Lignin genetic engineering

Although lignins play important roles in plants, they often represent an obstacle to the utilization of plant biomass in different areas: pulp industry, forage digestibility. The recent characterization of different lignification genes has stimulated research programmes aimed at modifying the lignin profiles of plants through genetic engineering (antisense and sense suppression of gene expression). The first transgenic plants with a modification of monomeric composition of lignins and lignin content have been recently obtained. Down regulation of the OMT gene induces dramatic reduction of syringyl units. CAD down regulated plants exhibit a unusual red phenotype associated with the developing xylem and several chemical modifications of their lignins including an increase in cinnamaldehydes in the polymer structure. Interestingly this novel lignin is removed more easily during the pulping process. In both OMT and CAD down regulated plants no changes in phenotypic characteristics such as growth architecture and morphology were observed. More recent experiments have shown that a reduction of CCR activity determines specific changes in the coloration of the xylem area suggesting significant chemical modifications which are currently being studied.These different results show that it is possible to manipulate lignins through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure. Future developments should probe the impact of down regulating the expression of other recently characterized lignification genes such as F5H and CCoAOMT and also of a combination of genes in order to tailor lignins more adapted to specific purposes. In addition to biotechnological applications which should provide important economical benefits for utilization of wood in the pulp industry, genetic engineering of lignins offer very promising perspectives for the understanding of lignin synthesis, structure and properties.     

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.     

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an alternative enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.     

Sequence analysis and functional characterization of the promoter of the PiceaglaucaCinnamylAlcoholDehydrogenase gene in transgenic white spruce plants

The enzyme Cinnamyl Alcohol Dehydrogenase (CAD) catalyses the last step of lignin monomer synthesis, and is considered as a molecular marker of cell wall lignification in different plants species. Here, we report the isolation and analysis of 5′ flanking genomic DNA regions upstream to the CAD gene, from two conifers, i.e. white spruce (Picea glauca (Moench) Voss) and loblolly pine (Pinus taeda L.). Sequence comparisons with available CAD gene promoters from angiosperms highlighted the conservation of cis-elements matching MYB, WRKY and bHLH binding sites. Functional characterization of the P. glauca CAD promoter used P. glauca seedlings stably transformed with a DNA fragment of 1,163 base pairs (PgCAD) fused to the β-glucuronidase (GUS) gene. Histochemical observations of different vegetative organs of the transgenic trees showed that this sequence was sufficient to drive GUS expression in lignifying tissues, and more specifically in differentiating xylem cells. Quantitative RT-PCR experiments also indicated that the native CAD gene was preferentially expressed in differentiating xylem both in stems and roots. In addition, GUS expression driven by the PgCAD promoter was wound-inducible which was consistent with the accumulation of CAD mRNA in response to jasmonate application and mechanical wounding. The spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Eucalyptus gunnii CCR and CAD2 promoters are active in lignifying cells during primary and secondary xylem formation in Arabidopsis thaliana

Cell-specific expression patterns of the Eucalyptus gunnii cinnamoyl coenzymeA reductase (EgCCR) and cinnamyl alcohol dehydrogenase (EgCAD2) promoters were analyzed by promoter-GUS histochemistry in the primary and secondary xylem tissues from floral stems and roots of Arabidopsis thaliana. Expression patterns indicated that the EgCCR and EgCAD2 genes were expressed in a coordinated manner in primary and secondary xylem tissues of the Arabidopsis floral stem and root. Both genes were expressed in all lignifying cells (vessel elements, xylem fibers and paratracheal parenchyma cells) of xylem tissues. The capacity for long-term monolignol production appeared to be related to the cell-specific developmental processes and biological roles of different cell types. Our results suggested that lignification of short-lived vessel elements was achieved by a two-step process involving (i) monolignol production by vessel elements prior to vessel programmed cell death and (ii) subsequent monolignol production by vessel-associated living paratracheal parenchyma cells following vessel element cell death. EgCCR and EgCAD2 gene expression patterns suggested that the process of xylem cell lignification was similar in both primary and secondary xylem tissues in Arabidopsis floral stems and roots.     

Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.

Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.