WOOD STRUCTURE and Topochemistry of Juniperus Excelsa

IAWA Journal, Vol. 32 (1), 2011: 67–76

WOOD STRUCTURE and topochemistry of Juniperus excelsa

Stergios Adamopoulos1* and Gerald Koch2

SUMMARY Wood structure and topochemical distribution of lignin and phenolic extractives in Juniperus excelsa Bieb. were investigated using a mature specimen, aproximately 80 years of age, from the Rhodope mountains, Greece. The wood of J. excelsa was found to possess the same qualita- tive anatomical features as those reported for other Juniperus species of the Western Hemisphere. Quantitative anatomical characteristics recorded for mature wood (heartwood and sapwood) included earlywood and latewood tracheid length, double wall thickness of earlywood and latewood tracheids, lumen diameter of earlywood tracheids and ray height. Scanning UV microspectrophotometry revealed a pronounced lignification of J. excelsa tracheids with detected absorbance values of the secondary cell wall layers being much higher in comparison to all other softwoods studied using this technique. The cell corners and compound middle lamellae were characterised by relative high UV absorbance values as compared to the S2 layers. The phenolic compounds deposited in the axial and ray parenchyma cells possessed higher absorbance values than cell wall associated lignins and had a different spectral behaviour due to the presence of chromophoric groups. According to the obtained UV absorbance spectra, more condensed phenolic compounds were deposited in the heartwood than in the sapwood. Key words: Juniperus excelsa Bieb., wood anatomy, scanning UV microspectrophotometry, lignin distribution, phenolic extractives.

Introduction

Juniperus (Cupressaceae) is the second largest genus of the conifers and consists of approximately 60 species distributed almost exclusively in the Northern Hemisphere. Because of its site-insensitivity and ability to grow on shallow and stony soils in severe environments the genus is extremely diverse with species forming prostrate mats above the timberline, to large trees up to 50–60 m in height nearer to sea level (Florin 1963). 1) Technological Educational Institute of Larissa, Department of Forestry and Management of Natu- ral Environment, 43100 Karditsa, Greece. 2) Institute for Wood Technology and Wood Biology, Federal Research Institute of Rural Areas, Forestry and Fisheries (vTI), Leuschnerstr. 91, 21031 Hamburg, Germany. *) corresponding author [E-mail: [email protected]]. Associate Editor: Alex Wiedenhoeft

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Juniperus excelsa Bieb. extends from the central and south Balkans through Ana- tolia to Crimea, central and southwest Asia and east Africa (Boratynsky et al. 1992; Christensen 1997). Juniperus excelsa creates extended forests in Balouchistan of Pakistan and in Turkey. Moreover, it is the dominant woody plant species above 2,100 m altitude in almost all the northern mountains of Oman (Ahmed et al. 1990; Gardner & Fisher 1996; Carus 2004). It is considered a slow growing species and can attain a height of 20 m (Ahmed et al. 1990). In Greece, the species is commonly found on rocky regions at altitudes of 50 to 1,600 m as a component of degraded shrublands, as scattered individual trees or as small groups of trees in open forests and rarely as larger units of mixed or pure stands (Athanasiadis 1986). Because of the high natural durability of the heartwood, dimensional stability, good machinability, and moderate strength as well as decorative colour and texture, J. excelsa timber is highly desirable for furniture, building components, flooring, and poles (Tsoumis 1991). Successive intense anthropogenic disturbances, grazing, and illegal cuttings have led to the degradations of J. excelsa old-growth stands and have limited the availability of large-diameter timbers (Milios et al. 2007). Therefore, nowadays its wood is mainly used for turnery, carvings, novelties and small agricultural construc- tions. The wood anatomy and chemistry of J. excelsa has not been studied in detail, un- like many Juniperus in the Western Hemisphere (Phillips 1968; Herbst 1978; Panshin & deZeeuw 1980; ter Welle & Adams 1998; Bauch et al. 2004). Recently, degraded J. excelsa stands in northeast Greece have received attention in terms of their growth ecology, structure, and regeneration patterns (Milios et al. 2007, 2009) while utilisation potentials might also arise. A better insight of J. excelsa wood anatomy and chemistry at the cell wall level could serve as a basis for understanding its wood properties and improve the wise use of its timber. The present study reports the wood anatomical characteristics and scanning UV microspectrophotometric analyses on the distribution of lignin and phenolic extractives in the heartwood and sapwood of J. excelsa.

