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Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus I. preferential lignin degradation prior to hemicelluloses in Norway spruce wood
International Biodeterioration & Biodegradation 105 (2015) 30e40
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Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus I. preferential lignin degradation prior to hemicelluloses in Norway spruce wood Jong Sik Kim, Jie Gao, Geoffrey Daniel* Wood Science, Department of Forest Products, Swedish University of Agricultural Sciences, P.O. Box 7008, SE-750 07 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 June 2015 Received in revised form 13 August 2015 Accepted 13 August 2015 Available online 28 August 2015
Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) in Norway spruce wood (softwood) by the white rot fungus Pycnoporus sanguineus was investigated using transmission electron microscopy coupled with immunocytochemistry. P. sanguineus produced selective decay in xylem cells including tracheids, ray tracheids and ray parenchyma cells. Lignin was preferentially removed first from cell walls with hemicelluloses (xylan and mannan) and cellulose remaining. Lignin in compound middle lamella (CML) regions of tracheids was also preferentially degraded prior to degradation of xyloglucan (hemicellulose). This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. In contrast, no clear preferential degradation of lignin prior to pectins and hemicelluloses was observed in middle lamella cell corner (MLcc) regions of tracheids which were the last regions to be attacked by the fungus. Micromorphologically, prominent cell separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls was observed as evidence of selective decay. Variations in resistance to decay by the fungus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types were also observed. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Hemicelluloses Immunocytochemistry Lignin Norway spruce (softwood) Pycnoporus sanguineus Selective decay
1. Introduction The defining character of white rot fungi in the decomposition of lignocellulosic materials is their capacity to degrade lignin. This reflects their major role in carbon cycling in nature and commercial potential for various applications in areas such as biopulping, biobleaching of pulp, bioconversion of lignocellulosic materials and biodegradation of organopollutants (Kenealy and Jeffries, 2003; Hakala et al., 2004; Hatakka and Hammel, 2010). In this regard, the white rot fungus Pycnoporus sanguineus is widely studied (Eugenio et al., 2010; Uzan et al., 2010; Lomascolo et al., 2011; Rohr et al., 2013). For example, its ability to synthesize laccases capable of enduring high temperature (Litthauer et al., 2007) is particularly interesting for biobleaching of pulp and bioconversion of lignocellulosic materials (Levin et al., 2007; Eugenio et al., 2009; Lomascolo et al., 2011; Martin-Sampedro et al., 2015). Concerning
* Corresponding author. E-mail address: [email protected] (G. Daniel). http://dx.doi.org/10.1016/j.ibiod.2015.08.008 0964-8305/© 2015 Elsevier Ltd. All rights reserved.
decay patterns produced in wood by P. sanguineus, Luna et al. (2004) and Singh et al. (2012) reported selective (preferential) degradation of lignin without extensive loss of cellulose, whereas others reported simultaneous degradation of lignin and cellulose (Ferraz et al., 1998; Levin et al., 2007; Van Heerden et al., 2008; Rohr et al., 2013). Van Heerden et al. (2008) proposed that this difference may reflect the different strains and culture conditions used. With respect to white rot fungi and wood degradation, immunocytochemical probes, including antibodies and enzymeegold complexes have been frequently used to understand the temporal and spatial degradation of major cell wall components in combination with transmission electron microscopy (TEM) (Daniel et al., 1989; Daniel, 1994, 2003). However, little information is available about degradation of non-cellulosic polysaccharides (i.e. pectins and hemicelluloses) during white rot decay at the cellular level. Only a few studies using enzymeegold complexes have provided cellular information about degradation of non-cellulosic polysaccharides in white rot decayed wood (Ruel and Joseleau, 1984; Blanchette et al., 1989). In particular, it is poorly understood whether lignin decay occurs before hemicellulose degradation by
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white rot fungi, specifically during selective decay. Using antibodies specific to various polysaccharides, TEM immunocytochemistry can now provide more specific and diverse information on the distribution of polysaccharides than using enzymeegold complexes in sound and decayed wood. However, TEM immunocytochemistry using such antibodies has not been carried out for understanding fungal decay wood even though they have frequently been used with success on sound wood (e.g. Kim and Daniel, 2012). This approach can provide both morphological and chemical information at the same time for understanding fungal decayed wood at the cellular level.
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The main goal of this study series (part I, II) is to extend our understanding of the temporal and spatial degradation process of lignin and non-cellulosic polysaccharides by the white rot fungus P. sanguineus. In this study (part I), we investigated the decay process in Norway spruce wood (softwood) using various microscopy techniques. Correlated TEM and TEM immunogold labeling combined with cytochemical staining for lignin and monoclonal antibodies specific for pectins and hemicelluloses provide new insights on degradation of cell wall components by white rot fungi.
Fig. 1. Anatomical changes in decayed spruce wood stained with toluidine blue (TB). (a, b) Earlywood (EW) tracheids showing higher resistance in tangential (Ta)-than radial (Ra) cell walls and formation of prominent delignified zones stained purple (marked 1, insets in b). Delignification in compound middle lamella (CML) regions (marked 2, insets in b) occurred prior to complete degradation of secondary cell walls. Cell regions stained light-and dark blue (marked 3 and 4, inset in b) indicate lignified secondary cell wall and middle lamella cell corner (MLcc) regions, respectively. (c) EW tracheids at advance stages of decay showing non-delignified MLcc regions and removal of majority of lignin in secondary cell walls and CML regions. (d) MLcc regions of EW tracheids remained at late stages of decay. (e) LW tracheids showing higher resistance in Ra-than Ta cell walls (i.e. opposite to EW tracheids). Note formation of prominent delignified zones stained purple. (f, g) Uni/triseriate rays (R) in EW showing prominent degradation in cross-field pit regions (arrowheads in f) and adjacent ray parenchyma cell walls (arrowheads in g). T, tracheid. Bars ¼ 25 mm (a, b, g), 10 mm (c, d, e, f). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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2. Materials and methods 2.1. Fungal decay and microscopy Small wood blocks (20 20 5 mm3) of Norway spruce (Picea abies Karst., softwood) sapwood were exposed to the white rot fungus P. sanguineus (Culture collection, Department of Forest Products) for 10 weeks in soil-block cultures at 25 C and 75% relative humidity (AWPA E10-08, 2008). For microscopy, small pieces from uninoculated sterile wood blocks (control) and decayed samples were embedded in LR White resin. The mass loss of decayed samples used was around 31%. Transverse ultrathin sections (ca. 90 nm) prepared from embedded blocks were stained with 1% w/v KMnO4 in 0.1% sodium citrate for lignin (Donaldson,
1992). For immunogold labeling, sections were incubated with either LM6 (1,5-a-arabinan, Willats et al., 1998), LM10/LM11 (xylan, McCartney et al., 2005), LM15 (XXXG motif of xyloglucan, Marcus et al., 2008), LM20 (methyl-esterified homogalacturonan, Verhertbruggen et al., 2009) or LM21 (mannan, Marcus et al., 2010) monoclonal antibodies (PlantProbes, Leeds, UK), followed by incubation with 10-nm colloidal gold particles (BBInternational, Cardiff, UK) (Kim and Daniel, 2012). Sections were examined with Philips CM12 TEM (Philips, Eindhoven, Netherlands) after staining with either KMnO4 or 4% w/v uranyl acetate (in distilled water). Some semi-thin sections (ca. 1 mm) were also examined using light microscopy (Leica DMLB, Wetzlar, Germany) after staining with 1% w/v toluidine blue in 1% borax (w/v in H2O).
Fig. 2. Delignification of tracheid cell walls stained with KMnO4 for lignin. (a) Earlywood (EW) tracheids showing earlier delignification in radial (Ra)-than tangential (Ta) cell walls including compound middle lamella (CML) regions. Note uneven structure of the delignified innermost cell wall (arrowheads in inset). (b) Latewood (LW) tracheids showing earlier delignification in Ta-than Ra cell walls (i.e. opposite to EW tracheids). (c) Lignin distribution in control EW tracheid. (d) Early stages of delignification progressing from the lumen side outwards (arrow). (e) Progression of gradual delignification showing formation of prominent delignified zones (double headed arrows) in the cell walls. MLcc, middle lamella cell corner. Bars ¼ 2 mm (a, b), 500 nm (cee).
