JIPB
Journal of Integrative Plant Biology
Free Access
Research Article
Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.) €nni3, Tuomas H€anninen4, Marko Mononen1, Haiqing Ren2, Seppo Andersson1*, Yurong Wang2, Raili Po 1 5 Ritva Serimaa and Pekka Saranp€a€a 1
Department of Physics, University of Helsinki, 00560 Helsinki, Finland, 2Research Institute of Wood Industry, Chinese Academy of Forestry, 100091, Beijing, China, 3Department of Forest Products Technology, Aalto University, School of Chemical Technology, 00076 Aalto, Finland, 4 VTT Technical Research Centre of Finland, 02044 Espoo, Finland, 5Natural Resources Institute Finland, 01300 Vantaa, Finland. *Correspondence: seppo.andersson@helsinki.fi
Abstract We studied in detail the mean microfibril angle and the width of cellulose crystals from the pith to the bark of a 15year-old Maidenhair tree (Ginkgo biloba L.). The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using Xray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean microfibril angle, varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The microfibril angle
remained large over the 14 annual rings. The entire stem disc, with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.
INTRODUCTION
cycads and conifers. A similar conclusion was reached by Gong et al. (2009) based on the chemical composition of G. biloba wood. They also reported a higher lignin content in G. biloba than in other conifers. Timell (1986) demonstrated that the tracheid walls of G. biloba are morphologically similar to those of representative conifers. However, according to Timell (1978), the compression wood of G. biloba differs from the more recently evolved gymnosperms in the more angular shape of the cells, thinner cell walls, and lack of helical cavities. Burgert et al. (2004) compared the micromechanical properties of thin compression wood foils and isolated tracheids of four gymnosperm species (Ginkgo biloba L., Taxus baccata L., Juniperus virginiana L., and Picea abies (L.) Karst.). The compression wood of G. biloba was found to be stiffer than that of the other species, and the authors consequently concluded that there has been an evolutionary trend towards much more flexible compression wood. Properties of wood depend on the assembly mode of cellulose, other polysaccharides, and lignin in the cell wall. The plant cell wall provides the shape and mechanical stability of the cell, but also has a role in intercellular communication and defense against potential pathogens (Keegstra 2010). In the secondary cell walls of xylem cells, the cell wall typically
The Maidenhair tree (Ginkgo biloba L.) is a unique living species of the ancient lineage of Ginkgophyta (Zhou and Zheng 2003). Fossil studies have revealed that Ginkgoales originated in the Paleozoic Carboniferous period and flourished during the Jurassic period, comprising a diverse and widespread group, and it may be considered as a living fossil. Ginkgo biloba may be even older, because fossils displaying similar vegetative morphology have been found as early as the Permian period (Rothwell and Holt 1997). Major (1967) suggested that G. biloba has persisted unchanged over many millions of years because it has adapted to its environment and is resistant to various pests, such as bacteria, viruses, and fungi. Ginkgo biloba has a number of unspecialized and derived traits that make it a valuable tool for studies on the evolution of seed plants. The analysis conducted by Brenner and co-workers (2005) on an expressed sequence tag (EST) dataset from G. biloba revealed genes potentially unique to gymnosperms. Wang et al. (2011) demonstrated that the Maidenhair tree is extremely similar to cycads in terms of embryology, but more similar to conifers in macromorphology and vegetative anatomy, suggesting that the G. biloba lineage may have an intermediate phylogenetic position between April 2015 | Volume 57 | Issue 4 | 388–395
Keywords: Cellulose structure; cell wall; compression wood; lignin; Maidenhair tree; microfibril angle Citation: Andersson S, Wang Y, P€ onni R, H€anninen T, Mononen M, Ren H, Serimaa R, Saranp€a€ a P (2015) Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.). J Integr Plant Biol 57: 388–395 doi: 10.1111/jipb.12349 Edited by: Kurt Fagerstedt, University of Helsinki, Finland Received Sept. 30, 2014; Accepted Mar. 3, 2015 Available online on Mar. 4, 2015 at www.wileyonlinelibrary.com/ journal/jipb © 2015 Institute of Botany, Chinese Academy of Sciences
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Properties of the Ginkgo biloba cell wall has three layers, an outer S1 with transversely oriented microfibrils, a thick S2 layer with axially oriented microfibrils, and an inner S3 layer also with transversely oriented microfibrils, in an S-Z-S helical organization as reviewed by Donaldson (2008). Microfibril orientation in S2 layer is relatively uniform (Donaldson and Xu 2005). Terashima et al. (2009) conducted a field-emission scanning electron microscopy study of the tracheids of G. biloba and proposed a schematic model of assembly of cell wall polymers in the tracheid. According to their model, bundles of cellulose microfibrils are surrounded by tubular hemicellulose–lignin modules, which keep the microfibril bundles apart. The thickness of the microfibril bundle is approximately 12 2 nm and it could consist of 4 3 microfibrils. The thickness and length of individual tubular modules are approximately 3–4 nm and 160 3 nm, respectively. Lignification and the lignin structure in G. biloba have been reported to be similar to conifers (Terashima et al. 2009). The contents of cellulose, pentosan, and lignin of G. biloba are reported to be 42.43%, 10.32%, and 27.26%, respectively (Fei et al. 2000a), which are similar to the contents of the poplar (Fei et al. 2005). Interestingly, cell cultures, but not woody cell walls of G. biloba, have been found to synthesize syringyl (S) lignin; the S-lignin pathway in gymnosperms is generally thought to be absent (Uzal et al. 2009). Juvenile wood is produced in a tree during its first years of growth (e.g. Paul 1957; Zobel and Sprague 1998). Young trees solely consist of juvenile wood, while in older stems, juvenile wood is found in the central core (Lachenbruch et al. 2011). Based on structural studies conducted on mature stems, it has been observed that the cellular and cell wall structures of wood change as a function of the cambial age, namely, as a function of the number of annual rings from the pith to the bark. For instance, the diameter of the cells increases and at the same time the microfibril angle (MFA) decreases significantly from the pith to the bark (e.g. Bonham and Barnett 2001; Sar en et al. 2004). The juvenile wood zone has been estimated to cover approximately the first 5–20 annual rings, depending on species and growth rate (Lachenbruch et al. 2011). In G. biloba, this zone has been found to cover approximately 20 annual rings (Fei et al. 2000b). The annual rings are wider near the pith than in outer wood, then decrease in width, and remain at the same width in mature wood (Fei et al. 2000a, 2000b). The aim of our study was to investigate the variation in the mean MFA and crystalline size in G. biloba from the pith to the bark. The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using X-ray diffraction. Previous studies have only been conducted on single tracheids or random pieces of wood. We also aimed to compare compression wood and opposite or normal wood. Raman microscopy was used to compare the lignin distribution in the cell wall of opposite and compression wood. Based on micro- and nanostructural parameters, the goal was to define the juvenile wood in G. biloba. Our results are compared with the modern conifers Norway spruce (Picea abies (L.) Karst.), Scots pine (Pinus sylvestris L.), and common juniper (Juniperus communis L.). www.jipb.net
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RESULTS Microscopic structure The lumen diameter of G. biloba tracheids is relatively small and did not increase from the pith to the bark over the first 14 annual rings (Figures 2, 3; Table 1). The dark compression wood zones (Figure 1) did not show any round tracheids in cross-section or intercellular cavities. Slit-like bordered pit openings indicated the presence of mild compression wood (Figure 4), but there were no helical cavities in the cell wall, which are typical for severe compression wood. Crystallite width and length The mean crystallite width in normal, clear wood was either 3.1 nm or 3.2 nm and the mean crystallite length (Table 2) was
Figure 1. Stem disk of Ginkgo biloba showing where the samples for X-ray and Raman studies were taken For X-ray studies, a sample was cut from the pith to the bark from two cardinal directions (EAST and WEST). Eight samples (arrows 1–8) were tangentially cut for X-ray studies (NORTH). Five samples were prepared tangentially for Raman imaging (arrows, a–e).
