Trees (2001) 15:503517 DOI 10.

1007/s00468-001-0126-6

O R I G I N A L A RT I C L E

Claudia Grnwald Katia Ruel Jean-Paul Joseleau Matthias Fladung

Morphology, wood structure and cell wall composition of rolC transgenic and non-transformed aspen trees
Received: 11 June 2001 / Accepted: 12 September 2001 / Published online: 9 November 2001 Springer-Verlag 2001

Abstract Morphology, wood structure and cell wall composition of 35S-rolC transgenic hybrid aspen (P. tremulatremuloides) were compared with non-transformed control trees. The transgenics are characterised by stunted growth, altered physiological parameters and light green leaves of reduced size. Histometric measurements revealed thinner fibre walls as compared to the controls. UV microspectrophotometry of individual wall layers did not reveal distinctive differences in the lignification of xylem cells, but in the extremely thin-walled fibres of the transgenics the secondary walls were less lignified as revealed by KMnO4 staining in transmission electron microscopy. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to only a few rows. Immunocytochemical analyses revealed the deposition of lignins in less differentiated xylem cells as compared to the controls. The first labelling of condensed lignin appeared in cell corners and of non-condensed lignin in secondary walls near cell corners during the deposition of S1 polysaccharides. Because of alterations in the formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful for studies on molecular factors controlling the differentiation continuum.

Keywords Hybrid aspen rolC Wood formation UV spectroscopy Transmission electron microscopy Immunolabelling

Introduction
Wood formed by the vascular cambium during secondary thickening is one of our most abundant renewable resources. Increasing pressure on land use resulted in discussion on increasing the productivity of wood farms to meet the growing demands for wood, fibre, and energy while reducing pressure on native forests (Strauss 1999). To increase the value of trees for specific purposes, such as the production of wood with reduced or altered lignin, the technique of genetic engineering is becoming increasingly important (e.g. Boudet and Grima-Pettenati 1996; Baucher et al. 1998; Hu et al. 1999; Sederoff et al. 1999; Halpin et al. 2000). However, changing wood characteristics by transfer of suitable genes requires a fundamental understanding of wood formation. Numerous papers have so far been published describing wood anatomy, wood chemistry and wood properties (e.g. Kollmann and Ct 1968; Panshin and de Zeeuw 1980; Fengel and Wegener 1984) as well as the cambium and the differentiation of xylem cells involving cell elongation and diameter increase, formation of secondary walls, lignification, deposition of secondary metabolites and autolysis of the protoplast (e.g. Catesson 1994; Larson 1994; Barnett 1995; Lachaud et al. 1999). However, most pieces of information are purely descriptive and knowledge about biosynthesis and deposition of the main cell wall components as well as biochemistry and molecular biology of cambial activity and the differentiating xylem remains rudimentary (Savidge 1996, 2000; Higuchi 1997; Chaffey 1999, 2000). Hybrid aspen (Populus tremula L. tremuloides Michx.) and other Populus species have become the model hardwood species to study the regulation of wood formation in the northern hemisphere because of their small genomes which are only three to five times larger than that of Arabidopsis (Bennett and Leitch 1997). A

C. Grnwald () Institute for Wood Biology, University of Hamburg, Leuschnerstrasse 91, 21031 Hamburg, Germany e-mail: gruenwald@holz.uni-hamburg.de Tel.: +49-40-73962444, Fax: +49-40-428912835 K. Ruel Centre de Recherches sur les Macromolecules Vgtales (CERMAV)-CNRS, BP 53, 38041 Grenoble, cedex 9, France J.-P. Joseleau Centre de Recherches sur les Macromolecules Vgtales (CERMAV)-CNRS, BP 53, 38041 Grenoble, cedex 9, France M. Fladung Institute for Forest Genetics and Forest Tree Breeding, Federal Research Centre for Forestry and Forest Products, Sieker Landstrasse 2, 22927 Grohansdorf, Germany

