Trees DOI 10.1007/s00468-014-1025-y
ORIGINAL PAPER
Antioxidative response patterns of Norway spruce bark to low-density Ceratocystis polonica inoculation Andreja Urbanek Krajnc • Metka Novak • Mateja Felicijan • Nada Krasˇevec • Mario Lesˇnik Neja Zupanec • Radovan Komel
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Received: 19 February 2014 / Revised: 7 April 2014 / Accepted: 28 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message Based on time courses of individual antioxidant compounds, bark phenolic metabolism has been recognised to integrate ascorbate–glutathione system as a redox hub in Norway spruce defence against Ceratocystis polonica infection. Abstract Temporal courses of individual phenolics, thiols and ascorbate were studied in Norway spruce phloem over a 5-month period after inoculation at low density with Ceratocystis polonica. The initial reaction of Norway spruce 3 days after inoculation was characterised by significantly increased isorhapontin and taxifolin concentrations, accompanied by significantly lowered catechin contents. On later sampling dates, catechin concentrations within infected bark increased until September. The slightly accumulated astringin contents in April and May diminished at later sampling dates in response to infection. The isorhapontin levels strongly raised in April and were
slightly lowered from June onwards. Compared to the controls, taxifolin concentrations were higher in the infected samples showing a double peak with maxima in April and June. The taxifolin values eased later but remained above the control levels. The initial response of the ascorbate–glutathione system to fungal infection was characterised by a significantly more oxidised glutathione pool, slightly more reduced ascorbate system and by higher glutathione reductase activity. Three weeks later an accumulation of thiols was observed, whereas total ascorbate was significantly lowered and the ascorbate redox state shifted towards more oxidised values. Until the middle of July a gradual increase of total glutathione was determined within the infected bark, which was accompanied by significantly increased cysteine contents, higher glutathione reductase activity, but significantly lowered total ascorbate contents. The increased pressure on the ascorbate system reflects its interaction with phenolics, as ascorbate is needed for reducing the phenoxyl radicals formed during pathogen defence.
Communicated by W. Osswald.
Electronic supplementary material The online version of this article (doi:10.1007/s00468-014-1025-y) contains supplementary material, which is available to authorized users. A. Urbanek Krajnc (&) M. Felicijan M. Lesˇnik Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hocˇe, Slovenia e-mail:
[email protected] M. Novak N. Krasˇevec N. Zupanec R. Komel National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia e-mail:
[email protected] N. Zupanec R. Komel Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
Keywords Antioxidant defence system Ascorbate Glutathione Phenolics Picea abies Sap flow
Introduction Although bark beetles and their fungal associates are integral parts of forest ecosystems (Klepzig et al. 2009), the European spruce bark beetle (Ips typographus L.) and the associated pathogenic blue stain fungus Ceratocystis polonica (Siem.) C. Moreau are the most devastating pests regarding Norway spruce (Krokene and Solheim 1999; Wermelinger 2004; Linnakoski et al. 2012; Novak et al. 2014). Conifers have a wide range of effective defence
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mechanisms that are based on the bark anatomy and physiological state of the tree. The basic function of bark defences is to protect the nutrient- and energy-rich phloem, the vital meristematic region of the vascular cambium, and the transpiration flow in the sapwood. Pathogens and insects must overcome three types of conifer defence strategies as they penetrate into the bark and enter the sapwood (Franceschi et al. 2005). The first and basic defence strategy, constitutive defence, involves the secretion of oleoresin from preformed resin ducts as well as accumulation of phenolics within the concentric layers of the polyphenolic parenchyma (PP) cells, the preformed sclerenchyma and calcium oxalate crystals. Inducible defences are activated following an initial attack by pests and they include the de novo formation of a wound periderm and traumatic resin ducts, which coincide with increased terpene production and resin flow. Furthermore, swelling and proliferation of PP cells occurs, which is associated with qualitative and quantitative changes in phenolics (Beckman 2000; Krekling et al. 2000; Hudgins and Franceschi 2004; Franceschi et al. 2005; Schmidt et al. 2005; Witzel and Martin 2008). Both constitutive and inducible defences may deter beetle invasion, impede fungal growth and close entrance wounds. During