Figure 2. Micrograph of a cross-section of Ginkgo biloba ring no. 5 Note the round shape of tracheids and only a few latewood tracheids at the ring border. April 2015 | Volume 57 | Issue 4 | 388–395
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Figure 3. Mean diameter of the tracheid lumen as a function of the annual ring number from the pith in Ginkgo biloba (o) and Picea abies (L.) Karst (*) Reference samples.
Figure 5. Azimuthal intensity profile of a normal wood sample and a compression wood sample The profile of the normal wood sample is marked with a blue thick line and that of the compression wood sample with a green thin line.
28.7 nm with a standard deviation of 0.9 nm. In a compression wood sample, the width was 3.1 nm and the length was only 20.5 nm. The diffraction pattern of the compression wood sample differed from that of the clear wood sample in having slightly broader diffraction peaks. Neither the width nor the length of crystallites showed any correlation with the ring number from the pith (Table 2).
Figure 4. Micrograph of the radial section of Ginkgo biloba showing slit-like openings of bordered pits and indicating presence of compression wood No helical cavities were observed in the cell wall.
MFA distributions Figure 5 presents the azimuthal intensity profiles of a normal wood sample (sample 2, annual ring 11) and a compression wood sample (sample 4, annual ring 5), and Figure 6 presents the fitted MFA distributions for both samples. The intensity profiles display two pairs of maxima, which were interpreted to arise from the helical arrangement of microfibrils in the S2 and S1 layers. The contribution of the S3 layer cannot be resolved. It is assumed to be too thin or have MFA in the
Table 1. Results for anatomical measurements from cross-sections cut with a microtome
Sample
Annual ring no. from the pith
Cardinal direction
No. of measured cells (n)
Mean area of lumen (A) mm2
Standard derivation (sA) mm2
Mean diameter of lumen (d) mm
Standard deviation (sd) mm
Ginkgo biloba L. G. biloba L. G. biloba L. G. biloba L. G. biloba L. G. biloba L. Picea abies L. P. abies L. P. abies L. P. abies L. P. abies L. P. abies L.
5 5 10 11 13 14 3 3 3 15 15 21
N W W N W N Not Not Not Not Not Not
1,054 1,277 864 982 842 788 447 454 322 167 181 147
361 247 346 331 350 385 1,162 1,317 1,489 3,650 3,435 4,149
261 224 315 349 320 360 476 629 564 1,630 1,689 2,314
20 16 19 18 19 20 37 39 42 66 63 68
7 7 8 9 8 9 8 10 8 15 19 23
known known known known known known
Norway spruce (Picea abies L.) samples were used as reference samples. Samples were prepared from two cardinal directions: north (N) and west (W). April 2015 | Volume 57 | Issue 4 | 388–395
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same range as the other two layers. In the profile of the compression wood sample there is also a maximum at an MFA of 0°, indicating that the sample contains a small number of microfibrils aligned parallel to the cell axis. There is a possibility that this peak increases from the S2(L) layer. The mean angles and the standard deviations determined from the fitted MFA distributions are provided in Table 2. Figure 7 presents the microfibril angles of the S2 and S1 layers as a function of the annual ring number. The mean MFA of the S2 layer shows a declining trend only after the 12th year ring. The mean MFA of the S1 layer does not vary significantly from the pith to the bark. The mean MFA values for compression wood of both layers were large, the mean MFA for the S2 layer being 42° and that of the S1 layer 89°.
Figure 6. Microfibril angle (MFA) distribution of a normal wood sample and a compression wood sample of Ginkgo biloba The distribution of the normal wood sample is marked with a blue thick line and that of the compression wood sample with a green thin line.
Raman imaging Raman images of the samples taken from the juvenile wood near the pith (Figure 8) clearly display a layer with a high lignin content in the outer layer of the secondary cell wall. Such a layer is characteristic of compression wood and is commonly known as the S2L layer. This layer was only visible very near the pith (2nd growth ring), and none of the rest of the Raman images taken from the outer parts of the stem showed the S2L layer, as illustrated in Figure 8 for the 8th growth ring.
DISCUSSION Microstructure
Figure 7. Microfibril angle (MFA) of the S2 and S1 layers of Ginkgo biloba as a function of the annual ring number from the pith in two cardinal directions (EAST and WEST) The red squares represent the tangentially cut samples (see Figure 1).
Mild compression wood with slit-like bordered pit openings and a large mean MFA was detected in the sample of G. biloba L. According to Timell (1986), compression wood in the ancient G. biloba differs from that in most of the younger gymnosperms in the more angular outline of its tracheids, their thinner walls, and their lack of helical cavities. He studied samples from a band of pronounced compression wood on one side of an eccentric stem and branch wood which both should contain a severe type of compression wood. Both normal and compression wood of G. biloba contain two types of tracheids, one wide and having a thin wall, and another narrow with a thicker wall. In all other respects, the compression wood tracheids in G. biloba are ultrastructurally similar to those in other gymnosperms. Helical cavities probably developed relatively late in the evolution of
Table 2. Results of tangential cut samples Annual ring no. from the pith 5* 5 8 11 11 13 14 14
Cardinal direction
Mean MFA of S2-layer (°)
Standard deviation of MFA of S2-layer (°)
Mean MFA of S1-layer (°)
Standard deviation of MFA of S1-layer (°)
Length of cellulose crystallites (nm)
Width of cellulose crystallites (nm)
W* N W W N W W N
39 29 33 27 31 22 19 23
8 26 10 10 10 10 12 10
89 80 83 76 80 81 88 79
11 27 9 12 8 15 21 12
20.5 19.0 28.8 28.8 26.1 28.3 30.0 27.5
3.1 3.1 3.2 3.1 3.1 3.1 3.2 3.1
The first sample marked with the asterisk (*) was compression wood. Samples were prepared from two cardinal directions: north (N) and west (W). MFA, microfibril angle. www.jipb.net
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Figure 8. Raman images of cross-sections of Ginkgo biloba samples from the growth rings 2 (A) and 8 (B) The S2L layer is indicated by an arrow. The direction of laser polarization is indicated with a white line. The images are constructed according to band area of the characteristic bands (1,095/cm for cellulose and 1,600/cm for lignin). This is indicated by the intensity of the color (light color ¼ large band area, dark color ¼ small band area). The size of the images is 25 mm 25 mm.