MaterialS and MethodS

The study material originates from Juniperus excelsa stands located in moderate most south facing slopes of the Pascalia public forest (41° 11'–41° 15' N, 24° 33'–24° 41' E). The forest lies in the central part of the Nestos valley at the Rhodope mountains, Greece. The elevation ranges from 100 to 350 m. The annual rainfall of the area is 676 mm and the mean yearly temperature is 13.4 °C. The substrate is limestone and the soils are sandy-clay and rocky. A cross-cut, with mean diameter 20.1 cm, was taken at breast height from a dominant tree approximately 80 years of age and 7.9 m in height to carry out wood anatomical and topochemical studies. The mean diameter of heartwood was 7.0 cm while the mean width of sapwood 10.1 cm. Our tree was a healthy mature individual, representative of J. excelsa stands in the area. Moreover, it was selected among other trees with minimal lean as to avoid compression wood. The cross-cut at breast height did not show any marked eccentric growth.

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Light microscopy Transverse, radial, and tangential sections (15–20 µm thick) were sequentially prepared from cambium to pith using a sliding microtome. Several sections of heartwood and sapwood were both unstained and double stained with safranin and astra blue, and embedded with Euparal for normal light microscopy. Furthermore, wood material was reduced to slivers separately for heartwood and sapwood and macerated in a mixture of equal parts of acetic acid and hydrogen peroxide (20 vol) in an oven at 60 °C for 48 hours (Tsoumis 1991). The macerations were mounted in glycerine for tracheid length measurements. We selected five successive growth rings with moderate width in each of three radial positions for determining tracheid length values in heartwood and sapwood. Heartwood material was taken more peripheral than ring 25 to avoid juvenile wood, at approximately 2.5 cm from the pith. Sapwood being wider than heartwood was sampled at approximately 5.5 cm and 8.5 cm from the pith. Microscopic observations and analysis were carried out with an Olympus AX 70 microscope and a digitized image analysis system (analySIS®, Olympus). Descriptive terminology followed the IAWA List of Microscopic Features for Softwood Identifica- tion (IAWA Committee 2004). About 100 measurements for tracheid length and 50 measurements for all other microscopic features (cell wall thickness, lumen diameter) were taken (Hapla & Saborowski 1987). Cell wall thickness and lumen diameter were measured in the tangential direction.

Ultra-violet (UV) microspectrophotometry (UMSP) The topochemical distribution of lignin and phenolic extractives was investigated on a subcellular level using scanning UV microspectrophotometry (Zeiss UMSP 80 microspectrophotometer) as in Koch and Kleist (2001) and Koch and Grünwald (2004). Small heartwood and sapwood blocks (1 × 1 × 5 mm) were directly embedded in Spurr’s epoxy resin (Spurr 1969) under mild vacuum with several cycles of evacuation and ventilation as described by Kleist and Schmitt (1999) to avoid chemical changes of the extractives caused by reactions with solvents. Transverse sections of 1 µm in thickness were cut with an ultramicrotome equipped with a diamond knife. The sections were transferred to quartz microscope slides, embedded in non-UV absorbing glycerine and covered with a quartz cover slip. The topochemical analyses were carried out with a UMSP 80 microspectrophotometer (Zeiss) equipped with a scanning stage which enables the determination of image profiles at defined wavelengths with the scan softwareAPAM OS® (Zeiss). The topochemical distribution of lignin and extractives was recorded at a defined wavelength of 280 nm (representing the absorbance maximum for softwood lignin). The scan program digitises rectangular fields on the tissue with a local geometrical resolution of 0.25 × 0.25 µm and a photometrical scale resolution of 4096 grey scale level, which are converted to 14 basic colours to visualize the UV absorbance intensities. The scans were depicted as two-dimensional image profiles, including a statistical evaluation of the UV absorbance values. The photometric characterisation of individual cell wall layers and tissues impreg- nated with extractives was additionally carried out by point measurements with a

Downloaded from Brill.com09/24/2021 06:19:30PM via free access 70 IAWA Journal, Vol. 32 (1), 2011 spot size of 1 µm2 between 240 and 560 nm wavelengths. For quantitative studies, 15 spectra were taken from each cell wall layer and cell type, respectively and evaluated with the program LAMWIN® (Zeiss). The lignin concentration was estimated semi- quantitatively according to Lambert-Beer’s law: UV absorbance = ε·C·d where ε is the extinction coefficient, C the volume concentration and d the thickness of the absorbing layer. Considering the cell wall in a cross section prepared of 1 µm thickness, the incident UV light intensity I0 is reduced to the intensity Icell wall emerging from the cell wall due to the absorbance by the constituent lignin. Measurement of I0 is facilitated by the passage of the incident radiation unchanged through the embedding medium in the cell lumen. I0 may therefore be replaced by Ilumen, the intensity of UV light emerging from the lumen:

UV absorbance = log Ilumen/Icell wall Thus, the prepared sections of 1 µm thickness are individually calibrated (high sensi- tivity of the UV light transmission) and the detected absorbance values should be evaluated on a semi-quantitative level.

