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1 Botany Department, Washington State University, Pullman, Washington 99164-4238 USA; 2 Norwegian Forest Research Institute, Høgskoleveien 12, N-1432 Ås, Norway; 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
The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined. Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid-Schiff's staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation. In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce. These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.
Key Words: bark beetles • phenolics • phloem • Picea abies • Pinaceae • plant defense • resin • traumatic ducts
The bark of conifers, which includes the periderm and secondary phloem, provides a sophisticated defensive barrier to invading organisms. This barrier includes static and constitutive defenses such as suberized and/or lignified periderm derivatives, sclerified cells, or cell layers (Wainhouse, Cross, and Howell, 1990
), cells with calcium oxalate crystals (Kartuch, Kartuch, and Weilgong, 1991
), parenchyma cells filled with secondary products such as phenolics (Cheniclet, Bernard-Dagan, and Pauly, 1988
; Franceschi et al., 1998
), and multicellular ducts or canals with constitutive resins and other products (Wu and Hu, 1997
). The defensive roles of these structures are deduced from their anatomy and chemical composition. The constitutive defense mechanisms provide an immediate resistance to invasion of the bark, but may be overcome by organisms that have become adapted to these structures. Thus, of equal importance to a defensive strategy are inducible defense systems. In conifers, secondary resin production (Raffa and Berryman, 1982
; Croteau et al., 1987
; Klepzig et al., 1995
), synthesis of new phenolics (Brignolas et al., 1995a, b
; Klepzig et al., 1995
), traumatic resin duct formation (Reid, Whitney, and Watson, 1967
; Berryman, 1969
; Werner and Illman, 1994
; Alfaro, 1995
; Tomlin et al., 1998
; Nagy et al., 1999), and initiation of a wound periderm (Oven and Torelli, 1994
) are known induced responses to wounding or bark beetle and fungus attack. Some of these responses, such as induction of phenolic synthesis, require a change in metabolism of existing cells (Brignolas et al., 1995a, b
; Bois and Lieutier, 1997
; Franceschi et al., 1998
), while other responses such as formation of traumatic ducts (Kuroda and Shimaji, 1983
; Christiansen et al., 1999a
; Nagy et al., 2000) and wound periderm (Fahn, 1990
) involve complex changes in cell division activity/patterns and differentiation.
A number of inducible defense responses have been identified in conifer bark, but the spatial pattern and time frame of induction and the cells responsible for signal reception and transduction have not been well characterized. We have used Norway spruce as a model system to study the constitutive and inducible defense mechanisms of conifer stems. By using fungal inoculations and wounding, it was demonstrated that two primary inducible anatomical responses in this species are changes in phloem parenchyma cells, called polyphenolic parenchyma cells (PP cells; Franceschi et al., 1998
), and induction of formation of traumatic resin ducts (Nagy et al., 2000;
Christiansen et al., 1999a
). One or both of these mechanisms may also be involved in the acquired resistance shown to be inducible in experimentally inoculated Norway spruce (Christiansen et al., 1999b
). Induction of change in activity of the PP cells appears to be relatively rapid, with visible changes seen within 6 d of treatment (Franceschi et al., 1998
), while traumatic duct formation is a longer process, requiring ~2 wk for formation of structures with secretory capacity (Christiansen et al., 1999a
; Nagy et al., 2000). We have suggested that the PP cells represent a primary site of both constitutive and induced defenses based upon their phenolic contents, high constitutive expression of phenylalanine ammonia lyase (PAL, a key enzyme in phenolic biosynthesis), and dynamic nature with respect to cellular changes after wounding (Franceschi et al., 1998
). However, while PP cells are also commonly found in a number of other conifers (Abbe and Crafts, 1939
; Holdheide, 1951
; Murmanis and Evert, 1967
; Alfieri and Evert, 1973
), little is known about their general function in the bark.