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3. Results 3.1. Anatomical and histochemical changes in decayed spruce wood Overall, earlywood (EW) tracheids showed more progressive degradation in radial (Ra)-than tangential cell walls (Ta) (Fig. 1a, b), while latewood (LW) tracheids showed the opposite pattern (i.e. less degradation in Ta than Ra) (Fig. 1e). However, the decay process in tracheid cell walls was similar between EW and LW. Decay of tracheid cell walls occurred by progressive thinning from the lumen surface (Fig. 1a, b, e). During decay, a thin layer stained purple by toluidine blue was apparent in tracheid cell walls (marked 1, insets in Fig. 1b). Since toluidine blue stains the lignified cell wall blue and non-lignified cell walls purple, this layer indicates presence of completely/or prominently delignified cell wall regions caused by fungal attack. As decay progressed, separation of compound middle lamella (CML) regions in adjacent tracheids was frequently observed, no matter radial or tangential cell walls
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(Fig. 1aec, e). This separation was only detectable in tracheids that showed loss of toluidine blue staining in CML regions (marked 2, insets in Fig. 1b). In more advance stages, notable lignin staining was only detected in middle lamella cell corner (MLcc) regions of tracheids (Fig. 1c). At late stages of decay, MLcc regions with thin cell corner regions generally only remained in tracheids (Fig. 1d). With ray parenchyma cells, at early stages of decay, prominent degradation was detected in cross-field pits (i.e. pits between tracheids and rays) (arrowheads in Fig. 1f) and adjacent ray cell walls of biseriate rays (arrowheads in Fig. 1g). 3.2. Degradation of lignin in tracheids Observations using light microscopy were further advanced by TEM. Fig. 2a, b shows EW and LW tracheids stained with KMnO4 for lignin. In the same EW tracheid, delignification of Ra cell walls including CML regions was faster than Ta cell walls (Fig. 2a). In contrast, degradation and delignification in Ra cell walls of LW
Fig. 3. Delignification of compound middle lamella (CML) regions of tracheids stained with KMnO4 for lignin. (aef) Delignification of CML regions started following delignification of secondary cell walls (bed). Separation of CML regions occurred following delignification in both tangential (Ta, e) and radial (Ra, f) cell walls. Note progressive thinning of secondary cell wall concomitant with progressive delignification (b, c). (g, h) Delignification of CML regions by attack from pit chambers (arrowheads in g, h). Note earlier delignification of bordered pit membranes (M) than pit borders (g). Bars ¼ 1 mm.
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tracheids was slower than Ta cell walls (Fig. 2b). These temporal variations in delignification were consistent with differences in resistance to decay depending on cell wall orientations in EW and LW described above (Fig. 1a, e). The innermost layer (i.e. lumen wall) of delignified tracheid cell wall also frequently showed an uneven structure during progressive thinning of the wall (arrowheads, inset in Fig. 2a; Figs. 3 and 6). This may indicate how thinning of cell walls progresses in tracheid secondary cell wall following delignification. Delignification of secondary cell wall progressed gradually from the lumen side outwards (Fig. 2c, d). During decay, prominent delignified zones were detected in the secondary cell wall (double headed arrows in Fig. 2e). Fig. 3 shows the delignification process of CML regions in tracheids which was similar for Ra-and Ta cell walls. The only difference was that delignification of CML regions occurred through pit regions in Ra cell walls (Fig. 3g, h). Delignification of CML regions started after delignification of secondary cell walls (Fig. 3c, d). Thinning of secondary cell walls characterized by uneven structure of the innermost tracheid cell wall layers was also
observed before delignification of CML regions was initiated (Fig. 3aec). Separation of tracheids was frequently apparent after complete delignification of CML regions, no matter cell wall orientation (Fig. 3e, f). At late stages of decay, lignin staining was only detected in MLcc regions (Fig. 4a) with the secondary cell walls and CML regions completely delignified at this stage (Fig. 4a). The thickness of delignified secondary cell walls remaining varied between tracheids. In very advanced stages, MLcc regions generally remained in tracheids, followed by delignification (Fig. 4b). However, some tracheids showed delignification of MLcc regions prior to complete delignification of secondary cell walls because of the variation in decay rate between neighboring tracheids (arrowheads in Fig. 4c). Some tracheids also showed severe delignification of MLcc regions by attack from CML regions before delignification in neighboring tracheid cell walls was completed (arrowheads in Fig. 4d). Unlike secondary cell walls (Fig. 2e), a prominent delignified zone was not detected in MLcc regions of tracheids (Fig. 4b), presumably because of no/or very limited cellulose in MLcc regions.
Fig. 4. Late stages of decay in tracheids stained with KMnO4 for lignin. (a) Non-delignified middle lamella cell corner (MLcc) regions after delignification of secondary cell walls and compound middle lamella (CML) regions. (b) MLcc regions remained after degradation of entire cell walls including CML regions. Note no prominent delignified zone in MLcc regions compared to secondary cell walls (Fig. 2e). (c, d) Delignification of MLcc regions prior to complete delignification of secondary cell walls because of the variation in decay rate between neighboring tracheids (arrowheads in c) and by attack from CML regions (arrowheads in d). Bars ¼ 2 mm (a), 1 mm (c, d), 500 nm (b).
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Fig. 5. Immunolocalization of mannan and xylan epitopes in tracheid cell walls, followed by KMnO4 staining for lignin. (a, b) Delignified cell walls (double headed arrows) showing abundant xylan and mannan epitopes like lignified cell walls. (c, d) Cell walls remained after complete delignification of compound middle lamella (CML) regions showing abundant mannan (c) and xylan (d) epitopes. MLcc, middle lamella cell corner. Bars ¼ 500 nm.
3.3. Immunolocalization of non-cellulosic polysaccharides in tracheids Fig. 5 shows immunogold labeling of mannan and xylan epitopes in decayed tracheids. Abundant epitopes were detected in both delignified (double headed arrows in Fig. 5a, b) and lignified regions (Fig. 5a, b). Abundant epitopes were also detected in delignified tracheid cell walls remaining after complete delignification of CML regions (Fig. 5c, d). These results demonstrate that lignin is preferentially degraded prior to complete degradation of hemicelluloses by P. sanguineus in the course of decay. HG and xyloglucan which represent major chemical components in CML regions of tracheids were detected in CML regions at early stages of delignification (Fig. 6a, c). However, in more advanced stages of delignification, HG epitopes were not detectable in CML regions (Fig. 6b), suggesting that HG may be simultaneously degraded with lignin in CML regions. In contrast, xyloglucan epitopes were detected in CML regions during the entire delignification process of CML regions, even after complete delignification (Fig. 6d, e). This result like for secondary cell walls demonstrates preferential degradation of lignin prior to degradation of xyloglucan (hemicellulose) by the fungus. At late stages of decay, MLcc regions showed
abundant arabinan, HG, xyloglucan and xylan epitopes (Fig. 7aed) similar to that of controls (not shown). However, it was unclear whether lignin is preferentially degraded from MLcc regions before removal of pectins (i.e. arabinan and HG) and hemicelluloses (i.e. xyloglucan and xylan). 3.4. Degradation of ray tracheids and ray parenchyma cells Norway spruce has 1e3 layers of ray tracheids that form part of rays. Ray tracheids are distinct from ray parenchyma cells by presence of small bordered pits in their cell walls in transverse sections (Fig. 8a). Ray tracheids showed either similar or slightly lower resistance to decay compared to axial tracheids (tracheids) (Fig. 8a, c). Although the full process of delignification in ray tracheids was unclear in this study due to their thin cell walls in transverse sections, it is expected that progressive delignification occurs from the lumen towards ML regions like in axial tracheids since considerable lignin staining was still detectable in ML regions when ray tracheid cell walls had completely lost lignin staining (inset in Fig. 8c). Distribution of abundant xylan and mannan epitopes in delignified ray tracheids cell walls after complete delignification in ML regions also supported that decay patterns in
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Fig. 6. Immunolocalization of homogalacturonan (HG) and xyloglucan epitopes in compound middle lamella (CML) regions of tracheids, followed by KMnO4 staining for lignin. (a, b) At early stages of delignification in CML regions, HG epitopes were detected in CML regions (a), but were absent at late stages (b). (cee) Xyloglucan epitopes were detected in CML regions during the course of delignification of CML regions (c, d) even after complete delignification (e). Bars ¼ 500 nm.
