Secondary Cell Wall Structure Research Articles

Morphological Characterization and Transcriptome Analysis of New Dwarf and Narrow-Leaf (dnl2) Mutant in Maize

Lodging is the primary factor limiting high yield under a high plant density. However, an optimal plant height and leaf shape can effectively decrease the lodging risk. Here we studied an ethyl methanesulfonate (EMS)-induced dwarf and a narrow-leaf mutant, dnl2. Gene mapping indicated that the mutant was controlled by a gene located on chromosome nine. Phenotypic and cytological observations revealed that dnl2 showed inhibited cell growth, altered vascular bundle patterning, and disrupted secondary cell wall structure when compared with the wild-type, which could be the direct cause of the dwarf and narrow-leaf phenotype. The phytohormone levels, especially auxin and gibberellin, were significantly decreased in dnl2 compared to the wild-type plants. Transcriptome profiling of the internodes of the dnl2 mutant and wild-type revealed a large number of differentially expressed genes enriched in the cell wall biosynthesis, remodeling, and hormone biosynthesis and signaling pathways. Therefore, we suggest that crosstalk between hormones (the altered vascular bundle and secondary cell wall structure) may contribute to the dwarf and narrow-leaf phenotype by influencing cell growth. These results provide a foundation for DNL2 gene cloning and further elucidation of the molecular mechanism of the regulation of plant height and leaf shape in maize.

Expression of the Trichoderma reesei expansin-like protein, swollenin, in poplar results in biomass with improved sugar release by enzymatic hydrolysis

Ethanol production from lignocellulosic biomass has been considered an alternative to corn-based ethanol and fossil fuel. However, the secondary cell wall structure prevents efficient degradation of cellulose. Expression of cell wall degrading enzymes has been suggested as one solution to generate suitable feedstocks for lignocellulosic ethanol production. Swollenin (SWO), originating from Trichoderma reesei, disrupts cellulose structure with low-level hydrolytic activity to the glucan chain. Previously, characterization of T. reesei SWO has been carried out in multiple microorganisms and tobacco. This study heterologously overexpressed T. reesei SWO in hybrid poplar (Populus tremula × alba), a promising feedstock for biofuel production, under the control of either the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter or the putative vascular tissue specific Subterranean Clover Stunt Virus S7 (SCSV) promoter. Transgenic poplar lines with high or moderate SWO expression level showed abnormal growth and reduced biomass production, along with increased glucose and reduced lignin contents. Increased cellulose crystallinity was observed in two transgenic events. This study also investigated the effect of in planta SWO expression on cellulose saccharification efficiency and found that transgenic events improved glucan conversion rates compared to wild type poplar. The results suggest SWO-overexpressing transgenic poplar lines could be a potential feedstock for ethanol production.

Distribution of lignin, pectins and hemicelluloses in tension wood fibers of European ash (Fraxinus excelsior)

ABSTRACT Although there is considerable information on the chemistry of gelatinous (G) layers in tension wood (TW) fibers consisting of S1 + S2 + G cell wall structure (poplar), little is known on the chemistry of G-layers in TW fibers organized with S1+ G structure. This study investigated the distribution of lignin and non-cellulosic polysaccharides in ash TW fibers (S1+ G) using histochemistry and immunolocalization methods. TW fibers studied were fully developed (mature fibers) and obtained from two (TW-1, TW-2) mature European ash trees (Fraxinus excelsior L.). Based on differences in microfibril angle (MFA) and TW trees used, TW fibers were mainly classified into three types; 1) Type-1 fibers with MFA almost parallel to the fiber axis that were found in TW-1, 2) Type-2 fibers with 12° MFA that were abundant at the end of growth rings of TW-1 and 3) Type-3 fibers with 10° MFA that were found in TW-2. The S3 layer was absent in all TW fibers. In this study, the secondary cell wall structure of Type-1 and Type-2/Type-3 fibers were defined as G and GL (gelatinous-like) layers, respectively. Lignin with syringyl (S) units was detected in G/GL-layers, in which intensity and patterns of lignin staining likely related to the difference in MFA between G- and GL-layers. With hemicelluloses, heteroxylan and heteromannan epitopes were detected in G/GL-layers but these were much less abundant than those in S2 layers of normal wood (NW) fibers. Like lignin, distribution patterns of heteromannan epitopes in G/GL-layers likely related to differences in MFA between fiber types. Sparse xyloglucan epitopes were also detected in G/GL-layers. Homogalacturonan epitopes were absent in G/GL-layers. All fiber types showed abundant a-1, 5-arabinan epitopes in G/GL-layers. Overall results indicate that the chemistry of ash TW fibers studied differs significantly from that of other species reported previously, specifically TW fibers composed of S1 + S2 + G structure.