The PP cells must play an important role in inducible bark defense since they represent a major proportion of living cells in the secondary phloem (Abbe and Crafts, 1939
; Holdheide, 1951
; Franceschi et al., 1998
). Organisms such as bark beetles are able to break through the periderm layers and interact with the consecutive rows of PP cells. We have hypothesized that wounding by the activity of the bark beetles or pathogenic fungi germinating from the spores they carry is likely to initiate a local response from PP cells in the direct vicinity of the attack (Franceschi et al., 1998
), as well as induce a response in more distant cells via radial and circumferential transmission of a signal through the PP cells and interconnected radial rays (Krekling et al., in press). The purpose of this study was to determine the structural effects induced in PP cells and surrounding secondary phloem by pathogenic fungal inoculation and by bark-beetle attack and to test for the potential of transmission of a signal from PP cells and rays to give rise to defense responses in other tissues some distance from the site of an attack. The results demonstrate that the PP cells of Norway spruce are involved in local and distant defense responses and further indicate the importance of this cell type in conifers.
MATERIALS AND METHODS
Field procedure for experimental fungal inoculations
Three Norway spruce (Picea abies (L.) Karst.) trees, 30 yr old, were selected from a stand at Hogsmark, Ås, Norway for experimental fungal inoculations. Two trees, designated F2 and F3, belonged to the same clone, while one tree, designated F1, belonged to a separate clone that was a half sibling to F2 and F3. Circumferences at inoculation height for the different trees were 76, 86, and 73 cm for F1, F2, and F3, respectively. Trees were inoculated with the pathogenic blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau, an associate of the spruce bark beetle, Ips typographus L. Inoculum consisted of 5-mm diameter plugs of malt agar (2% malt, 1.5% agar) colonized by the fungus. Inoculation with sterile agar produces more limited reactions than do fungal infections (Solheim, 1988
). Since the objective of the present report is to elucidate reactions to fungal inoculation and bark-beetle attack, sterile inoculations were not included.
The inoculations were performed to three different depths in the phloem: ~1 mm inside the periderm, and halfway to the cambium using a 4-mm drill bit, and all the way into the cambium/sapwood with a 5-mm cork borer. In addition, inoculum was placed on the bark surface without wounding the bark. For this treatment, inoculum also consisted of 5-mm diameter plugs of malt agar (2% malt, 1.5% agar colonized by the fungus. The four treatments plus an untreated site for sampling of intact bark and sapwood were evenly spaced in a ring around the bole, with three rings per tree (at 1.4, 2.2, and 3.5 m above ground). Strips of parafilm were wrapped around the tree, covering the inoculation points, to reduce contamination and prevent excessive drying of the inoculations. At the time of the inoculations on 16 June 1997 (wk 0), a sample of fresh bark and sapwood was taken from each tree. Three, 6, and 9 wk later, one ring per tree was sampled, starting with the uppermost ring. Samples with bark and sapwood (~5 cm long and 1.6 cm wide) centered around the inoculation point were excised with a hammer and chisel and immediately immersed in a fixative of 3% glutaraldehyde in 100 mmol/L Hepes buffer, pH 7.0.
Field procedure for bark-beetle samples
Two Norway spruce trees, designated B1 and B2, were selected from the same stand for bark-beetle attack. Both trees were half-siblings to each other and to the three inoculated trees. On 26 May 1997, the trees were baited with one bark-beetle pheromone dispenser each (Ipslure®, Borregaard, Norway) to induce mass attack by I. typographus. This was 3 wk prior to artificial inoculation described above. The earlier time point was chosen to ensure a mass attack during the main flight period of the beetles. The trees were most likely mass attacked on 31 May (day 0), since this was the first day after baiting with temperatures above 18°–20°C, the flight threshold temperature for I. typographus. Five samples containing bark and sapwood were excised from each tree on each of days 3, 6, 13, and 19 after mass attack and immediately immersed in fixative consisting of 3% glutaraldehyde in 100 mmol/L Hepes buffer (pH 7.0). Samples (2–10 cm long, 1 cm wide, 1 cm deep) were taken with a hammer and chisel upward from the beetle entrance hole and in the early samples included the whole length of the beetle gallery.
On each sampling day, 2–4 bark beetle galleries on each tree were exposed and the gallery length was measured in order to estimate the attack stage. Lengths and general characteristics for galleries sampled were as follows. On day 3 (N = 4), gallery lengths were 13–26 mm (tree B1; one gallery with eggs), and 9–17 mm (tree B2), corresponding to attack phase 3–5 (Birgersson et al., 1984
). On day 6 (N = 3) gallery lengths were 36–77 mm (tree B1), and 31–42 mm (tree B2), with eggs in all galleries, i.e., attack phase 5–6. On day 19 (N = 2) gallery lengths were 105–140 mm (tree B1), and 150 mm (tree B2), with eggs in all galleries, i.e., attack phase 7. Both trees were mass attacked and eventually killed by the bark beetles.