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large number of genes have already been discovered by the production of expressed sequence tags (ESTs) in the wood-forming tissue of hybrid aspen (e.g. Sterky et al. 1998). Hybrid aspen is fast growing, can be genetically transformed and regenerated, thus enabling analyses of gene functions in vivo (Chaffey 1999). Further, transgenic trees can be regarded as mutants showing stable secondary effects such as morphological alterations, which can be useful to characterise the molecular mechanisms responsible for these effects by comparing gene or protein expression patterns with non-transformed controls. Among the genes available for the modification of morphological traits in hardwood trees the rol genes from the T-DNA of Agrobacterium rhizogenes have attracted attention (e.g. Fladung et al. 1996; Nilsson and Olsson 1997; Tzfira et al. 1998). Our study focused on 35S-rolC transgenic aspen trees (Populus tremula L. tremuloides Michx.) characterised by dwarfed phenotype, precocious bud break/leaf development, and smaller wrinkled leaves as compared with non-transformed control trees (Fladung et al. 1996, 1997a). Transgenic plants carrying the 35S-rolC gene construct show primarily alterations in the content of cytokinins (Nilsson et al. 1993; Schmlling et al. 1993; Fladung et al. 1997a;). However, in leaves, buds, apices and stems of different 35S-rolC transgenic aspen lines the levels of different other plant hormones, such as abscisic acid (ABA) and indole-3-acetic acid (IAA), are altered as well (Fladung et al. 1997a). Wood formation of the 35S-rolC transgenic aspen trees shows stable alterations as compared with non-transformed controls, such as delayed formation of cells, the occurrence of thin-walled and less lignified fibres and the lack of typical latewood. In addition, discolorations and tyloses have been found in the roots and lower stems (Grnwald et al. 2000). The objective of our present study was to obtain deeper insights into correlations of the effects of 35S-rolC gene expression to plant morphology, wood structure, and chemistry of rolC-transgenic aspen trees. To characterise the differences in the deposition of cell wall components between transgenic and control trees, PATAg (PA = periodic acid, T = thiocarbohydrazide, Ag = silver proteinate) staining and immunolabelling of the most represented lignin sub-units in combination with transmission electron microscopy (TEM) as well as UV microspectrophotometry of individual fibre and vessel wall layers were carried out. The results are discussed in relation to morphological and structural differences between the two independent transgenic lines, and to the known insertion loci of the 35S-rolC gene construct.

rolC-#1 and Esch5:35S-rolC-#5) were available. The hybrid aspen clone Esch5 was transformed with the rolC gene under the control of the constitutive cauliflower-35S promoter as described by Fladung et al. (1996). Evidence for the integration of 35S-rolC into the aspen genome, as well as for the presence and expression of the rolC transcript, has been provided by Fladung et al. (1997b). Transgenic and control trees were cultivated in a greenhouse (5 l containers, changing natural daylight and temperature conditions with a temperature minimum during winter around 810C and maximum during summer around 2530C) as well as in the field. The greenhouse plants were watered daily and fertilised 23 times a year. Analysis of morphological data Tree height, stem diameter and leaf width to length ratios were obtained from 32 individuals of the control clone Esch5 and of the two transgenic clones Esch5:35S-rolC-#1 and Esch5:35S-rolC-#5 each. The field trial with these 96 trees was established in 1996 with 1-yearold plants of similar height. Morphological data from greenhouse grown trees have already been published (Fladung et al. 1996). Light microscopy and histometric analysis of fibre wall thickness For the light microscopy and histometric analyses stem sections of 14 individuals of the control clone Esch5, 17 individuals of Esch5:35S-rolC-#1 and 16 individuals of Esch5:35S-rolC-#5 were investigated. All the trees used in these studies were 3 years old and had been grown in a greenhouse. Several 555 mm3 samples containing bark, cambium and developing xylem were dissected from each tree at a stem height of 50 cm, immediately fixed in a phosphate-buffered solution of 37% formaldehyde, dehydrated in propanol and embedded in glycol methacrylate (Technovit 7100). A rotary microtome was used to produce 8-m-thick transversesections that were stained with a standard Giemsa solution and mounted on glass slides with Euparal. Light microscopy revealed distinctive differences between the tissue formed at the beginning of the growth ring (referred to as earlywood) and the tissue at the end of the growth ring (referred to as latewood). Therefore 100500 double fibre walls (middle lamella and secondary walls of two neighbouring fibres) of the earlywood and the latewood each were measured in transverse sections of all stems using a colour image analysis system (Olympus CUE-3, Germany). Immunocytochemistry and transmission electron microscopy Samples (114 mm3) containing bark, cambium and developing xylem were fixed in a freshly prepared mixture of 5% glutaraldehyde and 8% paraformaldehyde, washed in 0.1 M cacodylate buffer, dehydrated through a graded series of acetone and embedded in Spurrs (1969) epoxy resin. Ultrathin transverse sections were stained with KMnO4 according to Donaldson (1992) and examined with a Philips CM12 TEM at an accelerating voltage of 60 kV. PATAg staining (Thiry 1967 as modified by Ruel et al. 1977, 1984) and immunolabelling were carried out with single Esch5 and Esch5:35S-rolC-#5 trees from the greenhouse. The samples were fixed in a freshly prepared mixture of 0.2% glutaraldehyde and 4% paraformaldehyde in 0.05 M phosphate buffer (pH 77.2), rinsed in phosphate buffer, dehydrated through a graded series of ethanol, infiltrated and embedded in LR White resin (hard mixture, TAAB). Immunolabelling was done on ultrathin transverse sections (500 ) floating on plastic rings according to Ruel et al. (1999). Polyclonal lignin antibodies raised in rabbit against condensed guaiacylsyringyl (GS), non-condensed GS and condensed guiaiacyl (G) synthetic dehydrogenative polymers were used for the first incubation. The antisera were assayed for their specificity towards the different types of lignin by using an electron microscopy affinity test (Ruel et al. 1994; Joseleau and Ruel 1997). The sections were floated on each antiserum diluted 1/501/80. Protein A-gold, pA 5