compression wood, because they are lacking not only in G. biloba but also in the Taxales and the Araucariaceae. The occurrence of compression wood in G. biloba indicates that this tissue has probably existed since the Devonian period. Very likely, the arborescent habit of the gymnosperms has always been dependent on their ability to form compression wood (Timell 1978). Nanostructure The width of cellulose crystallites in G. biloba of 3.1 nm and in two cases 3.2 nm is approximately the same as that in the modern conifers Norway spruce (3.2 nm), Scots pine (3.1 nm) (Andersson et al. 2003, 2004), and Sitka spruce (3.0 nm) (Vainio et al. 2002). This indicates that cellulose microfibril formation follows the same pattern as 170 million years ago. Cellulose crystallites are formed of hydrogen-bonded sheets of cellulose chains. The value of 3.1 nm indicates that an average crystallite contains roughly eight parallel sheets. As discussed by Fernandes et al. (2011); among others, one cannot reliably determine the width of the sheets or the shape of the crystallite from the diffraction pattern. The length of cellulose crystallites in G. biloba, 27.5– 30.0 nm, is slightly shorter than that in Norway spruce (up to April 2015 | Volume 57 | Issue 4 | 388–395
40.0 nm) (Pirkkalainen et al. 2012). The value of 20.5 nm obtained for compression wood of G. biloba is similar to that of common juniper (J. communis) and compression wood of Norway spruce (H€anninen et al. 2012). Although the length of cellulose crystallites varied between 20.5 and 30.0 nm, their width was almost constant. The mean microfibril angle of G. biloba was large in the whole range of annual rings, from 1 to 14 years, compared with Norway spruce grown in Nordic countries, in which the mean MFA already decreases below 10° at the sixth to the 10th ring from the pith (e.g. Andersson et al. 2003). Terashima et al. (2009) determined a mean MFA value of 30° in individual tracheids of G. biloba. For juvenile saplings of spruce, pine, and birch, mean MFA values of approximately 30° have been € m et al. 2012). The mean MFA also shows reported (Svedstro large genetic variation in Sitka spruce (Picea sitchensis (Bong.) Carr.) (Vainio et al. 2002). The mean MFA of Sitka spruce was approximately 22° close to the pith and decreased to approximately 11° in the outer rings. The mean MFA varied considerably, not only as a function of the annual ring and the distance from the pith, but also as a function of the seed origin. Trees of California and Queen Charlotte Islands provenance had a larger mean MFA in general than trees of www.jipb.net
Properties of the Ginkgo biloba cell wall Washington and Oregon provenance. Transplants were grown in Ireland (Shillelagh Forest) (Vainio et al. 2002). Sitka spruce from Washington and Oregon provenance showed a similar behavior of MFA to Norway spruce (e.g. Andersson et al. 2003; Sar en et al. 2004). The microfibril angle distributions of G. biloba resembled those obtained for common juniper (H€anninen et al. 2012). In both cases, it is reasonable to assume that the layer with large MFA values is S1. As can be seen in Figures 5 and 6, the MFA distributions of normal wood and compression wood differ significantly from each other. The mean MFA values are larger in compression wood, and furthermore, the area under the second peak at 70–90° (presumably S1) is larger in the MFA distribution of the compression wood sample, indicating that the volume fraction of the S1 layer is larger in compression wood than in normal wood (Table 2). Donaldson and Xu (2005) have studied in detail the MFA variation for the S1, S2, and S3 layers from juvenile and mature wood of Pinus radiata. They applied polarized light microscopy and transmission electron microscopy to measure the orientation. According to their results, microfibrils in the S1 layer are usually arranged in the S-helix varying from 79° to 117°. However, within individual tracheids the MFA in S1 can be quite variable, sometimes changing from S- to Z-helix (Donaldson and Xu 2005). Raman images of the samples near the pith resemble those of compression wood previously reported in the published work, showing a layer with a high lignin content in the outer layer of the secondary cell wall (Gierlinger et al. 2010; H€anninen et al. 2012). In opposite wood or normal wood samples (Figure 8), the distribution of the lignin was even and no residual layers could be distinguished from the images. The uneven distribution of cellulose in the region of the S1 layer is caused by the orientation of microfibrils when using a polarized excitation laser. When fibrils in the sample are aligned parallel to the polarization of the laser, the intensity of cellulose Raman bands is significantly stronger than when the fibrils are aligned perpendicularly. This effect has been discussed in the published work (Gierlinger et al. 2010; H€anninen et al. 2012). Juvenile wood Ginkgo biloba showed no significant increase in the tracheid lumen diameter over the first 14 annual rings, and the mean MFA also only decreased over the few outer annual rings in the sample. Fei and co-workers (2000b) only found a decrease in the MFA in G. biloba after 20 annual rings from the pith. Thus, the juvenile wood of G. biloba shows similar properties to that of other conifers, for example, Norway spruce (Sar en et al. 2004), and according to the definition, almost the whole sample of 18 cm in diameter consisted of juvenile wood.
CONCLUSIONS Ginkgo biloba showed relative large mean MFA (values) varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm, which corresponds to modern conifers. Mild compression wood without any intercellular spaces or helical cavities was also observed. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Juvenile wood www.jipb.net
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showed no increase in tracheid diameter and the mean MFA remained large over the 10 first annual rings. The properties of juvenile and compression wood and the cellulose orientation and crystalline width of G. biloba indicate that wood formation and especially cellulose biosynthesis is very similar to modern conifers.