Light microscopy
The samples in fixative were taken to the laboratory and a strip 20 mm long by 4 mm wide by ~10 mm deep containing the inoculation site or bark-beetle gallery was dissected out and placed in fresh fixative. Small subsamples of 4 by 2 mm were cut at ~2 mm from the inoculation hole or at the end of the bark beetle gallery. These were fixed overnight in fresh fixative, then dehydrated with ethanol series (70, 80, 90, 96, and 4 x 100%), embedded in L. R. White resin, and polymerized for 24 h at 60°C. Serial cross and radial sections 1.5 µm thick were cut on glass knives, dried onto gelatin-coated slides, and stained with Stevenel's blue (del Cerro, Cogen, and del Cerro, 1980
) for general observation of tissue structure and cell changes. Serial sections were also stained with the periodic acid-Schiff's reagent procedure for sugar residues with vicinal hydroxyl groups. Reagents were from Sigma Chemical Co., St. Louis, Missouri, USA. The sections were incubated for 30 min in 1% w/v periodic acid at room temperature, washed with distilled water, and air dried. Some sections were incubated in distilled water instead of periodic acid, as "non-oxidized" controls. The sections were then incubated in Schiff's reagent in the dark at room temperature for 1 h, rinsed with distilled water, dried, and coverslipped with immersion oil as a mounting medium. Sections were examined and photographed with a Leitz Aristoplan photomicroscope.
Some of the serial sections were dried down onto gelatin-coated slides and coverslipped with immersion oil, unstained, for examination of phenolic body autofluorescence (blue excitation; filter block I3) patterns and intensity, and to check whether phenolics could be seen to accumulate in the walls of tissues after treatment. Ultraviolet light excitation (filter block A) was used to see whether suberization or lignification was occurring in the samples in response to the treatments. A Leitz Aristoplan microscope set up for epifluorescence imaging was used. Previous studies (Franceschi et al., 1998
) showed that auto-fluorescence of the phenolic contents of bark samples can be readily visualized in non-osmicated L. R. White resin embedded samples.
Scanning electron microscopy (SEM)
Large pieces of the original samples (stored in fixative at 4°C) from the bark-beetle experiment containing galleries sliced longitudinally (radial section of bark and sapwood) were selected for SEM observations. Some samples had bark-beetles within the galleries. The larger samples were cut in half, and a few thin pieces were cut from the middle for examination of the galleries and surrounding tissues in cross section (transverse section of wood and bark) as well as longitudinal section. The samples were dehydrated with ethanol series as before, and critical point dried from CO2· Selected samples were mounted on aluminum stubs, sputter coated with palladium/platinum, and examined with a JEOL JSM 840 SEM.
RESULTS
Structural changes induced by fungal inoculations
Examination of surface inoculation samples and fresh bark samples taken from each tree indicated that all three trees had a bark/sapwood anatomy similar to untreated tissues previously examined (Franceschi et al., 1998
), with circumferential layers of PP cells separated by blocks of sieve cells and radial rays, occasionally containing a radial resin duct (Figs. 1, 2, 4, 7, and 10). Sapwood axial resin ducts were generally absent from sections examined or, when present, were small, few, and scattered (see Fig. 4). Most of the PP cells had light, even-staining phenolic bodies. In the present study there were no mature sieve cells below the most recent PP cell layer, and the older sieve cells, between present and previous years layer of PP cells, looked typical, being compressed but not completely collapsed in the first eight layers or so from the cambium. The cambial zone was quite large in terms of numbers of undifferentiated cells in a radial file, which is typical for the time of year the sampling started.