a successful attack, systemic acquired resistance (SAR) becomes effective and represents a third defence strategy (Evensen et al. 2000; Percival 2001; Nagy et al. 2004; Franceschi et al. 2005; Bonello et al. 2006; Witzel and Martin 2008). It gradually develops throughout the plant and provides a systemic change within the whole tree’s metabolism, which is maintained over a longer period of time. The broad range of defence mechanisms that contribute to the appearance of SAR, includes antioxidants and antioxidant enzymes (Foyer and Rennenberg 2000; RiedleBauer 2000), which are generally linked to the action of reactive oxygen species (ROS). While several studies have demonstrated that the induction of phenolics occurs on a local scale (Evensen et al. 2000; Franceschi et al. 2000, 2005), several recent studies have also found evidence of the systemic induction of soluble low molecular weight phenolics (Bonello et al. 2001, 2006; Wallis et al. 2008). A SAR hypothesis postulated by Bonello et al. (2006) illustrated the interplay between SAR and induced susceptibility in trees against microbes and herbivores. The authors postulated that the time course of pathogen infection may have a bell-shaped effect on the strength of SAR that is similar to spatial variation of constitutive secondary metabolites generated by resource availability. In the earliest stages of pathogen infection, SAR responses are predicted to rapidly and systemically increase concentrations of compounds involved in defence against pathogens and insects. However, if the inducing pathogen is able to grow despite the deployment of localised defence responses, the
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infection will progress, and the three will become increasingly stressed by the resulting resource limitations (e.g. reduction of sap flow caused by blue stain fungi, limited nutrient and water absorption, decreased carbon assimilation and growth). Consequently, a degradation of molecules involved in SAR will follow. Although attempts have been made to use phenolic metabolites as predictors of resistance (Brignolas et al. 1995, 1998; Lieutier et al. 2003; Nagy et al. 2005; Witzell and Martin 2008), the spatial and temporal gradations of single phenolic compounds in relation to a specific defence response, capable of stopping the invading pathogen, need to be studied more carefully. The detection of definite trends is complicated by the fact that the impacts of the plants’ phenolic status may vary considerably amongst pathogens with different life strategies. In addition, the mode of infection biology of the fungus is critical to determine its interaction with plant phenolics. In vitro tests are frequently used to complement studies with intact plant–parasite interactions (Nagy et al. 2005; Schmidt et al. 2005; Witzel and Martin 2008; Mala et al. 2011). Nagy et al. (2005) studied the anatomical changes in the PP cells of spruce barks’ callus cultures, initiated from trees with different resistances, after inoculation with C. polonica and Heterobasidion annosum. Callus cells from resistant clones had more polyphenolic bodies in comparison to callus cultures with weak resistances. The latter were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistances. Nagy et al. (2005) concluded that callus from trees of varying resistances seems to reflect the relative resistances of the trees from which they are derived. However, the biological significance of any information obtained from artificial systems has to be interpreted with caution, since the effects of plant chemicals against fungi in vitro may differ considerably from their effects or modes of action in vivo, and vice versa. Furthermore, correlations between phenolic concentrations and disease symptoms in the field are unlikely to be reliable unless confirmed by systematic, long-term studies. Phenolics range from simple, low molecular weight, single aromatic-ringed compounds to large and complex tannins and derived polyphenols that provide different chemical and structural defence strategies. The more important phenolics in the phloem of Norway spruce are stilbenes such as astringin and isorhapotin, acetophenone picein, flavonoids such as taxifolin glucoside and (?)-catechin, as well as tannins (e.g. Lieutier et al. 2003; Schmidt et al. 2005; Witzell and Martin 2008; Hammerbacher et al. 2011). In general, phenolics fulfil different defencive functions. Phenolics can act as antioxidants by donating electrons to guaiacol-type (class III) peroxidases that have been implicated, for example, in lignification and