Ginseng-derived patatin-related phospholipase PgpPLAIIIβ alters plant growth and lignification of xylem in hybrid poplars

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIβ derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIβ plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

Investigation of the structure of ramie fibers by enzymatic peeling

Ramie offers excellent wearability compared to other fibrous material; however, since the inner structure of the fibers remains largely unexplored, this excellent wearability lacks a reasonable explanation. In this study, enzymatic peeling, using xylanase and laccase coupled with PM, FLSM, and SEM, was confirmed to be an effective and efficient method to characterize the inner structure of ramie fibers. The surface of bio-degummed ramie fibers is smooth, has a fibrillar structure and contains dislocations including kinks, nodes, and scales. The transverse structure is multilayered (designated as L1, L2, and L3 layers) and has a big compressed lumen. The L1 layer is the outermost layer, which shows bright indigo fluorescent signals that indicate xylan. After removal of the L1 layer via enzymatic treatment, fibers become twisted and cracked, and the fibrillar and buckled secondary cell wall structure clarifies. Under laccase treatment, visible pores can be exposed on the secondary wall, including pit-like pores on the bulk of the cell walls and irregular pores lined at the dislocation region. L3 was identified as the innermost layer. Almost the complete L3 layer was separated from the secondary wall, using an enzymatic peeling method, and showed a closed, round, and blunt tube end and a honeycomb-like inner structure. The mesopores were filled with pectin and the surface of the tube emitted a blue fluorescent signal. The honeycomb-like innermost layer together with the pore structure of the secondary wall forms a physical basis for the cause of special wearabilities such as the shrinkage, absorbency, and scratchiness of ramie fibers.

Comprehensive transcriptome analysis of developing xylem responding to artificial bending and gravitational stimuli in Betula platyphylla.

Betula platyphylla Suk (birch) is a fast-growing woody species that is important in pulp industries and the biofuels. However, as an important pulp species, few studies had been performed on its wood formation. In the present study, we investigated the molecular responses of birch xylem to artificial bending and gravitational stimuli. After trunks of birch trees were subjected to bending for 8 weeks, the cellulose content was significantly greater in tension wood (TW) than in opposite wood (OW) or normal wood (NW), whereas the lignin content in TW was significantly lower than that in OW and NW. In addition, TW grew more rapidly than OW and generated TW-specific fibers with an additional G-layer. Three transcriptome libraries were constructed from TW, OW and NW of B. platyphylla, respectively, after the plants were subjected to artificial bending. Overall, 80,909 nonredundant unigenes with a mean size of 768 nt were assembled. Expression profiles were generated, and 9,684 genes were found to be significantly differentially expressed among the TW, OW and NW libraries. These included genes involved in secondary cell wall structure, wood composition, and cellulose or lignin biosynthesis. Our study showed that during TW formation, genes involved in cellulose synthesis were induced, while the expression of lignin synthesis-related genes decreased, resulting in increased cellulose content and decreased lignin levels in TW. In addition, fasciclin-like arabinogalactan proteins play important role in TW formation. These findings may provide important insights into wood formation at the molecular level.