ray tracheids were similar to axial tracheids (Fig. 8d). Interestingly, bordered pit membranes of ray tracheids which stained strongly with KMnO4 for lignin showed higher resistance to decay than (ray/ axial) tracheid cell walls (Fig. 8aec). This contrasted with decay in bordered (Fig. 3g)- and cross-field pit (Fig. 1f and 9c) membranes of axial tracheids that showed lower decay resistance than tracheid cell walls even though the membranes were lignified in controls (not shown). Other factors, such as accumulation of extractives (rate and type) and degree of lignification in pit membranes may differ between axial- and radial tracheids, thereby producing different decay resistance. However, at present chemical differences in pit membranes between axial- and radial tracheids are unclear. Overall, ray parenchyma cells showed higher resistance to decay than tracheids (Fig. 9a). Several decay patterns were observed in ray parenchyma cell walls; 1) Progression of decay from tracheids to rays across the ML regions (Fig. 9b), 2) Progression of decay from cross-field pit regions towards ray cell walls including ML/MLcc regions (Fig. 9c and inset), 3) Degradation of ML regions in adjacent ray cell walls of biseriate rays (Figs. 1g and 9d) and 4) Local degradation of ray cell walls initiated either from the lumen or ML regions of adjacent ray cell walls of biseriate rays (Figs. 1g and 9e). The former two decay patterns were more prominent in ray
parenchyma cells than the latter patterns during decay. Ray parenchyma cell walls in contact with axial tracheids were generally degraded earlier than adjacent ray cell walls of biseriate rays (i.e. ray cell wall not in contact with tracheid cell walls). Degradation of ray parenchyma cell walls facing the lumen (except for local degradation as outlined in pattern 4) was rarely observed even though considerable degradation progressed in ray parenchyma cell walls facing tracheids and ML regions (Fig. 9b, d, f, g). Mannan and xylan epitopes were detectable in delignified ray cell wall regions (Fig. 9f, g and insets) like tracheids (Fig. 5).
4. Discussion Using light-and TEM microscopy techniques, this study characterizes micromorphological and chemical decay patterns of Norway spruce wood decayed by the white rot fungus P. sanguineus. TEM immunocytochemistry using antibodies specific for pectins and hemicelluloses provides new insight on degradation of cell wall components by white rot fungi. To our knowledge this study is the first report demonstrating temporal controlled degradation of lignin and non-cellulosic polysaccharides in wood by P. sanguineus at the cellular level.
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Fig. 7. Immunolocalization of pectin and hemicellulose epitopes in middle lamella cell corner (MLcc) regions of tracheids, followed by uranyl acetate (UA) staining. (aed) MLcc regions remaining at late stages of decay showing distribution of arabinan (a), homogalacturonan (HG, b), xyloglucan (c) and xylan (d) epitopes. Bars ¼ 500 nm.
4.1. Decay type in tracheids Results demonstrate that lignin is selectively degraded without extensive loss of hemicelluloses (probably also cellulose) in tracheid cell walls by P. sanguineus (i.e. selective decay). These results are consistent with previous studies even though lignin and cellulose were mainly focused in the studies (Luna et al., 2004; Singh et al., 2012). In contrast, results are inconsistent other studies that proposed simultaneous degradation of lignin and cellulose by P. sanguineus (Ferraz et al., 1998; Levin et al., 2007; Van Heerden et al., 2008; Rohr et al., 2013). Selective removal of lignin without extensive degradation of xyloglucan (hemicellulose) in CML regions also supports selective delignification by P. sanguineus. Micromorphologically, separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls also indicate selective decay of lignin induced by P. sanguineus. Tracheid/fiber separation is generally considered as evidence of selective decay (Blanchette, 1984). Prominent cell separation has also been reported in poplar fibers degraded by P. sanguineus (Luna et al., 2004). As described for other selective white rot fungi (Blanchette and Reid, 1986; Daniel, 2003), characteristic features of simultaneous decay, such as progressive thinning of cell walls and MLcc regions as the last areas to be attacked by P. sanguineus were also observed. Consequently, results indicate that P. sanguineus produces selective decay in spruce tracheids. Hemicelluloses are degraded after removal of lignin in tracheid cell walls and CML regions (i.e. considerable hemicelluloses remains after complete delignification). Although further studies are
needed on other selective white rot fungi, these results differ from previous concepts that hemicelluloses are degraded concomitantly with lignin in wood cell wall by selective white rot fungi and that only modified cellulose remains (Goodell et al., 2003). Concerning the mechanism of selective attack of lignin in tracheid cell walls, we consider that activity of ligninolytic enzymes and free radicals are likely to be more aggressive than hemicellulolytic enzymes (probably also cellulolytic enzymes) during decay by P. sanguineus. Formation of a prominent delignified zone in tracheids cell wall during decay may reflect differences in activity between ligninolytic (and free radicals) and hemicellulolytic/ cellulolytic enzymes since the progressive thinning of delignified cell wall (i.e. degradation of hemicelluloses and cellulose) occur concomitantly with delignification in the same tracheid cell wall (i.e. concurrent attack by ligninolytic, hemicellulolytic and cellulolytic enzymes). In similar delignified zones (decay zones), ligninand Mn peroxidase were concentrated in the interface degraded and non-degraded cell walls (Srebotnik et al., 1988; Daniel et al., 1989, 1991, 2004). 4.2. Different resistance between radial-and tangential cell walls of tracheids Results show that decay differs between radial (Ra) and tangential (Ta) cell walls in tracheids with opposite pattern between EW (Ra < Ta) and TW (Ra > Ta). Daniel et al. (1989) also found higher resistance in Ra-than Ta cell walls in Scots pine LW tracheids by Phanerochaete chrysosporium. They suggested that this
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Fig. 8. Degradation of ray tracheids (RT) stained with KMnO4 for lignin. (aec) Ray tracheids in latewood (LW; a, b) and earlywood (EW, c) showing either similar or slightly lower resistance to decay compared to axial tracheids (T). Bordered pit membranes (between RT and T) showed higher resistance than ray/axial tracheid cell walls (b, arrowheads in a, c). Note delignification of middle lamella (ML) regions after complete delignification of (ray/axial) tracheid cell walls (inset in c). (d) Localization of xylan epitopes showing abundant epitopes in delignified ray tracheid cell walls. Note absence of lignin staining in ML regions. Bars ¼ 2 mm (a), 1 mm (b), 500 nm (c, d).
