Topochemical Studies on Modified Lignin Distribution in the Xylem of Poplar (Populus spp.) after Wounding

Abstract

• Background and Aims Information on the influence of wounding on lignin synthesis and distribution in differentiating xylem tissue is still scarce. The present paper provides information on cell modifications with regard to wall ultrastructure and lignin distribution on cellular and subcellular levels in poplar after wounding.

• Methods Xylem of Populus spp. close to a wound was collected and processed for light microscopy, transmission electron microscopy and cellular UV microspectrophotometry. Cell wall modification with respect to lignin distribution was examined at different stages of wound tissue development. Scanning UV microspectrophotometry and point measurements were used to determine the lignin distribution.

• Key Results Xylem fibres within a transition zone between differentiated xylem laid down prior to wounding and the tissues formed after wounding developed distinctively thickened secondary cell walls. Those modified walls and cell corners showed, on average, a higher lignin content and an inhomogeneous lignin distribution within the individual wall layers.

• Conclusions The work presented shows that wounding of the xylem may induce a modified wall architecture and lignin distribution in tissues differentiating at the time of wounding. An increasing lignin content and distinctively thickened walls can contribute to improved resistance as part of the compartmentalization process.

INTRODUCTION

In trees, wounding induces specific reactions which have been the subject of numerous investigations at macroscopic and microscopic levels (e.g. Sharon, 1973; Mullick, 1977; Shortle, 1979; Shigo, 1984; Schmitt and Liese, 1990, 1992a, b; Liese and Dujesiefken, 1996; Pearce, 2000). Depending on the depth of wounding, the reactions can be restricted to the bark or they can also extend to the cambium and xylem. The wound reactions can either occur close to the wound or affect larger portions of tissue adjacent to the wound. This response depends on the extent of wounding, the season and tree vitality (Schmitt and Liese, 1992a). Lesions affecting the cambial region often lead to the formation of a wound callus and subsequently modified xylem and phloem (e.g. Liese and Dujesiefken, 1989; Fink, 1999; Grünwald et al., 2002). Within the xylem a combination of different reactions taking place compartmentalizes the inflicted area (Sharon, 1973; Shigo and Marx, 1977; Bauch et al., 1980; Rademacher et al., 1984; Shigo, 1984; Lowerts et al., 1986; Liese and Dujesiefken, 1996). The macro- and microscopically most prominent mechanism involved in compartmentalization is the formation of a so called boundary layer. This layer is mainly characterized by the synthesis of phenolic compounds in parenchyma cells with their ensuing extrusion into neighbouring cells and vessel occlusions (e.g. Schmitt and Liese, 1990; Pearce, 2000).

Research on wound reactions in poplar has been mostly carried out by conventional light microscopy (e.g. Buntrock, 1989). Earlier studies mainly focused on wound reactions of the bark, such as the formation of the ligno-suberized zone, of new periderms, and new bark (Kaufert, 1937; Soe, 1959; Trockenbrodt and Liese, 1991). The influence of pressure on tissue differentiation on longitudinal bark strips, separated from the bole, has been examined in detail (Brown and Sax, 1962). Bucciarelli et al. (1999) compared, over a 96-h period, the wound responses of the bark of resistant and susceptible Populus tremuloides genotypes, inoculated with Entoleuca mammata, using histochemical and microspectrophotometric analyses.

The objective of the present study was to provide detailed information on the modification of xylem elements within a transition zone between differentiated xylem laid down prior to wounding and the tissues laid down after wounding. The xylem elements in this transition zone often develop distinct wall thickenings. A particular emphasis is given to the lignin distribution on a cellular and subcellular level. For this purpose, the unaffected xylem and modified xylem were investigated by transmission electron microscopy (TEM) and cellular UV microspectrophotometry.

