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1 Norwegian Forest Research Institute, Høgskoleveien 12, N-1432 Ås, Norway; 2 Botany Department, Washington State University, Pullman, Washington 99164; and 3 Laboratory for Analytical Chemistry, Agricultural University of Norway, N-1432 Ås, Norway
Received for publication January 5, 1999. Accepted for publication July 2, 1999.
ABSTRACT
Wounding of Norway spruce by inoculation with sterile agar, or agar containing the pathogenic fungus Ceratocystis polonica, induced traumatic resin duct formation in the stem. Visible anatomical responses occurred in the cambium 6–9 d post-inoculation. Near the inoculation site cellular proliferation, polyphenolic accumulation, and lignification were induced as a wound reaction to seal the damaged area. Five centimetres from the inoculation site cells in the cambial zone swelled and divided to form clusters. By 18 d post-inoculation, these cells began to differentiate into resin duct epithelial cells surrounding incipient schizogenous lumens. Mature axial traumatic ducts appeared by 36 d as a row of ducts in the xylem centripetal to the cambium. The ducts formed an interconnected network continuous with radial resin ducts. Parenchyma cells surrounding the ducts accumulated polyphenols that disappeared as the cells differentiated into tracheids. These polyphenols appeared to contain fewer sugar residues compared to those accumulating in the secondary phloem, as indicated by the periodic acid—Schiff's staining. The epithelial cells did not accumulate polyphenols but contained immunologically detectable phenylalanine ammonia lyase (EC 4.3.1.5), indicating synthesis of phenolics as a possible resin component. These findings may represent a defense mechanism in Norway spruce against the pathogenic fungus Ceratocystis polonica.
Key Words: Picea abies • Pinaceae • plant defense • polyphenol • traumatic resin duct • wound response
The family Pinaceae, which has existed since at least early Cretaceous, is the largest modern conifer family and forms the most widespread coniferous forests of the Northern Hemisphere (see Biswas and Johri, 1997
). The success of these long-lived plants may partly be ascribed to their ability to resist a constant pressure of attacks by insects and microorganisms.
Bark beetles and their associated pathogenic fungi represent one of the most serious pests to coniferous forests (Berryman, 1972
). An important defense response of conifers to insect/fungus attacks is production and mobilization of resin (Berryman, 1972
; Raffa and Berryman, 1983
), a mixture of monoterpenes, sesquiterpenes, and diterpene resin acids (Croteau and Johnson, 1985
; Gershenzon and Croteau, 1991
). The defensive effects of conifer resin are attributed to its toxic and sticky properties, to massive resin flow that can flush out or repel invading insects, and to inhibitory effects on pathogenic fungi (Reid, Whitney, and Watson, 1967
; Shrimpton and Whitney, 1968
; Bordasch and Berryman, 1977
; Gijzen et al., 1993
).
Conifer resin is synthesized and accumulated in specialized secretory structures, such as resin ducts, resin blisters, and resin cavities. Bannan (1936)
made a classification of the conifers based on the anatomy of these structures. In the stem of fir (Abies), cedar (Cedrus), hemlock (Tsuga), and golden larch (Pseudolarix) the resin-producing cells form blisters, which are sac-like structures lined by epithelial cells. These cells are short-lived, and their walls become lignified during development. More complex structures are the tube-like resin ducts, found in wood and bark of spruce (Picea), pine (Pinus), larch (Larix), and Douglas-fir (Pseudotsuga). In these genera, thin-walled, long-lived secretory epithelial cells synthesize resin.
Resin ducts may appear as a normal feature of development in tissues and organs, or their formation may be induced by external factors. In response to wounding, conifers such as spruces develop axial resin ducts in the secondary xylem, which normally are absent or occur in low numbers in unwounded trees. In contrast to "normal" axial ducts, which generally are single and have a scattered distribution, these so-called "traumatic ducts" are found in tangential series of one or two rows within an annual ring of xylem (Thomson and Sifton, 1926
; Bannan, 1936
). The induction of traumatic resin ducts and resin synthesis represents a widespread defense mechanism in the Pinaceae, and may have developed as an evolutionary adaptation to defend against bark-beetle attack (Berryman, 1972
).