MATERIALS AND METHODS Samples The Maidenhair tree was cut down from a garden located in the village of Xingxiang, located in the county of Tancheng, Shangdong Province, China. Its height at the time of felling was approximately 12 m. Two sample disks were taken at breast height (1.3 m). Their diameter at breast height was 18 cm and their thickness was 20 mm. The disks contained 15 annual rings. The upper disk was used in X-ray and Raman microscopy studies and the lower disk in optical microscopy studies. Eight tangential samples and two radial stripes from the pith to the bark were taken for X-ray studies. The thickness of tangential samples was 1 mm and the thickness of the radial stripes 2 mm. The positions from which the samples were taken are indicated in Figure 1. Samples 1–8 were cut in tangential directions. Samples from the cardinal directions W (west) and E (east) were cut in a radial direction. Figure 1 also presents the positions where five samples were prepared tangentially for Raman imaging (arrows, a–e). For optical microscopy, six cross-section slices were cut with a microtome. Three slices were cut from the north side from the fifth, 11th, and 14th annual rings, and the other three from the west side from the fifth, ninth, and 13th annual rings. Light microscopy The sample dimensions were 10 mm in cross, radial, and tangential directions. The samples were softened in a glycerol and alcohol mixture for 15 d. Cross-sections with a thickness of 20 mm were cut by using a sliding microtome. The sections were dehydrated through an alcohol series and stained with safranin and mounted in Canada balsam. The sections were examined and photographed using an Olympus BX61 light microscope and an Olympus DP71 digital camera (Olympus, Tokyo, Japan). The lumen areas were determined from the images using the Image Processing Toolbox of Matlab. Because the average area of the lumen is an unillustrative quantity, the average diameter of the lumen was selected in place of it.qIfffiffiffithe area of the lumen is A, the diameter of the lumen d ¼ Ap. Thus, the diameter is equal to the diameter of a round lumen of which the area is A. For comparison, six Norway spruce samples taken from three stems were studied. Wide-angle X-ray scattering experiments to determine the crystalline size and MFA Wide-angle X-ray scattering measurements were carried out using a Huber 420/511 four circle goniometer and a sealed copper anode X-ray tube. A ground and bent germanium monochromator (the reflection 111) was used to select CuKa1 radiation (wavelength ¼ 0.1541 nm). An NaI(Tl) scintillation counter was used as the detector. The instrumental April 2015 | Volume 57 | Issue 4 | 388–395
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broadening was determined from the full width at half maximum of the reflections of hexamethyltetramine (C6H12N4) (Lotfy 1974; Tanaka 1981). Because of the fiber texture in solid wood samples, both symmetrical reflection and transmission geometry were needed. Symmetrical reflection geometry was advantageous for determining the crystallite width, because the cellulose reflection 200 is particularly strong in this geometry. The diffraction pattern was determined in the scattering angle range 10–50° with a step of 0.4°. The intensity profile of the cellulose reflection 004 was measured using the symmetrical transmission mode for the determination of the crystallite length according to Andersson et al. (2003). This measurement geometry was also used to determine the MFA distribution from the azimuthal intensity profile I (f) of the reflection 004. Crystallite size The average width and length of the cellulose crystallites was determined using the Scherrer formula: Kl Bhkl ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2ffi 2 fwhm þ inst cosu where K is a constant, fwhm is the full width at half maximum of the reflection, 2u is the position of the reflection in the scattering angle axis, and inst is the instrumental broadening of the reflection. When determining the crystallite width, constant K was 0.9 (the 200 reflection) and for the length it was 1.0 (the 004 reflection). For determination of the full width at half maximum of the cellulose reflection 200, Gaussian functions presenting the diffraction peak and amorphous background were fitted to the measured intensity. The amorphous background was measured from a sulfate lignin sample. For determination of the full width at half maximum of the cellulose reflection 004, Gaussian functions presenting the peak and linear background were fitted to the measured intensity (Andersson et. al. 2003). The accuracy of the width and length of the crystallites is estimated to be 0.1 nm and 2 nm, respectively. MFA distribution To avoid the effect of the cell shape, the MFA distribution was determined from the azimuthal intensity of the cellulose reflection 004 measured using the symmetrical transmission geometry (Cave 1997a, 1997b). If the MFA distribution is assumed to be Gaussian and the cell wall consists of only one layer, the azimuthal intensity profile I (f) can be modeled as:
IðfÞ ¼ Ae
ðfðm180 ÞÞ2 2s 2
þ Ae
ðfðmþ180 ÞÞ2þ Ae
2s 2
ðfðmÞÞ2 2s 2
þ Ae
ðfðþmÞÞ2 2s 2
þB
The azimuthal angle f varied between 180° and þ180°. The model consists of four Gaussians, of which the positions are m180°, m, þm, and mþ180°. The height of the Gaussian is A and the standard deviation is s. The background B is assumed to be constant. The fitting of this model to the April 2015 | Volume 57 | Issue 4 | 388–395
measured azimuthal intensity profile gives the MFA distribution (w) of one layer: MFAðfÞ ¼ Ce
ðmfÞ2 2s 2
in which m is the mean MFA hMFAi of the layer, s is the standard deviation, and C normalized constant. If the cell wall consists of two or three layers, the intensity profile can be modeled as the sum of two or three groups of Gaussians. The MFA distribution MFAðfÞ then consists of the sum of two or n and Serimaa three Gaussians (Andersson et al. 2003; Sare 2006; Wang et al. 2012). Raman imaging Embedding and sectioning of the samples was carried out according to H€anninen et al. (2011). The samples were analyzed with an alpha300 R Confocal Raman microscope (Witec, Ulm, Germany) under ambient conditions. The Raman spectra were obtained by using a frequency-doubled Nd:YAG laser (532.35 nm, 10 mW) and a Nikon 100 (NA ¼ 0.95) air objective. The Raman system was equipped with a DU970NBV EMCCD camera behind a grating of 600 lines/mm (Andor Technology, Belfast, Northern Ireland). The excitation laser was horizontally polarized. For each of the Raman images, the integration time and the excitation laser power was varied, depending on how prone the samples were to burning. The size of one pixel in the image is 0.1 mm. The baselines of the spectra were corrected with WiTec Project 1.94 (WiTec) by using a fifth order equation in the wave number area from 600–2,000/cm. A constant background was not subtracted from the spectra due to the varying baseline of the individual measurements. Spectral regions for imaging representing different compounds were selected according to H€anninen et al. (2011).
ACKNOWLEDGEMENTS The authors thank the National Natural Science Foundation (31370562) for financial support and Mr Tapio J€arvinen for skillful sample preparation.