MATERIALS AND METHODS

Investigations were carried out with four 25-year-old Populus tremula L. × Populus tremuloides Michx. trees and one 35-year-old Populus tremula L. tree, growing in the east of Hamburg. Rectangular wounds of 10 × 10 cm² were set on 7 Jul. 2002 and 23 Jun. 2003 by removing the bark from the stems using a saw and a chisel. The edges of the debarked areas were smoothed and the wound surfaces scraped with a razor blade to remove remnants of the cambium and the differentiating xylem. The trees were wounded at stem heights between 50 and 100 cm and the wounds were facing different directions.

For all microscopic work, samples were collected from the lateral wound edge 2, 4, 10, 17, 62 and 95 weeks after wounding. Callus tissue and the adjacent modified xylem were removed with chisel and razor blades. Unaffected xylem of the same poplar trees served as reference material.

Light microscopy

For light microscopy, samples were cut into 10 × 10 × 8 mm³ pieces, fixed for 3 d in 4 % buffered formol (Lillie, 1954) washed in distilled water, and embedded in polyethylene-glycol. Transverse sections, 10 μm thick, were cut with a sliding microtome, stained with a standard safranine/astra blue solution and mounted onto glass slides.

Transmission electron microscopy

For TEM, the samples from the same wounds as used for light microscopy were cut to a final size of 3 × 3 × 8 mm³ (Fig. 1), fixed for 1 d in a buffered mixture of glutaraldehyde and paraformaldehyde according to Karnovsky (1965), partly postfixed with a 1% osmium tetroxide solution, washed in 0·1 m cacodylate buffer (pH 7·3), serially dehydrated in a graded series of acetone and embedded in Spurr's epoxy resin (Spurr, 1969).

Fig. 1.

Diagram of a transverse section through a lateral wound callus showing the area containing thick-walled xylem fibres (hatching) within the transition zone (arrows) and the position of tissue sampling for TEM and UV microspectrophotometry.

Ultrathin (80–100 nm) transverse sections were cut with diamond knives, placed on copper grids and either double-stained with uranyl acetate and lead citrate or with potassium permanganate. The samples were examined with a Philips CM 12 TEM at an accelerating voltage of 60 or 80 kV.

UV microspectrophotometry

Samples for UV microspectrophotometry were prepared in the same way as for TEM. Semi-thin (1-μm) transverse sections were cut on an ultramicrotome using a diamond knife. The semi-thin sections were transferred to quartz slides, immersed in a drop of non-UV absorbing glycerine and covered with a quartz cover slip. For the microscopic investigations the ultrafluar objectives 32 : 1 and 100 : 1 were used.

Scanning UV microspectrophotometry was carried out using a ZEISS UMSP 80 microspectrophotometer equipped with an Osram high-pressure xenon lamp, an ultrafluar quartz condenser and a scanning stage enabling the determination of image profiles at a constant wavelength of 280 nm using the scan program APAMOS (Automatic-Photometric-Analysis of Microscopic Objects by Scanning, Zeiss). This wavelength represents the typical absorbance maximum of lignified cell walls. The scan program digitizes rectangular tissue portions with a local geometrical resolution of 0.25 μm² and a photometrical resolution of 4096 grey scale levels, converted into 14 basic colours representing the measured absorbance intensities (Koch and Kleist, 2001).

The specimens were also analysed by UV microspectrophotometry point measurements with a spot size of 1 μm². UV-spectra were taken at wavelengths from 240 to 400 nm in 2-nm steps using the program LAMWIN (Zeiss) (Takabe, 2002). These point measurements, which were used for a semi-quantitative determination of lignin content, were automatically repeated 50 times at each spot for individual wall layers: (a) compound middle lamella, (b) S2 layers of the secondary wall and (c) cell corners.