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pathogen defence (Perez et al. 2002). The phenoxyl radicals that are formed can then be reduced by the ubiquitous cell antioxidant, ascorbate (Sakihama et al. 2002). However, phenoxyl radicals may also exhibit pro-oxidant activity under conditions that prolong the radical lifetime (e.g. microbial attack) and catalyse cellular DNA degradation in the presence of transitive metals, such as iron (Bhat et al. 2007). Most of the phenolics contribute to resistance indirectly. For instance, some low molecular weight phenolic compounds may function as precursors for other defensive compounds (e.g. lignin; Bonello and Blodgett 2003), or they may confer resistance as a group, rather than as individual compounds (Wallis et al. 2008). Catechin is a building block of condensed tannins for which antimicrobial activity via protein precipitation and iron depletion has been suggested (Witzell and Martin 2008). Changes in the concentration of stilbenes in Norway spruce in response to injury or fungal infection are considered to be an active defence response (Brignolas et al. 1995, 1998; Viiri et al. 2001; Schmidt et al. 2005; Witzell and Martin 2008; Li et al. 2012, Hammerbacher et al. 2013). Stilbenes are known to inhibit fungal growth by interfering with microtubule assembly (Woods et al. 1995; Adrian et al. 1997), disrupting plasma membranes and uncoupling electron transport within fungal spores and germ tubes (Pont and Pezet 2008; Adrian and Jeandet 2012). The mechanisms of stilbene toxicity towards fungi are not well understood (Chong et al. 2009), but fungal cytochromes P450 are frequently involved in detoxification of benzoic acid and other phenolics (Podobnik et al. 2008). Recently, the CYP53D subfamily from basidiomycete Postia placenta was identified, whose members converted stilbenes through O-demethylation (Ide et al. 2012). Stilbenes have also been shown to protect plants against oxidative stress (He et al. 2008), to deter herbivores (Torres et al. 2003), and to inhibit the growth of competing plants (Fiorentino et al. 2008). Besides phenolics, there is no research available on the ascorbate–glutathione system within blue stain fungusaffected Norway spruce. The ascorbate–glutathione cycle is considered to be the main pathway for ROS removal, and both ascorbate and glutathione are recognised as the heart of the redox hub within the cell (Foyer and Noctor 2011). Previously, thiols and ascorbate have been studied regarding Norway spruce bark exposed to various degrees of bark beetle attack (Urbanek Krajnc 2009; Urbanek Krajnc et al. 2011). A sequence of changes in the endogenous levels of antioxidant molecules within the bark of beetle-affected Norway spruce showed consistency with the general ecophysiological stress-response concept, and provided important avenues for evaluating the roles and
effectiveness of antioxidants during SAR against bark beetle attack (Urbanek Krajnc 2009). The presented investigation was aimed in understanding the antioxidant defence mechanisms of Norway spruce [Picea abies (L.) Karst.] against low-density C. polonica infection by providing a time course analysis on the levels and relative compositions of single antioxidative compounds (phenolics, cysteine, glutathione, ascorbic acid) over a 5-month period, regarding patterns of sap flow and weather conditions.
Materials and methods Study site The experiment was carried out within a second growth Norway spruce even-aged stand with dense patches of small diameter trees (35-year-old clonal Norway spruce trees: tree height: 20 ± 2 m; crown length: 8 ± 0.7 m; DBH: 30 ± 2 cm) in Pivola, Hocˇe, Pohorje in Slovenia (N 46°300 2200 , E 15°C360 1800 ). Twenty trees were randomly selected within the parcel, the distances between the trees ranging from 7 to 12 m. Half of them were inoculated with fungus whilst the other half served as control. Seven inoculated trees and seven controls were considered as test sets, whilst periodical sap flow measurements were performed on three of the inoculated trees and three of the controls. Inoculation of test trees with Ceratocystis polonica In regard to the inoculations of the trees, a culture of blue stain fungus, C. polonica strain ZLVG349 isolated from Ips typographus (deposited at the fungus collection of Slovenian Forestry Institute; Repe et al. 2013), was maintained in Petri dishes for 1 week on MBFA media (Malt extract agar-Blakeslee formula: 2 % (w/v) malt extract agar (Merck, Germany), 0.1 % (w/v) Bacto Peptone (BD, USA) and 1.5 % (w/v) agar (Merck, Germany) (Table 1). On the April 15th 2011, ten of the selected trees were inoculated at the lower parts of their trunks with C. polonica at a low-density, using a 7-mm cork borer, and the bark plug was reinserted into the hole according to the method originally described by Wright (1933). Each of these trees received 20 inoculations between 0.8 and 1.5 m above ground, in five rings of four inoculations along the stem circumference, two on the east and two on the west side. The horizontal and vertical distances between the two inoculations at E and W side of the trunk were 15 cm. Ten trees used for control were left untreated (uninfected trees).