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Pirkkalainen K, Peura M, Lepp€ anen K, Salmi A, Meril€ainen A, Saranp€a€a P, Serimaa R (2012) Simultaneous x-ray diffraction and x-ray fluorescence microanalysis on secondary xylem of Norway spruce. Wood Sci Technol 46: 1113–1125 Rothwell GW, Holt B (1997) Fossils and phenology in the evolution of Ginkgo biloba. In: Hori T, Ridge RW, Tulecke W, Del Tredici P, Tremouillaux-Guiller J, Tobe H, eds. Ginkgobiloba - A Global Treasure. Tokyo, Springer-Verlag. pp. 223– 230 Sar en MP, Serimaa R (2006) Determination of microfibril angle distribution by X-ray diffraction. Wood Sci Technol 40: 445– 460 Sar en MP, Serimaa R, Andersson S, Saranp€aa€ P, Keckes J, Fratzl P (2004) Effect of growth rate on mean microfibril angle and crosssectional shape of the tracheids of Norway spruce. Trees 18: 354– 362 Svedstr€ om K, Lucenius J, Van den Bulcke J, Van Loo D, Immerzeel P, Suuronen JP, Brabant L, Van Acker J, Saranp€ a€ a P, Fagerstedt K, Mellerowicz E, Serimaa R (2012) Hierarchical structure of juvenile hybrid aspen xylem revealed using xray scattering and microtomography. Trees-Struct Funct 26: 1793–1804 Tanaka F, Koshijima T, Okamura K (1981) Characterization of cellulose in compression and opposite woods of a Pinus densiflora tree grown under the influence of strong wind. Wood Sci Technol 15: 265–273 Terashima N, Kitano K, Kojima M, Yoshida M, Yamamoto H, Westermark U (2009) Nanostructural assembly of cellulose, hemicellulose, and lignin in the middle layer of secondary wall of ginkgo tracheid. J Wood Sci: 409–416 Timell TE (1978) Ultrastructure of compression wood in Ginkgo biloba. Wood Sci Technol 12: 89–103 Timell TE (1986) Compression Wood in Gymnosperms. Vol. 1. SpringerVerlag, New York. pp. 604–613 Uzal EN, Ros LVG, Pomar F, Bernal MA, Paradela A, Albar JP, Barcelo AR (2009) The presence of sinapyl lignin in Ginkgo biloba cell cultures changes our views of the evolution of lignin biosynthesis. Physiol Plant 135: 196–213 Vainio U, Andersson S, Serimaa R, Paakkari T, Saranp€a€a P, Treacy M, Evertsen J (2002) Variation of microfibril angle between four provenances of Sitka spruce (Picea sitchensis [Bong.] Carr.). Plant Biol 4: 27–33 Wang L, Wang D, Lin MM, Lu Y, Jiang XX, Jin B (2011) An embryological study and systematic significance of the primitive gymnosperm Ginkgo biloba. J Syst Evol 49: 353–361 Wang Y, Lepp€ anen K, Andersson S, Serimaa R, Ren H, Fei B (2012) Studies on the nanostructure of the cell wall of bamboo using Xray scattering. Wood Sci Technol 46: 317–332 Zhou Z, Zheng S (2003) The missing link in Ginkgo evolution. Nature 423:821–822 Zobel B, Sprague J (1998) Juvenile Wood in Forest Trees. Springer Series in Wood Science. Springer-Verlag, New York. pp. 300
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Journal of Integrative Plant Biology
Free Access
Research Article
Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.) €nni3, Tuomas H€anninen4, Marko Mononen1, Haiqing Ren2, Seppo Andersson1*, Yurong Wang2, Raili Po 1 5 Ritva Serimaa and Pekka Saranp€a€a 1
Department of Physics, University of Helsinki, 00560 Helsinki, Finland, 2Research Institute of Wood Industry, Chinese Academy of Forestry, 100091, Beijing, China, 3Department of Forest Products Technology, Aalto University, School of Chemical Technology, 00076 Aalto, Finland, 4 VTT Technical Research Centre of Finland, 02044 Espoo, Finland, 5Natural Resources Institute Finland, 01300 Vantaa, Finland. *Correspondence: seppo.andersson@helsinki.fi
Abstract We studied in detail the mean microfibril angle and the width of cellulose crystals from the pith to the bark of a 15year-old Maidenhair tree (Ginkgo biloba L.). The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using Xray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean microfibril angle, varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The microfibril angle
remained large over the 14 annual rings. The entire stem disc, with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.
INTRODUCTION
cycads and conifers. A similar conclusion was reached by Gong et al. (2009) based on the chemical composition of G. biloba wood. They also reported a higher lignin content in G. biloba than in other conifers. Timell (1986) demonstrated that the tracheid walls of G. biloba are morphologically similar to those of representative conifers. However, according to Timell (1978), the compression wood of G. biloba differs from the more recently evolved gymnosperms in the more angular shape of the cells, thinner cell walls, and lack of helical cavities. Burgert et al. (2004) compared the micromechanical properties of thin compression wood foils and isolated tracheids of four gymnosperm species (Ginkgo biloba L., Taxus baccata L., Juniperus virginiana L., and Picea abies (L.) Karst.). The compression wood of G. biloba was found to be stiffer than that of the other species, and the authors consequently concluded that there has been an evolutionary trend towards much more flexible compression wood. Properties of wood depend on the assembly mode of cellulose, other polysaccharides, and lignin in the cell wall. The plant cell wall provides the shape and mechanical stability of the cell, but also has a role in intercellular communication and defense against potential pathogens (Keegstra 2010). In the secondary cell walls of xylem cells, the cell wall typically
The Maidenhair tree (Ginkgo biloba L.) is a unique living species of the ancient lineage of Ginkgophyta (Zhou and Zheng 2003). Fossil studies have revealed that Ginkgoales originated in the Paleozoic Carboniferous period and flourished during the Jurassic period, comprising a diverse and widespread group, and it may be considered as a living fossil. Ginkgo biloba may be even older, because fossils displaying similar vegetative morphology have been found as early as the Permian period (Rothwell and Holt 1997). Major (1967) suggested that G. biloba has persisted unchanged over many millions of years because it has adapted to its environment and is resistant to various pests, such as bacteria, viruses, and fungi. Ginkgo biloba has a number of unspecialized and derived traits that make it a valuable tool for studies on the evolution of seed plants. The analysis conducted by Brenner and co-workers (2005) on an expressed sequence tag (EST) dataset from G. biloba revealed genes potentially unique to gymnosperms. Wang et al. (2011) demonstrated that the Maidenhair tree is extremely similar to cycads in terms of embryology, but more similar to conifers in macromorphology and vegetative anatomy, suggesting that the G. biloba lineage may have an intermediate phylogenetic position between April 2015 | Volume 57 | Issue 4 | 388–395
Keywords: Cellulose structure; cell wall; compression wood; lignin; Maidenhair tree; microfibril angle Citation: Andersson S, Wang Y, P€ onni R, H€anninen T, Mononen M, Ren H, Serimaa R, Saranp€a€ a P (2015) Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.). J Integr Plant Biol 57: 388–395 doi: 10.1111/jipb.12349 Edited by: Kurt Fagerstedt, University of Helsinki, Finland Received Sept. 30, 2014; Accepted Mar. 3, 2015 Available online on Mar. 4, 2015 at www.wileyonlinelibrary.com/ journal/jipb © 2015 Institute of Botany, Chinese Academy of Sciences