RESULTS

Light microscopy revealed that wounding induced the formation of xylem cells with modified characteristics in the transition zone between the xylem laid down prior to wounding and the tissues laid down after wounding (Figs 1 and 2). Within this zone the xylem cells were still differentiating at the time of wounding. The formation of distinctively thick-walled fibres was the most prominent structural pattern within this zone. Electron microscopy showed that these fibres deposited additional secondary wall (S2) material leading to extremely thick walls compared with regular xylem cells (Figs 3–5). This wound-induced reaction resulted in three modes of wall-thickening. One group of xylem fibres showed the presence of a supplementary S2 layer (Fig. 3); the second group of fibres developed a continuously thickened secondary cell wall (Fig. 4) and in the third, smaller group, fibre walls developed a sclereid-like sublayering (Fig. 5). Cell corners within the transition zone predominantly consisted of material with a high electron density, which is unusual for poplar (Fig. 4).

Fig. 2.

Transverse section (light microscopy) through a wound callus and the modified xylem that is located within a transition zone between xylem laid down prior to and after wounding containing fibres with wound-induced wall thickenings (arrows). Scale bar = 10 mm.

Fig. 3.

Transverse section (TEM) through a modified xylem fibre with very thick, lamellate secondary wall (arrows), ray parenchyma cell (RP). Scale bar = 5 μm. Duration of wound response 10 weeks.

Fig. 4.

Transverse section (TEM) through modified xylem of the transition zone. Fibres with continuous secondary cell wall thickenings (without lamellation); cell corners contain material with extremely high electron density (arrow). Scale bar = 2.5 μm. Duration of wound response 4 weeks.

Fig. 5.

Transverse section (TEM) through wound-induced fibres with a distinct polylamellate structure of the secondary wall (arrows). Scale bar = 2.5 μm. Duration of wound response 4 weeks.

To determine the lignin distribution within different wall layers of unmodified and modified tissues, these were first analysed by scanning UV microspectrophotometry at a constant wavelength (λ) of 280 nm. Figures 6 and 7 show representative two- and three-dimensional (2D and 3D, respectively) UV image profiles of unaffected mature secondary poplar xylem. Figure 6 shows the distribution pattern of lignin in earlywood fibres and vessel elements characterized by thin S2 wall layers of relatively low uniform absorbance values (Abs_280nm 0·09–0·16) and increasing values in the compound middle lamella regions (Abs_280nm 0·16–0·23). The highest absorbencies of Abs_280nm 0·35–0·67 were found in some cell corners of vessel elements. Terminal latewood fibres (Fig. 7) showed slightly higher absorbance values especially in the compound middle lamella (Abs_280nm 0.23–0.35), as compared with earlywood. The broader S2 layer revealed absorbance values of Abs_280nm 0·09–0·23, the cell corners of Abs_280nm 0·35–0·67.

Fig. 6.

UV micrograph and a 3D UV microscopic scanning profile of unaffected xylem tissue. The coloured pixels represent different UV-absorbance values within the cell wall layers of vessel elements (V) and fibres (F) at 280 nm wavelength.

Fig. 7.

UV micrograph and a 3D image profile of unaffected latewood fibres (F) at the annual ring border.

The influence of lesions on the lignin distribution within walls of modified xylem cells of the transition zone is presented in Figs 8–11. As early as 4 weeks after wounding, many fibres and vessel elements displayed different stages of secondary wall thickening (Fig. 8). The secondary wall of the scanned modified fibres showed microscopically invisible concentric sublayers with increasing absorbance values from Abs_280nm 0·09 to 0·48. The lowest values always occurred in the wall regions adjacent to lumen. High absorbance values were commonly recorded in the compound middle lamella (Abs_280nm 0·55–0·68) between fibres and also in the cell corners (Abs_280nm 0·81–0·94). The absorbance values of vessel walls also indicated a heterogeneous lignin distribution with unusually high values of Abs_280nm 0·67 up to 0·87 in the compound middle lamella between vessels and neighbouring fibres (Fig. 8).

Fig. 8.

UV micrograph and a 3D image profile of a small vessel element (V) and fibres (F) in the transition zone 4 weeks after wounding.

Fig. 9.

UV micrograph and a 3D image profile of fibres showing strongly developed secondary wall thickenings within a band of fibres close to the former wound surface, 62 weeks after wounding. Some fibre lumina are filled with phenolic compounds (arrow).

