Founder effect in a Belgian-Dutch fragile X population

For many years, the high prevalence of the fragile X syndrome was thought to be caused by a high mutation frequency. The recent isolation of the FMR1 gene and identification of the most prevalent mutation enable a more precise study of the fragile X mutation. As the vast majority of fragile X patients show amplification of an unstable trinucleotide repeat, DNA studies can now trace back the origin of the fragile X mutation. To date, de novo mutations leading to amplification of the CGG repeat have not yet been detected. Recently, linkage disequilibrium was found in the Australian and US populations between the fragile X mutation and adjacent polymorphic markers, suggesting a founder effect of the fragile X mutation. We present here a molecular study of Belgian and Dutch fragile X families. No de novo mutations could be found in 54 of these families. Moreover, we found significant (P < 0.0001) linkage disequilibrium in 68 unrelated fragile X patients between the fragile X mutation and an adjacent polymorphic microsatellite at DXS548. This suggests that a founder effect of the fragile X mutation also exists in the Belgian and Dutch populations. Both the absence of new mutations and the presence of linkage disequilibrium suggest that a few ancestral mutations are responsible for most of the patients with fragile X syndrome.     

p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G₁ phase, although recent studies have identified a novel ER stress G₂ checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G₂ delay involving CHK1 and a later induction of G₁ arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G₁ checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G₂ progression prior to ultimate G₁ arrest.     

Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem

Lignin is an aromatic heteropolymer, abundantly present in the walls of secondary thickened cells. Although much research has been devoted to the structure and composition of the polymer to obtain insight into lignin polymerization, the low-molecular weight oligolignol fraction has escaped a detailed characterization. This fraction, in contrast to the rather inaccessible polymer, is a simple and accessible model that reveals details about the coupling of monolignols, an issue that has raised considerable controversy over the past years. We have profiled the methanol-soluble oligolignol fraction of poplar (Populus spp.) xylem, a tissue with extensive lignification. Using liquid chromatography-mass spectrometry, chemical synthesis, and nuclear magnetic resonance, we have elucidated the structures of 38 compounds, most of which were dimers, trimers, and tetramers derived from coniferyl alcohol, sinapyl alcohol, their aldehyde analogs, or vanillin. All structures support the recently challenged random chemical coupling hypothesis for lignin polymerization. Importantly, the structures of two oligomers, each containing a gamma-p-hydroxybenzoylated syringyl unit, strongly suggest that sinapyl p-hydroxybenzoate is an authentic precursor for lignin polymerization in poplar.     

Unravelling cell wall formation in the woody dicot stem

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.     

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.     