Results and discussion Wood anatomy Selected quantitative data (average ± standard deviation; minimum and maximum values in parentheses) of wood anatomical structures of Juniperus excelsa are listed in Table 1. Figure 1 shows various anatomical characteristics in heartwood and sapwood of J. excelsa.

Table 1. Anatomical characteristics of mature wood of Juniperus excelsa.

Anatomical characteristic Mature wood (heartwood + sapwood) Earlywood tracheid double wall thickness (μm) 7 (5–10) ± 0.9 Latewood tracheid double wall thickness (μm) 10 (7–13) ± 1.6 Earlywood tracheid lumen diameter (μm) 14 (9–17) ± 2.3 Earlywood tracheid length (mm) 1.4 (1.0–1.9) ± 0.2 Latewood tracheid length (mm) 1.8 (1.5–2.3) ± 0.2 Ray height (number of cells) 4.0 (1–9) ± 1.7 Note: cell wall thickness and lumen diameter were measured in the tangential direction.

Sapwood is distinctly lighter in colour compared to the yellowish-brown to dark brown heartwood, locally with a purplish or reddish tinge and dark colour stripes. The heartwood darkens slightly under prolonged exposure. Boundaries of growth rings are distinct. The prominent latewood zones produce a decorative figure on tangential (V-shaped markings) and radial (fine striping) faces. Transition from earlywood to latewood is gradual. Latewood zones are relatively narrow and composed of up to 5 cells. Resin canals are absent. False rings are present (Fig. 1a). Tracheid outline is polygonal or rounded (Fig. 1b). Intercellular spaces exist occasionally (Fig. 1b).

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a b

c d

Figure 1. Microtome sections of heartwood and sapwood of Juniperus excelsa. – a: Transverse unstained section of heartwood showing a false ring (arrows) and brown substances in ray and axial parenchyma. – b: Much lower presence of brown substances in sapwood (transverse unstained section). – c: Uniseriate tracheid pits and homocellular rays with cupressoid cross-field pitting (radial stained section of sapwood). – d: Uniseriate rays composed of circular cells filled with substances. The arrow indicates a nodular axial parenchyma end wall (tangential stained section of heartwood). — Scale bars in a & b = 200 µm; in c = 40 µm; in d = 50 µm.

Tracheids are 1.4 (1.0–1.9) mm and 1.8 (1.5–2.3) mm long in earlywood and latewood, respectively. The double wall thickness of earlywood tracheids average 7 (5–10) µm. Latewood tracheids are relatively thin-walled with the double wall thickness being 10 (7–13) µm. Earlywood tracheids possess a lumen diameter of 14 (9–17) µm. Tracheid pitting on radial walls of both earlywood and latewood is uniseriate (Fig. 1c). Organic deposits and helical thickenings in tracheids are absent. Axial parenchyma cells are present and filled with brown extractives (Fig. 1a). The concentration of brown extractives is much lower in sapwood (Fig. 1b). Axial parenchyma is solitary or grouped into loosely tangential bands, especially in the latewood (Fig. 1a). The end walls of axial parenchyma cells are slightly sinuous (Fig. 1d). Rays are homocellular (Fig. 1c), uniseriate (Fig. 1d) and rarely partly biseriate. Ray tracheids are absent. Cross-field pitting is cupressoid (Fig. 1c). Ray cells are circular (Fig. 1d) and filled with a higher concentration of extractives in heartwood (Fig. 1a, 1d) than in sapwood (Fig. 1b, 1c). Ray height ranges from 1 to 9 cells. Horizontal walls of ray parenchyma cells are smooth, and end walls are nodular (Fig. 1c).

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Figure 2. Representative 2D profiles of UMSP analysis of heartwood (a, c, e) and sapwood (b, d, f) tissue in Juniperus excelsa. The colour pixels represent different UV absorption values of the cell wall layers and phenolic extractives measured at 280 nm.

Juniperus excelsa possesses the basic wood anatomical attributes of the genus as described by ter Welle and Adams (1998). In general, the wood structure of this genus is homogeneous and the differences between the various species are mostly quantitative (Jacquiot 1955). Also, Ayaz and Nasir (1992) indicated similar results for J. excelsa grown in Pakistan.