Materials and methods

Plant material and growth conditions One non-transformed (control) hybrid aspen clone Populus tremula L. P. tremuloides Michx. (referred to as Esch5) and two independent 35S-rolC transgenic hybrid aspen clones Populus tremula L. P. tremuloides Michx. (referred to as Esch5:35S-

505 (Amersham) was used as a secondary marker. The gold particles were further enhanced with a silver enhancing Amersham kit. Finally the thin sections were transferred to copper grids, poststained with 2.5% aqueous uranyl acetate and examined with a Philips CM12 TEM at an accelerating voltage of 80 kV. Determination of lignin in specific cell wall layers by UV microspectrophotometry UV spectroscopic measurements were carried out on three greenhouse trees, one each of Esch5, Esch5:35S-rolC-#1 and Esch5:35S-rolC-#5. The samples containing differentiated xylem were embedded in Spurrs epoxy resin as described above. Transverse sections of 1 m thickness were mounted on quartz slides, immersed in glycerine and covered with a quartz cover slip. UV spectra were taken at wavelengths from 240 to 400 nm in 2-nm steps using a Zeiss UMSP 80 microspectrophotometer. Measurements of the lignin content within individual cell wall layers were repeated 50 times at each step for a point measured in 10 normal and gelatinous fibres as well as vessel walls each.

Results
Morphology of 35S-rolC transgenics and controls The morphological parameters of 35S-rolC transgenic aspen trees were compared with those of non-transformed control trees, both grown in the field. Tree height

Fig. 1 Morphological parameters

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was measured annually and was similar between transgenics and controls when transferred to the field in 1996 (not shown). In the following years, however, tree height revealed lower values in the transgenic lines than in nontransformed controls (Fig. 1a). Further, the dynamics of growth expressed as increase of tree height per year (Fig. 1b) was significantly different with decreasing values in the transgenic trees. Measurements of the stem diameter at 10 cm height revealed a similar tendency. Starting with the same values in 1996 when transferred to the field (not shown) stem diameter was lower in 35SrolC transgenic trees than in controls (Fig. 1c). The increase of stem diameter per year was much lower in transgenic trees with increasing values during the first 3 years but without any change in the last year (Fig. 1d). Further, the leaves of the 35S-rolC transgenics formed

Fig. 2 a Mean value and variation of double fibre wall thickness. b Frequency distribution of double fibre wall thickness

