Gene expression in tension wood and bast fibres

Tension wood is produced in the xylem of some angiosperm trees, such as poplar (Populus spp.), whereas bast fibers are phloem-derived cells best known from annual crops, such as flax (Linum usitatissimum L.). Despite their different origins, secondary walls of both tension wood and bast fibers share distinctive properties, including an abundance of axially oriented, crystalline cellulose produced in a distinctive gelatinous-type layer. Because of these unique properties, tension wood and phloem fibers have separately been the subject of at least nine previously published gene or protein profiling studies. Here we review these experiments with a focus on those genes, whose expression distinguishes both tension wood and bast fibers from the more predominant types of xylem found elsewhere in the stem. Notable among these is an evolutionarily distinctive group of fasciclin-like arabinogalactan proteins (FLA) and a putative rhamnogalacturonan lyase.     

Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO₄-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical woody plant lignin, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.     

Engineering of PHB synthesis causes improved elastic properties of flax fibers

Flax stem is a source of fiber used by the textile industry. Flax fibers are separated from other parts of stems in the process called retting and are probably the first plant fibers used by man for textile purposes (1). Nowadays flax cultivation is often limited because of its lower elastic property compared to cotton fibers. Thus the goal of this study was to increase the flax fiber quality using a transgenic approach. Expression of three bacterial genes coding for beta-ketothiolase (phb A), acetoacetyl-CoA reductase (phb B), and PHB synthase (phb C) resulted in poly-beta-hydroxybutyrate (PHB) accumulation in the plant stem. PHB is known as a biodegradable thermoplastic displaying chemical and physical properties similar to those of conventional plastics (i.e., polypropylene). The fibers isolated from transgenic flax plants cultivated in the field and synthesizing PHB were then studied for biomechanical properties. All measured parameters, strength, Young's modulus, and energy for failure of flax fibers, were significantly increased. Thus the substantial improvement in elastic properties of fibers from the transgenic line has been achieved. Since the acetyl CoA, substrate for PHB synthesis, is involved not only for energy production but also for synthesis of many cellular constituents, the goal of this study was also the analysis of those metabolites, which interfere with plant physiology and thus fiber quality. The analyzed plants showed that reduction in lignin, pectin, and hemicellulose levels resulted in increased retting efficiency. A significant increase in phenolic acids was also detected, and this was the reason for improved plant resistance to pathogen infection. However, a slight decrease in crop production was detected.     

Composition and Distribution of Cell Wall Phenolic Compounds in Flax (Linum usitatissimum L.) Stem Tissues

Cell wall phenolic compounds were analysed in xylem and bastfibre-rich peels of flax stems by biochemical, histochemicaland ultrastructural approaches. Localization of cell wall phenolicsby the enzyme-gold method using laccase revealed several goldparticle distribution patterns. One of the major types (an evendistribution of single gold particles) was present mainly inxylem, while the other (compact branched groups of ten–40gold particles) was found both in xylem and fibre cells. Thelignin content of the stem parts was estimated by the Klasonprocedure and by the thioglycolic acid assay, and the phenolicproducts recovered after alkaline cupric oxide oxidation ofcell walls were analysed by GC. By combining chemical analysisdata and the frequency of various gold particle types withinthe tissues, different patterns of gold particle distributioncould be ascribed to certain cell wall phenolics; lignin wasstained as evenly distributed single gold particles, while branchedclusters represented hydroxycinnamic acids. The Klason proceduredid not remove all the non-lignin components from flax fibres,known for their highly crystalline cellulose, and considerablyoverestimated the lignin content. The thioglycolic acid assayresults were consistent with GC and microscopic observations.Copyright 2000 Annals of Botany Company Linum usitatissimum L., bast fibres, cell wall, lignin, hydroxycinnamic acids     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Cloning and Expression Analysis of Two Wheat cDNAs Encoding Cinnamoyl-CoA Reductase

Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lignin biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat ( Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.     

小麦中两个肉桂酰辅酶A还原酶基因的分离和表达分析

肉桂酰辅酶A还原酶（CCR）负责催化木质素单体生物合成中最重要的代谢反应,它将类苯丙酸类代谢物转移到木质素的合成途径中,为了更好地了解木质素在小麦生长发育中的作用,从小麦（Triticum aestivum L.ev,H4564)中克隆了两个肉桂酰辅酶A还原酶的cDNA,相似性和进化关系的分析表明这两个cDNA片段分别属于不同的肉桂酰辅酶A还原酶,这两个cDNA片段分别命名为W－cr6和W-cr19,RT-PCR和Northen杂交结果证明,W－cr6基因主要在小麦的茎和叶中表达,W－cr19基因主要在根和茎中表达,上述结果表明在小麦的基因组中至少存在两类肉桂酰辅酶A还原酶基因。     