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Properties of the Ginkgo biloba cell wall has three layers, an outer S1 with transversely oriented microfibrils, a thick S2 layer with axially oriented microfibrils, and an inner S3 layer also with transversely oriented microfibrils, in an S-Z-S helical organization as reviewed by Donaldson (2008). Microfibril orientation in S2 layer is relatively uniform (Donaldson and Xu 2005). Terashima et al. (2009) conducted a field-emission scanning electron microscopy study of the tracheids of G. biloba and proposed a schematic model of assembly of cell wall polymers in the tracheid. According to their model, bundles of cellulose microfibrils are surrounded by tubular hemicellulose–lignin modules, which keep the microfibril bundles apart. The thickness of the microfibril bundle is approximately 12 2 nm and it could consist of 4 3 microfibrils. The thickness and length of individual tubular modules are approximately 3–4 nm and 160 3 nm, respectively. Lignification and the lignin structure in G. biloba have been reported to be similar to conifers (Terashima et al. 2009). The contents of cellulose, pentosan, and lignin of G. biloba are reported to be 42.43%, 10.32%, and 27.26%, respectively (Fei et al. 2000a), which are similar to the contents of the poplar (Fei et al. 2005). Interestingly, cell cultures, but not woody cell walls of G. biloba, have been found to synthesize syringyl (S) lignin; the S-lignin pathway in gymnosperms is generally thought to be absent (Uzal et al. 2009). Juvenile wood is produced in a tree during its first years of growth (e.g. Paul 1957; Zobel and Sprague 1998). Young trees solely consist of juvenile wood, while in older stems, juvenile wood is found in the central core (Lachenbruch et al. 2011). Based on structural studies conducted on mature stems, it has been observed that the cellular and cell wall structures of wood change as a function of the cambial age, namely, as a function of the number of annual rings from the pith to the bark. For instance, the diameter of the cells increases and at the same time the microfibril angle (MFA) decreases significantly from the pith to the bark (e.g. Bonham and Barnett 2001; Sar en et al. 2004). The juvenile wood zone has been estimated to cover approximately the first 5–20 annual rings, depending on species and growth rate (Lachenbruch et al. 2011). In G. biloba, this zone has been found to cover approximately 20 annual rings (Fei et al. 2000b). The annual rings are wider near the pith than in outer wood, then decrease in width, and remain at the same width in mature wood (Fei et al. 2000a, 2000b). The aim of our study was to investigate the variation in the mean MFA and crystalline size in G. biloba from the pith to the bark. The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using X-ray diffraction. Previous studies have only been conducted on single tracheids or random pieces of wood. We also aimed to compare compression wood and opposite or normal wood. Raman microscopy was used to compare the lignin distribution in the cell wall of opposite and compression wood. Based on micro- and nanostructural parameters, the goal was to define the juvenile wood in G. biloba. Our results are compared with the modern conifers Norway spruce (Picea abies (L.) Karst.), Scots pine (Pinus sylvestris L.), and common juniper (Juniperus communis L.). www.jipb.net
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RESULTS Microscopic structure The lumen diameter of G. biloba tracheids is relatively small and did not increase from the pith to the bark over the first 14 annual rings (Figures 2, 3; Table 1). The dark compression wood zones (Figure 1) did not show any round tracheids in cross-section or intercellular cavities. Slit-like bordered pit openings indicated the presence of mild compression wood (Figure 4), but there were no helical cavities in the cell wall, which are typical for severe compression wood. Crystallite width and length The mean crystallite width in normal, clear wood was either 3.1 nm or 3.2 nm and the mean crystallite length (Table 2) was
Figure 1. Stem disk of Ginkgo biloba showing where the samples for X-ray and Raman studies were taken For X-ray studies, a sample was cut from the pith to the bark from two cardinal directions (EAST and WEST). Eight samples (arrows 1–8) were tangentially cut for X-ray studies (NORTH). Five samples were prepared tangentially for Raman imaging (arrows, a–e).
Figure 2. Micrograph of a cross-section of Ginkgo biloba ring no. 5 Note the round shape of tracheids and only a few latewood tracheids at the ring border. April 2015 | Volume 57 | Issue 4 | 388–395
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Figure 3. Mean diameter of the tracheid lumen as a function of the annual ring number from the pith in Ginkgo biloba (o) and Picea abies (L.) Karst (*) Reference samples.
Figure 5. Azimuthal intensity profile of a normal wood sample and a compression wood sample The profile of the normal wood sample is marked with a blue thick line and that of the compression wood sample with a green thin line.
28.7 nm with a standard deviation of 0.9 nm. In a compression wood sample, the width was 3.1 nm and the length was only 20.5 nm. The diffraction pattern of the compression wood sample differed from that of the clear wood sample in having slightly broader diffraction peaks. Neither the width nor the length of crystallites showed any correlation with the ring number from the pith (Table 2).
Figure 4. Micrograph of the radial section of Ginkgo biloba showing slit-like openings of bordered pits and indicating presence of compression wood No helical cavities were observed in the cell wall.
MFA distributions Figure 5 presents the azimuthal intensity profiles of a normal wood sample (sample 2, annual ring 11) and a compression wood sample (sample 4, annual ring 5), and Figure 6 presents the fitted MFA distributions for both samples. The intensity profiles display two pairs of maxima, which were interpreted to arise from the helical arrangement of microfibrils in the S2 and S1 layers. The contribution of the S3 layer cannot be resolved. It is assumed to be too thin or have MFA in the
Table 1. Results for anatomical measurements from cross-sections cut with a microtome
Sample
Annual ring no. from the pith
Cardinal direction
No. of measured cells (n)
Mean area of lumen (A) mm2
Standard derivation (sA) mm2
Mean diameter of lumen (d) mm
Standard deviation (sd) mm
Ginkgo biloba L. G. biloba L. G. biloba L. G. biloba L. G. biloba L. G. biloba L. Picea abies L. P. abies L. P. abies L. P. abies L. P. abies L. P. abies L.
5 5 10 11 13 14 3 3 3 15 15 21
N W W N W N Not Not Not Not Not Not
1,054 1,277 864 982 842 788 447 454 322 167 181 147
361 247 346 331 350 385 1,162 1,317 1,489 3,650 3,435 4,149
261 224 315 349 320 360 476 629 564 1,630 1,689 2,314
20 16 19 18 19 20 37 39 42 66 63 68
7 7 8 9 8 9 8 10 8 15 19 23
known known known known known known
Norway spruce (Picea abies L.) samples were used as reference samples. Samples were prepared from two cardinal directions: north (N) and west (W). April 2015 | Volume 57 | Issue 4 | 388–395
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same range as the other two layers. In the profile of the compression wood sample there is also a maximum at an MFA of 0°, indicating that the sample contains a small number of microfibrils aligned parallel to the cell axis. There is a possibility that this peak increases from the S2(L) layer. The mean angles and the standard deviations determined from the fitted MFA distributions are provided in Table 2. Figure 7 presents the microfibril angles of the S2 and S1 layers as a function of the annual ring number. The mean MFA of the S2 layer shows a declining trend only after the 12th year ring. The mean MFA of the S1 layer does not vary significantly from the pith to the bark. The mean MFA values for compression wood of both layers were large, the mean MFA for the S2 layer being 42° and that of the S1 layer 89°.
Figure 6. Microfibril angle (MFA) distribution of a normal wood sample and a compression wood sample of Ginkgo biloba The distribution of the normal wood sample is marked with a blue thick line and that of the compression wood sample with a green thin line.
Raman imaging Raman images of the samples taken from the juvenile wood near the pith (Figure 8) clearly display a layer with a high lignin content in the outer layer of the secondary cell wall. Such a layer is characteristic of compression wood and is commonly known as the S2L layer. This layer was only visible very near the pith (2nd growth ring), and none of the rest of the Raman images taken from the outer parts of the stem showed the S2L layer, as illustrated in Figure 8 for the 8th growth ring.
DISCUSSION Microstructure
Figure 7. Microfibril angle (MFA) of the S2 and S1 layers of Ginkgo biloba as a function of the annual ring number from the pith in two cardinal directions (EAST and WEST) The red squares represent the tangentially cut samples (see Figure 1).