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The surface inoculation samples from 3 wk after treatment (Figs. 1, 10) looked like the original fresh bark sample taken at the start of the experiment (Fig. 2). However, there were dramatic anatomical and cellular changes visible in the sections where inoculations were made below the surface. Even the samples from inoculation just inside the periderm ("subperidermal") showed a strong reaction. For the inoculations that were subperidermal (Fig. 11) and halfway ("1/2"; Figs. 3, 12) into the secondary phloem, the layers of PP cells were swollen and contained very dense phenolic bodies compared to fresh or surface-inoculated samples. The sieve cells in layers older than 2 yr were completely collapsed due to the increase in size of the PP cells. A particularly surprising observation was the presence of traumatic resin ducts in the samples with only subperidermal or 1/2 inoculation (Figs. 3, 11, 12). Well-developed traumatic ducts with evidence of secretory activity (lumen forming and materials seen in lumen) were present in the cambial zone 3 wk after phloem inoculation (Figs. 3, 11). Mature tracheids had not differentiated between the traumatic ducts and the cambial zone at this stage. Another interesting observation made near these shallow inoculation sites 3 wk after treatment was that an additional PP cell layer was forming, with a few scattered PP cells present, ~2–3 mature sieve cells centripetal to the original present year's PP cell layer. In the 1/2 inoculation, proliferation of PP cells in the outer layers was often apparent, which, as will be shown later, is the initiation of a wound periderm. Fungus was not observed in any of the samples from any of the trees with subperidermal or 1/2 inoculations. In contrast, after inoculation to the cambium, part of the cambial zone was destroyed by the fungus, but the infected tissues appeared to be "walled off" by cellular proliferation in the intact part of the cambial zone (not shown, but see Nagy et al., 2000
). This was seen in all three trees. Most of the cambial cell derivatives in this region contained phenolic materials. No fungus could be seen in the region of cellular proliferation or in the sapwood centripetal to it, but could be found in the sapwood centripetal to the destroyed cambial zone. Examination of autofluorescence from these samples indicated that cells closest to the fungal-infected cambium had become partially or completely lignified as indicated by blue-green autofluorescence (not shown). Even some of the mature PP cells directly above the fungal-destroyed cambium had thickened lignified walls. This lignification did not appear to reach beyond the first few layers of cells above the cambium.
Samples from 6 wk after subsurface inoculation showed symptoms similar to the 3-wk samples, but more advanced, while surface inoculation had no apparent effect on anatomy of the bark and cambium (Fig. 4). Subperidermal inoculation samples had a row (in cross section) of mature traumatic ducts with 10–12 mature tracheids between them and the cambial zone (Fig. 5). The PP cells were greatly swollen and had dense phenolic bodies, while the intervening blocks of sieve cells were completely collapsed. An additional PP cell layer was developing 2–3 sieve cells centripetal to the normal PP cell layer for the year of sampling. Inoculations halfway into the secondary phloem also induced formation of mature traumatic ducts, with 9–12 tracheids between them and the cambial zone (Fig. 6). A second row of traumatic ducts was forming in the cambial zone of the sample shown here, with small lumens already present (Fig. 6). Multiple rows of traumatic ducts were not seen in the samples from the other two trees, but are not uncommon in samples examined from very long time points in other studies (see 
Fig. 31). The PP cells were greatly swollen, with dense phenolic bodies, and in some areas of the sample evidence of proliferation of PP cells in the outer layers was seen. An additional PP cell layer was differentiating 2–3 sieve cells centripetal to the original layer. No fungus could be seen in these samples. Tissue samples from the site of inoculation at the cambium were completely destroyed by fungal growth (see Fig. 9), as indicated by the abundance of hyphae in the sections and general loss of cytoplasm from cells. The PP cells appeared to have lost much of their phenolic contents.