Fig. 10.

UV micrograph and a 3D image profile of fibres (F) and vessel elements (V), close to the wound surface, 95 weeks after wounding.

Fig. 11.

UV micrograph and a 3D image profile of radially flattened fibres with secondary wall thickenings and ray parenchyma cells (Rp), some of them filled with phenolic compounds (arrow), 95 weeks after wounding.

Figure 8 displays the lignin distribution in modified fibres with extremely thickened secondary walls close to a wound surface after 62 weeks of response. The lumina in most of these fibres were extremely reduced by the thickened wall and in some cases the remaining lumina were filled with strongly UV-absorbing deposits (Fig. 9). The inhomogeneously distributed absorbance values within the thickened S2 varied between Abs_280nm 0·09 and increased maxima of Abs_280nm 0·42, both within the S2 of an individual fibre and between fibres. Compound middle lamella absorbance values ranged from Abs_280nm 0·61 to 0·81 and in cell corners from Abs_280nm 0·81 up to Abs_280nm over 0·94.

Figures 10 and 11 show the lignin distribution patterns for vessel elements, fibres and ray parenchyma cells within the transition zone after nearly 2 years of wound response. The image profiles display distinctly high absorbance values across the entire thickened walls of fibres and vessels (Fig. 10). The S2 layer of vessels showed absorbance values between Abs_280nm 0·23 and Abs_280nm 0·42. For compound middle lamella regions UV absorbance ranged from Abs_280nm 0·48 to 0·68 and for the cell corners from Abs_280nm 0·55 to 0·94. The absorbance values of the S2 in fibre walls varied between Abs_280nm 0·16 and Abs_280nm 0·35. The distribution of the values was inhomogeneous. The absorbance values for the compound middle lamella and the cell corners were Abs_280nm 0·23–0·55 and Abs_280nm 0·42–0·94, respectively (Figs 10 and 11).

To characterize the UV-absorbance behaviour of previously scanned tissue portions, point measurements with a spot size of 1 μm and a wavelength between 240 and 400 nm were carried out. In Fig. 12, representative UV absorbance spectra of unaffected and modified fibre wall layers are shown. Those point measurements of wall layers from unaffected early- and latewood fibres were characterized by mean absorption maxima at a wavelength of 270/272 nm for the S2 and the compound middle lamella, and of 278 nm for the cell corners (Fig. 12A). In distinctively thickened secondary wall regions of modified fibres, absorption maxima at wavelengths between 272 and 274 nm were regularly detected. The spectra taken in middle lamella regions of modified fibres also shifted towards higher wavelengths of 274–276 nm when compared with unaffected fibres. The absorbance maxima of cell corner regions of transition zone fibres with wavelengths of 276–280 nm were not notably different from cell corners of fibres from the unaffected references (Fig. 12B–F).

Fig. 12.

Representative UV absorbance spectra of fibre cell wall layers of unaffected controls (A) and after different periods of wound response (B–F); (B) 2, (C) 4, (D) 17, (E) 62 and (F) 95 weeks after wounding. Vertical line indicates the 272 nm wavelength.

The ratio of UV absorbance at 280 nm to that at 260 nm (A₂₈₀ : A₂₆₀) for the S2 layer, compound middle lamella and cell corner areas of the modified tissue and the reference material are listed in Table 1. This ratio combined with the characteristic wavelength of the lignin absorption maxima (Fig. 12) can be used as an indicator for the proportion of the different lignin moieties and p-hydroxy benzoic acid residues associated with them.

Table 1.