difference may reflect the different cell wall thickness between Raand Ta cell walls (i.e. thicker in Ra than Ta). Ollinmaa (1961) reported that Ra cell walls of Norway spruce LW tracheids are thicker than Ta cell walls. However, this explanation cannot be applied to EW tracheids that showed lower decay resistance in Ra-than Ta cell walls by P. sanguineus in this study (i.e. opposite to LW) since the Ra wall is thicker than Ta cell walls in Norway spruce EW tracheids like LW (Ollinmaa, 1961). This indicates that some other factors, particularly presence of bordered pits may also be important for inducing different decay resistance between cell walls in EW tracheids. In Norway spruce EW tracheids, the majority of bordered € m, 2001). Bordered pits are present on the Ra cell walls (Br€ andstro pit membranes were degraded at early stages of decay, mostly earlier than tracheid cell walls by P. sanguineus in this study. Delignification of CML regions through the pit regions was also observed in this study. Although Ra cell walls of LW tracheids also contain bordered pits, it may play only a minor role in affecting decay compared to cell wall thickness since the number of bordered pits in Ra cell walls of LW tracheids is much less than in EW tra€ m, 2001). The diameter of bordered pits is also cheids (Br€ andstro € m, 2001). much smaller in LW than EW (Br€ andstro
4.3. Degradation of ray parenchyma cells Ray parenchyma cells showed higher resistance to decay by P. sanguineus than tracheids. Higher resistance in ray parenchyma cell walls isolated from tracheids (i.e. central adjacent two ray cell walls of biseriate rays) than ray parenchyma cell walls in contact with tracheids may reflect a lower resistance of tracheids than rays. This is consistent with differences in lignin concentration between ray parenchyma cells and tracheids. Westermark et al. (1988) reported that lignin concentration of ray parenchyma cells is higher than tracheids in spruce. Ray parenchyma cells also showed stronger lignin staining than tracheids in control (not shown) like decayed samples (Fig. 9a) in this study. Fungal colonization generally occurs more frequently through ray parenchyma cells than tracheids since ray parenchyma cells contain storage materials that can accelerate colonization and penetration into the wood structure. This general concept also supports the positive correlation between lignin concentration and fungal resistance in this study, i.e. ray parenchyma cells provide space for fungal colonization at early stages of decay, but are not degraded due to high concentration of lignin. This type of fungal resistance is frequently
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Fig. 9. Degradation of ray parenchyma cells (R) stained with KMnO4 for lignin. (a) Ray cell walls connected to tracheids (T) showing higher resistance to decay than tracheid cell walls at early stages of decay (double headed arrows indicate delignified zones in tracheids). (b) Progression of decay from tracheids to rays across the middle lamella (ML) regions (arrow). (c) Preferential decay of ML/middle lamella cell corner (asterisk) regions (between rays and tracheids) through degradation of cross-field pits (arrowheads). Inset shows ray cell walls degraded together with ML regions through cross-field pits. (d) Preferential decay of ML regions in adjacent ray cell walls of biseriate rays. (e) Local degradation of ray cell walls (arrow). (f, g) Localization of mannan (f) and xylan (g) epitopes showing distribution of epitopes in completely/or considerably delignified ray cell walls. Bars ¼ 1 mm (aee), 500 nm (f, g).
observed in vessels of hardwoods. High lignin concentration in vessels is generally considered one factor causing their greater resistance to decay than fibers in hardwoods (Blanchette et al., 1988; Schwarze, 2007). The only exception is that some ray parenchyma cells showed degradation of ML/MLcc regions (between tracheids and rays or between ray and ray in biseriate rays) through cross-field pits and bore holes prior to degradation of ray cell walls even though lignin concentration in ML/MLcc regions is generally higher than cell walls (Figs 1f, 9c and d). 5. Conclusions Correlated TEM and TEM-immunocytochemistry demonstrates that the white rot fungus P. sanguineus produces selective decay in
Norway spruce tracheids including secondary cell walls and CML regions. Lignin was preferentially removed from tracheids with hemicelluloses and cellulose remaining. This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. These results may also reflect potential of P. sanguineus for various biological applications as outlined in the introduction. Results also show variations in resistance to decomposition by P. sanguineus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types including axial tracheids, ray tracheids and ray parenchyma cells. Finally, this study shows the potential of TEM immunocytochemistry to characterize the temporal degradation process of cell wall components in wood decayed by white rot fungi.
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Acknowledgements The authors gratefully acknowledge funding provided by Formas projects 2008-1399, 2009-582 & 2011-416 and the EcoInnovation initiative-EU project TV4NEWOOD Eco/12/333079. References AWPA Technical Subcommittee P6, 2008. Standard Method of Testing Wood Preservatives by Laboratory Soil-block Culture. AWPA E10-08. American Wood Preservers Association, Birmingham, Ala. Blanchette, R.A., 1984. Screening wood decayed by white rot fungi for preferential lignin degradation. Appl. Environ. Microbiol. 48, 647e653. Blanchette, R.A., Reid, I.D., 1986. Ultrastructural aspects of wood delignification by Phlebia (Merulius) tremellosus. Appl. Environ. Microbiol. 52, 239e245. Blanchette, R.A., Obst, J.R., Hedges, J.I., Weliky, K., 1988. Resistance of hardwood vessels to degradation by white rot Basidiomycetes. Can. J. Bot. 66, 1841e1847. Blanchette, R.A., Abad, A.R., Cease, K.R., Lovrien, R.E., Leathers, T.D., 1989. Colloidal gold cytochemistry of endo-1,4-b-glucanase, 1,4-b-D-glucan cellobiohydrolase, and endo-1,4-b-xylanase: ultrastructure of sound and decayed birch wood. Appl. Environ. Microbiol. 55, 2293e2301. € m, J., 2001. Micro-and ultrastructural aspects of Norway spruce tracheids: Br€ andstro a review. IAWA J. 22, 333e353. Daniel, G., Nilsson, T., Pettersson, B., 1989. Intra-and extracellular localization of lignin peroxidase during the degradation of solid wood and wood fragments by Phanerochaete chrysosporium by using transmission electron microscopy and immuno-gold labeling. Appl. Environ. Microbiol. 55, 871e881. ski, A., Crawford, R., 1991. Use of monoDaniel, G., Jellison, J., Goodell, B., Paszczyn clonal antibodies to detect Mn (II)-peroxidase in birch wood degraded by Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 35, 674e680. Daniel, G., 1994. Use of electron microscopy for aiding our understanding of wood biodegradation. FEMS Microbiol. Rev. 13, 199e233. Daniel, G., 2003. Microview of wood under degradation by bacteria and fungi. In: Goodell, B., Nicholas, D.D., Schultz, T.P. (Eds.), Wood Deterioration and Preservation: Advances in Our Changing World, ACS Symposium Series. American Chemical Society, Washington, DC, pp. 34e72. Daniel, G., Volc, J., Niku-Paavola, M.-L., 2004. Cryo-FE-SEM & TEM immunotechniques reveal new details for understanding white-rot decay of lignocelluloses. Comptes Rendus Biol. 327, 861e871. Donaldson, L.A., 1992. Lignin distribution during latewood formation in Pinus radiata D. Don. IAWA Bull. 13, 381e387. Eugenio, M.E., Carbajo, J.M., Martín, J.A., Gonz alez, A.E., Villar, J.C., 2009. Laccase production by Pycnoporus sanguineus under different culture condition. J. Basic Microbiol. 49, 433e440. lez, A.E., Eugenio, M.E., Santos, S.M., Carbajo, J.M., Martín-Sampedro, R., Conza Villar, J.C., 2010. Kraft pulp biobleaching using an extracellular enzymatic fluid produced by Pycnoporus sanguineus. Bioresour. Technol. 101, 1866e1870. Ferraz, A., Esposito, E., Bruns, R.E., Bur an, N., 1998. The use of principal component analysis (PCA) for pattern recognition in Eucalyptus grandis wood biodegradation experiments. World J. Microbiol. Biotechnol. 14, 487e490. Goodell, B., Nicholas, D.D., Schultz, T.P. (Eds.), 2003. Wood deterioration and Preservation: Advances in Our Changing World. Oxford University Press, Washington, DC. Hakala, T.K., Maijala, P., Konn, J., Hatakka, A., 2004. Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme Microb. Technol. 34, 255e263. Hatakka, A., Hammel, K.E., 2010. Fungal biodegradation of lignocelluloses. In: Hofrichter, M. (Ed.), The Mycota-Industrial Applications, second ed., vol. 10. Springer-Verlag, Berlin-Heidelberg, pp. 319e340.