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Sap flow and temperature measurements
Phenolic extraction and analyses
Three days before, and up to 5 months after inoculation, the sap flows of two of the inoculated trees (trees no. 1 and 2) and two controls (trees no. 3 and 4) were measured at the outermost sapwood (0–3 cm depth) using the thermal dissipation method (Granier 1985). On each tree, a standardlength (3 cm) sap flow TDP-30 (Dynamax, USA) was installed 1.5 m above the ground within the inoculated area, between the last two inoculations. Those parts of the trunk housing sensors were insulated using 4-cm-thick cell foam wrappings covered by an external aluminium shielding with a width of 0.5 m. The insulations were protected against rainfall using plastic aprons. The sensor outputs were logged every minute and stored as 10-min averages (CR1000 datalogger, Campbell Scientific Inc., Logan, UT, USA). The air temperatures and relative humidity (CS215 Temperature and Relative Humidity Sensor, Campbell Scientific Inc., USA) were measured during the period of sampling together with the soil temperatures 20 cm below the surface (107 Temperature Probe sensors, Campbell Scientific Inc., USA). The results were informative and were not statistically evaluated because of the low number of replicates (2 per inoculation/control).
Phenolic extractions were performed on the lyophilised phloem samples. 150 mg of each sample was analysed separately. All extractions were done at 4 °C over two steps, as previously described (Brignolas et al. 1995; Lieutier et al. 2003). In brief, the resinous compounds were removed using pentane and soluble phenolics extracted with 50 % methyl alcohol containing 2.5 mM vanillyl alcohol as the internal standard. The phenols were analysed using a reverse-phase HPLC (Waters 2695, Dual Absorbance detector, Waters 2487) on a YMC-Pack ODS-AQ, C18 reversed-phase silica-based HPLC column [5 lm (150 9 4.6 mm)], YMC Europe GmbH, Germany. The samples were analysed using a gradient program with a two solvent system (solvent A: 95 % w/v ACN, 0.2 % w/v formic acid; solvent B: 5 % w/v ACN, 0.2 % w/v formic acid). Solvent A (95 %) was used as the mobile phase for the initial 7 min, then lowered to 80 % (7–13 min), then changed to 65 % of solvent A for 3 min and to 5 % for 4 min, and for the final 3 min changed back to 95 %. The flow rate was 0.8 ml per min, and the injection volume was 10 ll. The signals were detected at 280 nm. The peak identifications were based on the standard catechin, based on comparing the results from previous studies on the same tree species (Brignolas et al. 1995, 1998). Extract formations were analysed on the LC Agilent 1100 Series system (Agilent Technologies, USA) using a YMC-ODS-AQ chromatographic column coupled to an Applied Biosystems 4000 Q TRAP mass spectrometer (AB, USA). The results were expressed as milligrams of catechin equivalent per gram of lyophilised phloem samples. The total phenolic compounds (tPH) were determined spectrophotometrically, according to Ainsworth and Gillespie (2007) and the absorbance was measured at 765 nm. The total phenolics were expressed as milligrams gallic acid equivalents per gram of lyophilized phloem samples.