Mild compression wood with slit-like bordered pit openings and a large mean MFA was detected in the sample of G. biloba L. According to Timell (1986), compression wood in the ancient G. biloba differs from that in most of the younger gymnosperms in the more angular outline of its tracheids, their thinner walls, and their lack of helical cavities. He studied samples from a band of pronounced compression wood on one side of an eccentric stem and branch wood which both should contain a severe type of compression wood. Both normal and compression wood of G. biloba contain two types of tracheids, one wide and having a thin wall, and another narrow with a thicker wall. In all other respects, the compression wood tracheids in G. biloba are ultrastructurally similar to those in other gymnosperms. Helical cavities probably developed relatively late in the evolution of
Table 2. Results of tangential cut samples Annual ring no. from the pith 5* 5 8 11 11 13 14 14
Cardinal direction
Mean MFA of S2-layer (°)
Standard deviation of MFA of S2-layer (°)
Mean MFA of S1-layer (°)
Standard deviation of MFA of S1-layer (°)
Length of cellulose crystallites (nm)
Width of cellulose crystallites (nm)
W* N W W N W W N
39 29 33 27 31 22 19 23
8 26 10 10 10 10 12 10
89 80 83 76 80 81 88 79
11 27 9 12 8 15 21 12
20.5 19.0 28.8 28.8 26.1 28.3 30.0 27.5
3.1 3.1 3.2 3.1 3.1 3.1 3.2 3.1
The first sample marked with the asterisk (*) was compression wood. Samples were prepared from two cardinal directions: north (N) and west (W). MFA, microfibril angle. www.jipb.net
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Figure 8. Raman images of cross-sections of Ginkgo biloba samples from the growth rings 2 (A) and 8 (B) The S2L layer is indicated by an arrow. The direction of laser polarization is indicated with a white line. The images are constructed according to band area of the characteristic bands (1,095/cm for cellulose and 1,600/cm for lignin). This is indicated by the intensity of the color (light color ¼ large band area, dark color ¼ small band area). The size of the images is 25 mm 25 mm.
compression wood, because they are lacking not only in G. biloba but also in the Taxales and the Araucariaceae. The occurrence of compression wood in G. biloba indicates that this tissue has probably existed since the Devonian period. Very likely, the arborescent habit of the gymnosperms has always been dependent on their ability to form compression wood (Timell 1978). Nanostructure The width of cellulose crystallites in G. biloba of 3.1 nm and in two cases 3.2 nm is approximately the same as that in the modern conifers Norway spruce (3.2 nm), Scots pine (3.1 nm) (Andersson et al. 2003, 2004), and Sitka spruce (3.0 nm) (Vainio et al. 2002). This indicates that cellulose microfibril formation follows the same pattern as 170 million years ago. Cellulose crystallites are formed of hydrogen-bonded sheets of cellulose chains. The value of 3.1 nm indicates that an average crystallite contains roughly eight parallel sheets. As discussed by Fernandes et al. (2011); among others, one cannot reliably determine the width of the sheets or the shape of the crystallite from the diffraction pattern. The length of cellulose crystallites in G. biloba, 27.5– 30.0 nm, is slightly shorter than that in Norway spruce (up to April 2015 | Volume 57 | Issue 4 | 388–395
40.0 nm) (Pirkkalainen et al. 2012). The value of 20.5 nm obtained for compression wood of G. biloba is similar to that of common juniper (J. communis) and compression wood of Norway spruce (H€anninen et al. 2012). Although the length of cellulose crystallites varied between 20.5 and 30.0 nm, their width was almost constant. The mean microfibril angle of G. biloba was large in the whole range of annual rings, from 1 to 14 years, compared with Norway spruce grown in Nordic countries, in which the mean MFA already decreases below 10° at the sixth to the 10th ring from the pith (e.g. Andersson et al. 2003). Terashima et al. (2009) determined a mean MFA value of 30° in individual tracheids of G. biloba. For juvenile saplings of spruce, pine, and birch, mean MFA values of approximately 30° have been € m et al. 2012). The mean MFA also shows reported (Svedstro large genetic variation in Sitka spruce (Picea sitchensis (Bong.) Carr.) (Vainio et al. 2002). The mean MFA of Sitka spruce was approximately 22° close to the pith and decreased to approximately 11° in the outer rings. The mean MFA varied considerably, not only as a function of the annual ring and the distance from the pith, but also as a function of the seed origin. Trees of California and Queen Charlotte Islands provenance had a larger mean MFA in general than trees of www.jipb.net
Properties of the Ginkgo biloba cell wall Washington and Oregon provenance. Transplants were grown in Ireland (Shillelagh Forest) (Vainio et al. 2002). Sitka spruce from Washington and Oregon provenance showed a similar behavior of MFA to Norway spruce (e.g. Andersson et al. 2003; Sar en et al. 2004). The microfibril angle distributions of G. biloba resembled those obtained for common juniper (H€anninen et al. 2012). In both cases, it is reasonable to assume that the layer with large MFA values is S1. As can be seen in Figures 5 and 6, the MFA distributions of normal wood and compression wood differ significantly from each other. The mean MFA values are larger in compression wood, and furthermore, the area under the second peak at 70–90° (presumably S1) is larger in the MFA distribution of the compression wood sample, indicating that the volume fraction of the S1 layer is larger in compression wood than in normal wood (Table 2). Donaldson and Xu (2005) have studied in detail the MFA variation for the S1, S2, and S3 layers from juvenile and mature wood of Pinus radiata. They applied polarized light microscopy and transmission electron microscopy to measure the orientation. According to their results, microfibrils in the S1 layer are usually arranged in the S-helix varying from 79° to 117°. However, within individual tracheids the MFA in S1 can be quite variable, sometimes changing from S- to Z-helix (Donaldson and Xu 2005). Raman images of the samples near the pith resemble those of compression wood previously reported in the published work, showing a layer with a high lignin content in the outer layer of the secondary cell wall (Gierlinger et al. 2010; H€anninen et al. 2012). In opposite wood or normal wood samples (Figure 8), the distribution of the lignin was even and no residual layers could be distinguished from the images. The uneven distribution of cellulose in the region of the S1 layer is caused by the orientation of microfibrils when using a polarized excitation laser. When fibrils in the sample are aligned parallel to the polarization of the laser, the intensity of cellulose Raman bands is significantly stronger than when the fibrils are aligned perpendicularly. This effect has been discussed in the published work (Gierlinger et al. 2010; H€anninen et al. 2012). Juvenile wood Ginkgo biloba showed no significant increase in the tracheid lumen diameter over the first 14 annual rings, and the mean MFA also only decreased over the few outer annual rings in the sample. Fei and co-workers (2000b) only found a decrease in the MFA in G. biloba after 20 annual rings from the pith. Thus, the juvenile wood of G. biloba shows similar properties to that of other conifers, for example, Norway spruce (Sar en et al. 2004), and according to the definition, almost the whole sample of 18 cm in diameter consisted of juvenile wood.