Topochemical characterisation of lignin and extractives Figure 2 shows representative UV scanning two-dimensional (2D) profiles measured at a defined wavelength of 280 nm forJ. excelsa heartwood and sapwood. The colour scale indicates the semi-quantitative UV absorbance representing the distribution of lignin (absorbance maximum at 280 nm) and phenolic extractives. A distinct lignification of tracheids was observed in both heartwood and sapwood (Fig. 2a, b). In detail, the cell corners and compound middle lamellae are characterised by comparatively high UV absorbance values (abs280nm 0.8 to 1.0) as compared to the adjacent S2 layers with a lower, slightly varying lignin distribution (abs280nm 0.5 to 0.7). These results coin- cide with past findings by Fergus et al. (1969), Koch and Kleist (2001), Koch and Grünwald (2004), Bauch et al. (2004), and Jungnikl et al. (2008), who all demon- strated the applicability of this technique for the topochemical detection of lignin within

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individual cell wall layers. However, the detected absorbance values of the individual cell wall layers of J. excelsa were significantly higher in comparison to other softwoods studied (Koch & Kleist 2001; Koch & Grünwald 2004). The evaluated mean absorbance values (including the entire cell walls) of the scanned tracheids of J. excelsa amount in the range of 0.55. In comparison, the mean absorbance values of spruce tracheids (Picea abies) are in a range of 0.30 (e.g. Koch & Kleist 2001) representing the sig- nificant higher lignification ofJ. excelsa. Comparative supplementary point measurements from 250 nm to 560 nm show typical softwood lignin spectra of the individual wall layers with a distinct maximum at 280 nm and a local minimum at about 250 nm (Fig. 3a, b). The maximum absorbance at 280 nm usually indicates the presence of the strongly absorbing guaiacyl-lignins (Fergus et al. 1969).

a 1.6 calibration-curve 1.4 CML-layer CML-layer 1.2 CML-layer S2-layer 1.0 S2-layer S2-layer 0.8 extractive-ray extractive-ray 0.6 extractive-ray

0.4

0.2

0.0 240 290 340 390 440 490 540 wavelength (nm) b 1.6 calibration-curve 1.4 CML-layer CML-layer 1.2 CML-layer S2-layer 1.0 S2-layer S2-layer 0.8 extractive-ray extractive-ray

absorbance 0.6 extractive-ray

0.4

0.2

0.0 240 290 340 390 440 490 540 wavelength (nm)

Figure 3. UV absorbance spectra of compound middle lamella (CML-layer) and secondary wall (S2-layer) of tracheids, and extractives in ray cell lumen (extractive-ray) of heartwood (a) and sapwood (b) in Juniperus excelsa.

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Scanning UV microspectrophotometry can also be used to detect and quantify phenolic extractives associated with the woody tissue. The presence of extractives can be easily visualised as spherical conglomerations of high absorbance as compared with the surrounding tissue. In Figure 2d and 2e local depositions of extractives in the lumina of axial and ray parenchyma cells of J. excelsa are emphasised by a significantly higher absorbance (abs280nm 0.8 to overflow) as compared to the cell wall associated lignins. Phenolic compounds are generally synthesised by parenchyma cells in situ and are highly condensed. The spectral behaviour of the detected compounds is illustrated in Figure 3. Their absorbance maxima display a bathochromic shift to a wavelength of 285 to 290 nm and wide shoulder at a wavelength range of 390 to 460 nm. This spectral behaviour can be explained by the presence of chromophoric groups, e.g., conjugated double bonds. The higher degree of conjugation stabilises π-π* transitions resulting in absorbance bands shifted to higher wavelengths (Goldschmid 1971) which can be detected by UV microspectrophotometry. However, the technique does not allow the chemical identification of the extractives. The measurements reveal the deposition of higher condensed phenolic compounds in heartwood tissue of J. excelsa than in sapwood (Fig. 3b).

Conclusion

The present study reports on the wood anatomy of Juniperus excelsa and topochemical detection of lignin and phenolic extractives at subcellular level. Juniperus excelsa was found to be very similar in its wood anatomy with other Juniperus species. Cellular UMSP analysis revealed a pronounced lignification of both the heartwood and sapwood. Interestingly, the results showed much higher UV absorbance values in the individual cell wall layers compared to other softwoods studied before using the UMSP analysis. It should be mentioned that previous studies reveal a very good correlation between the topochemical analyses of the lignin content using UMSP and the determination of e.g. Klason lignin content with wet-lab chemistry (Koch et al. 2003). Deposits of extractives could be clearly detected in both ray and axial parenchyma cells and at higher concentrations in heartwood than in sapwood. The study provided knowledge on wood anatomy and topochemistry of J. excelsa required for its utilisation also in applications (e.g. fibre and glued products), other than construction timber, which presumes availability of large dimensions.

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