We have used even-aged clonal stands of Norway spruce [Picea abies (L.) Karst.] to investigate stem defense reactions to simulated attack of a bark-beetle-transmitted pathogenic fungus. Recent studies have shown that polyphenolic parenchyma cells in the secondary phloem are particularly responsive to wounding and fungal inoculation (Franceschi et al., 1998
) and that resistance to pathogenic fungi can be induced by pretreatment with mechanical wounding or fungal infection (Christiansen et al., 1999b
). Formation of axial traumatic resin ducts in the sapwood, seem involved in spruce resistance to bark-beetle attack and fungal mass-inoculation (Christiansen et al., 1999a
), and preliminary evidence suggest that traumatic resin is more fungistatic than normal resin (Solheim, 1991
), probably due to inclusions of substances other than monoterpenes, such as phenolics. However, very little information exists on the precise site, mechanism, and timing of traumatic resin duct development. The purpose of this study was to characterize the basic temporal and spatial features of traumatic resin duct formation in Norway spruce and to explore cytochemical features relevant to the defense potential of the traumatic resin ducts and the surrounding tissues.
MATERIALS AND METHODS
Plant material
The field experiment was carried out June to August 1996, using 28-yr-old Norway spruce trees from a clonal stand at the Hogsmark plantation of the Norwegian Forest Research Institute, Ås, Norway. This stand is described by Franceschi et al. (1998)
. Two clones, one relatively resistant (clone 168) and one susceptible (clone 495) to the blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau, were used. The fungus is an important associate of the spruce bark beetle Ips typographus L. and a pathogen to Norway spruce (Horntvedt et al., 1983
; Solheim, 1986
). Colonies of C. polonica were provided from the Culture Collection of the Norwegian Forest Research Institute.
The stems of four trees (two of each clone) were experimentally wounded into the cambium by removing a plug of bark (5 mm diameter) with a cork borer (Wright, 1935
). The wounds were inoculated with agar, either sterile or colonized by C. polonica, before the bark plugs were reinserted. A total of ten inoculation wounds were made in two rings around the trunk (0.50 m apart with the lowest 1.5 m above ground), each ring with alternating fungal and sterile wounds.
Tissue samples were collected 0, 3, 6, 9, 18, and 36 d after inoculation, by cutting out a rectangular strip (1.6 cm width x 10 cm height x 0.5 cm depth) with the inoculation site at the lower end. From each tissue strip, samples were dissected 0.3, 5, and 10 cm above the inoculation sites. All the samples included phloem, vascular cambium, and xylem and were cut to produce both cross, radial, and tangential surfaces. The samples were further processed for microscopy, as described below.
Resin embedding
The samples were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde in 50 mmol/L L-piperazine-N-N’-bis (2-ethane sulfonic) acid buffer (pH 7.2) for 12 h at room temperature. Fixed tissue was rinsed with the same buffer, dehydrated in ethanol series (70-80-90-96-4 x 100%), infiltrated with L. R. White acrylic resin (TAAB Laboratories, Aldermason, Berkshire, UK), and polymerized at 60°C for 24 h.
Anatomy
For light microscopy (LM) study, semi-thin (1 µm) sections were cut with a diamond knife on an ultramicrotome, dried onto silanated slides (Digene Diagnostic, Beltsville, Maryland, USA), and stained with Stevenel's blue (del Cerro, Cogen, and del Cerro, 1980
). The sections were mounted with immersion oil and imaged in a Leitz Aristoplan light microscope.
