Wout Boerjan

Lignin Biosynthesis and Structure

Lignin is the generic term for a large group of aromatic polymers resulting from the oxidative combinatorial coupling of 4-hydroxyphenylpropanoids ([Boerjan et al., 2003][1]; [Ralph et al., 2004][2]). These polymers are deposited predominantly in the walls of secondarily thickened cells, making them

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.

Field and pulping performances of transgenic trees with altered lignification

The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar

The growth of perennial plants in the temperate zone alternates with periods of dormancy that are typically initiated during bud development in autumn. In a systems biology approach to unravel the underlying molecular program of apical bud development in poplar (Populus tremula × Populus alba), combined transcript and metabolite profiling were applied to a high-resolution time course from short-day induction to complete dormancy. Metabolite and gene expression dynamics were used to reconstruct the temporal sequence of events during bud development. Importantly, bud development could be dissected into bud formation, acclimation to dehydration and cold, and dormancy. To each of these processes, specific sets of regulatory and marker genes and metabolites are associated and provide a reference frame for future functional studies. Light, ethylene, and abscisic acid signal transduction pathways consecutively control bud development by setting, modifying, or terminating these processes. Ethylene signal transduction is positioned temporally between light and abscisic acid signals and is putatively activated by transiently low hexose pools. The timing and place of cell proliferation arrest (related to dormancy) and of the accumulation of storage compounds (related to acclimation processes) were established within the bud by electron microscopy. Finally, the identification of a large set of genes commonly expressed during the growth-to-dormancy transitions in poplar apical buds, cambium, or Arabidopsis thaliana seeds suggests parallels in the underlying molecular mechanisms in different plant organs.

Biosynthesis and Genetic Engineering of Lignin

Lignin, a complex heteropolymer of cinnamyl alcohols, is, second to cellulose, the most abundant biopolymer on Earth. Lignification has played a determining role in the adaptation of plants to terrestrial life. As all extracellular polymers, lignin confers rheological properties to plant tissues and participates probably in many other functions in cell and tissue physiology or in cell-to-cell communication. Economically, lignin is very important because it determines wood quality and it affects the pulp and paper-making processes as well as the digestibility of forage crops. For all these reasons the lignin biosynthesis pathway has been the subject of many studies. At present, most genes encoding the enzymes involved in the biosynthesis of lignin have been cloned and characterized. Various recent studies report on the alteration of the expression of these genes by genetic engineering, yielding plants with modified lignin. In addition, several mutants have been analyzed with changes in lignin content or lignin composition resulting in altered properties. Thanks to these studies, progress in the knowledge of the lignin biosynthesis pathway has been obtained. It is now clear that the pathway is more complex than initially thought and there is evidence for alternative pathways. A fine manipulation of the lignin content and/or composition in plants is now achievable and could have important economical and environmental benefits.

Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula × 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.

Lignin: genetic engineering and impact on pulping.

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.

Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells.

Jasmonates (JAs) are plant-specific signaling molecules that steer a diverse set of physiological and developmental processes. Pathogen attack and wounding inflicted by herbivores induce the biosynthesis of these hormones, triggering defense responses both locally and systemically. We report on alterations in the transcriptome of a fast-dividing cell culture of the model plant Arabidopsis thaliana after exogenous application of methyl JA (MeJA). Early MeJA response genes encoded the JA biosynthesis pathway proteins and key regulators of MeJA responses, including most JA ZIM domain proteins and MYC2, together with transcriptional regulators with potential, but yet unknown, functions in MeJA signaling. In a second transcriptional wave, MeJA reprogrammed cellular metabolism and cell cycle progression. Up-regulation of the monolignol biosynthesis gene set resulted in an increased production of monolignols and oligolignols, the building blocks of lignin. Simultaneously, MeJA repressed activation of M-phase genes, arresting the cell cycle in G2. MeJA-responsive transcription factors were screened for their involvement in early signaling events, in particular the regulation of JA biosynthesis. Parallel screens based on yeast one-hybrid and transient transactivation assays identified both positive (MYC2 and the AP2/ERF factor ORA47) and negative (the C2H2 Zn finger proteins STZ/ZAT10 and AZF2) regulators, revealing a complex control of the JA autoregulatory loop and possibly other MeJA-mediated downstream processes.