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.     

Morphology, wood structure and cell wall composition of rolC transgenic and non-transformed aspen trees

Morphology, wood structure and cell wall composition of 35S-rolC transgenic hybrid aspen (P. tremula2tremuloides) were compared with non-transformed control trees. The transgenics are characterised by stunted growth, altered physiological parameters and light green leaves of reduced size. Histometric measurements revealed thinner fibre walls as compared to the controls. UV microspectrophotometry of individual wall layers did not reveal distinctive differences in the lignification of xylem cells, but in the extremely thin-walled fibres of the transgenics the secondary walls were less lignified as revealed by KMnO₄ staining in transmission electron microscopy. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to only a few rows. Immunocytochemical analyses revealed the deposition of lignins in less differentiated xylem cells as compared to the controls. The first labelling of condensed lignin appeared in cell corners and of non-condensed lignin in secondary walls near cell corners during the deposition of S1 polysaccharides. Because of alterations in the formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful for studies on molecular factors controlling the differentiation continuum.     

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background  

Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions.     

Lignin composition in cambial tissues of poplar

The cambial tissues of a Populus balsamifera, Balsam poplar clone were studied during a growth season. The Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and the protein content. Both the phloem and the xylem sides of the cambial region were examined. The samples were analyzed by thioacidolysis and structures of dimeric products were determined by mass spectrometry after desulphuration. Chemical analysis of samples during the growth season was combined with microscopy of embedded specimens that showed the state of cell differentiation at the time of sampling. In spring and early summer, growth is very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. The Klason lignin, protein content and carbohydrate monomer distribution showed that all the specimens from the cambial tissues sampled during a growth season contained predominantly middle lamella and primary walls; except for the developing xylem sampled in August where the carbohydrate composition showed that secondary walls were present. Thioacidolysis showed that the lignin from the cambial tissues had more condensed structures than the lignin from the reference balsam poplar clone wood. More guaiacyl than syringyl units were detected and mass spectrometry showed that the cambial tissues contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place predominantly in a bulk fashion during the summer.     

Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana

In the Arabidopsis mutant irx3, truncation of the AtCesA7 gene encoding a xylem-specific cellulose synthase results in reduced cellulose synthesis in the affected xylem cells and collapse of mature xylem vessels. Here we describe spectroscopic experiments to determine whether any cellulose, normal or abnormal, remained in the walls of these cells and whether there were consequent effects on other cell-wall polysaccharides. Xylem cell walls from irx3 and its wild-type were prepared by anatomically specific isolation and were examined by solid-state NMR spectroscopy and FTIR microscopy. The affected cell walls of irx3 contained low levels of crystalline cellulose, probably associated with primary cell walls. There was no evidence that crystalline cellulose was replaced by less ordered glucans. From the molecular mobility of xylans and lignin it was deduced that these non-cellulosic polymers were cross-linked together in both irx3 and the wild-type. The disorder previously observed in the spatial pattern of non-cellulosic polymer deposition in the secondary walls of irx3 xylem could not be explained by any alteration in the structure or cross-linking of these polymers and may be attributed directly to the absence of cellulose microfibrils which, in the wild-type, scaffold the organisation of the other polymers into a coherent secondary cell wall.     

Defense response of pine stem phloem to wounding and treatment with mycelial extracts from <Emphasis Type="Italic">Ceratocystis laricicola</Emphasis>

Ophiostomatoid fungi colonize the conducting tissues of conifer stems, the phloem and the xylem. These pathogenic fungi penetrate into the stem through injuries made by xylophagous insects vectoring these pathogens. In this study the response of the phloem of Scotch pine (Pinus sylvestris L.) to wounding (treatment 1) was compared with the response to wounding combined with application of high-molecular-weight compounds isolated from the mycelium of the ophiostomatoid fungus Ceratocystis laricicola Redfern & Minter (treatment 2). Both treatments induced the appearance of necrosis in the inner bark, the formation of periderm separating living and dead tissues, and formation of the callus alongside the wound perimeter. In addition, the bark accumulated lignin, bound proanthocyanidins, and resins, with a parallel decrease in the content of free proanthocyanidins, low-molecular-weight carbohydrates, and non-lignin components of the cell wall (P > 0.95). The size of necrotic spots, as well as changes in the content of most substances, were significantly higher in the treatment 2 than in the treatment 1 (P > 0.95). The accumulation of lignin in cell walls of phloem sieve cells was delayed in the treatment 2 as compared with that in the treatment 1. This suggested that the mycelial extract temporarily inhibited lignification at the early stage of the wound response. This disturbance of the cell wall protective transformation led to the hypothesis that the fungal suppressors retard the repair of inner bark injured by insects, thereby favoring the invasion of conifer tissues by ophiostomatoid fungi.     

