Lloyd Donaldson

Restricted access of proteins to mannan polysaccharides in intact plant cell walls

How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.

Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata.

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a “bonsai tree-like” appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated β-aryl ether (β-O-4 unit) and spirodienone (β-1) levels, accompanied by lower phenylcoumaran (β-5), resinol (β-β), and dibenzodioxocin (5-5/β-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.

Lignin distribution in coppice poplar, linseed and wheat straw

Summary Lignin distribution was determined by interference microscopy, and by confocal laser scanning microscopy (CLSM) for a range of agricultural residues including coppice poplar, linseed, and wheat straw. Interference microscopy was used to determine the lignin concentration in the middle lamella at the cell corner, and for the secondary wall of libriform fibres in the secondary xylem of poplar and linseed. Wheat was examined in the same way for cortical fibres. In addition the secondary wall of vessel elements was examined for poplar. Confocal microscopy was used to confirm the results from interference microscopy by providing semiquantitative information based on lignin autofluorescence, and by staining with acriflavine. Wheat had the lowest level of lignification, with 31 % lignin in the middle lamella of cortical fibres and 9% lignin in the secondary wall. Poplar had a lignin concentration of 63% in the middle lamella and 6% in the secondary wall of libriform fibres, while linseed had corresponding values of 69 % and 13 %. The secondary wall of poplar vessel elements had a lignin concentration of 25 %. In all three species most of the stem tissue was lignified except for phloem and bark, where present. In linseed the pith was unlignified. In wheat, most of the parenchyma cells were lignified except for a few cells lining the stem cavity. Libriform fibres in poplar and linseed sometimes had an unlignified gelatinous layer in samples containing tension wood. In linseed, lignification was greater in xylem fibres compared to bast fibres. Ray parenchyma cells of poplar and linseed appeared to be lignified to the same extent as xylem fibres.

Localization of Cell Wall Polysaccharides in Normal and Compression Wood of Radiata Pine: Relationships with Lignification and Microfibril Orientation

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.

Quantification of compression wood severity in tracheids of Pinus radiata D. Don using confocal fluorescence imaging and spectral deconvolution.

Confocal fluorescence microscopy was used to examine the spectral characteristics of lignin autofluorescence in secondary cell walls of normal and compression wood from Pinus radiata. Using UV excitation, fluorescence spectra of normal and compression wood sections showed significant differences, especially in the outer secondary cell wall of tracheids, with a shift in maxima from violet to blue wavelengths between normal and compression wood. A comparison of normal wood, mild and severe compression wood, showed that the wavelength shift was intermediate in the mild compression wood compared to the severe compression wood, thus offering the possibility of quantifying the severity by measuring ratios of fluorescence at violet and blue wavelengths. Fluorescence induced by blue light, rather than UV, was less well differentiated amongst wood types. Spectral deconvolution indicated the presence of a minimum of five discrete lignin fluorophores in the cell walls of both normal and compression wood tracheids. Comparison with lignin model compounds suggest that the wavelength shift may correspond in part to increased levels of p-hydroxy type lignin in the compression wood samples. The combination of confocal fluorescence imaging and related spectral deconvolution therefore offers a novel technique for characterising cell wall lignin in situ.

Wood Formation from the Base to the Crown in Pinus Radiata: Gradients of Tracheid Wall Thickness, Wood Density, Radial Growth Rate and Gene Expression

Wood formation was investigated at five heights along the bole for two unrelated trees of Pinus radiata. Both trees showed clear gradients in wood properties from the base to the crown. Cambial cells at the base of the tree were dividing 3.3-fold slower than those at the crown, while the average thickness of cell walls in wood was highest at the base. Cell wall thickness showed an overall correlation coefficient of >0.7 with wood density in both genotypes. Microscopic examination of developing tracheids showed that 33% of cells had formed secondary cell walls at the base of the tree, reducing to 3% at the crown. In total, 455 genes differentially expressed in developing xylem tissue from either the base or the crown were identified using modified differential display. RT-PCR analysis of 156 genes confirmed differential expression for 77%. Of the genes tested, 73% showed gradients in transcript abundance either up or down the bole of the tree, although the steepness of the gradients differed between genes. Genes involved in cell division and expansion tended to be more highly expressed in the crown of the tree, and two putative cell-cycle repressor genes were expressed 2-fold higher at the base. Conversely, transcripts of genes involved in secondary wall thickening were more abundant at the base of the tree. These results suggest that differences in the rate of cambial cell division, differences in the rate and duration of tracheid wall thickening, and differences in gene expression underpin the gradients of wood properties found in pines.