Kenealy, W.R., Jeffries, W., 2003. Enzyme processes for pulp and paper: a review of recent developments. In: Goodell, B., Nicholas, D.D., Schultz, T.P. (Eds.), Wood Deterioration and Preservation: Advances in Our Changing World, ACS Symposium Series. American Chemical Society, Washington, DC, pp. 210e239. Kim, J.S., Daniel, G., 2012. Distribution of glucomannans and xylans in poplar xylem and their changes under tension stress. Planta 236, 35e50. Levin, L., Villalba, L., Da Re, V., Forchiassin, F., Papinutti, L., 2007. Comparative studies of loblolly pine biodegradation and enzyme production by Argentinean white rot fungi focused on biopulping processes. Process Biochem. 42, 995e1002. Litthauer, D., van Vuuren, M.J., van Tonder, A., Wolfaardt, F.W., 2007. Purification and kinetics of a thermostable laccase from Pycnoporus sanguineus (SCC 108). Enzyme Microb. Technol. 40, 563e568. €l-Gimbert, I., Sigoillot, J.-C., LesageLomascolo, A., Uzan-Boukhris, E., Herpoe Meessen, L., 2011. Peculiarities of Pycnoporus species for applications in biotechnology. Appl. Microbiol. Biotechnol. 92, 1129e1149. ~ o, M.E., 2004. Patterns of decay caused by Luna, M.L., Murace, M.A., Keil, G.D., Otan Pycnoporus sanguineus and Ganoderma lucidum (Aphyllophorales) in poplar wood. IAWA J. 25, 425e433. ndez, M., Arias, M.E., Martin-Sampedro, R., Miranda, J., García-Fuentevilla, L.L., Herna Diaz, M.J., Eugenio, M.E., 2015. Influence of process variables on the properties of laccase biobleached pulp. Bioprocess Biosyst. Eng. 38, 113e123. , C., Ordaz-Ortiz, J., Farkas, V., Pedersen, H.L., Marcus, S.E., Verhertbruggen, Y., Herve Willats, W.G.T., Knox, J.P., 2008. Pectic homogalactronan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC Plant Biol. 8, 60. Marcus, S., Blake, A.W., Benians, T.A.S., Lee, K.J.D., Poyser, C., Donaldson, L., Leroux, O., Rogowski, A., Petersen, H.L., Boraston, A., Gilbert, H.J., Willats, W.G.T., Knox, J.P., 2010. Restricted access of proteins to mannan polysaccharides in intact plant cell walls. Plant J. 64, 191e203. McCartney, L., Marcus, S.E., Knox, J.P., 2005. Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J. Histochem. Cytochem. 53, 543e546. Ollinmaa, P.J., 1961. Study on reaction wood. Acta For. Fenn. 72, 1e54. Rohr, C.O., Levin, L.N., Mentaberry, A.N., Wirth, S.A., 2013. A first insight into Pycnoporus sanguineus BAFC 2126 transcriptome. PLoS ONE 8, e81033. Ruel, K., Joseleau, J.-P., 1984. Use of enzyme-gold complexes for the ultrastructural localization of hemicelluloses in the plant cell wall. Histochemistry 81, 573e580. Schwarze, F.W.M.R., 2007. Wood decay under the microscope. Fungal Biol. Rev. 21, 133e170. Singh, P., Sulaiman, O., Hashim, R., Peng, L.C., singh, R.P., 2012. Biodegradation study of Pycnoporus sanguineus and its effects on structural and chemical features on oil palm biomass chips. Lignocellulose 1, 210e227. Srebotnik, E., Messner, K., Foisner, R., 1988. Penetrability of white rot-degraded pine wood by the lignin peroxidase of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 54, 2608e2614. Uzan, E., Nousiainen, P., Balland, V., Sipila, J., Piumi, F., Navarro, D., Asther, M., Record, E., Lomascolo, A., 2010. High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and application. J. Appl. Microbiol. 108, 2199e2213. Van Heerden, A., Le Roux, N.J., Swart, J., Gardner-Lubbe, S., Botha, A., 2008. Assessment of wood degradation by Pycnoporus sanguineus when co-cultured with selected fungi. World J. Microbiol. Biotechnol. 24, 2489e2497. Verhertbruggen, Y., Marcus, S.E., Haeger, A., Ordaz-Ortiz, J.J., Knox, J.P., 2009. An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydr. Res. 344, 1858e1862. Westermark, U., Lidbrandt, O., Eriksson, I., 1988. Lignin distribution in spruce (Picea abies) determined by mecurisation with SEM-EDXA technique. Wood Sci. Technol. 22, 243e250. Willats, W.G.T., Marcus, S.E., Knox, J.P., 1998. Generation of monoclonal antibody specific to (1/5)-a-ʟ-arabinan. Carbohydr. Res. 308, 149e152.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus I. preferential lignin degradation prior to hemicelluloses in Norway spruce wood Jong Sik Kim, Jie Gao, Geoffrey Daniel* Wood Science, Department of Forest Products, Swedish University of Agricultural Sciences, P.O. Box 7008, SE-750 07 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 June 2015 Received in revised form 13 August 2015 Accepted 13 August 2015 Available online 28 August 2015
Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) in Norway spruce wood (softwood) by the white rot fungus Pycnoporus sanguineus was investigated using transmission electron microscopy coupled with immunocytochemistry. P. sanguineus produced selective decay in xylem cells including tracheids, ray tracheids and ray parenchyma cells. Lignin was preferentially removed first from cell walls with hemicelluloses (xylan and mannan) and cellulose remaining. Lignin in compound middle lamella (CML) regions of tracheids was also preferentially degraded prior to degradation of xyloglucan (hemicellulose). This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. In contrast, no clear preferential degradation of lignin prior to pectins and hemicelluloses was observed in middle lamella cell corner (MLcc) regions of tracheids which were the last regions to be attacked by the fungus. Micromorphologically, prominent cell separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls was observed as evidence of selective decay. Variations in resistance to decay by the fungus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types were also observed. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Hemicelluloses Immunocytochemistry Lignin Norway spruce (softwood) Pycnoporus sanguineus Selective decay
1. Introduction The defining character of white rot fungi in the decomposition of lignocellulosic materials is their capacity to degrade lignin. This reflects their major role in carbon cycling in nature and commercial potential for various applications in areas such as biopulping, biobleaching of pulp, bioconversion of lignocellulosic materials and biodegradation of organopollutants (Kenealy and Jeffries, 2003; Hakala et al., 2004; Hatakka and Hammel, 2010). In this regard, the white rot fungus Pycnoporus sanguineus is widely studied (Eugenio et al., 2010; Uzan et al., 2010; Lomascolo et al., 2011; Rohr et al., 2013). For example, its ability to synthesize laccases capable of enduring high temperature (Litthauer et al., 2007) is particularly interesting for biobleaching of pulp and bioconversion of lignocellulosic materials (Levin et al., 2007; Eugenio et al., 2009; Lomascolo et al., 2011; Martin-Sampedro et al., 2015). Concerning
* Corresponding author. E-mail address: [email protected] (G. Daniel). http://dx.doi.org/10.1016/j.ibiod.2015.08.008 0964-8305/© 2015 Elsevier Ltd. All rights reserved.
decay patterns produced in wood by P. sanguineus, Luna et al. (2004) and Singh et al. (2012) reported selective (preferential) degradation of lignin without extensive loss of cellulose, whereas others reported simultaneous degradation of lignin and cellulose (Ferraz et al., 1998; Levin et al., 2007; Van Heerden et al., 2008; Rohr et al., 2013). Van Heerden et al. (2008) proposed that this difference may reflect the different strains and culture conditions used. With respect to white rot fungi and wood degradation, immunocytochemical probes, including antibodies and enzymeegold complexes have been frequently used to understand the temporal and spatial degradation of major cell wall components in combination with transmission electron microscopy (TEM) (Daniel et al., 1989; Daniel, 1994, 2003). However, little information is available about degradation of non-cellulosic polysaccharides (i.e. pectins and hemicelluloses) during white rot decay at the cellular level. Only a few studies using enzymeegold complexes have provided cellular information about degradation of non-cellulosic polysaccharides in white rot decayed wood (Ruel and Joseleau, 1984; Blanchette et al., 1989). In particular, it is poorly understood whether lignin decay occurs before hemicellulose degradation by
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white rot fungi, specifically during selective decay. Using antibodies specific to various polysaccharides, TEM immunocytochemistry can now provide more specific and diverse information on the distribution of polysaccharides than using enzymeegold complexes in sound and decayed wood. However, TEM immunocytochemistry using such antibodies has not been carried out for understanding fungal decay wood even though they have frequently been used with success on sound wood (e.g. Kim and Daniel, 2012). This approach can provide both morphological and chemical information at the same time for understanding fungal decayed wood at the cellular level.