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on both shoots from 1997 and 1998 were much smaller as compared with the controls (Fig. 1e) according to measurements carried out in 1998, and also the lengthto-width ratio was different (Fig. 1f). Histometric measurements of wood fibre wall thickness of the transgenic aspen trees revealed reduced mean values and variations as compared with the non-transformed controls, even though the differences were statistically not significant (Fig. 2a). The frequency distribution of the measurements showed the lowest mean value and range between maximum and minimum in the transgenic trees of Esch5:35S-rolC-#5, followed by Esch5:35S-rolC-#1 and non-transformed trees of Esch5 which are characterised by the occurrence of double fibre walls up to 9.95 m in the earlywood and 11.75 m in the latewood (Fig. 2b). Light and electron microscopy of transverse sections containing bark, cambium and differentiating wood revealed a narrower cambium and differentiation zone in the 35S-rolC transgenics as compared with the nontransformed controls (Fig. 3). The architecture of xylem cell walls in 35S-rolC transgenic and non-transformed aspen trees was quite similar. In the normal wood, fibre and parenchyma cell walls were characterised by a sequence of middle lamella (ML), primary wall (P) and secondary walls S1 and S2. Vessel walls consisted of ML/P, S1, S2 and S3 (Fig. 4 a). Apart from the normal xylem cell wall architecture, a clear layering of fibre walls characterised as a sequence of middle lamella and primary wall, S1 and S2, and an additional gelatinous layer was observed due to the formation of tension wood. The gelatinous fibres were arranged in tangential belts or solitarily among normal fibres and occurred more often in the transgenic than in the control trees (Fig. 4b). Localisation of cell wall components in 35S-rolC transgenics and controls General staining of polysaccharides by PATAg in transmission electron microscopy General staining of polysaccharides by PATAg revealed no differences between 35S-rolC transgenics and controls except for the abundant occurrence of gelatinous fibres in the transgenic trees (Fig. 4). According to the small differentiation zone in the transgenics the deposition of wall material increased from cell to cell more rapidly than in the non-transformed controls possibly due to less frequent cambial cell divisions (Fig. 5). Thus, in the transgenics fully developed cells could be observed very close to the cambium (Fig. 5d). For instance, there was no secondary wall formation in the first differentiating cell adjacent to the cambium (Fig. 5e), whereas in the second cell a complete secondary wall and gelatinous layer could be seen in a transgenic aspen tree (Fig. 5f). In contrast, in the non-transformed controls the deposition of secondary wall material slowly increased from the first cell adjacent

to the cambium (Fig. 5b) to the sixth cell (Fig. 5c) according to their broad differentiation zone (Fig. 5a). General staining of lignins by KMnO4 in transmission electron microscopy In general, KMnO4 staining did not reveal distinctive differences in the lignification of normal and tension wood cell walls of 35S-rolC transgenics and non-transformed controls. Most fibres showed a regular fibrillar structure and lignification (Fig. 6a). Only the secondary walls of extremely thin-walled fibres occurring in the transgenics had a loose fibrillar appearance and were less lignified than the normally developed fibre tissue (Fig. 6d). The gelatinous fibres of transgenics and controls looked similar. Some of the innermost gelatinous layers were distinctly less, some slightly less stained than the adjacent S2 layer (Fig. 6c). Lignification of xylem cell walls as revealed by UV microspectrophotometry Microspectrophotometry at a cellular level gave an insight into the lignification of different fibre and vessel wall layers of transgenic and non-transformed aspen trees. No distinctive differences could be seen between the transgenic and control trees, as illustrated by the comparison of the sample spectra of one Esch5 (a), Esch5:35S-rolC-#1 (b) and Esch5:35S-rolC-#5 (c) aspen tree (Fig. 7). The spectra measured in middle lamella regions between vessels and fibres exhibited an absorption maximum at a wavelength of about 276 nm. In all fibre wall layers the absorption maximum was located at about 272 nm. All spectra taken from the same wall layer of numerous cells were characterised by synchrony. However, between 260 and 300 nm, the spectra differed in their absorption values, depending on the lignin content. Middle lamella regions between vessels and fibres showed the highest absorption levels (Fig. 7: a3, b3, c3) followed by middle lamella regions between normal (Fig. 7: a1, b1, c1) and gelatinous fibres (Fig. 7: a4, b4, c4). Outer secondary walls (adjacent to the middle lamellae) were less lignified. In all trees a tendency to slightly higher absorption levels in middle lamella regions and outer secondary walls of normal fibres as compared to gelatinous fibres could be seen. Nearly no or very low absorptions were analysed in gelatinous layers, indicating that they are only slightly lignified or not at all (Fig. 7: a6, b6, c6). Topochemistry of lignins as revealed by immunolabelling in transmission electron microscopy Figure 8 illustrates the abundance of non-condensed mixed guaiacyl-syringyl (GS) lignin sub-units in both the wood of the non-transformed control (Fig. 8a) and

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Fig. 3 Cambial differentiation zones of a non-transformed Esch5 (a) and two transgenic Esch5:35S-rolC-#1 (b) and Esch5:35S-rolC-#5 (c) aspen trees. Bar 100 m