Periodic acid—Schiff's (PAS) staining of polysaccharides
LM sections were oxidized with periodic acid (1%) for 1 h, then washed with distilled water, and treated with Schiff's reagent (Sigma Chemical Co., St. Louis, Missouri, USA) for 1 h in darkness. Control slides were incubated in distilled water prior to treatment with Schiff's reagent. After several washes the sections were dried, mounted, and imaged as above. PAS is selective for carbohydrate residues with vicinal-hydroxyl groups, which in the first step is oxidized to aldehyde groups by periodic acid, and then reacts with Schiff's reagent to give a pink-colored complex (Hotchkiss, 1948
; McManus, 1948
).
Immunocytochemistry
To investigate the possible role of traumatic resin ducts in phenol synthesis the enzyme phenylalanine ammonia lyase (PAL) was examined by immunolocalization technique. Semi-thin sections (1 µm) of LR White resin embedded tissue on silanated slides were blocked for non-specific binding of antibodies by incubation for 1 h in 1% bovine serum albumin (BSA) and 0.05% polyvinyl pyrrolidone (PVP, 10 000 molecular mass) in tris-buffered saline with Tween 20 (TBST; 10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween 20, pH 7.2). The sections were then incubated for 4 h at room temperature with a rabbit anti-PAL polyclonal antibody diluted 1:25 with 1% BSA in TBST. The antibody was made against PAL isolated from Forsythia suspensa stems (Dr. N. Lewis, Institute of Biochemistry, Washington State University), and the specificity in Norway spruce bark was verified by Franceschi et al. (1998)
. After incubation in primary antibody the sections were rinsed with 1% BSA in TBST 4 x 15 min, and incubated in 10 nm protein A gold (Janssen Biotech, Olen, Belgium) for 1 h at room temperature, diluted 1:50 with 1% BSA in TBST. After sequential rinsing with 1% BSA in TBST, TBST, and distilled water (15 min in each), the sections were silver enhanced (Amersham Silver Enhancement kit, Amersham Life Science Ltd., Buckinghamshire, UK) and stained with 0.5% safranin O. Finally the sections were mounted with immersion oil and imaged with a laser scanning confocal microscope (BioRad 1024, Hercules, California, USA), using a combination of reflected light and transmitted imaging.
RESULTS
Normal anatomy
Prior to inoculation (0 d), the tissue appeared typical of Norway spruce (see Franceschi et al., 1998
). The vascular cambium contained small, rectangular, flattened cells as viewed in cross section (see 
Figs. 5, 6). The secondary phloem consisted of rows of sieve cells, axial phloem parenchyma cells containing polyphenolic bodies, and radial ray parenchyma cells. The xylem region consisted of regular rows of tracheids and xylem mother cells close to the cambium. Occasionally radial resin ducts in the phloem and scattered axial resin ducts in the xylem were observed in untreated samples.
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). The zone, extending from secondary phloem, through the cambium and into the secondary xylem, contained thin-walled axial and ray parenchyma cells as well as irregularly shaped thick-walled tracheids (Figs. 2, 3). No hyphae were seen in fungal-inoculated material sampled 5 and 10 cm above the wound site. Both treatments, however, resulted in the appearance of a tangentially oriented band of axial traumatic resin ducts in the newly formed xylem 18 d after inoculation (Fig. 4). Traumatic resin ducts seemed more developed in resistant clones than susceptible clone at this time. Otherwise cells and tissues at these distances from the wound appeared similar to that seen in untreated material (Fig. 5).
Development of axial traumatic resin ducts
The traumatic ducts were more developed and formed longer bands at a distance 5 cm compared to 10 cm above the inoculation site. Otherwise, tissues from the two sampling sites appeared similar. Samples taken at 5 cm, however, are located well within the reaction zone known to form after wounding (Reid, Whitney, and Watson, 1967
; Berryman, 1972
; Horntvedt et al., 1983
; Shigo, 1984
) and were used for characterization of general features of traumatic duct development described below.