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The main goal of this study series (part I, II) is to extend our understanding of the temporal and spatial degradation process of lignin and non-cellulosic polysaccharides by the white rot fungus P. sanguineus. In this study (part I), we investigated the decay process in Norway spruce wood (softwood) using various microscopy techniques. Correlated TEM and TEM immunogold labeling combined with cytochemical staining for lignin and monoclonal antibodies specific for pectins and hemicelluloses provide new insights on degradation of cell wall components by white rot fungi.
Fig. 1. Anatomical changes in decayed spruce wood stained with toluidine blue (TB). (a, b) Earlywood (EW) tracheids showing higher resistance in tangential (Ta)-than radial (Ra) cell walls and formation of prominent delignified zones stained purple (marked 1, insets in b). Delignification in compound middle lamella (CML) regions (marked 2, insets in b) occurred prior to complete degradation of secondary cell walls. Cell regions stained light-and dark blue (marked 3 and 4, inset in b) indicate lignified secondary cell wall and middle lamella cell corner (MLcc) regions, respectively. (c) EW tracheids at advance stages of decay showing non-delignified MLcc regions and removal of majority of lignin in secondary cell walls and CML regions. (d) MLcc regions of EW tracheids remained at late stages of decay. (e) LW tracheids showing higher resistance in Ra-than Ta cell walls (i.e. opposite to EW tracheids). Note formation of prominent delignified zones stained purple. (f, g) Uni/triseriate rays (R) in EW showing prominent degradation in cross-field pit regions (arrowheads in f) and adjacent ray parenchyma cell walls (arrowheads in g). T, tracheid. Bars ¼ 25 mm (a, b, g), 10 mm (c, d, e, f). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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2. Materials and methods 2.1. Fungal decay and microscopy Small wood blocks (20 20 5 mm3) of Norway spruce (Picea abies Karst., softwood) sapwood were exposed to the white rot fungus P. sanguineus (Culture collection, Department of Forest Products) for 10 weeks in soil-block cultures at 25 C and 75% relative humidity (AWPA E10-08, 2008). For microscopy, small pieces from uninoculated sterile wood blocks (control) and decayed samples were embedded in LR White resin. The mass loss of decayed samples used was around 31%. Transverse ultrathin sections (ca. 90 nm) prepared from embedded blocks were stained with 1% w/v KMnO4 in 0.1% sodium citrate for lignin (Donaldson,
1992). For immunogold labeling, sections were incubated with either LM6 (1,5-a-arabinan, Willats et al., 1998), LM10/LM11 (xylan, McCartney et al., 2005), LM15 (XXXG motif of xyloglucan, Marcus et al., 2008), LM20 (methyl-esterified homogalacturonan, Verhertbruggen et al., 2009) or LM21 (mannan, Marcus et al., 2010) monoclonal antibodies (PlantProbes, Leeds, UK), followed by incubation with 10-nm colloidal gold particles (BBInternational, Cardiff, UK) (Kim and Daniel, 2012). Sections were examined with Philips CM12 TEM (Philips, Eindhoven, Netherlands) after staining with either KMnO4 or 4% w/v uranyl acetate (in distilled water). Some semi-thin sections (ca. 1 mm) were also examined using light microscopy (Leica DMLB, Wetzlar, Germany) after staining with 1% w/v toluidine blue in 1% borax (w/v in H2O).
Fig. 2. Delignification of tracheid cell walls stained with KMnO4 for lignin. (a) Earlywood (EW) tracheids showing earlier delignification in radial (Ra)-than tangential (Ta) cell walls including compound middle lamella (CML) regions. Note uneven structure of the delignified innermost cell wall (arrowheads in inset). (b) Latewood (LW) tracheids showing earlier delignification in Ta-than Ra cell walls (i.e. opposite to EW tracheids). (c) Lignin distribution in control EW tracheid. (d) Early stages of delignification progressing from the lumen side outwards (arrow). (e) Progression of gradual delignification showing formation of prominent delignified zones (double headed arrows) in the cell walls. MLcc, middle lamella cell corner. Bars ¼ 2 mm (a, b), 500 nm (cee).
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3. Results 3.1. Anatomical and histochemical changes in decayed spruce wood Overall, earlywood (EW) tracheids showed more progressive degradation in radial (Ra)-than tangential cell walls (Ta) (Fig. 1a, b), while latewood (LW) tracheids showed the opposite pattern (i.e. less degradation in Ta than Ra) (Fig. 1e). However, the decay process in tracheid cell walls was similar between EW and LW. Decay of tracheid cell walls occurred by progressive thinning from the lumen surface (Fig. 1a, b, e). During decay, a thin layer stained purple by toluidine blue was apparent in tracheid cell walls (marked 1, insets in Fig. 1b). Since toluidine blue stains the lignified cell wall blue and non-lignified cell walls purple, this layer indicates presence of completely/or prominently delignified cell wall regions caused by fungal attack. As decay progressed, separation of compound middle lamella (CML) regions in adjacent tracheids was frequently observed, no matter radial or tangential cell walls
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(Fig. 1aec, e). This separation was only detectable in tracheids that showed loss of toluidine blue staining in CML regions (marked 2, insets in Fig. 1b). In more advance stages, notable lignin staining was only detected in middle lamella cell corner (MLcc) regions of tracheids (Fig. 1c). At late stages of decay, MLcc regions with thin cell corner regions generally only remained in tracheids (Fig. 1d). With ray parenchyma cells, at early stages of decay, prominent degradation was detected in cross-field pits (i.e. pits between tracheids and rays) (arrowheads in Fig. 1f) and adjacent ray cell walls of biseriate rays (arrowheads in Fig. 1g). 3.2. Degradation of lignin in tracheids Observations using light microscopy were further advanced by TEM. Fig. 2a, b shows EW and LW tracheids stained with KMnO4 for lignin. In the same EW tracheid, delignification of Ra cell walls including CML regions was faster than Ta cell walls (Fig. 2a). In contrast, degradation and delignification in Ra cell walls of LW
Fig. 3. Delignification of compound middle lamella (CML) regions of tracheids stained with KMnO4 for lignin. (aef) Delignification of CML regions started following delignification of secondary cell walls (bed). Separation of CML regions occurred following delignification in both tangential (Ta, e) and radial (Ra, f) cell walls. Note progressive thinning of secondary cell wall concomitant with progressive delignification (b, c). (g, h) Delignification of CML regions by attack from pit chambers (arrowheads in g, h). Note earlier delignification of bordered pit membranes (M) than pit borders (g). Bars ¼ 1 mm.