Sample preparation for biochemical analysis To obtain a temporal sequence of defence chemicals, the test trees were sampled five times from the 18th of April to the 9th September 2011 [3 days post-inoculation (3 dpi), and then 3 weeks, and 2, 3 and 5 months after fungal inoculations]. Samples containing bark and secondary phloem (4 cm 9 4 cm) were collected from each of the 14 test-set trees (seven inoculated and seven controls) for biochemical analysis. Two samples were collected on the west- and two on the east-facing side of each tree about 5 cm above the inoculation point. The phloem samples from the control trees were taken at the same heights and locations as from the fungus-inoculated trees. The wounded sites were protected by commercially available resin (Kambisan, Agrorusˇe, Slovenia). The bark was removed, immediately frozen in liquid N2 and stored in a freezer at -80 °C. All the samples were collected on clear days between 11.00 and 14.00 solar time to avoid effects from diurnal patterns of metabolite accumulation (Tausz et al. 2003). After 5 months, at the end of the experiment, all the infected trees were felled. To check and measure the growth of the fungus into the sapwood, seen as a dark blue stain, each tree was sliced into five discs (between the fungal inoculation sites). The dark blue symptoms of fungal infection within the discs of the test trees have been previously evaluated and described in Novak et al. (2014).
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Determination of low molecular weight thiols and glutathione reductase activity Glutathione (oxidized and reduced) was determined as described by Tausz et al. (2003). Thiols were extracted from 60 mg of lyophilised plant material in 2 mL of 0.1 M HCl with 60 mg of polyvinylpolypyrrolidone added to remove phenolics. 280 lM of this extract was incubated with 420 lL of CHES buffer [200 mM 2-(N-cyclohexylamino) ethanesulfonic acid, pH 9.0] and 70 lL of 5 mM dithiothreitol for 1 h at room temperature (RT) to reduce the thiol groups. The SH groups were labelled with monobromobimane (BmBr) by incubating with 50 lL 8 mM BmBr in the dark at RT for 15 min. Derivatisation
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was stopped by adding a 760 lL aliquot of 0.25 % (v/v) methanesulfonic acid. For the determination of the content of oxidised thiols 30 lL of 50 mM N-ethylmaleimide and 600 lL of 200 mM CHES buffer were added to 400 lL of the thiol extract, and the mixture was then incubated at RT for 15 min to block the SH groups. The excess of N-ethylmaleimide was removed by extraction with toluene, and the remaining oxidised thiols were reduced by 50 lL dithiothreitol and derivatised with BmBr, as described above. The separations and determinations of the derivatised thiols were done using a gradient high-pressure liquid chromatography system (HPLC): Waters 2695 HPLC system, Waters 2475 Multi Fluorescence Detector (excitation: 380 nm wavelength; emission: 480 nm wavelength), column Spherisorb S5 ODS2 25 9 4.6 mm. Solvent A: 0.25 % (v/v) acetic acid in water containing 5 % methanol, pH 3.9. Solvent B: 90 % (v/v) methanol in water; gradient: 5 % solvent B to 15 % solvent B in 20 min, 100 % solvent B for 6 min, and 5 % solvent B for another 8 min. The flow rate was 1 mL min-1. Glutathione reductase activity was assayed using the spectrophotometric method modified according to Kranner (1998) at 340 nm, by following the decrease in absorbance as NADPH was oxidised. The GR activities within the bark samples were expressed as mU/g DW. Determination of ascorbic acid Total ascorbic acid (tASC) and dehydroascorbic acid (DHA) were analysed using an isocratic reversed-phase chromatography method modified according to Tausz et al. (2003) and Herbinger et al. (2005). Aliquots (80 mg) of powdered bark material were homogenised in 3 mL 3 % (w/v) metaphosphoric acid containing l mM ethylenediamine tetraacetic acid disodium salt dihydrate and 60 mg of polyvinylpolypyrrolidone to remove phenolics. The obtained extracts were either analysed directly to determine ascorbic acid (reduced form) alone or derivatised to carry out simultaneous determination of both the oxidised and reduced forms of ascorbate. When determining the tASC (reduced and oxidised forms), an aliquot (600 lL) of the sample extract was adjusted to neutral pH = 6.2 by adding 280 ll of 0.4 M Tris (in aqua bidest). 50 lL of 0.26 M dithiothreitol was added to a sample extract (600 lL) and the samples were then incubated for 10 min at 25 °C in the dark, to reduce the DHA. The reaction was stopped with 100 lL of orthophosphoric acid (1:10). When determining the ascorbic acid (reduced form), 280 lL of 0.4 M Tris buffer and 50 lL of ddH2O were added to the 600 lL sample extract, incubated and stopped as described above. Separation and determination of the ascorbate were done on a gradient HPLC: Waters 2695 HPLC system, Waters
996 PDA detector (excitation: 245 nm). Column Spherisorb S5 ODS2 25 9 4.6 mm. Solvent: 50 mM NaH2PO4. Run time: 30 min. Flow rate was 0.5 mL min-1. Statistics The results of biochemical analyses represented the medians and median deviations (MD) of seven replica samples. They were statistically evaluated by one-way analysis of variance (P \ 0.05) using the SPSS 12 software (SPSS Inc., Chicago, IL, USA). The differences between the controls and C. polonica-infected trees were tested using the Duncan test at the 0.05 significance level. Significant differences were indicated by different letters (a–e). Decision rule: P \ 0.05 was regarded as significant.