Effects of nitrogen fertilization on global xylem transcript profiling of Eucalyptus urophylla x grandisevaluated by RNA-seq technology

Background Eucalyptus species are the most widely planted hardwood trees in the world representing more than 4.75 million ha in Brazil. Their high productivity, valuable wood properties and wide adaptability could allow sustainable and cost-efficient production of lignocellulosic bioenergy. The main limitation to this objective is wood recalcitrance to degradation which is linked to the structure and composition of lignified secondary cell walls. Lignin, for example, impairs the accessibility of cellulose during kraft pulping as well as during saccharification, a key step of bioethanol production. The application of nitrogen fertilizers is one strategy to increase growth rates and productivity since nitrogen is one of the most limiting nutrient for tree growth and carbon sequestration. However, the effects of nitrogen availability on wood properties and related gene expression are poorly understood. In poplar, it was recently reported that N fertilization increased aerial biomass, while in wood, fibre morphology and secondary cell wall structure and composition were modified. An increase in cellulose coupled with a decrease in lignin was observed and the mRNA profiles evaluated by microarray showed that nitrogen and tension wood have overlapping effects [1]. Moreover, a highly significant genetic correlation was observed between plant growth and lignin/cellulose composition. Quantitative trait loci co-localization identified the genomic position of potential pleiotropic regulators [2]. In order to get an insight on the regulation of nitrogen availability on wood formation in Eucalyptus, we have studied the effects of nitrogen fertilization on xylem transcriptome profiles using RNA-seq technology.

The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.

Biochemical control of xylan biosynthesis — which end is up?

Xylans are major components of land plant secondary cell walls and are required for normal plant growth and development. Secondary walls also account for the bulk of lignocellulosic biomass, a potential feedstock for large-scale production of biofuels. Glucuronoxylan and arabinoxylan affect the conversion of lignocellulosic biomass to fermentable sugar, a crucial and expensive step in biofuel production. Thus, knowledge of xylan biosynthesis may provide tools to modify secondary cell wall structure and thereby improve the bioprocessing characteristics of biomass. Recent studies have shown that glucuronoxylan structure and biosynthesis are far more complex than previously appreciated and the number of glycosyltransferases implicated in this process continues to increase. New hypotheses regarding the mechanisms of glucuronoxylan biosynthesis challenge some widely held views.

Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar

The application of nitrogen-containing fertilisers is one approach used to increase growth rates and productivity of forest tree plantations. However, the effects of nitrogen fertilisation on wood properties have not been systematically assessed. The aim of this work was to document the impacts of nitrogen fertilisation on wood formation and secondary xylem fibre properties. We used three fertilisation treatments in which the level of ammonium nitrate was adjusted to 0, 1 and 10 mM in a complete nutrient solution applied daily over a period of 28 days in standardised greenhouse experiments with clonal material of Populus trichocarpa (Torr and Gray) × deltoides (Bartr. ex Marsh). We showed that there was a short-term and repeatable response in which xylem fibre morphology and secondary cell wall structure adapt to a shift in N availability. Under high-nitrogen exposure, xylem fibres were 17% wider and 18% shorter compared to the adequate nitrogen treatment. A very significant thickening of the fibre cell walls was also observed throughout the stem of trees receiving the high-N treatment. It appeared that cell wall structure was greatly affected by the high-N treatment as fibres developed a modified inner cell wall layer. Histological observations indicated that the internal cell wall layer was enriched in cellulose and chemical determinations showed that wood contained more holocellulose. Together, these results indicate that the response of poplar to nitrogen availability may involve marked effects on secondary xylem formation.

