Gerald Koch

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.

Application of scanning UV microspectrophotometry to localise lignins and phenolic extractives in plant cell walls

Summary The localisation of lignin and phenolic extractives in woody tissue was determined using scanning UV microspectrophotometry. This improved cellular analytical technique enabled direct imaging of the topochemical lignin distribution within individual cell wall layers with a resolution of 0.25 μm2. Selected softwood (Picea abies), hardwood (Fagus sylvatica, Entandrophragma cylindricum, Prunus serotina) and monocotyledon (Phyllostachys edulis) sections of 1 μm thickness were scanned at a fixed wavelength and evaluated with the “APAMOS” software. This approach allowed the distribution pattern of lignins and aromatic extractives within the cell wall to be visualised simultaneously. The method was found to be ideally suited to the study of their subcellular distribution in plant cell walls.

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

BackgroundLignin 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.ResultsVascular bundles were more abundant in the rind, whereas parenchyma cells predominated in the pith region. UV measurements of untreated fiber cell walls gave absorbance spectra typical of grass lignin, with a band at 278 nm and a pronounced shoulder at 315 nm, assigned to the presence of hydroxycinnamic acids linked to lignin and/or to arabino-methylglucurono-xylans. The cell walls of vessels had the highest level of lignification, followed by those of fibers and parenchyma. Pith parenchyma cell walls were characterized by very low absorbance values at 278 nm; however, a distinct peak at 315 nm indicated that pith parenchyma cells are not extensively lignified, but contain significant amounts of hydroxycinnamic acids. Cellular UV image profiles scanned with an absorbance intensity maximum of 278 nm identified the pattern of lignin distribution in the individual cell walls, with the highest concentration occurring in the middle lamella and cell corners. Chlorite treatment caused a rapid removal of hydroxycinnamic acids from parenchyma cell walls, whereas the thicker fiber cell walls were delignified only after a long treatment duration (4 hours). Untreated pith samples were promptly hydrolyzed by cellulases, reaching 63% of cellulose conversion after 72 hours of hydrolysis, whereas untreated rind samples achieved only 20% hydrolyzation.ConclusionThe low recalcitrance of pith cells correlated with the low UV-absorbance values seen in parenchyma cells. Chlorite treatment of pith cells did not enhance cellulose conversion. By contrast, application of the same treatment to rind cells led to significant removal of hydroxycinnamic acids and lignin, resulting in marked enhancement of cellulose conversion by cellulases.

Topochemical characterisation of phenolic extractives in discoloured beechwood (Fagus sylvatica L.)

Summary The topochemical distribution of phenolic extractives in steamed and kiln-dried beechwood with discolourations was investigated on a cellular level by using scanning UV microspectrophotometry (UMSP). For the chemical characterisation of accessory compounds, acetone and methanol extracts of the discoloured beechwood were separated by accelerated solvent extraction (ASE) and analysed with high performance liquid chromatography (HPLC). The UV microscopic investigations reveal that the accessory compounds responsible for the discolouration of beechwood are mainly restricted to the longitudinal and ray parenchyma cells and the lumen of vessels. The detected extractives are characterised by high UV absorbance values and an absorbance maximum in a wavelength range between 280 and 290 nm. The separation of the acetone and methanol extracts of discoloured beechwood shows the presence of different low molecular phenols such as catechin and 2,6-dimethoxybenzochinon, which are transformed into high condensation compounds during steaming and kiln-drying.

Stiffness gradients in vascular bundles of the palm Washingtonia robusta

Palms can grow at sites exposed to high winds experiencing large dynamic wind and gust loads. Their stems represent a system of stiff fibrous elements embedded in the soft parenchymatous tissue. The proper design of the interface of the stiffening elements and the parenchyma is crucial for the functioning of the stem. The strategy of the palm to compromise between stiff fibre caps and the soft parenchymatous tissue may serve as a model system for avoiding stress discontinuities in inhomogeneous and anisotropic fibre-reinforced composite materials. We investigated the mechanical, structural and biochemical properties of the fibre caps of the palm Washingtonia robusta at different levels of hierarchy with high spatial resolution. A gradual decrease in stiffness across the fibre cap towards the surrounding parenchymatous tissue was observed. Structural adaptations at the tissue level were found in terms of changes in cell cross sections and cell wall thickness. At the cell wall level, gradients across the fibre cap were found in the degree of orientation of the microfibrils and in the lignin level and composition. The impact of these structural variations in the local material stiffness distribution is discussed.

Topochemical investigations of wood extractives and their influence on colour changes in American black cherry (Prunus serotina Borkh.)