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Changes in periodic acid-Schiff's (PAS) staining in inoculated samples
In the sections prepared with a general stain as described above, it appeared that the PP cell phenolic bodies were often more darkly stained after inoculation. Thus, we were interested to see whether this change in general staining might be related to chemical changes in the phenolics. The periodic acid-Schiff's staining procedure (PAS) preferentially stains glycans with vicinal hydroxyl groups and has been successfully used to show chemical changes in PP cell phenolic bodies after more severe wounding (Nagy et al., 2000
). PAS staining of surface-inoculated samples (Fig. 13) and fresh bark samples from wk 0 (Fig. 14) gave intense coloration of cell walls and starch grains, which were abundant in radial rays and PP cells. However, the PP cell phenolic bodies were mostly of pale color, with a few of the PP cells in the outer layers having darker stained phenolic bodies. In contrast, after subperidermal inoculations (Fig. 15) and inoculations halfway through the phloem (Fig. 16) there was a dramatic change in the PAS reactivity of the PP cell phenolic bodies. The PP cell phenolic bodies of these samples stained intensely dark. Where proliferation of PP cells could be seen in the outer phloem, these new cells also had dark-staining phenolic globules in them. Starch was still abundant in these sections, except that little or no starch was present in the epithelial cells of the traumatic ducts. Note that the PAS-stained sections shown are serial sections from the same blocks illustrated in Figs. 1, 2, 3, and 12. Comparison of periodic acid oxidized sections to non-oxidized sections demonstrated that the staining patterns seen, and which will be described below, are not an artifact or non-specific binding of the Schiff's reagent to phenolic compounds, but represent real chemical changes to the PP cell phenolics after wounding (not shown here, but see Nagy et al., 2000
). Only PAS staining results of the 3-wk samples are illustrated. The 6- and 9-wk samples gave the same pattern of changes in PAS staining as seen in the 3-wk samples and thus are not shown here.
PP cells and wound periderm formation
In some of the samples with subperidermal or 1/2 inoculations, there was evidence of PP cell proliferation in the outer, older layers after 3 wk. Examination of many sections indicates this is a stage in wound periderm formation, and thus the PP cells are instrumental in this process. An early stage of this process is the proliferation of cells within a PP cell layer by division of existing cells (Fig. 17), which eventually leads to organization of a line of flattened rectangular cells in cross section (Fig. 18). In our samples, this seemed to occur in PP cell layers that were 8–10 yr old (8–10 rows from the cambial zone). These cells eventually form a well-organized wound periderm with a distinct phellogen (Figs. 19, 20). Within the time frame of our experiments, the phellogen produces a phellem consisting of 2–3 layers of irregularly shaped lignified cells and, interior to that, a layer of thin-walled cells (Fig. 20). This layer of thin-walled cells showed strong blue autofluorescence along the entire wall when viewed with UV light, indicating suberization (Fig. 21). Suberization was not apparent in the lignified cells nor in any other cell type except in PP cells that produce calcium oxalate crystals, which is a normal feature of spruce bark (Kartuch, Kartuch, and Weilgong, 1991
). Phelloderm cells are also produced by the phellogen and occur as files of thin-walled parenchyma, many of which accumulate phenolic compounds (Fig. 20). In the example of a well-developed periderm shown in Fig. 19, the PP cells outside of the periderm had lost most of their phenolic contents and the orange rather than the typical green autofluorescence of walls that was seen in these samples indicated the phenolics had been deposited in the walls.
Bark-beetle attack and defense responses
To determine whether anatomical reactions induced by experimental inoculations are similar to reactions induced by a bark-beetle attack, the anatomy of bark near bark-beetle galleries from two different trees of the same age as those used for inoculations was characterized. Samples from 23 different bark-beetle galleries were sectioned and examined (seven from day 3, eight from day 6, four from day 13, and four from day 19; 11 from tree B1 and 12 from tree B2). Samples taken 2–4 mm away from the tip of beetle galleries 3 d (Fig. 22) and 6 d (Fig. 23) after attack by I. typographus showed only minor changes, consisting of slight swelling of cells of the inner 3–5 PP cell layers and some increase in sieve cell collapse around these layers. There was no obvious visible sign of traumatic duct development in the 15 different samples examined from these time points. Samples from 13 d after initiation of an attack in both trees showed anatomical features identical to those seen in the experimental inoculations (Figs. 24, 25). The PP cells were swollen and contained dense phenolic bodies, and the surrounding sieve cells were collapsed. Traumatic ducts were developing in the cambial zone but were at a very early stage, and only a few of them already had small lumens containing secretory products. Phenolics were also present in the vacuoles of the cells surrounding the developing traumatic ducts, as seen in early stages of traumatic duct development induced by more severe wounding (Nagy et al., 2000
). PAS staining patterns were consistent with results of experimental inoculations (Fig. 25). Where phenolic bodies were present after beetle attack, they stained dark, except in the cells surrounding the developing traumatic ducts, which stained much lighter.
