Xylem vessels, lignin, wood

Xylem vessels, lignin, wood

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I learnt that lignin impregnates xylem cells, causing the cytoplasm to die due to the inability of water and nutrients to pass freely, hence creating hollow tubes adapted to transport materials. This means that in order for the cytoplasm to die and the tubes to be hollow, lignin needs always be present… However lignin is not present in all xylem vessels… Lignin is the woody tissue that only forms the trunk of a tree. So how do these cells hollow out in other parts of the plant/tree to create empty transport vessels… ??

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides x P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofluorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.

Enzymes, Industrial (overview)

Lignin Conversion

Lignin is a water-insoluble, long-chain heterogeneous polymer composed largely of phenylpropane units which are most commonly linked by ether bonds. The conversion of cellulose and hemicellulose into fuels and chemicals leaves lignin as a byproduct. In recent years, removal of lignin from lignin–carbohydrate complex has received much attention because of potential application in the pulp and paper industry. The lignin barrier can be disrupted by a variety of pretreatments rendering the cellulose and hemicellulose more susceptible to enzymatic attack. The basidiomycete, Phanerochaete chrysosporium, is able to degrade lignin in a H2O2-dependent process catalyzed by extracellular peroxidases (lignin peroxidase and manganese peroxidase). Due to extreme complexity of the problem, a great deal of research remains to reveal the essential factors involved in lignin biodegradation.

The Location of Guaiacyl and Syringyl Lignins in Birch Xylem Tissue

Introduction The ratio of syringyl to guaiacyl residues in isolated hardwood lignins can vary widely depending on the extraction process. These ratios are usually determined by the relative amounts of syringaldehyde and vanillin (S: V) isolable on alkaline nitrobenzene oxidation of the liquors (Stone/Blundell 1951). Both Stone (1955) and Marth (1959) have found that the initial lignin fraction obtained from the neutral sulphite cooking of aspen was deficient in syringyl residues when compared with the overall ratio of S : V for aspen wood. Furthermore, Kyogoku and Hachihama (1961, 1962) obtained lignosulphonate fractions from beech wood which were heterogeneous with respect to the yields of syringaldehyde and vanillin on alkaline nitrobenzene oxidation. One of the more astonishing results in the literature is that birch xylem tissue yields a value of S:V = 3:1 (Leopold/Malmstr m 1952) whereas, for birch native lignin, a ratio of : u is obtained (de Stevens/Nord 1953). It has never been demonstrated whether isolated hardwood lignins are mixtures of separate guaiacyl and syringyl macromolecules or guaiacyl-syringyl copolymers. Similarly, it is not known for certain whether the guaiacyl or syringyl residues predominate in a particular morphological region of the xylem tissue although this has been


Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood &ndash de Gruyter

Xylem conducts water and dissolved minerals from the roots to all the other parts of the plant. In angiosperms, most of the water travels in the xylem vessels. These are thick-walled tubes that can extend vertically through several feet of xylem tissue. Their diameter may be as large as 0.7 mm. Their walls are thickened with secondary deposits of cellulose and are usually further strengthened by impregnation with lignin. The secondary walls of the xylem vessels are deposited in spirals and rings and are usually perforated by pits.

Xylem vessels arise from individual cylindrical cells oriented end to end. At maturity the end walls of these cells dissolve away, and the cytoplasmic contents die. The result is the xylem vessel, a continuous nonliving duct. Xylem also contains tracheids. These are individual cells tapered at each end so the tapered end of one cell overlaps that of the adjacent cell. Like xylem vessels, they have thick, lignified walls and, at maturity, no cytoplasm. Their walls are perforated so that water can flow from one tracheid to the next. The xylem of ferns and conifers contains only tracheids. In woody plants, the older xylem ceases to participate in water transport and simply serves to give strength to the trunk. Wood is xylem. When counting the annual rings of a tree, one is counting rings of xylem.