Six and nine days after inoculation, small clusters of swollen, irregularly shaped, periclinal and anticlinal dividing cells appeared in the region of the cambial zone containing what we interpret as xylem mother cells (Figs. 7, 8). These cell clusters are here considered to represent initial stages in the development of traumatic resin ducts. Eighteen days after inoculation, the clustered cells had begun differentiation into traumatic duct epithelial cells, and gaps produced schizogenously appeared between them (Figs. 9, 10). The epithelial cells were distinguishable from other cambial derivatives by their dense cytoplasm containing many plastids and a conspicuously enlarged nucleus (Fig. 10). Frequently, a zone of thin-walled parenchyma cells with polyphenolic bodies and starch grains appeared around the developing traumatic resin ducts (see Fig. 19). Polyphenol accumulations were also a feature of ray parenchyma cells adjacent to the ducts, but never occurred in ray parenchyma or derivatives of the cambial initials in unwounded samples.
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Three-dimensional structure of axial traumatic resin ducts
Examination of samples in cross, tangential, and radial sections showed that the traumatic ducts formed an anastomosing reticulum (Figs. 13, 14, 17). In cross sections, the adjacent traumatic ducts often fused into more elongated structures (Fig. 13), due to longitudinal enlargement of the lumen and schizogeny of cell layers between single ducts. Lumenal interconnections between the axial traumatic resin ducts and radial resin ducts were occasionally observed in both cross sections (Fig. 13) and tangential sections (Figs. 15, 16). In radial sections, mature traumatic resin ducts appeared as epithelial lined tubes in close connection to ray parenchyma (Fig. 17). Serial sections indicated that the lumen of the ducts was often deflected around the radial rays and thus the ducts are not simple, short, resin-filled tubes. It was also observed that many radial rays in the reorganized cambial zone did not extend centripetally to the region of the traumatic ducts and were seen only in the most recent rows of xylem and phloem (Fig. 11), indicating they were initiated at, or after, traumatic duct formation.
Polyphenols and starch
The PAS reagent stained cell walls and starch for carbohydrates, but also polyphenolic bodies of phloem parenchyma cells and xylem parenchyma cells associated with the traumatic resin ducts. The reaction is visualized by grey to black intensity in Figs. 18–24. At day 0, staining properties of polyphenolic bodies of phloem parenchyma cells ranged from very faint staining in the youngest layers (Fig. 18) to a stronger coloration of older cell layers. Newly accumulating polyphenolic bodies of thin-walled parenchyma cells and ray parenchyma reacted weakly with PAS and had a light staining compared to phenols in the old phloem parenchyma cells that stained intensely dark (Fig. 19). By day 36, the loss of polyphenols in the traumatic duct region (comparing Figs. 19–20) resulted in an intense PAS staining of thick-walled cells now surrounding the traumatic ducts (Figs. 20, 22). Furthermore, there was an enhanced PAS staining of polyphenolic bodies in phloem parenchyma on days 18 and 36 (Figs. 19–20) compared to day 0 (Fig. 18).
All parenchyma cells in the phloem contained abundant and conspicuous PAS-stained starch grains at the time of inoculation. The older axial parenchyma cells contained large starch granules, while younger cells contained smaller grains (Figs. 18, 23). Starch grains also occurred in cytoplasm of ray parenchyma outside the cambial zone and in small amounts in traumatic duct epithelial cells (Figs. 21, 22). PAS-positive materials was seen in the lumen of developing (Fig. 21) and mature (Fig. 22) traumatic ducts as well, but not in the intercellular spaces between other cell types. Comparison of PAS staining of serial sections with and without oxidation (negative control) illustrates that the PAS procedure is selective for polyphenols, cell walls, and starch in all samples only after oxidation (Figs. 23, 24).
Cytochemical localization of PAL
Phenylalanine ammonia lyase (PAL) was localized in the ray parenchyma cells and the polyphenol-containing parenchyma cells both in the xylem parenchyma surrounding developing traumatic ducts and in the secondary phloem (data not shown), in accordance with recent work of Franceschi et al. (1998)
. A particularly interesting observation was that significant labeling for PAL also occurred in the traumatic duct epithelial cells (Figs. 25, 27) compared to the pre-immune control (Fig. 26). PAL was mainly distributed in the cytoplasm and along the inner surface of the plasma membranes lining the walls of these cells.