By 19 d after bark-beetle attack the samples were either partially killed (Fig. 26) or completely destroyed (Fig. 27) by fungal colonization. In those samples that were not destroyed by fungal growth, immature traumatic ducts were present (Fig. 26). The reactions of the PP cells were more variable than in the experimental inoculations. In some regions the PP cells were almost devoid of phenolic bodies or only had small globules in the vacuole (Fig. 27), while in other areas dense phenolic bodies completely filled the vacuole. This variability is likely due to the imprecision of determining the actual age of a point along an individual gallery.
SEM of bark-beetle galleries and surrounding tissues
Since a major elicitor of the defense reactions in spruce bark will be the bark beetles (and the fungi they carry), it was of interest to determine the extent of damage caused by its activity in the bark. The male bark beetle, I. typographus, bores a slanted entrance tunnel from the bark surface to the cambium and, in the inner phloem, forms a mating chamber (Fig. 28). After mating, in which 1–4 females take part, maternal galleries are bored by the females in the axial direction up and down from the mating chamber (Figs. 28, 29). Cross sections through the galleries clearly demonstrate that the boring activity of the beetle destroys the younger layers of phloem, the cambium, and some of the differentiating tracheids (Fig. 30). Thus, the sapwood is directly exposed to the blue-stain fungus spores carried by the beetle. The gallery wall was found to be remarkably smooth (Fig. 30). Swelling of PP cells and collapse of the layers of sieve cells could also be seen in some of these samples (see Fig. 30), although most were from early stages (day 6–13) of attack. Examination of samples from old bark-beetle attacks that had subsequently failed gives a further indication of the extent of the reaction in surrounding tissues formed after the attack. As viewed in cross section, there was typically a resin-soaked lesion in the sapwood surrounded centrifugally by a ball of xylem, which gradually is followed by a more organized layer of xylem (Fig. 31). To either side of this area is one or more rows of tangentially arranged traumatic duct layers starting at the point where the lesion occur (Fig. 31). These lines of traumatic ducts are clearly induced by the beetle attack as only a few scattered axial ducts can be found in the wood below this zone and the wood formed 2–3 yr after the attack (Fig. 31). The observation that traumatic ducts occur in annual rings formed 1 or 2 yr after the initial attack indicate that close to the wound, there continues to be some stimulus for their formation.
DISCUSSION
The PP cells of Norway spruce secondary phloem are shown here to play a complex role in inducible defense responses to wounding and infection of the stem. While the stored phenolics are considered to be part of a constitutive defense strategy (Franceschi et al., 1998
), here we show that changes in phenolic synthesis occur in response to bark-beetle attack or fungal inoculation. These changes include an increase in the size of the PP cells and their phenolic bodies and changes in glycosylation of the phenolics in the PP cells as indicated by PAS staining. A previous analysis of soluble phenolics produced after wounding in Norway spruce indicated qualitative and quantitative changes over a similar time period as examined here (Brignolas et al., 1995a, b
). An increase in bark phenolic content was also seen in wounded and fungal-inoculated red pine (Klepzig et al., 1995
), though the actual cells responsible for phenolic accumulation or synthesis were not identified. We have found that the ray cells do not accumulate phenolics except in the region of developing traumatic ducts (Nagy et al., 2000
), thus it is almost certain that most of the new phenolic compounds seen in Norway spruce in response to wounding by Brignolas et al. (1995a)
were produced by the PP cells, since they are the major living cell type in the secondary phloem. The significance of the increased PAS staining of phenolics after defense induction is not clear, but could be related to changes in the solubility and/or toxicity of phenolic compounds. It will be interesting to determine whether the PP cells in response to a stimulus simply accumulate phenolic compounds in their vacuoles or secrete some of them to the wall space. We have found that there is a considerable amount of PAL associated with the plasma membrane of the PP cells, so perhaps there is localized production of selected phenolics at this site for subsequent transport to the wall space. This would be a useful strategy in terms of antifungal activity since the fungus used here grows through the walls as well as within the living parenchyma cell cytoplasm and sieve tube sap where it will use the nutrient content for growth (Franceschi et al., 1998
).