Lignification and cell wall thickening in nodes of Phyllostachys viridiglaucescens and Phyllostachys nigra

BACKGROUND AND AIMS: Bamboos are among the most important plants in the world. The anatomical structure and mechanical properties of the culm internode are well documented. Fewer details are available of the culm node. The aim of this study was a topochemical investigation on lignification and cell wall thickening in developing and maturing bamboo nodes. The deposition sequence and distribution of lignin structural units and cell wall thickening in different anatomical regions of the node of Phyllostachys viridiglaucescens and Phyllostachys nigra are discussed. METHODS: Cell wall thickening and lignification are investigated in the outer part of the nodal region and in the diaphragm of developing and maturing P. nigra culms and in maturing culms of P. viridiglaucescens of different age classes. The lignification during ageing was studied topochemically by means of cellular UV microspectrophotometry. A combination of light microscopy and image analysis techniques were used to measure cell wall thickness. KEY RESULTS: The fibre and parenchyma cell wall thickness does not significantly increase during ageing. In the diaphragm, the cell walls are thinner and the cell diameter is larger than in the outer part of the node. In shoots, the lignin content in the epidermis, hypodermis and in both fibre and parenchyma cells of the diaphragm is relatively low compared with older culms. The fibre and parenchyma cells of the diaphragm have higher values of p-coumaric and ferulic acids than fibre and parenchyma cells of the outer part of the node. CONCLUSIONS: It was hypothesized that the combination of more hydroxycinnamic acids and of thinner cell walls in combination with higher cell diameters (lower density and lower stiffness) in the diaphragm than in the outer part of the node may play an important role in the biomechanical function of the node by acting as a spring-like joint to support the culm by bending forces.     

Wound-induced lignin and suberin deposition in a woody angiosperm (Eucalyptus gunnii Hook.): Histochemistry of early changes in young plants

Summary A simple system was developed to investigate the deposition of lignin and synthesis occurring in response to mechanical wounding in the woody angiospermEucalyptus gunnii Hook. The spatiotemporal deposition of these phenolic polymers was histochemically characterized in stem tissue through a combination of fluorescent microscopy and specific stains. Lignin and suberin deposition was detectable 24 h post wounding in the xylem wound zone and by 3 days post wounding in the bark wound zone where a welldeveloped necrophylactic (wound) periderm could be observed by 7 days post wounding. Close examination suggests that the spatial reinforcement of cell walls with lignin and/or suberin is carefully orchestrated so as to rapidly produce an effective protective barrier. Specific lignin colour reactions indicate that the lignin formed in response to wounding in both the bark and xylem wound zones is relatively poor in syringyl monomers as compared to that of developmental xylem lignin.Abbreviations P-HCl phloroglucinol-HCl - RH relative humidity - NP necrophylactic periderm     

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。     

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.     

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.     

Correlation between the distribution of lignin and pectin and distribution of sorbed metal ions (lead and zinc) on coir (Cocos nucifera L.)

Plant fibres are capacious for sorption of metal ions, and can be used in water cleaning. Knowledge about the sorption will help in selection of the fibre and optimisation of its chemical modification, if any. The aim of this paper is to investigate the connection, if any, between the distribution of lignin and pectin and the loading of Pb and Zn on coir (mesocarp fibres from Cocos nucifera L.). The coir consisted mainly of xylem and a fibre sheath. The lignin was evenly distributed in the cell walls of the fibre sheath, but in the xylem, there was no detectable content in the compound middle lamella, and a smaller content of lignin in the secondary walls than in the walls of the fibre sheath. The only detectable content of pectin in the fibre sheath walls was in the middle lamella, cell corners and extracellular matrix, while in the xylem, the pectin was almost evenly distributed in the wall, with a higher concentration in the middle lamella and cell corners. All cell walls facing the lacuna had a high content of pectin. The metal ions were mainly loaded on the xylem and cell walls facing the lacuna, maybe with an additional trend to be loaded on the large fibres. Lead was distributed on and across the whole secondary wall. Zinc was loaded on the secondary walls, but there was no information about the distribution across the wall. If there is a simple correlation between the loading of metal ions and the distribution of lignin or pectin, these investigations point at no correlation with lignin and a positive correlation with pectin. It has to be stressed that these conclusions are made on limited material and are therefore preliminary in nature.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Anatomical features, monomer lignin composition and accumulation of phenolics in 1-year-old branches of the Mediterranean Cistus ladanifer L.