Fig. 4 Architecture of xylem cell walls in a non-transformed Esch5 aspen tree (a) and a transgenic Esch5:35S-rolC-#5 aspen tree (b) as revealed by PATAg staining. F Fibre, GF gelatinous fibre, V vessel. Bar 1 m

the wood of the transgenic with high tension wood content (Fig. 8d). S1, S2, S3 and G-layers reacted strongly, whereas middle lamellae and cell corners did not show any labelling, indicating that this type of lignin is totally absent in these cell wall regions. Condensed GS and G lignin sub-units were less abundantly represented than non-condensed GS lignin sub-units. In the differentiated wood the labelling of both condensed GS (Fig. 8b) and

Fig. 5 Differentiating xylem of a non-transformed Esch5 aspen tree (a) and a transgenic Esch5:35S-rolC-#5 aspen tree (d) as revealed by PATAg staining (1,800). In the first cell adjacent to the cambium both control (b) and transgenic (e) do not show deposition of secondary wall polysaccharides. In the control first secondary wall polysaccharides are visible in the sixth cell from the cambium (c), whereas in the transgenic the secondary walls and gelatinous layers are already completed in the second cell from the cambium (f). Bar 5 m (a, d), 0.2 m (b, c, e, f)

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511 Fig. 7 Absorption spectra on sections of different wall layers of normal fibres (12), vessels (3) and gelatinous fibres (46) of a non-transformed Esch5 (a) and two transgenic Esch5:35SrolC-#1 (b) and Esch5:35SrolC-#5 (c) aspen trees

Fig. 6 Normal and gelatinous fibres in a non-transformed Esch5 aspen tree (ac) and a transgenic Esch5:35S-rolC-#5 aspen tree (df) as revealed by KMnO4 staining. In the transgenic there were areas of extremely thin-walled less lignified fibres (d, e) as compared with the normal situation in the controls (a, b). Gelatinous fibres looked similar in controls (c) and transgenics (f). They can be associated with normal fibres (f). F Fibre, GF gelatinous fibre, V vessel. Bar 2.5 m (a, c, d, f), 0.2 m (b, e)

Fig. 8 Topochemistry of lignin sub-units in a non-transformed Esch5 (ac) and a transgenic Esch5:35S-rolC-#5 (df) aspen tree. Immunolabelling of non-condensed GS (a, d), condensed GS (b, e) and condensed G (c, f) lignin sub-units did not show distinctive differences between the transgenic and control. F Fibre, GF gelatinous fibre, V vessel. Bar 0.5 m

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Fig. 8 Legend see page 511

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Fig. 9 Onset of lignification in a non-transformed Esch5 (a-c) and a transgenic Esch5:35S-rolC-#5 (df) aspen tree as revealed by immunolabelling of G condensed (a, d), GS condensed (b, e) and GS non-condensed (c, f) lignin sub-units. Bar 0.5 m

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condensed G lignin sub-units (Fig. 8c) was denser in vessel walls than in fibre walls, and denser in secondary walls than in cell corners and middle lamellae. Regarding the onset of lignin deposition, immunolabelling of condensed G (Fig. 9a) and condensed GS lignin sub-units (Fig. 9b) did not reveal significant differences between the transgenic and the control. The first labelling appeared in cell corners and adjacent secondary walls of very low developed cells near the cambium. In contrast, immunolabelling of non-condensed GS lignin sub-units showed the first deposition of this type of lignin in earlier stages of development in the transgenic than in the control tree (Fig. 9c). It occurred in secondary walls in the vicinity of cell corners during the deposition of S1 polysaccharides (Fig. 9f). In contrast, in the control the first labelling could be seen later, around the completion of S1 polysaccharide deposition (Fig. 9c).