PP cell activity was increased in a similar pattern by both bark-beetle attack and fungal inoculations. Thus, it is clear that the PP cells are quickly activated by organisms breaking through the periderm. The subperidermal and shallow inoculations also show that PP cell activation does not require cambial zone wounding, which our previous experimental study used (Franceschi et al., 1998
). The swelling of the PP cells seen may be partly due to accumulation of osmotically active phenolic compounds, which is also consistent with the increase in staining intensity of the phenolic bodies. As a consequence of PP cell swelling, the surrounding dead sieve cells are collapsed into a dense layer of cell walls. This would provide a further resistance to growth of invading fungi since the empty sieve cell lumen that is typically used would no longer be a free path for hyphal growth, particularly in the axial direction.
There appeared to be a difference in the amount of PP cell swelling, which was related to the severity of treatment, though we did not attempt to quantify this difference. Generally, PP cells from inoculations halfway through the phloem showed more swelling than those from subperidermal inoculations. It is also likely that distance from the inoculation site is important to the severity of the response seen. Comparison of samples indicates that very close to a severe wound, such as that induced by the bark beetle or cambial inoculation, many PP cells may completely lose their phenolic bodies and particularly in the PP cells that are 3 yr old or older. This is consistent with our previous experiments showing swelling of PP cells and loss of phenolic contents in resistant clones of Norway spruce (Franceschi et al., 1998
). In addition, autofluorescent patterns indicate that the phenolics or their derivatives that are lost from the PP cells are deposited in the cell walls. This probably accounts for the increased browning often seen in the bark samples and the reaction zone around an inoculation site (personal observations). A difference in loss or accumulation of polyphenolics in general with distance from a wound was also noted in the bark of Abies alba (Oven and Torelli, 1994
).
It was interesting to find that in fungal inoculations that did not go to the cambium, fungal growth was arrested in most samples, while in cambial inoculations the fungus rapidly spread through the cambium and adjacent tissues. This further supports our hypothesis that the PP cells can activate potent biochemical mechanisms for defense against phytopathogens. The cambial cells, which do not accumulate phenolics and have little detectable PAL enzyme, do not themselves appear to have much defensive capacity. However, their derivatives close to the fungal growth zone, which have PP-cell-like characteristics, are capable of producing phenolics and lignified walls, as are nearby PP cells. While PP cell phenolics are certainly one important source of defense, we hypothesize that these cells are also capable of synthesis of a range of defense enzymes and chemicals, which is an aspect of PP cell biology in need of further study.
The dynamic nature of the PP cells with respect to defense is further substantiated by the observation that they are the cells responsible for initiation and development of a wound periderm. This is a very important defense response, which, if developed rapidly enough at some distance from the site of attack, can completely isolate the invading organism and stop its spread. The fact that this periderm seems to be produced along one or two layers of PP cells for some length indicates that there is circumferential transmission of a signal that can coordinate activity of the dividing PP cells to produce the new periderm. The extensive symplasmic contact between PP cells in a layer, shown in our developmental studies (Krekling et al., in press), would accommodate this need for intracellular signaling and coordination of cellular events. We have previously found that up to 70 intact, living PP cell layers can be found in older trees, indicating that the potential for periderm formation extends throughout much of the bark volume.
Previous studies have demonstrated that wounding can initiate traumatic resin duct formation in the cambial zone of Norway spruce stems (Christiansen et al., 1999a
; Nagy et al., 2000
). Here it is shown that bark-beetle attack induces the same reaction. However, we also made the surprising discovery that very shallow single inoculations into the secondary phloem can induce traumatic duct development in the cambial zone. This is consistent with a report that a series of shallow "scrapes" around the circumference of spruce trees used to prevent deer damage also induces traumatic duct and periderm formation by 8 wk after scraping (Lo and Schütt, 1980
). Taken together, these observations further implicate the PP cells and associated radial ray cells in signal perception and transport of some signal to other tissues and cell types, which can elicit defense responses such as formation of traumatic ducts.
As a consequence of traumatic duct formation there is a short-term disruption of normal cambium activity. Once the cambium is reorganized above the developing traumatic ducts, it appears that it "resets" its developmental programming to the beginning of the growth season with respect to generation of a new PP cell layer. This further indicates the importance of the PP cells; when cambial activity is resumed after a dormant period or disruption of normal function, PP cells are among the first cell types to be differentiated.