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tracheids was slower than Ta cell walls (Fig. 2b). These temporal variations in delignification were consistent with differences in resistance to decay depending on cell wall orientations in EW and LW described above (Fig. 1a, e). The innermost layer (i.e. lumen wall) of delignified tracheid cell wall also frequently showed an uneven structure during progressive thinning of the wall (arrowheads, inset in Fig. 2a; Figs. 3 and 6). This may indicate how thinning of cell walls progresses in tracheid secondary cell wall following delignification. Delignification of secondary cell wall progressed gradually from the lumen side outwards (Fig. 2c, d). During decay, prominent delignified zones were detected in the secondary cell wall (double headed arrows in Fig. 2e). Fig. 3 shows the delignification process of CML regions in tracheids which was similar for Ra-and Ta cell walls. The only difference was that delignification of CML regions occurred through pit regions in Ra cell walls (Fig. 3g, h). Delignification of CML regions started after delignification of secondary cell walls (Fig. 3c, d). Thinning of secondary cell walls characterized by uneven structure of the innermost tracheid cell wall layers was also
observed before delignification of CML regions was initiated (Fig. 3aec). Separation of tracheids was frequently apparent after complete delignification of CML regions, no matter cell wall orientation (Fig. 3e, f). At late stages of decay, lignin staining was only detected in MLcc regions (Fig. 4a) with the secondary cell walls and CML regions completely delignified at this stage (Fig. 4a). The thickness of delignified secondary cell walls remaining varied between tracheids. In very advanced stages, MLcc regions generally remained in tracheids, followed by delignification (Fig. 4b). However, some tracheids showed delignification of MLcc regions prior to complete delignification of secondary cell walls because of the variation in decay rate between neighboring tracheids (arrowheads in Fig. 4c). Some tracheids also showed severe delignification of MLcc regions by attack from CML regions before delignification in neighboring tracheid cell walls was completed (arrowheads in Fig. 4d). Unlike secondary cell walls (Fig. 2e), a prominent delignified zone was not detected in MLcc regions of tracheids (Fig. 4b), presumably because of no/or very limited cellulose in MLcc regions.
Fig. 4. Late stages of decay in tracheids stained with KMnO4 for lignin. (a) Non-delignified middle lamella cell corner (MLcc) regions after delignification of secondary cell walls and compound middle lamella (CML) regions. (b) MLcc regions remained after degradation of entire cell walls including CML regions. Note no prominent delignified zone in MLcc regions compared to secondary cell walls (Fig. 2e). (c, d) Delignification of MLcc regions prior to complete delignification of secondary cell walls because of the variation in decay rate between neighboring tracheids (arrowheads in c) and by attack from CML regions (arrowheads in d). Bars ¼ 2 mm (a), 1 mm (c, d), 500 nm (b).
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Fig. 5. Immunolocalization of mannan and xylan epitopes in tracheid cell walls, followed by KMnO4 staining for lignin. (a, b) Delignified cell walls (double headed arrows) showing abundant xylan and mannan epitopes like lignified cell walls. (c, d) Cell walls remained after complete delignification of compound middle lamella (CML) regions showing abundant mannan (c) and xylan (d) epitopes. MLcc, middle lamella cell corner. Bars ¼ 500 nm.
3.3. Immunolocalization of non-cellulosic polysaccharides in tracheids Fig. 5 shows immunogold labeling of mannan and xylan epitopes in decayed tracheids. Abundant epitopes were detected in both delignified (double headed arrows in Fig. 5a, b) and lignified regions (Fig. 5a, b). Abundant epitopes were also detected in delignified tracheid cell walls remaining after complete delignification of CML regions (Fig. 5c, d). These results demonstrate that lignin is preferentially degraded prior to complete degradation of hemicelluloses by P. sanguineus in the course of decay. HG and xyloglucan which represent major chemical components in CML regions of tracheids were detected in CML regions at early stages of delignification (Fig. 6a, c). However, in more advanced stages of delignification, HG epitopes were not detectable in CML regions (Fig. 6b), suggesting that HG may be simultaneously degraded with lignin in CML regions. In contrast, xyloglucan epitopes were detected in CML regions during the entire delignification process of CML regions, even after complete delignification (Fig. 6d, e). This result like for secondary cell walls demonstrates preferential degradation of lignin prior to degradation of xyloglucan (hemicellulose) by the fungus. At late stages of decay, MLcc regions showed
abundant arabinan, HG, xyloglucan and xylan epitopes (Fig. 7aed) similar to that of controls (not shown). However, it was unclear whether lignin is preferentially degraded from MLcc regions before removal of pectins (i.e. arabinan and HG) and hemicelluloses (i.e. xyloglucan and xylan). 3.4. Degradation of ray tracheids and ray parenchyma cells Norway spruce has 1e3 layers of ray tracheids that form part of rays. Ray tracheids are distinct from ray parenchyma cells by presence of small bordered pits in their cell walls in transverse sections (Fig. 8a). Ray tracheids showed either similar or slightly lower resistance to decay compared to axial tracheids (tracheids) (Fig. 8a, c). Although the full process of delignification in ray tracheids was unclear in this study due to their thin cell walls in transverse sections, it is expected that progressive delignification occurs from the lumen towards ML regions like in axial tracheids since considerable lignin staining was still detectable in ML regions when ray tracheid cell walls had completely lost lignin staining (inset in Fig. 8c). Distribution of abundant xylan and mannan epitopes in delignified ray tracheids cell walls after complete delignification in ML regions also supported that decay patterns in
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Fig. 6. Immunolocalization of homogalacturonan (HG) and xyloglucan epitopes in compound middle lamella (CML) regions of tracheids, followed by KMnO4 staining for lignin. (a, b) At early stages of delignification in CML regions, HG epitopes were detected in CML regions (a), but were absent at late stages (b). (cee) Xyloglucan epitopes were detected in CML regions during the course of delignification of CML regions (c, d) even after complete delignification (e). Bars ¼ 500 nm.
ray tracheids were similar to axial tracheids (Fig. 8d). Interestingly, bordered pit membranes of ray tracheids which stained strongly with KMnO4 for lignin showed higher resistance to decay than (ray/ axial) tracheid cell walls (Fig. 8aec). This contrasted with decay in bordered (Fig. 3g)- and cross-field pit (Fig. 1f and 9c) membranes of axial tracheids that showed lower decay resistance than tracheid cell walls even though the membranes were lignified in controls (not shown). Other factors, such as accumulation of extractives (rate and type) and degree of lignification in pit membranes may differ between axial- and radial tracheids, thereby producing different decay resistance. However, at present chemical differences in pit membranes between axial- and radial tracheids are unclear. Overall, ray parenchyma cells showed higher resistance to decay than tracheids (Fig. 9a). Several decay patterns were observed in ray parenchyma cell walls; 1) Progression of decay from tracheids to rays across the ML regions (Fig. 9b), 2) Progression of decay from cross-field pit regions towards ray cell walls including ML/MLcc regions (Fig. 9c and inset), 3) Degradation of ML regions in adjacent ray cell walls of biseriate rays (Figs. 1g and 9d) and 4) Local degradation of ray cell walls initiated either from the lumen or ML regions of adjacent ray cell walls of biseriate rays (Figs. 1g and 9e). The former two decay patterns were more prominent in ray
parenchyma cells than the latter patterns during decay. Ray parenchyma cell walls in contact with axial tracheids were generally degraded earlier than adjacent ray cell walls of biseriate rays (i.e. ray cell wall not in contact with tracheid cell walls). Degradation of ray parenchyma cell walls facing the lumen (except for local degradation as outlined in pattern 4) was rarely observed even though considerable degradation progressed in ray parenchyma cell walls facing tracheids and ML regions (Fig. 9b, d, f, g). Mannan and xylan epitopes were detectable in delignified ray cell wall regions (Fig. 9f, g and insets) like tracheids (Fig. 5).
4. Discussion Using light-and TEM microscopy techniques, this study characterizes micromorphological and chemical decay patterns of Norway spruce wood decayed by the white rot fungus P. sanguineus. TEM immunocytochemistry using antibodies specific for pectins and hemicelluloses provides new insight on degradation of cell wall components by white rot fungi. To our knowledge this study is the first report demonstrating temporal controlled degradation of lignin and non-cellulosic polysaccharides in wood by P. sanguineus at the cellular level.
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Fig. 7. Immunolocalization of pectin and hemicellulose epitopes in middle lamella cell corner (MLcc) regions of tracheids, followed by uranyl acetate (UA) staining. (aed) MLcc regions remaining at late stages of decay showing distribution of arabinan (a), homogalacturonan (HG, b), xyloglucan (c) and xylan (d) epitopes. Bars ¼ 500 nm.