non-transformed controls. Leaf metabolite investigations revealed different patterns of sucrose, fructose, glucose, starch and water content in the transgenics as compared with the controls (Fladung et al., unpublished results). However, trees with a nutritional deficit normally tend to react by reducing size and number of cells or by shortening the vegetation period, but not by reduc ufar et al., unpublished ing secondary wall thickness (C data). Therefore, it can be assumed that the reduced formation of xylem cells is a secondary effect caused by the altered morphology and physiology of the transgenic trees, while the formation of extremely thin-walled fibres is a primary effect of 35S-rolC insertion. The fibre wall thickness was reduced most in transgenic trees from Esch5:35S-rolC-#5, followed by Esch5:35S-rolC#1 and non-transformed trees from Esch5. Different hypotheses may explain the differences between the two transgenic lines: 1. Genomic regions flanking the transgene insertion locus may play a role in downstream physiological reactions (position effect) leading to changes in anatomical characteristics. However, no features of coding regions have been identified in genomic transgene flanking sequences of Esch5:35S-rolC-#1, whereas in Esch5:35S-rolC-#5 the flanking sequence reveals a gene-like structure with a probability of 0.939. 2. Differences in the organisation of the transgene insertion locus between Esch5:35S-rolC-#1 and Esch5: 35S-rolC-#5 have been found (Fladung 1999; Kumar and Fladung 2000). In Esch5:35S-rolC-#5 a single TDNA copy was detected; however, Esch5:35S-rolC#1 contains two T-DNA copies organised in one locus as an inverted repeat. 3. An unrelated genomic change during plant regeneration from single cells known as somaclonal variation cannot be totally excluded (Larkin and Scowcroft 1981). However, the transformation system used includes direct organogenesis with minor callus formation. Microscopic analyses did not reveal significant differences in the architecture of xylem cell walls between 35S-rolC transgenic and non-transformed control trees. Apart from the normal wood, the perfectly straight stems showed high contents of gelatinous fibres with the wall layers ML/P, S1, S2 and an innermost gelatinous (G-) layer as observed in other Populus species (e.g. Norberg and Meier 1966; Mia 1968; Nobuchi and Fujita 1972). The gelatinous fibres looked similar in the transgenics and controls, but light and electron microscopy revealed a more frequent occurrence in the transgenics than in the controls. The large proportion of tension wood in greenhouse trees is conspicuous because these trees do not need to sustain severe biomechanical stresses. The abundant formation of gelatinous fibres in the transgenics may be explained as follows: 1. As revealed by histological measurements, the wood of the transgenics has thinner fibre walls than the

Discussion
Morphology of 35S-rolC transgenics and controls Whereas for transgenic aspen, expressing the rolC gene under the control of its native promoter an accelerated growth and altered stem production index was reported (Tzifira et al. 1999), transgenic aspen expressing the rolC gene under the constitutive 35S promoter shows stunted growth probably due to physiological alterations, such as differences in the content of phytohormones (Fladung et al. 1997a). The field-grown transgenic aspen trees are characterised by significantly reduced increases in tree height and stem diameter as well as smaller lanceolate leaves with less chlorophyll as compared with the nontransformed controls, which corresponds well with earlier investigations of greenhouse trees (Fladung et al. 1996). These specific characteristics have been found in a large number of different independent transgenic aspen lines expressing the 35S-rolC gene; therefore position effects are very unlikely (Fladung et al. 1996, 1997a). Surprisingly, different tissues of the aspen trees, such as leaves and wood, did not show significant differences in cell size, but there was evidence for a difference in the initiation of the cambium and the formation of xylem cells (Fladung and Ahuja 1996; Grnwald et al. 2000). The cambium and differentiation zone of the non-transformed controls was broad, which is typical for fast-growing aspen trees. In the transgenics it was reduced to only a few cell rows, indicating that their dwarfism is due to a reduced frequency of cambial cell divisions and therefore a reduced number of cells. Altered levels of phytohormones, which are known to play an important role in the regulation of wood formation (e.g. Aloni 1991, 1995), as well as an altered nutrient supply may be responsible for the suppressed cambial activity in the transgenic trees. However, there was no evidence for deficiency symptoms due to nutrient limitations of the trees. Histometric measurements showed thinner fibre cell walls in the 35S-rolC transgenics in comparison to the

