Jørgen Holst Christensen

Genome-Wide Characterization of the Lignification Toolbox in Arabidopsis

Lignin, one of the most abundant terrestrial biopolymers, is indispensable for plant structure and defense. With the availability of the full genome sequence, large collections of insertion mutants, and functional genomics tools, Arabidopsis constitutes an excellent model system to profoundly unravel the monolignol biosynthetic pathway. In a genome-wide bioinformatics survey of the Arabidopsis genome, 34 candidate genes were annotated that encode genes homologous to the 10 presently known enzymes of the monolignol biosynthesis pathway, nine of which have not been described before. By combining evolutionary analysis of these 10 gene families with in silico promoter analysis and expression data (from a reverse transcription-polymerase chain reaction analysis on an extensive tissue panel, mining of expressed sequence tags from publicly available resources, and assembling expression data from literature), 12 genes could be pinpointed as the most likely candidates for a role in vascular lignification. Furthermore, a possible novel link was detected between the presence of the AC regulatory promoter element and the biosynthesis of G lignin during vascular development. Together, these data describe the full complement of monolignol biosynthesis genes in Arabidopsis, provide a unified nomenclature, and serve as a basis for further functional studies.

A Systems Biology View of Responses to Lignin Biosynthesis Perturbations in Arabidopsis

The combination of metabolomics and transcriptomics on Arabidopsis thaliana lines mutated in 10 steps of the lignin pathway provides insight into monolignol biosynthesis and the metabolic network in which it is embedded. In addition, this work reveals novel pathways and genes associated with lignin biosynthesis. Lignin engineering is an attractive strategy to improve lignocellulosic biomass quality for processing to biofuels and other bio-based products. However, lignin engineering also results in profound metabolic consequences in the plant. We used a systems biology approach to study the plant’s response to lignin perturbations. To this end, inflorescence stems of 20 Arabidopsis thaliana mutants, each mutated in a single gene of the lignin biosynthetic pathway (phenylalanine ammonia-lyase1 [PAL1], PAL2, cinnamate 4-hydroxylase [C4H], 4-coumarate:CoA ligase1 [4CL1], 4CL2, caffeoyl-CoA O-methyltransferase1 [CCoAOMT1], cinnamoyl-CoA reductase1 [CCR1], ferulate 5-hydroxylase [F5H1], caffeic acid O-methyltransferase [COMT], and cinnamyl alcohol dehydrogenase6 [CAD6], two mutant alleles each), were analyzed by transcriptomics and metabolomics. A total of 566 compounds were detected, of which 187 could be tentatively identified based on mass spectrometry fragmentation and many were new for Arabidopsis. Up to 675 genes were differentially expressed in mutants that did not have any obvious visible phenotypes. Comparing the responses of all mutants indicated that c4h, 4cl1, ccoaomt1, and ccr1, mutants that produced less lignin, upregulated the shikimate, methyl-donor, and phenylpropanoid pathways (i.e., the pathways supplying the monolignols). By contrast, f5h1 and comt, mutants that provoked lignin compositional shifts, downregulated the very same pathways. Reductions in the flux to lignin were associated with the accumulation of various classes of 4-O- and 9-O-hexosylated phenylpropanoids. By combining metabolomic and transcriptomic data in a correlation network, system-wide consequences of the perturbations were revealed and genes with a putative role in phenolic metabolism were identified. Together, our data provide insight into lignin biosynthesis and the metabolic network it is embedded in and provide a systems view of the plant’s response to pathway perturbations.

Engineering traditional monolignols out of lignin by concomitant up-regulation of F5H1 and down-regulation of COMT in Arabidopsis

Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92% benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from 5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in future cell-wall engineering strategies.

Biotechnology in trees: towards improved paper pulping by lignin engineering

Lignin is a heterogenous phenolic polymer that plays crucial roles in the development and physiology of vascular plants. However, it needs to be removed from cellulose by toxic and energy-requiring processes for the production of high-quality paper. Therefore, a major biotechnological challenge is to obtain transgenic trees with modified lignin to improve the quality of wood for paper making. Here, we review the results obtained by alterating the expression of genes of the monolignol biosynthesis pathway in trees and the effect of these modifications on the lignin polymer and on pulping. The data reported show that lignin engineering is a promising strategy to improve wood quality for the pulp and paper industry.

Control of Lignin Biosynthesis

The ever increasing worldwide use of forest tree products, which coincides with the diminishing of natural forests, necessitates programs for efficient tree farming. For this purpose, there is a demand for accelerated tree improvement strategies that aim at developing trees as wood-producing crops with both improved trunk performance and specific exploitation characteristics. Objectives of primary importance to the forestry industry are the genetic control of traits such as growth, adaptation to environmental stress, disease resistance, wood uniformity, specific gravity and fiber quality. With classical breeding these demands will not be fulfilled within a reasonable time span because of the long generation time of trees. To meet these needs, forest trees are anticipated to become major targets for genetic engineering and molecular breeding in the coming years. Biotechnology now provides the necessary tools to solve many of the problems faced by conventional tree breeding programs, for example by the establishment of genetic maps of forest trees species such as poplar (Bradshaw et al., 1994) and the generation and potential use of DNA marker-assisted selection in breeding programs (e.g., Cervera et al., 1996). Furthermore, plant genetic transformation has now become a common technique for the introduction of novel traits into a wide range of tree species both for basic research and for applied purposes.

Applications of molecular genetics for biosynthesis of novel lignins

Abstract For the production of high-quality paper, lignin needs to be removed from cellulose. This process is energy requiring, expensive and toxic. Molecular biology offers tools to generate trees with a modified lignin composition to facilitate the extractability of lignin. We have cloned several genes encoding enzymes in the methylation of the lignin monomers [bi-specific caffeic acid/5-hydroxyferulic acid O -methyltransferase (COMT) and caffeoyl-CoA O -methyltransferase], and encoding enzymes specific for the lignin biosynthesis pathway [cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase (CAD)]. The antisense strategy is used to study the effect of a down-regulation of these enzymes on the lignin content and the lignin composition of poplar wood. Our results demonstrate that the monomeric composition of lignin was dramatically affected by down-regulation of COMT. Transgenic poplars with a reduced CAD activity have better pulping properties, due to a higher extractability of the lignin.

Xylem Peroxidases: Purification and Altered Expression

ABSTRACT The peroxidase-dependent oxidation of the lignin monomer analogue syringaldazine (SYR) shows an appealing co-localization with lignification in most species analyzed. We have isolated two SYR-oxidizing peroxidases from the xylem of poplar (P. trichocarpa ‘Trichobel’). We have shown that these were the only isoenzymes able to catalyze this reaction in the xylem, and that the corresponding activity correlates with actively lignifying cells within the poplar xylem. Furthermore, we have isolated a cDNA that codes for one of these enzymes (PXP 3-4) and demonstrated that the mRNA is expressed in the bark and the xylem of the stem and in the xylem of the roots. The cDNA encodes a peroxidase that is expressed as a preprotein with signalpeptides at both termini. The peroxidase cDNA PXP 3-4 was expressed in P. trenula × P. alba in sense orientation, using constructs encoding either the native enzyme or a version without the C-terminal propeptide. Also plants expressing this cDNA in antisense orientation were generated. Overexpressing lines with more than 800-fold higher peroxidase activity were identified. In the bark of these plants, half of the extracted proteins corresponded to PXP 3-4, which is more than 0.5 mg peroxidase per g of bark. All overexpressing lines were phenotypically normal. No alteration was observed in lignin amount, condensation or monolignol composition and the metabolic profiles and the redox state of these plants were unaltered.