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This study demonstrates how axial traumatic resin ducts are induced and develop in response to wounding and fungal inoculation of Norway spruce stems. Traumatic resin ducts are complex anatomical and biochemical structures whose formation puts heavy demands on plant resources and redirects the cambial activity for a brief period. The eventual benefit of this investment is enhanced defense potential by increased resin flow. However, as discussed below our observations indicate that the defense potential goes beyond enhanced resin production.
Wounding by inoculation with sterile agar or fungus-infected agar led to the induction of chemical and anatomical defenses that may isolate the injured tissue and thus prevent the spread of pathogen at different distances from the wounded site. Adjacent to a wound we observed abnormal tissue consisting of aggregations of thin- and thick-walled polyphenol-rich parenchyma cells merged with ray cells and deformed tracheids (Figs. 2, 3). In fungi-infected wounds this region was characterized by local cell death (Fig. 1), which may be related to a hypersensitive reaction (Berryman, 1972
). These anatomical characteristics of wounded and infected cambium is likely to be a massive response at the site of damage, and form a "barrier zone" to seal off the wound or the infective agent and protect new cells from decay (Wong and Berryman, 1977
; Shigo, 1984
; Blanchette, 1992
). Such a barrier zone is complemented by the more highly regulated traumatic duct induction observed at a greater distance from the wound leading to impregnation of beetle and fungus in dead resinous tissue.
Traumatic resin duct formation is not part of the normal activity of the vascular cambium and thus raises some interesting developmental questions as to its origin. Bannan (1936)
states that it is the fusiform initials and not the ray initials of the vascular cambium that give rise to the axial resin ducts by segmentation. An additional interpretation, based on a study of allepo pine, is that the resin ducts are derived from the xylem mother cells in the cambial zone (Werker and Fahn, 1969
). Our observations of early developmental stages indicate that the xylem mother cells are the primary source of traumatic duct development in Norway spruce. The strict distribution pattern of their initiation implies a combination of predisposition and a tangential spread of a stimulus resulting in coordinated induction and differentiation of the xylem mother cells. The closely spaced radial rays may play a role in the organization of traumatic duct formation, as well as in the transfer of inductive stimuli and nutrients from the active phloem (see Werker and Fahn, 1969
; Franceschi et al., 2000
). This could explain why the traumatic resin ducts most frequently are associated with or bordered by a radial ray.
Considering the structural and functional complexity of the traumatic ducts, the time of their development with secretory activity by 18 d post-inoculation is quite rapid. The duration of the response points to a mechanism that may deal with resistance to subsequent attacks as discussed by Franceschi et al. (2000)
. This defense response would be too slow to protect against a massive attack of bark beetles, which involves hundreds or thousands of attacks on a tree within a few days (Berryman, 1972
). However, in situations where the aggregation of bark beetles is slow, the formation of traumatic resin ducts and associated chemical components may enhance the defensive capacity of the tree and reduce further beetle and fungal propagation. This is consistent with observations that traumatic ducts are formed not only at the wounded sites but also at considerable distances away (Christiansen et al., 1999b
), and thus may contribute to produce a broad, resin-filled deterrent that may inhibit later attacks.
Our data indicate that resistant clones induced traumatic ducts somewhat earlier than susceptible clones and were less damaged by fungal infection at earlier points of time (day 18). These observations are consistent with a recent study by Tomlin et al. (1998)
, which shows that trees from resistant families of white spruce have a stronger and more rapid response than trees from susceptible families, by producing multiple rings of traumatic resin canals. Wounding by sterile agar caused no difference in timing of the traumatic duct development as compared to fungi-infected agar. Although based on limited data, this confirms that development of traumatic resin ducts is a general response that may be induced by various stimuli such as mechanical wounding, pathogen infection, and application of plant growth hormones (Bannan, 1936
; Fahn and Zamski, 1970
; Fahn, Werker, and Ben-Tzur, 1979
; Kuroda and Shimaji, 1983
; Cheniclet, 1987
).