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wood of the controls. Therefore, the formation of gelatinous layers may be due to the reduced stability of the tissue. 2. Another reason may be water deficiency stress, which is known to initiate tension wood formation in hardwood trees (Fink 1999) as a consequence of discolorations and tyloses occurring in all transgenic but not in non-transformed control trees (Grnwald et al. 2000). 3. Further, the altered hormone levels may have directly stimulated the formation of tension wood in the transgenic trees. Localisation of cell wall components in 35S-rolC transgenics and controls In general, all qualitative and semi-quantitative methods applied did not reveal distinctive differences in the composition of xylem cell walls between 35S-rolC transgenic and non-transformed control trees. Only in extremely thin-walled fibres occurring in the transgenics did the walls exhibit a loose fibrillar appearance and were less lignified than the normal fibre tissue, indicating that a somewhat abnormal differentiation of fibres occurred. As discussed previously (Grnwald et al. 2000) this may be put down to an incomplete development of the cells formed towards the end of the vegetation period. PATAg staining showed that in the gelatinous fibres of tension wood occurring most abundantly in the transgenics the G-layers were only slightly stained. The low reactivity to PATAg strengthens the hypothesis that the G-layer is made of highly crystalline cellulose (Satiat-Jeunemaite 1986). The middle lamellae showed the highest lignification followed by secondary walls as revealed by staining with KMnO4 and UV microspectrophotomety. Gelatinous layers were only slightly or not at all lignified, corroborating observations by other authors describing the G-layer as unlignified, partially or slightly lignified (Cronshaw and Morey 1965; Ct et al. 1969; Timell 1969). The immunolabelling of condensed and non-condensed GS as well as condensed G sub-units did not reveal distinctive differences in the topochemistry of lignins between the transgenics and the controls. Most abundant were non-condensed GS lignin sub-units localised in secondary walls but not in cell corners and middle lamellae. Condensed G and GS lignin sub-units were less abundantly represented, and the highest density of labelling could be seen in vessel walls. The abundance of non-condensed GS lignin sub-units may be responsible for the maxima of UV spectra measured in fibre secondary walls at about 272 nm wavelength, though it is known that the position of the maximum depends on the syringyl/guaiacyl ratio and the condensation of methoxyl groups (Musha and Goring 1975). Syringyl residues become increasingly predominant in fibres as the MeO/C9 ratio of the lignin increases. With increasing MeO/C9 values the peak positions shift towards lower wavelengths and the intensity of absorption decreases. Fur-

ther, the peaks become less distinct due to the absorbance characteristics of p-hydroxy benzoic acid groups occurring in aspen wood distorting the syringyl and guaiacyl spectra. Vessel walls and cell corner regions contain mostly guaiacyl residues (Wolter et al. 1974; Musha and Goring 1975; Terashima 2000). This was evidenced by the UV spectra measured in middle lamella regions and vessel walls, which had their maximum at wavelengths of about 276 nm and by immunolabelling showing that these regions contain more condensed G lignin sub-units. Surprisingly, in gelatinous layers showing no or only slight UV absorption, non-condensed lignin subunits were evidenced by immunolabelling (Ruel et al., unpublished results). The discrepancy in the results may be ascribed to the difference in sensitivity of the two methods. Since the differentiation zone in the transgenics consists of fewer cell rows than that in the controls, more advanced stages of xylem differentiation are located close to the cambium. PATAg staining implied that in the transgenics the deposition of secondary wall polysaccharides starts much closer to the cambium than in the nontransformed controls. In the same manner, lignification starts earlier than in the non-transformed controls as revealed by immunolabelling of lignin sub-units. The first deposition of condensed G and GS lignin sub-units could be visualised in cell corners and middle lamellae of very low differentiated xylem cells. This corroborates observations by, for example, Takabe (1984) and Terashima (2000), who reported that lignification preferably starts in these wall regions. First significant immunolabelling of non-condensed GS lignin sub-units occurred in secondary walls near cell corners simultaneously with the completion of S1 polysaccharide deposition. In agreement with these results, labelling of peroxidases in developing aspen xylem revealed that these enzymes, which are supposed to be involved in catalysing the final step of lignification, are present in the cell wall during polysaccharide deposition (Kim et al. 2001). From the above results, it can be stated that 35S-rolC transgenic aspen trees show stable chronological differences in the formation and differentiation of cells as well as differences in the dimension of fibre walls as compared to the non-transformed controls. Hence, they can be useful for further in vivo research on wood formation. By comparing the gene or protein expression patterns of differentiating xylem in both 35S-rolC transgenic and non-transformed control trees as described by Allona et al. (1998), Sterky et al. (1998), Regan et al. (1999), Baba et al. (2000) and Van der Mijnsbrugge et al. (2000) it could be possible to identify factors that regulate the differentiation continuum.
Acknowledgements We thank the EU for financial support of two COST E20 short-term scientific missions in CERMAV, France. The authors are indebted to Prof. Dr. D. Eckstein and Dr. U. Schmitt for discussions and to C. Waitkus for supporting the photographic work.

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