Plant materials

The full-length open reading frame of PdWND3A was amplified from Populus deltoides genotype WV94 and cloned into the pAGW560 binary vector for transformation into WV94. We followed the same procedure for growing and maintaining transgenic plants in the greenhouses as reported in a previous publication [38]. The growth conditions were set with constant 25 °C with 16 h/8 h photoperiod.

Amino acid sequence alignment and phylogenetic analysis

AtSND1 (AT1G32770) was subjected to Phytozome v12.0 ( [8] and BLAST ( [1] to identify NAC domain-containing proteins in the Populus (P. trichocarpa) and Arabidopsis (A. thaliana) genomes. The full-length amino acid sequence homologs of AtSND1 from each species were subsequently used to perform reciprocal sequence homolog search with > 30% amino acid similarity cutoff (e-value< 0.01). The collected proteins were used as subjects in the Pfam database to predict putative protein domains and functional motifs [7]. The phylogenetic tree was constructed by PhyML (a phylogeny software based on the maximum-likelihood principle) using Jones-Taylor-Thornton (JTT) model matrix of amino acid substitution with 1000 bootstrap replication [9]. Nearest-Neighbor-Interchange (NNI) algorithm was used to perform tree topology search.

Phloroglucinol-HCl staining

To obtain the image of xylem vessel formation from OXPdWND3A transgenic plants and WV94 wild-type plants, stem tissues were collected at a position 15 cm above the stem base of 6-month-old plants. Cross-section specimen were sliced at 100 μm thickness without any fixation by using Leica RM2255 microtome (Leica biosystems, IL). Each slice was directly stained in 2% Phloroglucinol (Sigma-Aldrich, St. Louis, MO) dissolved in 95% ethanol for 5 min in dark. The red color was developed by adding 2–3 drops of concentrated Hydrochloride (HCl). Images were captured using SteREO Discovery V8 dissecting microscope (ZEISS, Thornwood, NY). The total count of vessel in each image was determined by ImageJ1 open source program [30].

RNA extraction and RT-PCR

To measure relative transcript abundance of PdWND3A and secondary cell wall biosynthesis-related genes, total RNA was extracted from young stem tissue (1–3 internode) and mature leaf (4-6th from apex) of six-month-old Populus plants with Plant Spectrum RNA extraction kit with treatment of in-column DNase following manufacture’s manual (Sigma-Aldrich). We performed quantitative reverse transcription polymerase chain reaction (sq- or qRT-PCR) to determine relative transcript abundance of selected genes. The single strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA by 1 h incubation with RevertAid reverse transcriptase (Thermo Fisher Scientific, Hudson, NH) at 42 °C. One μl of two-times diluted cDNA was used for real time PCR reaction. PCR reaction was performed with Maxima SYBR Green/ROX qPCR master mix including uracyl DNA glycosylase (UDG) (Thermo Fisher Scientific). Gene-specific primers used for PCR reactions were listed in the Additional file 1. PCR reaction was started with UDG activation at 50 °C for 2 min, a pre-denaturation of 95 °C for 10 min, followed by 40 cycles of combined two steps of 95 °C for 15 s and 60 °C for 30 s. The relative gene expression was calculated by 2 –ΔΔCt equation [17]. Populus UBIQUITIN C (PdUBCc, Potri.006G205700) was used as an internal control for all relative quantification analyses.

Chemical composition analysis

Chemical composition, including carbohydrates and lignin of the OXPdWND3A transgenic lines, was analyzed and compared with the control (wild-type WV94) by two-step sulfuric acid hydrolysis according to the NREL procedure [32]. Wiley-milled, 6-month-old Populus stems were Soxhlet-extracted using ethanol/toluene (1:2, v/v) for 12 h. For the analysis of leaf tissues, additional 12 h ethanol/toluene extraction and 12 h acetone extraction were conducted. The extractives-free samples were air-dried and hydrolyzed by two-step acid method. Briefly, the biomass was hydrolyzed with 72% H2SO4 at 30 °C for 1 h and 4% H2SO4 at 121 °C for 1 h. The solid residues were filtered and washed with excessive amounts of deionized water and oven-dried at 105 °C for 24 h. Ash content was measured by muffle furnace at 575 °C for 12 h. Klason lignin content was calculated as below:

Carbohydrate contents were analyzed using a Dionex ICS-3000 ion chromatography system with external standards.

