The cell biology of lignification in higher plants

Background Lignin is a polyphenolic polymer that strengthens and waterproofs the cell wall of specialized plant cell types. Lignification is part of the normal differentiation programme and functioning of specific cell types, but can also be triggered as a response to various biotic and abiotic stresses in cells that would not otherwise be lignifying.Scope Cell wall lignification exhibits specific characteristics depending on the cell type being considered. These characteristics include the timing of lignification during cell differentiation, the palette of associated enzymes and substrates, the sub-cellular deposition sites, the monomeric composition and the cellular autonomy for lignin monomer production. This review provides an overview of the current understanding of lignin biosynthesis and polymerization at the cell biology level.Conclusions The lignification process ranges from full autonomy to complete co-operation depending on the cell type. The different roles of lignin for the function of each specific plant cell type are clearly illustrated by the multiple phenotypic defects exhibited by knock-out mutants in lignin synthesis, which may explain why no general mechanism for lignification has yet been defined. The range of phenotypic effects observed include altered xylem sap transport, loss of mechanical support, reduced seed protection and dispersion, and/or increased pest and disease susceptibility.     

Genetic and molecular basis of grass cell-wall degradability. I. Lignin-cell wall matrix interactions

Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most variations in lignin composition or structure. Genetic manipulation of lignification can improve grass cell-wall degradability, but the degree of success will depend on genetic background, plant modification techniques employed, and analytical methods used to characterize cell walls.     

Peroxidases as Indicators of Growth and Differentiation in Aspen Callus Cultures

Aspen (Populus tremuloides Michx.) callus tissue grown on a synthetic medium containing either an auxin (2,4-dichloro-phenoxyacetic acid) or cytokinin [6-(3-methyl-2-butenylamino) purine] differed in growth rate, total peroxidase activity, peroxidase isoenzyme expression, and in lignin, cell wall sugars and extractive content. Tissue treated with auxin increased more rapidly in fresh weight, but stopped growing sooner than did the cytokinin-treated tissues. Lignification also proceeded more rapidly, and lignin formed a greater fraction of the cell wall weight in auxin-treated tissue. For both treatments, peroxidase activity and growth rate were positively related (r = 0.96). Polyacrylamide gel electrophoresis showed some quantitative, but few qualitative, isoenzyme differences with hormonal treatment and growth rate.     

Relations polyphenols-croissance: Lignification et limitation de croissance chez Lycopersicum esculentum

Abstract Polyphenols and growth – Lignification and limitation of growth in Lycopersicum esculentum. When fed with quinic acid, young tomato seedlings (Lycopersicum esculentum Mill. cv. St. Pierre) exhibited reduced growth and marked changes in cell wall composition: cellulose concentrations decreased whereas those of lignin increased. Exogenously supplied growth substances (IAA, GA₃) affected both the size of the plants and the lignification process: IAA treatments resembled quinic acid induced modifications with regard to both size and lignification: GA₃ applied to quinate treated plants counteracted the effects of this compound by reducing the lignin content and improving the growth. The possible effect of abnormal lignification as a limiting factor in cell growth is supported by the characterization of lignins in tissues different from xylem.     

Morphological and transcript changes in the biosynthesis of lignin in oil palm (Elaeis guineensis) during Ganoderma boninense infections in vitro

Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4‐hydroxylase (EC 1.14.13.11), caffeic acid O‐methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ‐hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

Differentiation Between Tissues from Carnation (Dianthus caryophyllus) Stems by Pyrolysis-Mass Spectrometry

Small pieces of different tissues from stems of young and oldcarnation plants were analyzed for lignification (lignin/celluloseratios) and lignin composition by means of pyrolysis-(gas chromatography)-massspectrometry. The epidermis and phloem of young and old stemswere essentially non-lignified. Pith parenchyma was only lignifiedin mature and senescing tissues. The type of lignin in sclerenchymadiffered from that in xylem and pith. Lignification in the xylemof very young tissues was a mainly guaiacyl-type lignin, whichgradually changed into a mixed guaiacyl-syringyl lignin in oldertissues. In mature tissues, the sclerenchyma was more highlylignified than the xylem. All tissues yielded comparatively large amounts of dihydroferulicacid, a compound which may be specific for carnation. Carnation, Dianthus caryophyllus, epidermis, cortex, sclerenchyma, phloem, xylem, pith, lignification, aging, dihydroferulic acid, pyrolysis-(gas chromatography)-mass spectrometry     

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 orin 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.     

