that 38 E. grandis genes are located in bona fide lignification clades. Four multigene families (shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeate/5-hydroxyferulate O-methyltransferase (COMT) and phenylalanine ammonia-lyase (PAL)) are expanded by tandem gene duplication compared with other plant species. Seventeen of the 38 genes exhibited strong, preferential expression in highly lignified tissues, probably representing the E. grandis core lignification toolbox. The identification of major genes involved in lignin biosynthesis in E. grandis, the most widely planted hardwood crop world-wide, provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities. © 2015 The Authors New Phytologist © 2015 New Phytologist Trust.
    New Phytologist 02/2015; 206(4). DOI:10.1111/nph.13313 · 7.67 Impact Factor
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    • "innamoyl - CoA reductase catalyses the conversion of cinna - moyl - CoA esters to their corresponding cinnamaldehydes in monolignol biosynthesis ( Figure 2 ) . In the transgenic hybrid poplar ( P . tremula 9 alba ) when CCR was down - regulated by antisense , lignin content was reduced to 50% , and this transgenic had improved pulping efficiency ( Lepl e et al . , 2007 ) ."
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    ABSTRACT: Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin-carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall-degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat-stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.
    Plant Biotechnology Journal 10/2014; 12(9). DOI:10.1111/pbi.12273 · 5.75 Impact Factor
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    • "Down-regulation of the CCR gene in annual model plants significantly reduces lignin content (Goujon et al., 2003a; Jones et al., 2001; Piquemal et al., 1998). Similarly, down-regulation of CCR in woody plant poplar (Populus tremula 9 Populus alba) also results in up to 50% lignin reduction in greenhouse-grown plants; and the lignin reduction is associated with a patchy, red-brown coloration of the outer xylem of the transgenic plants (Leple et al., 2007). To translate the knowledge gained in the laboratory (greenhouse) to conditions closer to industrial exploitation, field trial becomes a necessary step. "
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    ABSTRACT: Increased global interest in a bio-based economy has reinvigorated the research on the cell wall structure and composition in plants. In particular, the study of plant lignification has become a central focus, with respect to its intractability and negative impact on the utilization of the cell wall biomass for producing biofuels and bio-based chemicals. Striking progress has been achieved in the last few years both on our fundamental understanding of lignin biosynthesis, deposition and assembly, and on the interplay of lignin synthesis with the plant growth and development. With the knowledge gleaned from basic studies, researchers are now able to invent and develop elegant biotechnological strategies to sophisticatedly manipulate the quantity and structure of lignin and thus to create economically viable bioenergy feedstocks. These concerted efforts open an avenue for the commercial production of cost-competitive biofuel to meet our energy needs.
    Plant Biotechnology Journal 09/2014; 12(9). DOI:10.1111/pbi.12250 · 5.75 Impact Factor
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