Results Sap flow and temperature measurements All the inoculated trees resisted the low-density inoculation with C. polonica and were characterised by intensive and sustained exudation of resin within the inoculated area. No obvious external symptoms of fungal infection within the crown region were seen in the C. polonica-infected spruce trees during the 2011 vegetation season. The diurnal course of sap flow on representative days (from 18th April to 15th August 2011) is presented in Supplemental Fig. 1 and shows a typical bell-shaped daytime variation. Sap flow increased quickly after sunrise, reaching a maximum in the late morning. This was followed by a relatively stable period during mid-day and a steep decline to very low flows at night. Weather conditions (Fig. 1) altered the sap flow rates of all the investigated trees independent of inoculations. The cold and rainy periods at the end of April and beginning of May reduced the maximum sap flow values by 34 %. During the first 10 days of the experiment, the maximum sap flow decreased by 23 % in both the C. polonica-inoculated trees, and by 30 and 41 % in the control trees no. 3 and 4, respectively (Fig. 2). When comparing the monthly diurnal sap flow curves and the 10-day maximum values (Fig. 2, Supplemental Fig. 1), the infected trees were characterised by time shifts in sap flow curves, but had higher sap flow maxima than the control trees in June and August. In July, the highest sap flow was determined in control tree no. 3. In June, the inoculated tree no. 1 was characterised by a 17 % increase in sap flow in comparison to the previous 10-day maximum. The maximum sap flow values remained stable in other trees. In the inoculated tree no. 2, a gradual increase in sap flow was measured, reaching a 17 cm/h sap flow
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Trees Fig. 1 The relative humidity (a) and average soil and air temperature (b) during the 5-month sampling period in 2011
Fig. 2 Time course of 10-day maximum sap flow values of Norway spruce trees infected with the fungus Ceratocystis polonica (trees no. 1 and 2) and controls (trees no. 3 and 4)
maximum by the end of June. In July, trees no. 1 and 2 were characterised by a gradual reduction (38 and 50 %, respectively) of the sap flow maximum. The lowest values in sap flow for all the trees were recorded in the second half of July. In August, another steep increase in maximum sap flow values was measured in both infected trees. In contrast, control tree no. 3 showed a slight sap flow reduction during the first half
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of July and then a 30 % increase in sap flow towards the end of the month. At this time, the infected trees were characterised by the lowest diurnal maximum. Control tree no. 4 was characterised by a marked reduction in sap flow until the end of May, then the maximal diurnal values remained more or less stable, with two peaks at the end of June and then August (Fig. 2, Supplemental Fig. 1).