Lignins and lignification: Selected issues

Lignin deposition in plant cell walls is one of the mechanisms which allowed the development of upright plants adapted to a terrestrial habitat. At the present time, lignins and lignification are the subject of very active research which has recently moved from chemical and biochemical aspects to more biological and developmental problems. In this review, three different topics will be addressed. (a) A first section will deal with recent advances related to the biosynthesis of lignins. It will be shown that a complex array of O-methyltransferases may control the production of differentially methylated monolignols, the precursors of lignins, but that the downstream enzymes in the synthesis of monolignols are probably not encoded by multigene families which would provide additional possibilities for fine-tuning the monomeric composition of lignins. In addition, recent results obtained on laccases will illustrate the difficulty in identifying the true nature of oxidases involved in the production of phenoxy radicals, the oxidation products of monolignols. (b) A second set of data will highlight the potential interest of Arabidopsis mutants for understanding lignin synthesis, deposition and function. Indeed, different classes of lignification mutants with modifications in lignin content or composition and alterations of vascular differentiation or global vascular pattern have already been characterized. The identification of the corresponding genes will undoubtedly give rise to new insights on key steps and regulation mechanisms in the lignification process. (c) The last section is dedicated to the future of lignin genetic engineering. It will be emphasized that, after a first period which has demonstrated the potential of the approach, it is necessary to consider in greater detail the unexpected side effects and compensation mechanisms associated with induced lignin modifications. New targets for future lignin genetic engineering experiments will be identified and the extension of the technology to new woody species, the advantages for the pulp industry and the problems associated with public perception of these new products will be envisaged. Lignification is a tightly regulated and dynamic process subject to modulation at different levels during normal development and in response to different stresses. Understanding these subtle mechanisms which also involve the other polymers of the cell wall is an important challenge facing plant biology as we enter the next century.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

FORMATION OF CALLOSE AND LIGNIN DURING LEAF ABSCISSION

Deblading of bean leaves promoted the formation of callose and lignin in the abscission zone. The abscission layer became evident three days after deblading. The greatest increase in callose occurred in about two layers of cells during the development of the abscission layer. Four days after deblading, only a few layers of cells on the distal side of the abscission layer showed an increase in lignin. Lignification continued to expand to 8–10 layers of cells at the time of separation. After separation, the lignified cells remained with the petiole. Sieve elements in the abscission zone were covered with callose plugs and the vessels were occluded with tyloses.     

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging

Wood cell wall consists of several structural components, such as cellulose, hemicelluloses and lignin, whose concentrations vary throughout the cell wall. It is a composite where semicrystalline cellulose fibrils, acting as reinforcement, are bound together by amorphous hemicelluloses and lignin matrix. Understanding the distribution of these components and their functions within the cell wall can provide useful information on the biosynthesis of trees. Raman imaging enables us to study chemistry of cell wall without altering the structure by staining the sample or fractionating it. Raman imaging has been used to analyze distributions of lignin and cellulose, as well as the functional groups of lignin in wood. In our study, we observed the distribution of cellulose and lignin, as well as the amount of coniferyl alcohol and aldehyde groups compared to the total amount of lignin in pine (Pinus sylvestris) and spruce (Picea abies) wood samples. No significant differences could be seen in lignin and cellulose distribution between these samples, while clear distinction was observed in the distribution of coniferyl alcohols and coniferyl aldehyde in them. These results could provide valuable insight on how two similar wood species control biosynthesis of lignin differently during the differentiation of cell wall.     

Lignification in poplar plantlets fed with deuterium-labelled lignin precursors

Lignification was investigated in wild-type (WT) and in transgenic poplar plantlets with a reduced caffeic acid O-methyl-transferase (COMT) activity. Coniferin and syringin, deuterated at their methoxyl, were incorporated into the culture medium of microcuttings. The gas chromatography-mass spectrometry (GC-MS) analysis of the thioacidolysis guaiacyl (G) and syringyl (S) lignin-derived monomers revealed that COMT deficiency altered stem lignification. GC-MS analysis proved that the deuterated precursors were incorporated into root lignins and, to a lower extent, in stem lignins without major effect on growth and lignification. Deuterium from coniferin was recovered in G and S lignin units, whereas deuterium from syringin was only found in S units, which further establishes that the conversion of G to S lignin precursors may occur at the level of p-OH cinnamyl alcohols.     