Ultraviolet absorbance ratio A₂₈₀ : A₂₆₀ of different cell wall layers

	A280 : A260
Sample	S2 layer	Compound middle lamella	Cell corner region
Unaffected fibres	1·02	1·09	1·29
Affected fibres after 2 weeks of wound response	1·20	1·25	1·29
Affected fibres after 4 weeks of wound response	1·18	1·20	1·25
Affected fibres after 17 weeks of wound response	1·20	1·29	1·26
Affected fibres after 62 weeks of wound response	1·16	1·19	1·25
Affected fibres after 95 weeks of wound response	1·34	1·33	1·33

The spectra of the detected accessory phenolic extractives in fibres and ray parenchyma cells had much higher absorbance values (Abs_280nm 1·38) than cell wall lignins. Furthermore, their absorbance maxima displayed a bathochromic shift to a wavelength of 284–286 nm and a slower decrease of the absorbance, when compared with lignin.

The ratios (A₂₈₀ : A₂₆₀) of S2 and compound middle lamella regions from transition zone fibres with thickened walls were higher when compared with unmodified fibre walls. For cell corner regions of affected tissue portions no such trend for the ratio (A₂₈₀ : A₂₆₀) could be obtained, because the values were slightly higher and also lower as in the references.

In summary, the microscopic analyses showed that xylem fibres close to a wound and within a transition zone between differentiated xylem laid down prior to and tissue laid down after wounding developed a distinctively thicker secondary wall. These modified fibre walls regularly showed a higher lignin content and an inhomogeneous lignin distribution in the middle lamella and the secondary wall.

DISCUSSION

Microscopy

Light and electron microscopy showed that cells that were differentiating at the time of wounding were affected in the deposition of wall material. These cells were localized in a transition zone consisting of differentiating xylem cells at the time of wounding. Depending on their stage of differentiation at the time of wounding the different cell types respond in a distinctive manner. Within phloem tissue the wound-induced formation of modified fibres and sclereids as well as the lignification of inflicted areas is commonly reported (e.g. Soe, 1959; Brown and Sax, 1962; Mullick, 1977; Trockenbrodt and Liese, 1991; Bucciarelli et al., 1999; Fink, 1999). The formation of a tangential band of thick-walled, flattened xylem fibres within a zone between the modified and regular xylem after wounding through ‘pinning’, was observed in beech wood by Schmitt et al. (2000). A distinctive feature of wound-associated wood of yellow poplar (Liriodendron tulipifera) is the presence of sclereids (Lowerts et al., 1986). It is assumed for poplar that besides modified fibres with a lamellar S2 some cells with a less axial extension and a sublayering within the S2 are sclereids. These structural alterations within the existing tissue, as observed in poplar and also in beech and yellow poplar xylem (Lowerts et al., 1986, Schmitt et al., 1999), supplement in some cases the well-known compartmentalization processes in these species (e.g. Shigo and Marx, 1977; Shortle, 1979; Shigo, 1984; Buntrock, 1989; Liese and Dujesiefken, 1996).

Topochemical characterization

UV microspectrophotometry has proven to be a reliable technique to study lignin distribution and its semiquantitative determination under various conditions and in different tree species (e.g. Bland and Hillis, 1969; Scott et al., 1969; Fergus and Goring, 1970a, b; Musha and Goring, 1975; Bauch et al., 1976; Fukazawa, 1992; Okuyama et al., 1998; Bucciarelli et al., 1999; Grünwald et al., 2001, 2002a, b; Koch and Kleist, 2001; Takabe, 2002; Koch and Grünwald, 2004). The following discussion is based on the observed UV absorbance behaviour of tissues in response to wounding.

Both guaiacyl and syringyl model compounds show a minimum absorbance at wavelengths from 250 to 260 nm and a maximum from 270 to 280 nm which is characteristic for hardwood lignin. This is in contrast to a strong maximum absorbance of p-hydroxy benzoic acid residues at 260 nm. With an increasing guaiacyl lignin content, the absorbance maximum shifts closer to 280 nm (Fergus and Goring, 1970a, b; Musha and Goring, 1975; Terashima et al., 1986b; Fukazawa, 1992; Koch and Kleist, 2001; Takabe, 2002).