This study shows that the mature traumatic resin duct system forms a complex, interconnected epithelial cell-lined network of resin-filled cavities in the tangential plane of the new sapwood. The network encompasses direct contact between traumatic duct epithelial cells and the ray parenchyma cells, as well as lumenal continuity between adjacent traumatic ducts and radial resin ducts. In this way, the two duct systems complement each other by spanning the trunk in the axial direction as well as radially from the sapwood to the outer bark where attacks are initiated. Such networks give a more elaborate protection than if the induced resin system was only present in a narrow axial plane, and they facilitate enhanced resin flow and transport of agents to outer portions of the bark. At the wounded site the volatile components of the resin evaporate on exposure to air, leaving a semisolid residue that provides a physical and chemical barrier against subsequent invasion by pathogens and insects. It is likely that this mechanism contributes to the formation of the resin-filled reaction zones, which have been shown to enclose unsuccessful bark-beetle attacks (Reid, Whitney, and Watson, 1967
; Berryman, 1972
; Horntvedt et al., 1983
; Shigo, 1984
).
Radial resin ducts are found in both untreated and wounded material. It is not clear, from our observations, whether mechanical injury or fungal inoculation has any impact on the formation of these ducts or their connections to axial resin duct lumina. Wounding has previously been shown to induce an overall increase in number of ray cells in other tree species (Lev-Yadun and Aloni, 1992
), but these rays may not necessarily differentiate into radial resin ducts. In loblolly pine, Blanche et al. (1992)
found a correlation between the amount of resin flow and axial resin duct density, but no correlations between resin flow and radial resin duct density. This indicates that the axial ducts are main sources for enhanced resin secretion, and an interrelation between axial and radial resin ducts, as shown here, would further enhance the spatial defensive capacity of the traumatic duct system.
The epithelial cells of the traumatic ducts are thought to synthesize and secrete resin into the lumen of the ducts (Bannan, 1936
). The numerous plastids observed in the cytosol of the epithelial cells are proposed to be the primary site for resin synthesis (Charon, Launay, and Carde, 1987
; see also Gershenzon and Croteau, 1990
). The resin secreted from traumatic ducts may differ from that of preformed resin by containing higher concentrations of compounds that are toxic and repellent to beetles and fungus (Shrimpton and Whitney, 1968
; Bordasch and Berryman, 1977
; Raffa and Berryman, 1983
; Solheim, 1991
; Gershenzon and Croteau, 1991
; Klepzig et al., 1995
). Since the toxicity of resins might be due to changes in the monoterpene and diterpene composition, as well as inclusions of polysaccharides and polyphenolic components, our anatomical and cytochemical observations on the appearance of polyphenols, carbohydrates, and proteins may have relevance to the defensive capacity of traumatic duct resin.
Changes in the physical appearance of polyphenolic bodies occurred during the development of traumatic ducts. The association of polyphenol-rich parenchyma cells in the phloem and their constitutive and inducible defense reactions in Norway spruce bark are described by Franceschi et al. (1998)
, however, the present study shows the appearance of polyphenolic parenchyma cells in xylem close to the developing traumatic resin duct. Wounding leads to accumulation and later to the disappearance of polyphenols as the parenchyma cell walls thicken, and these cells differentiate into tracheids (see McCann, 1997
). The polyphenolic content in the xylem parenchyma cells may contribute to a higher toxicity of the resin secreted. In addition, a centripetal transport and deposition of polyphenols may cause the wood to darken and lead to the appearance of a brown zone seen in discs from the xylem of wounded regions (data not shown). The physical barrier made by lignification and dispersion of polyphenolic compounds would enhance the resistance of the newly formed sapwood to pathogen infection and insect attack. Furthermore, several polyphenolic compounds have anti-fungal activity (see Nicholson and Hammerschmidt, 1992
) due to their toxicity and ability to immobilize enzymes produced by microorganisms (Bell, 1981
).