The observations on inoculated trees and bark-beetle-attacked trees indicate that the changes induced in PP cells and other tissues are relevant to defense against the aggressive bark beetle I. typographus and the pathogenic/virulent blue-stain fungus it carries, C. polonica, which is the agent that helps kill the tree through destruction of the cambium, the active phloem, and the transport capacity of the sapwood (Horntvedt et al., 1983
; Christiansen, 1985
). However, a question arises—under which circumstances are the responses observed here rapid enough to provide an effective defensive barrier to invasion/infection? Some information on the behavior of the bark beetle during an attack is relevant to this question. When weather conditions are favorable for flight and the ambient I. typographus population is high, a mass aggregation of thousands of beetles may take place on a tree within a short time due to aggregation pheromones (Bakke, Frøyen and Skattbøl, 1977
), and entry into the bark will also develop rapidly; within a week the maternal galleries may be several centimetres long (Birgersson et al., 1984
). Given this type of rapidly developing mass attack the changes in the PP cells and the formation of traumatic ducts will occur too late to alter the outcome of the attack. On the other hand, the weather may often be very changeable during the flight period in spring and early summer. Under Scandinavian conditions one or a few days may have maximum temperatures exceeding the ~20°C necessary for flight, followed by shorter or longer periods of cooler weather, until a new warm spell occurs. Interruption of the aggregation process will allow time for the defense reactions to develop, rendering the trees more resistant to the next bout of attack. In support of this suggestion are the observations that mechanical wounding, as well as artificial inoculation of Norway spruce with C. polonica, enhances the defensive capacity to subsequent mass inoculation with this fungus (Christiansen et al., 1999b
). It is also significant that the defensive response can travel some distance from the site of attack, for instance traumatic duct development can occur 10 cm away from a single inoculation point within 2–3 wk (Nagy et al., 2000
), and development of traumatic ducts a metre or more away from an inoculated region occurs within 3–4 mo (Christiansen et al., 1999a
).
The changes that occur in the phloem and xylem following wounding and infection as described here may also interfere with the host selection process by the bark beetle, of which little is known. After landing on a spruce tree, a male I. typographus, the sex that initiates boring and gallery construction, spends most of its time searching and inspecting crevices in the bark (Paynter, Anderbrandt, and Schlyter, 1990
). During this exploration it seems likely that the beetles come in contact with defensive chemicals of the tree, including volatiles but possibly also other materials, which have to be tasted by the beetle. Chemical changes induced in the site by previous non-lethal attacks may be sensed by the beetle during its exploration and inhibit further boring activity, a hypothesis we plan to test experimentally with pretreated trees that will be subsequently exposed to bark-beetle attack.
In summary, this study demonstrates that PP cells of the secondary phloem are involved in a range of inducible defense responses. Some of these defense responses may be directly derived from the PP cells, such as production of phenolic compounds and formation of wound periderms. However, as the major living component of the secondary phloem, the PP cells also appear to have the capacity to sense invading organisms and transport a signal to other cells at some distance from the site of attack, resulting in additional defenses being initiated. PP cells are found in a number of other conifer species where they likely play similarly important roles in bark defense. It is interesting to note, for example, that in conifers without a resin duct system, such as Taxus and Juniperus, PP cells appear to make up an even larger component of the secondary phloem (see Holdheide, 1951
). In the absence of resinous secretions their importance may be even greater than in Picea. Since experimental inoculations give a similar response as bark-beetle attack, the Norway spruce system used here, including availability of clonal material (Brignolas et al., 1995a
; Franceschi et al., 1998
; Christiansen et al., 1999b
), can be very valuable for further characterization of the range of defense responses residing in PP cells of conifers and how these responses are elicited and controlled.
FOOTNOTES
1 This work was supported by NFR grant number 113626/111 to E.C. The authors thank Dr. H. Solheim, NISK, for providing the C. polonica inoculum; Dr. A. Berryman, WSU, for helpful discussions on this work; and Ms. Elisabeth Eng for technical assistance with sample preparation. 
2 Author for correspondence (FAX: 47–64–942980; e-mail: Erik.Christiansen{at}nisk.no
).
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