The spectra obtained in the present study for thin S2 layers in unaffected fibres of poplar with a relative low absorbance maximum at 270–272 nm and a less distinct peak correspond to spectra obtained in earlier studies (Fergus and Goring, 1970a, b; Musha and Goring, 1975; Fukazawa, 1992), although the ratio A₂₈₀ : A₂₆₀ is slightly higher than detected in other poplar species. Thus, the measurements indicate a low content of predominant syringyl-type lignin associated with p-hydroxy benzoic acid residues. The relatively low lignin content in the S2 layer, as compared with compound middle lamella and cell corner regions, as well as the uniform distribution within this layer, are in good agreement with general concepts for cell wall compositions of hardwoods (e.g. Fergus and Goring, 1970a, b; Musha and Goring, 1975; Terashima et al., 1986a, b, 1993; Donaldson, 2001; Donaldson et al., 2001; Koch and Kleist, 2001; Grünwald et al., 2002a; Takabe, 2002). The peak absorbance at 272 nm in fibre secondary walls and the only slightly higher absorbance of the compound middle lamella indicate increased lignin content with a nearly unmodified lignin composition. Grünwald et al. (2002a) reported alterations in the lignin composition in middle lamella regions of xylem fibres with increasing distance from the cambium, as indicated by a shift of the peak absorbance towards 272 nm at the end of cell differentiation due to an increasing amount of syringyl and p-hydroxyphenyl subunits. More frequently, however, a higher relative abundance of guaiacyl lignin moieties in hardwood middle lamellae has been reported (e.g. Fergus and Goring, 1970b; Musha and Goring, 1975; Terashima, 1986a, b, 1993). The present scannings of middle lamella regions showed a more speckled heterogeneous lignin distribution, whereas in Hevea brasiliensis the intercorner and cell corner middle lamella showed a mostly mottled lignin distribution, as observed by Singh and Schmitt (2000). The spectra obtained for cell corner regions showed the highest lignin concentrations of predominantly guaiacyl lignin, which is in good agreement with general lignification concepts for soft- and hardwoods (e.g. Fergus and Goring, 1970b; Terashima et al., 1986b, 1993; Fukazawa, 1992; Donaldson, 2001; Donaldson et al., 2001).

The spectra obtained from cells located in the transition zone showed that wounding may induce modification in lignin composition and distribution. These alterations were mainly restricted to secondary wall and compound middle lamella regions, whereas cell corner lignin remained nearly unaffected. Within thickened secondary walls the average peak absorbance shifted slightly towards higher wavelength and the ratio A₂₈₀ : A₂₆₀ simultaneously increased, verifying a higher amount of guaiacyl moieties and a reduced amount of associated p-hydroxy benzoic acid residues. The same effect was evidenced by UV spectra from middle lamella regions. More detailed information on the microdistribution of lignin was provided by UV scannings which also displayed elevated lignin content with highly variable maximum values in fibre and vessel walls. It was found that at an early stage of wound response the lignin content increases in concentric sublayers within the S2-layer. It is more likely that this distribution resembles phloem sclereids in hardwoods (authors' observations) as well as in bamboo fibres as shown by Koch and Kleist (2001). Normal hardwood fibres have a uniform lignin distribution across the entire S2 layer (Saka and Goring, 1988; Koch and Kleist, 2001). During later stages of wound response the lignin distribution within the secondary wall appeared more inhomogeneous. The lignin content in compound middle lamella and cell corner regions, however, was also significantly higher in late stages of wound response.

As mentioned before, the syringyl : guaiacyl ratio and the condensation of methoxyl groups determines the position of the absorbance maximum and also the absorbance intensity. With decreasing MeO/C₉ values, the peak position shifts towards higher wavelength and the intensity of absorbance increases, whereas the overall lignin concentration may still be constant (Musha and Goring, 1975). This phenomenon therefore restricts semiquantitative interpretations of maxima differences between inflicted and unaffected cells.

From these results obtained for poplar, it can be stated that wounding induces an increased wall thickness and a modified lignin topochemistry in xylem fibres differentiating at the time of wounding. It is assumed that this wound response is part of the compartmentalization and adds a further mechanism contributing to an increased resistance.