Mediterranean shrub species are described as having phenology, habitus , reproductive biology and anatomical alterations in certain tissues, allowing their survival during the dry season and protecting them from herbivory. Anatomical and chemical analyses were conducted in 1-year-old branches of Cistus ladanifer L. in order to investigate the role played by shoot structure in the adaptive strategies of this species in the Mediterranean environment. Results showed that both xylem and pith underwent lignification. Pith parenchyma cells had thickened walls, higher lignin content than xylem and different monomer composition. Xylem presented features aiding safe water transport. A large accumulation of phenolic substances was found in xylem, pith and cortical parenchyma. Observations reported in this paper suggest the occurrence of adaptive strategies in 1-year-old branches of C. ladanifer whose structural features: (1) allow mechanical reinforcement of tissues to withstand drought without suffering permanent damage; (2) favour safety rather than efficiency in water transport; (3) defend the plants from animal predation and pathogens by accumulating phenolics in various tissues, and (4) protect inner tissues against UV-B radiation through deposition of phenolic compounds in cortical layers.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155 , 361–371.     

Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls

The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-of-function mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall.     

Influence of over-expression of the FLOWERING PROMOTING FACTOR 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.)

Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.     

Alfalfa Stem Tissues: Cell-wall Development and Lignification

Alfalfa stems contain a variety of tissues with different patternsof cell-wall development. Development of alfalfa cell wallswas investigated after histochemical staining and with polarizedlight using light microscopy and scanning electron microscopy.Samples of the seventh internode, from the base of stems grownon cut stems, were harvested at five defined stages of developmentfrom early internode elongation through to late maturity. Internodeseven was elongating up to the third sample harvest and internodediameter increased throughout the entire sampling period. Chlorenchyma,cambium, secondary phloem, primary xylem parenchyma and pithparenchyma stem tissues all had thin primary cell walls. Pithparenchyma underwent a small amount of cell-wall thickeningand lignification during maturation. Collenchyma and primaryphloem tissues developed partially thickened primary walls.In contrast to a recent report, the formation of a ring shaped,lignified portion of the primary wall in a number of cells inthe exterior part of the primary phloem was found to precedethe deposition of a thick, non-lignified secondary wall whichwas degradable by rumen microbes. In numerous xylem fibres fromthe fourth harvest date onwards, an additional highly degradablesecondary wall layer was deposited against a previously depositedlignified and undegradable secondary wall. The pattern of lignificationobserved in alfalfa stem tissues suggests that polymerizationof monolignols by peroxidases at the luminal border of the primarycell wall creates an impermeable zone which restricts lignificationof the middle lamella region of tissues with thick primary walls.Copyright1998 Annals of Botany Company Alfalfa,Medicago sativaL., stem tissue, cell wall, development, lignification, degradation.     

Characterization of polyphenols in cell walls of cultured Populus trichocarpa tissues

The amount and composition of cell wall-bound polyphenol (lignin) in cultured Populus trichocarpa tissues which formed numerous xylem elements (xylogenic) or no xylem (non-xylogenic) were compared. Polyphenol accounted for ca 15% of the dry wt of the cell wall and did not differ significantly in amount in xylogenic and non-xylogenic tissues. The syringic acid derivatives, 3,4.5-trimethoxybenzoic acid, was identified as one of the oxidation products of methylated cell walls and was recovered in similar amounts irrespective of xylem formation. In contrast, lignin from xylogenic cultures contained more p-coumaryl alcohol derivatives and less coniferyl alcohol derivatives than lignin from non-xylogenic cultures. In this respect the lignin composition of xylogenic tissues closely resembled that from stems.     

Regenerative Xylem in Inflorescence Stems of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

By inserting entomological needles into the lower parts of young inflorescence stems of three-month-old Arabidopsis thaliana (L.) Heynh var. Colombia plants, we studied the process of regenerative xylem production. Regenerative xylem was formed only in one- to two-day-old inflorescence stems but not in older ones. The regenerative vessels originated from re-differentiation of cortical parenchyma. To characterize the process of regenerative xylem formation, we conducted a histological study from the time of wounding to day 30 after wounding. In the first day after wounding the tissues showed no structural responses except for the wounding itself. After six days, regenerative vessel members were already differentiating in a basipetal pattern, forming a vascular bypass around the wound. Regenerative vessel member formation reached a maximal level on the twelfth day after wounding. Sixteen days after wounding the pith parenchyma started to become loose as if indicating tissue senescence. Altogether, vascular regeneration following wounding in inflorescence stems of Arabidopsis thaliana is similar to that in other dicotyledon plants. These findings provide the basis for the use of Arabidopsis thaliana as a model system to study the genetics, physiology and cell biology of wound healing and regenerative vascular tissue formation.