CONCLUSIONS Ginkgo biloba showed relative large mean MFA (values) varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm, which corresponds to modern conifers. Mild compression wood without any intercellular spaces or helical cavities was also observed. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Juvenile wood www.jipb.net
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showed no increase in tracheid diameter and the mean MFA remained large over the 10 first annual rings. The properties of juvenile and compression wood and the cellulose orientation and crystalline width of G. biloba indicate that wood formation and especially cellulose biosynthesis is very similar to modern conifers.
MATERIALS AND METHODS Samples The Maidenhair tree was cut down from a garden located in the village of Xingxiang, located in the county of Tancheng, Shangdong Province, China. Its height at the time of felling was approximately 12 m. Two sample disks were taken at breast height (1.3 m). Their diameter at breast height was 18 cm and their thickness was 20 mm. The disks contained 15 annual rings. The upper disk was used in X-ray and Raman microscopy studies and the lower disk in optical microscopy studies. Eight tangential samples and two radial stripes from the pith to the bark were taken for X-ray studies. The thickness of tangential samples was 1 mm and the thickness of the radial stripes 2 mm. The positions from which the samples were taken are indicated in Figure 1. Samples 1–8 were cut in tangential directions. Samples from the cardinal directions W (west) and E (east) were cut in a radial direction. Figure 1 also presents the positions where five samples were prepared tangentially for Raman imaging (arrows, a–e). For optical microscopy, six cross-section slices were cut with a microtome. Three slices were cut from the north side from the fifth, 11th, and 14th annual rings, and the other three from the west side from the fifth, ninth, and 13th annual rings. Light microscopy The sample dimensions were 10 mm in cross, radial, and tangential directions. The samples were softened in a glycerol and alcohol mixture for 15 d. Cross-sections with a thickness of 20 mm were cut by using a sliding microtome. The sections were dehydrated through an alcohol series and stained with safranin and mounted in Canada balsam. The sections were examined and photographed using an Olympus BX61 light microscope and an Olympus DP71 digital camera (Olympus, Tokyo, Japan). The lumen areas were determined from the images using the Image Processing Toolbox of Matlab. Because the average area of the lumen is an unillustrative quantity, the average diameter of the lumen was selected in place of it.qIfffiffiffithe area of the lumen is A, the diameter of the lumen d ¼ Ap. Thus, the diameter is equal to the diameter of a round lumen of which the area is A. For comparison, six Norway spruce samples taken from three stems were studied. Wide-angle X-ray scattering experiments to determine the crystalline size and MFA Wide-angle X-ray scattering measurements were carried out using a Huber 420/511 four circle goniometer and a sealed copper anode X-ray tube. A ground and bent germanium monochromator (the reflection 111) was used to select CuKa1 radiation (wavelength ¼ 0.1541 nm). An NaI(Tl) scintillation counter was used as the detector. The instrumental April 2015 | Volume 57 | Issue 4 | 388–395
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broadening was determined from the full width at half maximum of the reflections of hexamethyltetramine (C6H12N4) (Lotfy 1974; Tanaka 1981). Because of the fiber texture in solid wood samples, both symmetrical reflection and transmission geometry were needed. Symmetrical reflection geometry was advantageous for determining the crystallite width, because the cellulose reflection 200 is particularly strong in this geometry. The diffraction pattern was determined in the scattering angle range 10–50° with a step of 0.4°. The intensity profile of the cellulose reflection 004 was measured using the symmetrical transmission mode for the determination of the crystallite length according to Andersson et al. (2003). This measurement geometry was also used to determine the MFA distribution from the azimuthal intensity profile I (f) of the reflection 004. Crystallite size The average width and length of the cellulose crystallites was determined using the Scherrer formula: Kl Bhkl ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2ffi 2 fwhm þ inst cosu where K is a constant, fwhm is the full width at half maximum of the reflection, 2u is the position of the reflection in the scattering angle axis, and inst is the instrumental broadening of the reflection. When determining the crystallite width, constant K was 0.9 (the 200 reflection) and for the length it was 1.0 (the 004 reflection). For determination of the full width at half maximum of the cellulose reflection 200, Gaussian functions presenting the diffraction peak and amorphous background were fitted to the measured intensity. The amorphous background was measured from a sulfate lignin sample. For determination of the full width at half maximum of the cellulose reflection 004, Gaussian functions presenting the peak and linear background were fitted to the measured intensity (Andersson et. al. 2003). The accuracy of the width and length of the crystallites is estimated to be 0.1 nm and 2 nm, respectively. MFA distribution To avoid the effect of the cell shape, the MFA distribution was determined from the azimuthal intensity of the cellulose reflection 004 measured using the symmetrical transmission geometry (Cave 1997a, 1997b). If the MFA distribution is assumed to be Gaussian and the cell wall consists of only one layer, the azimuthal intensity profile I (f) can be modeled as:
IðfÞ ¼ Ae
ðfðm180 ÞÞ2 2s 2
þ Ae
ðfðmþ180 ÞÞ2þ Ae
2s 2
ðfðmÞÞ2 2s 2
þ Ae
ðfðþmÞÞ2 2s 2
þB
The azimuthal angle f varied between 180° and þ180°. The model consists of four Gaussians, of which the positions are m180°, m, þm, and mþ180°. The height of the Gaussian is A and the standard deviation is s. The background B is assumed to be constant. The fitting of this model to the April 2015 | Volume 57 | Issue 4 | 388–395
measured azimuthal intensity profile gives the MFA distribution (w) of one layer: MFAðfÞ ¼ Ce
ðmfÞ2 2s 2
in which m is the mean MFA hMFAi of the layer, s is the standard deviation, and C normalized constant. If the cell wall consists of two or three layers, the intensity profile can be modeled as the sum of two or three groups of Gaussians. The MFA distribution MFAðfÞ then consists of the sum of two or n and Serimaa three Gaussians (Andersson et al. 2003; Sare 2006; Wang et al. 2012). Raman imaging Embedding and sectioning of the samples was carried out according to H€anninen et al. (2011). The samples were analyzed with an alpha300 R Confocal Raman microscope (Witec, Ulm, Germany) under ambient conditions. The Raman spectra were obtained by using a frequency-doubled Nd:YAG laser (532.35 nm, 10 mW) and a Nikon 100 (NA ¼ 0.95) air objective. The Raman system was equipped with a DU970NBV EMCCD camera behind a grating of 600 lines/mm (Andor Technology, Belfast, Northern Ireland). The excitation laser was horizontally polarized. For each of the Raman images, the integration time and the excitation laser power was varied, depending on how prone the samples were to burning. The size of one pixel in the image is 0.1 mm. The baselines of the spectra were corrected with WiTec Project 1.94 (WiTec) by using a fifth order equation in the wave number area from 600–2,000/cm. A constant background was not subtracted from the spectra due to the varying baseline of the individual measurements. Spectral regions for imaging representing different compounds were selected according to H€anninen et al. (2011).
ACKNOWLEDGEMENTS The authors thank the National Natural Science Foundation (31370562) for financial support and Mr Tapio J€arvinen for skillful sample preparation.
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