Fluorescence lifetime imaging of lignin autofluorescence in normal and compression wood

Wood cell walls fluoresce as a result of UV and visible light excitation due to the presence of lignin. Fluorescence spectroscopy has revealed characteristic spectral differences in various wood types, notably normal and compression wood. In order to extend this method of characterising cell walls we examined the fluorescence lifetime of wood cell walls using TCSPC (Time‐Correlated Single Photon Counting) as a method of potentially detecting differences in lignin composition and measuring the molecular environment within cell walls. The fluorescence decay curves of both normal and compression wood from pine contain three exponential decay components with a mean lifetime of τm = 473 ps in normal wood and 418 ps in compression wood. Lifetimes are spatially resolved to different cell wall layers or cell types where individual lifetimes are shown to have a log‐normal distribution. The differences in fluorescence lifetime observed in pine compression wood compared to normal wood, are associated with known differences in cell wall composition such as increased p‐hydroxyphenyl content in lignin as well as novel deposition of β(1,4)‐Galactan. Our results indicate increased deposition of lignin fluorophores with shorter lifetimes in the outer secondary wall of compression wood. We have demonstrated the usefulness of fluorescence lifetime imaging for characterising wood cell walls, offering some advantages over conventional fluorescence imaging/spectroscopy. For example, we have measured significant changes in fluorescence lifetime resulting from changes to lignin composition as a result of compression wood formation that complement similar changes in fluorescence intensity.

Quantitative chemical indicators to assess the gradation of compression wood

Abstract A chemistry-based parameter has been sought for determining the gradation of compression wood (CW), i.e., the severity, in tissues of Pinus radiata wood. Fluorescence microscopy was the reference for characterisation of the tissues containing CW. The collected material contained CW of varying severity, beginning with normal wood (NW containing no CW), continuing with material with some features of CW (CW of mild severity, MCW) and ending up with a material with pronounced features of CW (CW of high severity, SCW). Matching opposite wood (OW) was also included in the study. The chemical analyses included lignin determination, sugar analysis in the acid hydrolysate, thioacidolysis, 31P-NMR spectroscopic analysis and steric exclusion chromatography of thioacidolysis products. As the severity of CW changed progressively from NW through MCW to SCW, all chemical parameters changed concurrently. In particular, levels of galactose and lignin increased, while those of glucose and mannose decreased. The amounts of p-hydroxyphenyl β-ethers released by chemical degradation and uncondensed p-hydroxyphenyl C-9 units also increased at elevated CW severity levels. The amounts of galactose and the p-hydroxyphenyl content of the lignin correlated linearly with lignin for CW samples. The chemical differences between CW and OW in the stem, branch and seedling were similar, i.e., they are independent of the morphological origin of the sample. Parameters based on the p-hydroxyphenyl unit content appear the most suitable chemical indicators of CW severity, as they are least sensitive to the samples morphological origin and their response to CW severity is high.

Visualization of plant cell wall lignification using fluorescence‐tagged monolignols

Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development.

Suppression of CCR impacts metabolite profile and cell wall composition in Pinus radiata tracheary elements

Suppression of the lignin-related gene cinnamoyl-CoA reductase (CCR) in the Pinus radiata tracheary element (TE) system impacted both the metabolite profile and the cell wall matrix in CCR-RNAi lines. UPLC–MS/MS-based metabolite profiling identified elevated levels of p-coumaroyl hexose, caffeic acid hexoside and ferulic acid hexoside in CCR-RNAi lines, indicating a redirection of metabolite flow within phenylpropanoid metabolism. Dilignols derived from coniferyl alcohol such as G(8-5)G, G(8-O-4)G and isodihydrodehydrodiconiferyl alcohol (IDDDC) were substantially depleted, providing evidence for CCR’s involvement in coniferyl alcohol biosynthesis. Severe CCR suppression almost halved lignin content in TEs based on a depletion of both H-type and G-type lignin, providing evidence for CCR’s involvement in the biosynthesis of both lignin types. 2D-NMR studies revealed minor changes in the H:G-ratio and consequently a largely unchanged interunit linkage distribution in the lignin polymer. However, unusual cell wall components including ferulate and unsaturated fatty acids were identified in TEs by thioacidolysis, pyrolysis-GC/MS and/or 2D-NMR in CCR-RNAi lines, providing new insights into the consequences of CCR suppression in pine. Interestingly, CCR suppression substantially promoted pyrolytic breakdown of cell wall polysaccharides, a phenotype most likely caused by the incorporation of acidic compounds into the cell wall matrix in CCR-RNAi lines.