Brittle stalk 2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls

A spontaneous maize mutant, brittle stalk-2 (bk2-ref), exhibits dramatically reduced tissue mechanical strength. Reduction in mechanical strength in the stalk tissue was highly correlated with a reduction in the amount of cellulose and an uneven deposition of secondary cell wall material in the subepidermal and perivascular sclerenchyma fibers. Cell wall accounted for two-thirds of the observed reduction in dry matter content per unit length of the mutant stalk in comparison to the wildtype stalk. Although the cell wall composition was significantly altered in the mutant in comparison to the wildtype stalks, no compensation by lignin and cell wall matrix for reduced cellulose amount was observed. We demonstrate that Bk2 encodes a Cobra-like protein that is homologous to the rice Bc1 protein. In the bk2-ref gene, a 1 kb transposon-like element is inserted in the beginning of the second exon, disrupting the open reading frame. The Bk2 gene was expressed in the stalk, husk, root, and leaf tissues, but not in the embryo, endosperm, pollen, silk, or other tissues with comparatively few or no secondary cell wall containing cells. The highest expression was in the isolated vascular bundles. In agreement with its role in secondary wall formation, the expression pattern of the Bk2 gene was very similar to that of the ZmCesA10, ZmCesA11, and ZmCesA12 genes, which are known to be involved in secondary wall formation. We have isolated an independent Mutator-tagged allele of bk2, referred to as bk2-Mu7, the phenotype of which is similar to that of the spontaneous mutant. Our results demonstrate that mutations in the Bk2 gene affect stalk strength in maize by interfering with the deposition of cellulose in the secondary cell wall in fiber cells.

Transcript profiling of Eucalyptus xylem genes during tension wood formation

Tension wood formed in response to gravitational force is a striking example of the plasticity of angiosperm wood. In this study our goal was to characterize the early changes in gene expression during tension wood formation in Eucalyptus. Using cDNA array technology, transcript profiling of 231 genes preferentially expressed in differentiating Eucalyptus xylem was followed from 6 h to 1 wk of a tension time course of artificially bent Eucalyptus trees. 196 genes were differentially regulated between control and bent trees, some exhibiting distinctive expression patterns related to changes in secondary cell wall structure and composition. For instance, expression of a cellulose synthase gene was well correlated with the appearance of the G-layers. Cluster correlation analysis revealed differential regulation of lignin biosynthetic genes and may also be used to help infer the function of unknown gene products. Eucalyptus wood transcriptome analysis during tension wood formation not only provided new clues into the transcriptional regulatory network of genes preferentially expressed in xylem, but also highlighted candidate genes responsible for the genetic and environmentally induced variation of wood quality traits.

Microfibril angle in wood of Scots pine trees ( Pinus sylvestris) after irradiation from the Chernobyl nuclear reactor accident

The secondary cell wall structure of tracheids of Scots pine ( Pinus sylvestris L.), especially the angle of microfibrils in the S 2 layer, was examined in wood deposited prior to and after the Chernobyl accident in 1986. Microscopic analysis was carried out on wood samples collected in October 1997 from breast height of three pine trees 16, 30 and 42 years old. The polluted site was located in a distance of 5 km south from the Chernobyl nuclear power plant where radioactive contamination in 1997 was 3.7 × 10 5 kBq m −2. Anatomical analysis showed that the structure of the secondary cell wall in tracheids formed after the Chernobyl accident was changed. Changes occurred both in S 2 and S 3 layers. The angle of microfibrils in S 2 layer in wood deposited after the Chernobyl accident was different in comparison to this measured in wood formed prior to the disaster. The intensity of the changes, i.e. alteration of the microfibrils angle in S 2 layer and unusual pattern of the S 3 layer, depended on the age of the tree and was most intensive in a young tree.

A Multilayered Secondary Wall in Fibres of Eucalyptus Obliqua L'hérit.

The secondary cell wall structure most commonly found in fibres and tracheids consists of three layers, as first proposed by Bailey and Kerr (1935) and recently reviewed by Wardrop (1964). The thin outermost and innermost layers, the S1 and Sa layers respectively, are characterized by the microfibrils being aligned at a large angle to the fibre axis. Between these two layers there is another, the S2layer, in which the microfibrils are aligned at a small angle to the fibre axis. These layers can be readily distinguished using a polarizing microscope, the S1 and Sa layers appearing bright and the S2 layer dark in transverse sections.