The chemical properties of the polyphenolic globules may vary during the induction of traumatic resin ducts. While Stevenel's blue or safranine O stained polyphenols of the various cell types with similar intensity, carbohydrate-specific PAS stained polyphenolic bodies in phloem parenchyma cells, xylem parenchyma cells, and associated ray cells differently. Variability in the PAS staining properties under different experimental conditions can be considered as evidence for chemical changes and indicates differences in the carbohydrate composition of polyphenols formed before or after wounding. Since plant phenolic compounds have the ability to form complex compounds with carbohydrates and proteins (Spencer et al., 1988
; Porter, 1989
), the differential PAS reactivity may be related to variations in glycosylation or other chemical modifications of the polyphenolic pool. In this regard it is interesting that Brignolas et al. (1995a, b)
measured radical changes in the soluble-phenol content in Norway spruce phloem in response to wounding and inoculations.
The lumen of traumatic ducts also stained positively with PAS, indicating presence of carbohydrate-containing components in the resin. As terpenoid components are soluble in organic solvents (McNair, 1932
) and mostly are extracted from the tissue samples during the ethanol dehydration used here for tissue processing, the compounds that stained in the traumatic duct lumen may be polysaccharides or reactive polyphenols secreted by the epithelial cells.
Secondary wall lignification and polyphenol synthesis are achieved by enzyme activity of phenylalanine ammonia lyase (PAL), which catalyzes the production of trans-cinnamic acid, the basic precursors for lignin and polyphenol compounds (Camm and Towers, 1973
; Bell, 1981
; Nicholson and Hammerschmidt, 1992
). Thus, PAL activity may have relevance to the propagation of the defensive responses and the defensive capacity of resin from traumatic ducts. PAL was found in both ray parenchyma cells and polyphenol-containing parenchyma cells in phloem and xylem. This is consistent with the synthesis and accumulation of polyphenolic substances in these cells and may be important for the differentiation of parenchyma cells into lignified tracheids. PAL was also found in epithelial cells of the traumatic resin ducts. Since these cells apparently neither accumulated polyphenolic bodies, nor developed secondary thickened lignified walls during the study period, the function of PAL may here be related to secretion of soluble phenols into the traumatic resin. In this regard it is interesting that some common features in structural organization and regulation of phenolic and terpenoid biosynthesis have recently been reported (Paseshnichenko, 1995
).
Our system for experimental induction of traumatic duct formation in Norway spruce allows characterization of basic spatial and temporal features of their development. This study indicates a complex defense response that requires a rapid and coordinated reprogramming of cambial zone cells, resulting in a complex resin-producing network within an 18-d period. With respect to defense, the results of traumatic duct development are: (1) massive accumulation of resin and presumably enhanced resin flow at the site of damage; (2) accumulation and deposition of polyphenolics in the sapwood surrounding the traumatic ducts, presumably with accompanying enhanced anti-fungal activity; (3) production of a physical barrier due to the complex anastomosing structure, which later becomes lignified; and (4) the likelihood of a more toxic resin-phenol mix being produced by traumatic duct epithelial cells. Together, the traumatic resin ducts and the surrounding polyphenol accumulating cells may provide a potent protective structure where chemical substances accumulate, and are ready to retard the longitudinal and inward spread of pathogenic organisms attempting invasion after wounding.
FOOTNOTES
1 This study was done at the Electron Microscopy Center, Washington State University, Pullman, USA, and the Electron Microscopy unit at Laboratory for Analytical Chemistry, Agricultural University of Norway. Support was in part by a Norwegian-Fulbright Foundation Fellowship to N. E. Nagy, which is gratefully acknowledged, and Norwegian Research Council grant number 113626/111 to E. Christiansen. The authors thank Dr. N. Lewis, Institute of Biological Chemistry, Washington State University, for the antibodies, and Dr. A. Lønneborg, NISK, and Dr. A. Berryman, WSU, for valuable comments to the manuscript. 
2 Author for correspondence (Fax: 47 64 94 29 80; e-mail: Erik.Christiansen{at}nisk.no
).
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