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

Lignin is an aromatic polymer derived from the combinatorial coupling of monolignol radicals in the cell wall. Recently, various glycosylated lignin oligomers have been revealed in Arabidopsis thaliana. Given that monolignol oxidation and monolignol radical coupling are known to occur in the apoplast, and glycosylation in the cytoplasm, it raises questions about the subcellular localization of glycosylated lignin oligomer biosynthesis and their storage. By metabolite profiling of Arabidopsis leaf vacuoles, we show that the leaf vacuole stores a large number of these small glycosylated lignin oligomers. Their structural variety and the incorporation of alternative monomers, as observed in Arabidopsis mutants with altered monolignol biosynthesis, indicate that they are all formed by combinatorial radical coupling. In contrast to the common believe that combinatorial coupling is restricted to the apoplast, we hypothesized that the aglycones of these compounds are made within the cell. To investigate this, leaf protoplast cultures were cofed with ¹³C₆-labeled coniferyl alcohol and a ¹³C₄-labeled dimer of coniferyl alcohol. Metabolite profiling of the cofed protoplasts provided strong support for the occurrence of intracellular monolignol coupling. We therefore propose a metabolic pathway involving intracellular combinatorial coupling of monolignol radicals, followed by oligomer glycosylation and vacuolar import, which shares characteristics with both lignin and lignan biosynthesis.     

Identification of tyrosinase inhibitors from Marrubium velutinum and Marrubium cylleneum

Tyrosinase is a key enzyme in the production of melanins in plants and animals. Forty-five secondary metabolites isolated from Marrubium velutinum and Marrubium cylleneum belonging to the classes of flavonoids, phenylethanoid glycosides, phenolic acids and lignan glycosides were screened for their inhibitory activity against mushroom tyrosinase. Flavonoids and phenylethanoid glycosides showed moderate inhibitory activity, while phenolic acids were less active than phenylethanoid glycosides, suggesting that both phenolic groups are important for the activity.     

Improving retting of fibre through genetic modification of flax to express pectinases

Flax (Linum usitatissimum L.) is a raw material used for important industrial products. Linen has very high quality textile properties, such as its strength, water absorption, comfort and feel. However, it occupies less than 1% of the total textile market. The major reason for this is the long and difficult retting process by which linen fibres are obtained. In retting, bast fibre bundles are separated from the core, the epidermis and the cuticle. This is accomplished by the cleavage of pectins and hemicellulose in the flax cell wall, a process mainly carried out by plant pathogens like filamentous fungi. The remaining bast fibres are mainly composed of cellulose and lignin. The aim of this study was to generate plants that could be retted more efficiently. To accomplish this, we employed the novel approach of transgenic flax plant generation with increased polygalacturonase (PGI ) and rhamnogalacturonase (RHA) activities. The constitutive expression of Aspergillus aculeatus genes resulted in a significant reduction in the pectin content in tissue-cultured and field-grown plants. This pectin content reduction was accompanied by a significantly higher (more than 2-fold) retting efficiency of the transgenic plant fibres as measured by a modified Fried’s test. No alteration in the lignin or cellulose content was observed in the transgenic plants relative to the control. This indicates that the over-expression of the two enzymes does not affect flax fibre composition. The growth rate and soluble sugar and starch contents were in the range of the control levels. It is interesting to note that the RHA and PGI plants showed higher resistance to Fusarium culmorum and F. oxysporum attack, which correlates with the increased phenolic acid level. In this report, we demonstrate for the first time that over-expression of the A. aculeatus genes results in flax plants more readily usable for fibre production. The biochemical parameters of the cell wall components indicated that the fibre quality remains similar to that of wild-type plants, which is an important pre-requisite for industrial applications. Magdalena Musialak and Magdalena Wróbel-Kwiatkowska participated equally in the preparation of this paper     

A Genomics Approach to Deciphering Lignin Biosynthesis in Switchgrass

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.     

Constituents from the bark of Tabebuia impetiginosa

The bark of Tabebuia impetiginosa afforded nineteen glycosides, consisting of four iridoid glycosides, two lignan glycosides, two isocoumarin glycosides, three phenylethanoid glycosides and eight phenolic glycosides. Their structures were determined using both spectroscopic and chemical methods. Iridoid glycosides, phenylethanoid glycosides and lignan glycosides had ajugol, osmanthuside H and secoisolariciresinol 4-O-beta-D-glucopyranoside as their structural elements, respectively, whereas the aglycone moieties of the isocoumarin glycosides were considered to be (-)-6-hydroxymellein. Phenolic glycosides had 4-methoxyphenol, 2,4-dimethoxyphenol, 3,4-dimethoxyphenol, 3,4,5-trimethoxyphenol and vanillyl 4-hydroxybenzoate as each aglycone moiety. Additionally, the sugar chains of these isocoumarin glycosides and phenolic glycosides were concluded to be beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside as well as those of osmanthuside H and above phenylethanoid glycosides.