Relations of cell wall bound peroxidases,phenols and lignin in needles of Serbian spruce Picea omorika (Pančić) Purkynĕ in the natural habitat

The habitat of Picea omorika (Pančić) Purkynĕ, Serbian spruce, is characterized by extremely variable environmental conditions. Relations between the activities of cell wall bound peroxidase (POD) and contents of lignin and cell wall bound phenols were studied in needles of Serbian spruce, in the natural habitat on Mountain Tara. We intended to see, by using Redundancy Analysis, if the yearly peaks of activities of cell wall bound enzymes correspond to the cell wall bound phenols and lignin contents, and whether this relation contributes to the adaptation of Serbian spruce to severe habitat. The highest lignin content was found in spring and it was in high positive correlation with the activities of five covalent POD isoforms. The contents of cell wall bound phenols were lowest in spring and higher in summer and autumn, being in high positive correlation with ionic POD isoforms. The results indicate that relation found between ionic POD isoforms and cell wall bound phenols, as well as between covalent isoforms and lignin may be related to the environmental conditions.     

On the Cytochemistry of Cell Wall Formation in Poplar Trees

Abstract: The ultrastructure of cell walls and the mechanisms of cell wall formation are still not fully understood. The objective of our study was therefore to obtain additional fine structural details on the deposition of cell wall components during the differentiation of xylem cells in hybrid aspen ( Populus tremula L. × P. tremuloides Michx.) we used as a model tree. At the electron microscope level, PATAg staining revealed a successive deposition of polysaccharides with increasing distance from the cambium. Staining with potassium permanganate and UV microspectrophotometry showed that the cell walls were lignified, with some delay to the deposition of polysaccharides. Immunogold labelling of three lignin types in developing cell walls varied with progressive deposition of cell wall layers. Condensed lignin subunits were localized in corners of cells adjacent to the cambium prior to S1 formation, whereas non-condensed lignin subunits became labelled only in later stages - in secondary walls near cell corners and simultaneously with the completion of S1 formation. As S2 polysaccharide deposition progressed, the labelling extended towards the lumen. Labelling of peroxidases revealed their presence in cell corner regions of young xylem cells, still lacking a secondary wall, implying that peroxidases are incorporated into the developing cell wall at early developmental stages. A weak labelling of middle lamella regions and secondary walls could also be seen at later stages. The results are discussed in relation to current knowledge on the succession of polysaccharide and lignin deposition in woody cell walls.     

Dynamic Changes in Distribution of Lignin and Hemicelluloses in Cell Walls During Differentiation of Secondary Xylem in Eucommia ulmoides (in English)

The dynamic changes in the distribution of lignin and hemicelluloses (xylans and xyloglucans) in cell walls during the differentiation of secondary xylem in Eucommia ulmoides Oliv. were studied by means of ultraviolet light microscopy and transmission electron microscopy combined with immunogold labelling. In the cambial zone and cell expansion zone, xyloglucans were localized both in the tangential and radial walls, but no xylans or lignin were found in these regions. With the formation of secondary wall S1 layer, lignin occurred in the cell corners and middle lamella, while xylans appeared in S1 layer, and xyloglucans were localized in the primary walls and middle lamella. In pace with the formation of secondary wall S2 and S3 layer, lignification extended to S1, S2 and S3 layer in sequence, showing a patchy style of lignin deposition. Concurrently, xylans distributed in the whole secondary walls and xyloglucans, on the other hand, still localized in the primary walls and middle lamella. The results indicated that along with the formation and lignification of the secondary wall, great changes had taken place in the cell walls. Different parts of cell walls, such as cell corners, middle lamella, primary walls and various layers of secondary walls, had different kinds of hemicelluloses, which formed various cell wall architecture combined with lignin and other cell wall components.     

杜仲次生木质部分化过程中木质素与半纤维素组分在细胞壁中分布的动态变化

利用紫外光显微镜、透射电子显微镜结合免疫胶体金标记,研究了杜仲（Eucommia ulmoides Oliv.)次生木质部分化过程中木质素与半纤维素组分（木葡聚糖和木聚糖）在细胞壁分布的动态变化。在形成层及细胞伸展区域,细胞壁具有木葡聚糖的分布,而没有木聚糖和木质素沉积,随着次生壁S1层的形成,木质素出现在细胞角隅和胞间层,木聚糖开始出现在S1层中,此时木葡聚糖则分布在初生壁和胞间层;随着次生,壁S2层及S3层的形成和加厚,木质逐逐步由细胞角隅和胞间层扩展到S1、S2和S3层,其沉积呈现出不均匀的块状或片状沉积模式,在次生壁各层形成与其木质化的同时,木聚糖逐渐分布于整个次生壁中,而木糖聚糖仍局限分布于初生壁和胞间层。结果表明,随着细胞次生壁的形成与木质化,细胞壁结构发生较大变化。细胞壁的不同区域,如细胞角隅、胞间层、初生壁和次生壁各层,具有不同的半纤维素组成,其与木质等细胞壁组分结构构成不同的细胞壁分子结构。