Abstract The topochemical distribution of accessory compounds responsible for wood colouration during heartwood formation and processing of black cherry (Prunus serotina) is restricted to the axial and ray parenchyma cells. (+)-Catechin, taxifolin, aromadendrin, eriodictyol, naringenin, 4′-methoxynaringenin and prunin were identified in acetone/water extracts. However, the colour of wood after extraction is still reddish-brown, indicating that the coloured material is polymeric (cross-linked, condensed). It was demonstrated that (+)-catechin plays a pivotal role in the development of heartwood colour. Its concentration at the sapwood/heartwood boundary decreases, presumably due to the formation of non-soluble polymeric proanthocyanidins. Heat treatment of heartwood during veneer production intensifies the reddish-brown heartwood colour, probably by promoting the polymerisation of (+)-catechin and other flavonoid monomers.

Topochemical investigation on tension wood fibres of Acer spp., Fagus sylvatica L. and Quercus robur L.

Abstract Topochemical detection of lignin and phenolic compounds was carried out at subcellular level in tension wood fibres of maple (Acer spp.), beech (Fagus sylvatica) and oak (Quercus robur) by means of cellular UV-microspectrophotometry (UMSP) and confocal Raman microscopy. UMSP field scans at 278 nm revealed the presence of aromatic compounds in the gelatinous layer (G-layer) in tension wood of all three species. Characterisation of the detected substances by point measurements from 240 nm to 400 nm revealed a plausible relationship to by-products from the lignin biosynthesis pathway. Raman spectra, recorded from different regions within the G-layer, showed a high affinity to spectra of lignins. The degree of aromatic compounds in the G-layer of oak tension wood fibres was approximately three-fold higher than that in maple and beech. Moreover, the oak G-layer showed an accumulation of aromatic compounds towards the cell lumen up to 50% higher than that detected in the secondary cell wall. UMSP and Raman microscopy are considered valuable complementary methods for topochemical investigation on a subcellular level.

SeaSonal ultraStructural changeS in the cambial zone of beech (Fagus sylvatica) grown at two different altitudeS

Seasonal structural changes of cambial cells in mature beech (Fagus sylvatica L.) trees growing at elevations of 400 m a.s.l. (lowland) and 1200 m a.s.l. (mountains) are presented on the basis of light (LM) and electron microscopy (TEM). For LM, samples from trees were collected at weekly intervals and for TEM at two-month intervals from March till September, 2008. LM enabled us to follow the production of new xylem and phloem cells that lasted for 16.5 ± 3.7 weeks at the lowland site and for 10.7 ± 1.3 weeks in the mountains. TEM revealed differences in ultrastructure of cambial cells in the phases of dormancy, reactivation, activity and transition to dormancy. The seasonal patterns of ultrastructural changes in cambial cells were similar at both sites but their timing was different. TEM revealed changes in the fine structure of cambial cells, indicating their activation in spring and the earliest stages of cell divisions and development of new cell walls. When using LM, the onset of cambial activity could be observed one month later, compared with TEM. LM therefore enabled us to follow cambial productivity but not the activity and related cytoplasmic modifications during reactivation.

Topochemical investigations of cell walls in developing xylem of beech (Fagus sylvatica L.).

Abstract Formation and lignification of xylem cells in mature beech (Fagus sylvatica L.) trees growing in a forest site in Slovenia (46° N, 14°40′ E, 400 m a.s.l.) were studied on the cellular and subcellular level. Samples containing the cambial zone and developing xylem were taken from six beech trees every week throughout the 2006 vegetation period. Cell wall thickening and lignification in individual cell wall layers and cell types were determined by light microscopy, cellular UV-microspectrophotometry and transmission electron microscopy, respectively. Cell division started between the 18th and the 24th of April 2006. Lignification began in the newly formed xylem tissue on the 2nd of May. After 1 month, the developing earlywood portion contained fully differentiated vessels with completed wall deposition and lignification, and differentiated fibres and axial parenchyma became visible after 2 months. At the end of cambial cell division on the 9th August, the differentiation of the most recently formed fibres in the terminal zone of the growth ring continued for approximately 4 weeks. This indicates that the process of lignification in earlywood is slower than in latewood. The high temporal resolution of the investigated processes and the combination of the above-mentioned microscopic techniques provides a detailed insight into the process of cell wall thickening and lignification of woody tissue in beech.

Topochemical investigation on phenolic deposits in the vessels of afzelia (Afzelia spp.) and merbau (Intsia spp.) heartwood

Abstract The topochemical distribution of phenolic deposits in the vessels of afzelia (Afzelia spp.) and merbau (Intsia spp.) heartwood was investigated by means of cellular UV microspectrophotometry (UMSP) to characterise the chemical composition and synthesis by pit membrane-associated enzymes. UV absorbance spectra of the deposits attached to the vessel walls of merbau are characterised by a distinct maximum at a wavelength of 368 nm representing the UV absorbance of pure robinetin (C15H10O7). Deposits in the vessels of afzelia display a typical spectrum of kaempferol (C15H10O2) with two distinct maxima at 270 and 350 nm. The pit membranes and pit canals of associated vessel and parenchyma cells are impregnated by these compounds. These results verify the assumption that the synthesis of deposits in afzelia and merbau is regulated by pit membrane-associated enzymes.