4.1. Decay type in tracheids Results demonstrate that lignin is selectively degraded without extensive loss of hemicelluloses (probably also cellulose) in tracheid cell walls by P. sanguineus (i.e. selective decay). These results are consistent with previous studies even though lignin and cellulose were mainly focused in the studies (Luna et al., 2004; Singh et al., 2012). In contrast, results are inconsistent other studies that proposed simultaneous degradation of lignin and cellulose by P. sanguineus (Ferraz et al., 1998; Levin et al., 2007; Van Heerden et al., 2008; Rohr et al., 2013). Selective removal of lignin without extensive degradation of xyloglucan (hemicellulose) in CML regions also supports selective delignification by P. sanguineus. Micromorphologically, separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls also indicate selective decay of lignin induced by P. sanguineus. Tracheid/fiber separation is generally considered as evidence of selective decay (Blanchette, 1984). Prominent cell separation has also been reported in poplar fibers degraded by P. sanguineus (Luna et al., 2004). As described for other selective white rot fungi (Blanchette and Reid, 1986; Daniel, 2003), characteristic features of simultaneous decay, such as progressive thinning of cell walls and MLcc regions as the last areas to be attacked by P. sanguineus were also observed. Consequently, results indicate that P. sanguineus produces selective decay in spruce tracheids. Hemicelluloses are degraded after removal of lignin in tracheid cell walls and CML regions (i.e. considerable hemicelluloses remains after complete delignification). Although further studies are
needed on other selective white rot fungi, these results differ from previous concepts that hemicelluloses are degraded concomitantly with lignin in wood cell wall by selective white rot fungi and that only modified cellulose remains (Goodell et al., 2003). Concerning the mechanism of selective attack of lignin in tracheid cell walls, we consider that activity of ligninolytic enzymes and free radicals are likely to be more aggressive than hemicellulolytic enzymes (probably also cellulolytic enzymes) during decay by P. sanguineus. Formation of a prominent delignified zone in tracheids cell wall during decay may reflect differences in activity between ligninolytic (and free radicals) and hemicellulolytic/ cellulolytic enzymes since the progressive thinning of delignified cell wall (i.e. degradation of hemicelluloses and cellulose) occur concomitantly with delignification in the same tracheid cell wall (i.e. concurrent attack by ligninolytic, hemicellulolytic and cellulolytic enzymes). In similar delignified zones (decay zones), ligninand Mn peroxidase were concentrated in the interface degraded and non-degraded cell walls (Srebotnik et al., 1988; Daniel et al., 1989, 1991, 2004). 4.2. Different resistance between radial-and tangential cell walls of tracheids Results show that decay differs between radial (Ra) and tangential (Ta) cell walls in tracheids with opposite pattern between EW (Ra < Ta) and TW (Ra > Ta). Daniel et al. (1989) also found higher resistance in Ra-than Ta cell walls in Scots pine LW tracheids by Phanerochaete chrysosporium. They suggested that this
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Fig. 8. Degradation of ray tracheids (RT) stained with KMnO4 for lignin. (aec) Ray tracheids in latewood (LW; a, b) and earlywood (EW, c) showing either similar or slightly lower resistance to decay compared to axial tracheids (T). Bordered pit membranes (between RT and T) showed higher resistance than ray/axial tracheid cell walls (b, arrowheads in a, c). Note delignification of middle lamella (ML) regions after complete delignification of (ray/axial) tracheid cell walls (inset in c). (d) Localization of xylan epitopes showing abundant epitopes in delignified ray tracheid cell walls. Note absence of lignin staining in ML regions. Bars ¼ 2 mm (a), 1 mm (b), 500 nm (c, d).
difference may reflect the different cell wall thickness between Raand Ta cell walls (i.e. thicker in Ra than Ta). Ollinmaa (1961) reported that Ra cell walls of Norway spruce LW tracheids are thicker than Ta cell walls. However, this explanation cannot be applied to EW tracheids that showed lower decay resistance in Ra-than Ta cell walls by P. sanguineus in this study (i.e. opposite to LW) since the Ra wall is thicker than Ta cell walls in Norway spruce EW tracheids like LW (Ollinmaa, 1961). This indicates that some other factors, particularly presence of bordered pits may also be important for inducing different decay resistance between cell walls in EW tracheids. In Norway spruce EW tracheids, the majority of bordered € m, 2001). Bordered pits are present on the Ra cell walls (Br€ andstro pit membranes were degraded at early stages of decay, mostly earlier than tracheid cell walls by P. sanguineus in this study. Delignification of CML regions through the pit regions was also observed in this study. Although Ra cell walls of LW tracheids also contain bordered pits, it may play only a minor role in affecting decay compared to cell wall thickness since the number of bordered pits in Ra cell walls of LW tracheids is much less than in EW tra€ m, 2001). The diameter of bordered pits is also cheids (Br€ andstro € m, 2001). much smaller in LW than EW (Br€ andstro
4.3. Degradation of ray parenchyma cells Ray parenchyma cells showed higher resistance to decay by P. sanguineus than tracheids. Higher resistance in ray parenchyma cell walls isolated from tracheids (i.e. central adjacent two ray cell walls of biseriate rays) than ray parenchyma cell walls in contact with tracheids may reflect a lower resistance of tracheids than rays. This is consistent with differences in lignin concentration between ray parenchyma cells and tracheids. Westermark et al. (1988) reported that lignin concentration of ray parenchyma cells is higher than tracheids in spruce. Ray parenchyma cells also showed stronger lignin staining than tracheids in control (not shown) like decayed samples (Fig. 9a) in this study. Fungal colonization generally occurs more frequently through ray parenchyma cells than tracheids since ray parenchyma cells contain storage materials that can accelerate colonization and penetration into the wood structure. This general concept also supports the positive correlation between lignin concentration and fungal resistance in this study, i.e. ray parenchyma cells provide space for fungal colonization at early stages of decay, but are not degraded due to high concentration of lignin. This type of fungal resistance is frequently
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39
Fig. 9. Degradation of ray parenchyma cells (R) stained with KMnO4 for lignin. (a) Ray cell walls connected to tracheids (T) showing higher resistance to decay than tracheid cell walls at early stages of decay (double headed arrows indicate delignified zones in tracheids). (b) Progression of decay from tracheids to rays across the middle lamella (ML) regions (arrow). (c) Preferential decay of ML/middle lamella cell corner (asterisk) regions (between rays and tracheids) through degradation of cross-field pits (arrowheads). Inset shows ray cell walls degraded together with ML regions through cross-field pits. (d) Preferential decay of ML regions in adjacent ray cell walls of biseriate rays. (e) Local degradation of ray cell walls (arrow). (f, g) Localization of mannan (f) and xylan (g) epitopes showing distribution of epitopes in completely/or considerably delignified ray cell walls. Bars ¼ 1 mm (aee), 500 nm (f, g).
observed in vessels of hardwoods. High lignin concentration in vessels is generally considered one factor causing their greater resistance to decay than fibers in hardwoods (Blanchette et al., 1988; Schwarze, 2007). The only exception is that some ray parenchyma cells showed degradation of ML/MLcc regions (between tracheids and rays or between ray and ray in biseriate rays) through cross-field pits and bore holes prior to degradation of ray cell walls even though lignin concentration in ML/MLcc regions is generally higher than cell walls (Figs 1f, 9c and d). 5. Conclusions Correlated TEM and TEM-immunocytochemistry demonstrates that the white rot fungus P. sanguineus produces selective decay in
Norway spruce tracheids including secondary cell walls and CML regions. Lignin was preferentially removed from tracheids with hemicelluloses and cellulose remaining. This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. These results may also reflect potential of P. sanguineus for various biological applications as outlined in the introduction. Results also show variations in resistance to decomposition by P. sanguineus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types including axial tracheids, ray tracheids and ray parenchyma cells. Finally, this study shows the potential of TEM immunocytochemistry to characterize the temporal degradation process of cell wall components in wood decayed by white rot fungi.
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