Biomedical subjects
Publications and source records attributed to Wout Boerjan.
Cross-coupling of sinapyl p-hydroxybenzoate and sinapyl alcohol produces an 8-8-cross-coupled product that is also detected in lignifying poplar tissues, implicating sinapyl p-hydroxybenzoate as a lignin precursor.
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.
Huge potential exists for improving plant raw materials and foodstuffs via metabolic engineering. To date, progress has mostly been limited to modulating the expression of single genes of well-studied pathways, such as the lignin biosynthetic pathway, in model species. However, a recent report illustrates a new level of sophistication in metabolic engineering by overexpressing one lignin enzyme while simultaneously suppressing the expression of another lignin gene in a tree, aspen. This novel approach to multi-gene manipulation has succeeded in concurrently improving several wood-quality traits.
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.
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.
The lignin biosynthetic pathway has been studied for more than a century but has undergone major revisions over the past decade. Significant progress has been made in cloning new genes by genetic and combined bioinformatics and biochemistry approaches. In vitro enzymatic assays and detailed analyses of mutants and transgenic plants altered in the expression of lignin biosynthesis genes have provided a solid basis for redrawing the monolignol biosynthetic pathway, and structural analyses have shown that plant cell walls can tolerate large variations in lignin content and structure. In some cases, the potential value for agriculture of transgenic plants with modified lignin structure has been demonstrated. This review presents a current picture of monolignol biosynthesis, polymerization, and lignin structure.
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.
The Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a crucial role during late seed development and has an additional function at the vegetative meristem, particularly during periods of growth-arresting conditions and quiescence. Here, we show that the ABI3 homolog of poplar (PtABI3) is expressed in buds during natural bud set. Expression occurs clearly after perception of the critical daylength that initiates bud set and dormancy in poplar. In short-day conditions mimicking natural bud set, the expression of a chimeric PtABI3::beta-glucuronidase (GUS) gene occurred in those organs and cells of the apex that grow actively but will undergo arrest: the young embryonic leaves, the subapical meristem, and the procambial strands. If PtABI3 is overexpressed or downregulated, bud development in short-day conditions is altered. Constitutive overexpression of PtABI3 resulted in apical buds with large embryonic leaves and small stipules, whereas in antisense lines, bud scales were large and leaves were small. Thus, PtABI3 influences the size and ratio of embryonic leaves and bud scales/stipules that differentiate from the primordia under short-day conditions. These observations, together with the expression of PtABI3::GUS in embryonic leaves but not in bud scales/stipules, support the idea that wild-type PtABI3 is required for the relative growth rate and differentiation of embryonic leaves inside the bud. These experiments reveal that ABI3 plays a role in the cellular differentiation of vegetative tissues, in addition to its function in seeds.
The committing step in Met and S-adenosyl-L-Met (SAM) synthesis is catalyzed by cystathionine gamma-synthase (CGS). Transgenic Arabidopsis plants overexpressing CGS under control of the cauliflower mosaic virus 35S promoter show increased soluble Met and its metabolite S-methyl-Met, but only at specific stages of development. The highest level of Met and S-methyl-Met was observed in seedling tissues and in flowers, siliques, and roots of mature plants where they accumulate 8- to 20-fold above wild type, whereas the level in mature leaves and other tissues is no greater than wild type. CGS-overexpressing seedlings are resistant to ethionine, a toxic Met analog. With these properties the transgenic lines resemble mto1, an Arabidopsis, CGS-mutant inactivated in the autogenous control mechanism for Met-dependent down-regulation of CGS expression. However, wild-type CGS was overexpressed in the transgenic plants, indicating that autogenous control can be overcome by increasing the level of CGS mRNA through transcriptional control. Several of the transgenic lines show silencing of CGS resulting in deformed plants with a reduced capacity for reproductive growth. Exogenous feeding of Met to the most severely affected plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250-fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM and that SAM may be a regulator of CGS expression.