Lignin S/G ratio analysis

Nuclear magnetic resonance (NMR) analysis was used to measure the lignin S/G ratio. Stem samples were extracted as described above. Cellulolytic enzyme lignin was isolated from the extractives-free biomass as described in a previous study [42]. The isolated lignin (

30 mg) was dissolved with DMSO-d6 in 5 mm NMR tube. A Bruker Avance III 400 MHz spectroscopy equipped with a 5 mm Broadband Observe probe and Bruker standard pulse sequence (‘hsqcetgpsi2’) was used for two-dimensional (2D) 1 H- 13 C heteronuclear single quantum coherence (HSQC) NMR analysis at 300 K. The spectral widths of 11 ppm ( 1 H, 2048 data points) and 190 ppm in F1 ( 13 C, 256 data points) were employed for the 1 H and 13 C-dimensions, respectively. The number of transients was 64 and the coupling constant ( 1 JCH) used was 145 Hz. Bruker Topspin software (v3.5) was used for data processing.

Saccharification efficiency assay

Stem tissues collected at a position 15 cm above the stem base of 6-month-old plants were dried and Wiley-milled to 40-mesh for sugar release measurement. Approximately 250 mg of sample was placed in 50 mM citrate buffer solution (pH 4.8) with 70 mg/g-biomass of Novozymes CTec2 (Novozymes, Franklinton, NC) loading. The enzymatic hydrolysis was conducted at 50 °C with 200 rpm in an incubator shaker for 48 h. Enzymes in the hydrolysate were deactivated in the boiling water for 5 min prior to the analysis of released sugars by using the Dionex ICS-3000 ion chromatography system. Each analysis was conducted in duplicates from single plant of each transgenic line.

Statistical analysis

T-test (against WV94) was performed at p < 0.01 by t-test function integrated in the Excel software (Microsoft, Redmond, WA) for all statistical analysis. Asterisk in each figure indicates significant difference from WV94 or control samples (p < 0.01).

Xylem vessels, lignin, wood - Biology

Foto: Fredrik Larsson

My research focuses on lignification and cell death of xylem elements and how these processes influence the chemical and physical properties of the secondary cell walls and woody tissues of vascular plants. We use two model systems. The roots and hypocotyls of Arabidopsis thaliana provide excellent models for understanding the molecular and genetic control of xylem differentiation, while the woody tissues of aspen (Populus tremula) trees are practical for high-resolution gene expression, genetic, genomic and functional assays.

Xylem elements mature by depositing cellulose-rich secondary cell walls until they die through programmed cell death. Cell death therefore controls the thickness of the secondary cell walls of the xylem by controlling the life time of the xylem elements. We have earlier shown that cell death also controls lignification of xylem elements. Work done in the Zinnia elegans tracheary element differentiation system revealed that lignin biosynthesis continues even after cell death and that lignin polymerization occurs only after cell death. This sequence of events needs to be strictly controlled in time and place. In my previous work I have characterized the cell death process and identified factors that control both lignification and cell death of the xylem elements. The current aim is to identify factors that initiate and execute xylem cell death. One of the focus areas is the signaling and functional characterisation of the Arabidopsis thaliana metacaspase gene family using reverse genetic, forward genetic and biochemical methods in intact plants and in ectopic, hormonally induced tracheary elements.

The fact that the lifetime of the xylem elements controls the thickness of the cell walls and hence the extent of biomass production within each cell implies that that the identification of cell death controlling factors could be used to modify overall biomass production in forest trees. We have taken two different approaches to investigate the relationships between xylem maturation, the chemical and physical properties of the secondary cell wall and the properties of wood. The first approach is to modify expression of selected candidate genes in transgenic aspen (Populus tremula) trees using cell-specific promoters, newest DNA editing technologies and tree phenotyping platform with the aim of delaying xylem cell death and thereby improving biomass properties. The second approach takes advantage of the natural variation within a Swedish aspen population with the aim to identify variation in the secondary cell wall and wood properties and the underlying molecular mechanism by genome-wide association mapping.