Modeling Lignin Polymerization. I. Simulation Model of Dehydrogenation Polymers

Lignin is a heteropolymer that is thought to form in the cell wall by combinatorial radical coupling of monolignols. Here, we present a simulation model of in vitro lignin polymerization, based on the combinatorial coupling theory, which allows us to predict the reaction conditions controlling the primary structure of lignin polymers. Our model predicts two controlling factors for the β-O-4 content of syringyl-guaiacyl lignins: the supply rate of monolignols and the relative amount of supplied sinapyl alcohol monomers. We have analyzed the in silico degradability of the resulting lignin polymers by cutting the resulting lignin polymers at β-O-4 bonds. These are cleaved in analytical methods used to study lignin composition, namely thioacidolysis and derivatization followed by reductive cleavage, under pulping conditions, and in some lignocellulosic biomass pretreatments.Lignins are aromatic polymers that are predominantly present in secondarily thickened cell walls. These polymers make the cell wall rigid and impervious, allowing transport of water and nutrients through the vascular system and protecting plants against microbial invasion. Lignins are heterogeneous polymers derived from phenylpropanoid monomers, mainly the hydroxycinnamyl alcohols coniferyl alcohol (G-monomer) and sinapyl alcohol (S-monomer) and minor amounts of p-coumaryl alcohol (H-monomer). These monolignols differ in their degree of aromatic methoxylation (-OCH₃ group; Fig. 1). The resulting units in the lignin polymer are the guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units. They are linked by a variety of chemical bonds (Fig. 2) that have different chemical properties (Boerjan et al., 2003; Ralph et al., 2004; Vanholme et al., 2008).Open in a separate windowFigure 1.Chemical structures of three monolignols. A, H-monomer (p-coumaryl alcohol). B, G-monomer (coniferyl alcohol). C, S-monomer (sinapyl alcohol). G- and S-monomers are considered in our simulations. The G-monomer is methoxylated (-OCH₃ group) on position 3, and the S-monomer is methoxylated on positions 3 and 5.Open in a separate windowFigure 2.Chemical structures resulting from the possible bonding between two monomers (A) or a monomer and the bindable end of an oligomer (B). X and Y in the monomers denote the absence (for a G-unit) or presence (for an S-unit) of a methoxyl group at position 5 (see Fig. 1). The red line indicates the bonds generated by couplings of the B position and B, 4, or 5 position.Lignification is the process by which monomers and/or oligomers are polymerized via radical coupling reactions and typically occurs after the polysaccharides have been laid down in the cell wall. Lignin composition varies among cell types and can even be different in individual cell wall layers (Ruel et al., 2009). Lignin composition is also influenced by environmental conditions; for example, lignin in compression wood is enriched in H-units (Timell, 1986). Hence, both developmental and environmental parameters influence the composition and thus the structure of the lignin polymer (Boerjan et al., 2003; Ralph et al., 2004).Lignin is one of the main negative factors in the conversion of lignocellulosic plant biomass into pulp and bioethanol (Lynd et al., 1991; Hill et al., 2006). In these processes, lignin needs to be degraded by chemical or mechanical processes that are expensive and often environmentally polluting. Hence, major research efforts are devoted toward understanding lignin biosynthesis and structure. It has already been shown that reducing lignin content and modifying its composition in transgenic plants can result in dramatic improvements in pulping efficiency (Pilate et al., 2002; Baucher et al., 2003; Huntley et al., 2003; Leplé et al., 2007) and in the conversion of biomass into bioethanol (Stewart et al., 2006; Chen and Dixon, 2007; Custers, 2009). These altered biomass properties are related to the alterations in lignin composition and structure in terms of the frequencies of the lignin units and the bond types connecting them and possibly also their interaction with hemicelluloses (Ralph et al., 2004; Ralph, 2006).To study the parameters that influence lignin structure, lignin polymerization has been mimicked in vitro by experiments with dehydrogenation polymers (DHPs; Terashima et al., 1995). Indeed, lignification can be mimicked by oxidizing monolignols using a peroxidase, such as horseradish peroxidase (HRP), and supplying its cofactor hydrogen peroxide, producing synthetic DHP lignins. Monolignol oxidation can also be achieved without enzymes (e.g. by using transition metal one-electron oxidants, such as copper acetate). Some of these biomimetic DHPs have been suggested to be better models for wood lignins than HRP-generated DHPs (Landucci, 2000).In DHP experiments, the monolignols are either added in bulk (Zulauf experiment) or dropwise (Zutropf experiment) to the reaction mixture, yielding lignin polymers with very different bond frequencies (Freudenberg, 1956). Zutropf experiments approach the in vivo formation of lignin, which depends on the slow introduction of monolignols into the wall matrix via diffusion to the site of incorporation (Hatfield and Vermerris, 2001). Because the exact reaction conditions are known, such in vitro experiments have provided insight into the lignification process in planta. In this way, numerous factors were shown to influence lignin structure, including the relative supply of the monolignols, the pH, the presence of polysaccharides, hydrogen peroxide concentrations, and cell wall matrix elements in general (Grabber et al., 2003; Vanholme et al., 2008).Computer simulations of lignin polymerization can help explain and predict lignin structure from low-level chemical kinetic factors, including subunit-coupling probabilities and monolignol synthesis rates. Such models are helpful in explaining the mechanism behind a range of controlling factors identified in the experimental work, including (1) the ratio of coniferyl versus sinapyl alcohol monolignols, (2) the monolignol supply rate, and (3) the abundance of alternative monomers present during lignin biosynthesis in mutants and transgenics. Thus, computer models will also help in suggesting new targets for controlled lignin biosynthesis.Here, we propose a simulation model of synthetic lignin polymerization that is based upon an emerging consensus from a variety of observations and derives from a series of previous models of lignin polymerization (Glasser and Glasser, 1974; Glasser et al., 1976; Jurasek, 1995; Roussel and Lim, 1995). Our model uses a symbolic grammar to describe a constructive dynamical system (Fontana, 1992) or a rule-based system (Feret et al., 2009) in which it is not necessary to define all possible products in advance. We assume that G- and S-monomers and newly formed oligomers couple in a well-mixed medium, depending on coupling rules and experimentally measured coupling probabilities. To develop the model, we have used information from DHP experiments rather than natural lignins, as they are formed in a well-mixed medium and their reaction conditions are well known (e.g. the influx rate of monomers). Using information from natural lignin would have further complicated our model, as the structures of natural lignin polymers are influenced by many factors, including the possible involvement of dirigent proteins (Davin and Lewis, 2005), steric hindrance by polysaccharides, spatiotemporal regulation, and modifications during isolation procedures (Boerjan et al., 2003; Ralph et al., 2004).Using our simulation models, we study how putative controlling factors of lignin primary structure, including the influx rate of monomers and the relative amount of S-monomers, affect in silico lignin synthesis, and we compare our predictions with in vitro experiments. To predict the degradability of lignins formed in our simulations, we apply an in silico thioacidolysis, which cleaves the polymers at their β-O-4 positions. This simulates the molecular action of two of the most used methods to analyze lignin composition, thioacidolysis (Lapierre, 1993; Baucher et al., 2003) and derivatization followed by reductive cleavage (Lu and Ralph, 1997). The G+S-monomer yield is often taken as a reflection of the fraction of units bound by β-O-4 bonds. Cleavage of β-O-4 bonds is also the most important reaction in kraft pulping of wood (Baucher et al., 2003). The model predicts from first principles (1) that DHP lignins formed under Zutropf conditions have a higher β-O-4 content than those formed under Zulauf conditions, (2) that DHP lignins formed with high S content have a higher β-O-4 content than those formed with high G content, and (3) that a higher β-O-4 content does not necessarily reduce the average length of lignin fragments generated during in silico thioacidolysis.