Xylem and Phloem

Plants don’t have blood vessels - instead they have xylem and phloem vessels. Xylem transports water whereas phloem transports glucose and dissolved amino acids. They are both specialised to carry out their function and arranged in such a way to give the plant as much structure and support as possible.

Vascular Bundles

Plants contain vessels which function to transport water and sugars from one part of the plant to another. Xylem vessels transport water and dissolved mineral ions from the roots to the rest of the plant and also provide structural support. Phloem vessels transport dissolved substances, such as sucrose and amino acids, from the leaves to the rest of the plant. Xylem and phloem vessels are grouped together within the plant stem and form vascular bundles. Sclerenchyma fibres are also found within vascular bundles and provide support to the stem.

Within the plant stem, xylem vessels are found right on the inside. Phloem tissue is located in the middle of the vascular bundle and sclerenchyma fibres are found on the outside. Having the stronger xylem vessels in the centre provides strength to the stem and acts like an internal ‘scaffolding’ to support the stem and prevent it from bending in the wind.

In the root, the xylem forms a cross-like structure in the centre which is surrounded by phloem vessels. This arrangement adds strength to the root as it pushed through the soil.

Within the leaf, the xylem vessels are found towards the top of the vascular bundle with the phloem vessels found underneath.


Xylem vessels transport water and mineral ions from the roots to the rest of the plant. They are made up of dead, hollow cells with no end cell walls. This forms one continuous tube when the xylem cells are stacked on top of each other. The cells have no organelles or cytoplasm, which creates more space inside the vessel for transporting water. The cell walls contain pits which allows water and mineral ions to move into and out of the vessel. The cell wall also contains a tough, woody substance called lignin, which strengthens the xylem vessel and provides structure and support to the plant.


Phloem vessels transport dissolved substances, such as sucrose and amino acids from parts of the plant where they are made (sources) to the parts of the plant where they are used (sinks). Leaves are sources because they produce glucose from photosynthesis and parts of the plant where sugar is stored, such as roots and bulbs, act as sinks.

Phloem vessels are made up of two types of cell - sieve tube elements and companion cells. The sieve tube elements are living cells and are joined end-to-end to form sieve tubes. The ends of each cell consist of a ‘sieve plate’ which contains lots of holes to allow solutes to move from one cell to the next. The sieve tube cells contain no organelles and very little cytoplasm to create more space for solutes to be transported. The absence of a nucleus and other organelles means that these cells cannot survive on their own, so each sieve tube element is associated with a companion cell, which contains a nucleus and is packed full of mitochondria. The mitochondria provide lots of energy for the active loading of sucrose into the sieve tube element. The sieve tube element and the companion cell are connected through plasmodesmata (channels in the cell wall) which allows the two cells to communicate.


Together with xylem and phloem vessels, sclerenchyma fibres are also found within vascular bundles and provide structural support to the plant. They are made up of bundles of long, dead cells. The cells have a hollow lumen and the cell walls are thickened with lignin. The cell walls also contain more cellulose than a typical plant cell which makes sclerenchyma fibres particularly strong.

Dissecting Plant Stems

To view vascular bundles under the microscope, you first need to dissect the plant stem and prepare a tissue sample. You would do this by following the method below:

Cut a thin section of the plant stem using a scalpel. Take care when using the sample and remember to cut away from you.

Place the tissue sample into water to prevent it from drying out.

Place the tissue sample into a small dish containing the stain. A common stain that is used to view vascular bundles is toluidine blue O (TBO) which stains lignin blue/green which will enable you to visualise the xylem and sclerenchyma fibres. The phloem cells and remaining tissue will appear a pink/purple colour.

Rinse the tissue samples in water and place each one onto a microscope slide.

Vascular bundle in a clover leaf viewed under the microscope. Image credit: Berkshire Community College Bioscience Image Library.

Lignification and Peroxidase Activity in Bamboo Shoots (Phyllostachys edulis A. et C. Riv.)

Introduction Bamboos are perennial grasses with lignified tissues in the culms which are distributed mostly in natural Vegetation in Asia. Bamboos have been used äs a main material for house construction, furnitures, handicraft articles, and for pulp and paper making, because of the unique properties (Liese 1987). Bamboos are the fastest growing plants. They reach their füll height of 15--30 m within a period of 2 to 4 months (Itoh 1990 Liese 1987). No annual thickening of xylem cells occur over the year. Füll lignification of the component cells completes within one growing season (Itoh 1990). Itoh (1990) extensively investigated the progress of lignification in the culm of Phyllostachys heterocyda. He showed that transverse and axial progresses of lignification in the component cells prceeded from outside to inside of the culm and upward from the basal internode to the top one, respectively, and that lignification of fibers preceded that of ground tissue parenchyma. Lignification did not occur in parenchyma cells at least at the culm of 5 m in height (Higuchi et al. 1953). In bamboo culms, the parenchyma cells, forming the basal matrix, appear mostly elongated but small, almost cube-like cells are also present, interspersed between the


Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood &ndash de Gruyter

Xylem vessels, lignin, wood - Biology

1. Transpiration is the inevitable loss of water vapour as a consequence of gas exchange in the leaf.

  • thick-walled, elongated vascular tissue cells
    • arranged end-to-end
    • connected by perforated end-plates
    • leaving a continuous microtube for transporting water and dissolved mineral ions
    • from the roots to the above-ground portions of the plant
    • lignified xylem:
      • vascular tissue cells reinforced with helical or ring-shaped thickenings of the cellulose cell wall impregnated with lignin
      • lignin makes the cell walls hard, providing resistance to pressure

      Cell Wall in Xylem [image] Science Daily. Retrieved June 1, 2012

      Transpiration Stream [image] TwyfordBiology. Retrieved June 1, 2012

      5. Active uptake of mineral ions in the roots causes absorption of water by osmosis.

      • because the concentration of mineral ions is usually lower in the soil than in the root, active transport is used to concentrate mineral ions in the root
      • because active transport requires ATP, root epidermal cells are rich in mitochondria and require a supply of oxygen for cellular respiration
      • active transport: ATP oxidation provides the energy for protons to be pumped from the inside to the outside of root epidermal cell membranes = chemiosmosis, producing a H+ membrane potential
      • cations, such as K+, are driven from the extracellular fluid into the intracellular fluid, through membrane channels, by their electrical charge repulsion from the H+’s concentrated in the extracellular fluid
      • anions, such as NO3-, move from the extracellular fluid into the intracellular fluid, through membrane channels, by co-transport with H+, which moves down its diffusion gradient

      Ion Uptake [image] Biology Forums. Retrieved June 1, 2012 from

      6. Guard cells can regulate transpiration by opening and closing stomata. The plant hormone abscisic acid causes the closing of stomata.

      7. The abiotic factors of light, temperature, wind and humidity, affect the rate of transpiration in a typical terrestrial plant


      8. Application: Adaptations of plants in deserts and in saline soils for water conservation.

      • reduced leaves: minimizes water loss by reducing leaf surface area
      • thickened waxy cuticle: minimizes water loss by limiting water loss through epidermis
      • reduced number of stomata: minimizes water loss through leaves
      • succulence: stems specialized for water storage maximizes retention of water available during infrequent rains

      Applications and skills:

      Application: Models of water transport in xylem using simple apparatus including blotting or filter paper, porous pots and capillary tubing.

      Skill: Drawing the structure of primary xylem vessels in sections of stems based on microscope images.

      Skill: Measurement of transpiration rates using potometers. (Practical 7)

      Skill: Design of an experiment to test hypotheses about the effect of temperature or humidity on transpiration rates.

      Watch the video: Xylem and Phloem - Transport in Plants. Biology. FuseSchool (January 2023).