Trees Fig. 3 Total phenolic contents in phloem tissue of control trees and Ceratocystis polonicainfected trees during the period of sampling. Each value represents the median and median deviations (MD) of seven replicate samples. Significant differences were indicated by different letters (a– d). Decision rule P \ 0.05 was regarded as significant
Phenolic compounds In the presented investigation, the sequence of phenolic responses in the phloem tissues of trees infected by C. polonica was evaluated in comparison with the phloem tissues of the uninfected (control) trees. Four monophenols were analysed: catechin, astringin, isorhapontin and taxifolin. Other stilbenes, including resveratrol, piceid, as well as the aglycones of astringin and isorhapontin, could be detected in the bark using liquid chromatography–mass spectrometry (LC–MS) but could not be accurately quantified due to their low concentrations. When analysing the phenolic compounds over a period of 5 months, a seasonal variation should be considered. The total phenolic concentrations increased slightly but gradually towards September (Fig. 3). High catechin levels were measured until the middle of June, the later catechin values being lowered until September. The highest accumulation of isorhapontin was measured in April, in May the levels dropped significantly. Later, a gradual and slight increase in isorhapontin concentration was measured towards September. A similar seasonal time course of changes was analysed for astringin. Taxifolin reached its maximum concentration in the beginning of May, easing later and then remaining almost constant until September (Fig. 4). On the 18th April 2011 (3 dpi), the initial reaction to C. polonica infection was characterised by a significant 30 % decrease in catechin content in comparison to the control bark, whereas the concentrations of taxifolin and isorhapontin increased significantly by 38 and 244 %, respectively. The concentrations of astringin and total phenolics
remained unaffected by the initial C. polonica infection (Figs. 3, 4; Supplemental Fig. 1a–e). On May 6th 2011, 3 weeks after C. polonica infection, the total phenolics accumulated significantly (Fig. 3) accompanied by a 20 % increase in taxifolin and a 45 % increase in isorhapontin contents. Catechin and astringin values were slightly above the controls (Fig. 4; Supplemental Fig. 1a–e). On June 17th 2011, significant increases in the concentrations of total phenolics (16 %), catechin (20 %) and taxifolin (40 %) were measured within the C. polonicainfected bark, whereas the astringin and isorhapontin contents had diminished by 10 and 23 %, respectively (Figs. 3, 4; Supplemental Fig. 2a–e). On the 15th July 2011, the concentrations of total phenolics had increased to a similar level to that of the previous sampling date (Fig. 3; Supplemental Fig. 2e). The catechin and taxifolin contents of C. polonica-infected trees had enhanced by 24 and 16 %, respectively, as compared to the control. However, both the catechin and taxifolin levels were lower than in June. The concentrations of astringin and isorhapontin were slightly below the control levels but more or less equal in comparison to the previous sampling date (Fig. 4; Supplemental Fig. 2a–d). On 9th September 2011, lower concentrations of measured catechin and taxifolin were identified in comparison to July, whereas the astringin and isorhapontin concentrations had remained more or less equal to those at the previous sampling date. Within the infected bark, the total phenolics had continued to increase but to a lower percentage than on the previous sampling date. The concentration of catechin was 40 % higher within the infected
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Fig. 4 Concentrations of four bark phenols [(a) catechin, (b) astringin, (c) isorhapontin, (d) taxifolin] in phloem tissue of control trees and Ceratocystis polonica-infected trees during the period of sampling. Each value represents the median and median deviations
(MD) of seven replicate samples. Significant differences were indicated by different letters (a–e). Decision rule P \ 0.05 was regarded as significant
samples than in the controls, whereas the astringin and isorhapontin concentrations had lowered by 20 and 27 %, respectively. The taxifolin contents of the infected bark samples were comparable to the controls (Figs. 3, 4; Supplemental Fig. 2a-e).
On May 6th 2011, 3 weeks after C. polonica infection, the total glutathione increased by 30 % and total cysteine by 86 % (Figs. 5b, 7b; Supplemental Fig 2f, h). The glutathione redox state and glutathione reductase activity remained unaffected by C. polonica infection (Figs. 5a, 6), whereas the percentage of cystine was lower in comparison to the control (Fig. 7a). The accumulation of thiols was accompanied by a significant degradation of total ascorbate (-22 %) and an increased ascorbate redox state (Fig. 8; Supplemental Fig. 1i). On June 17th 2011, the C. polonica-infected bark was characterised by 75 % accumulation of total glutathione in comparison to the controls, while the percentages of oxidised glutathione and glutathione reductase activities were moderately raised (Figs. 5, 6; Supplemental Fig. 2f, g). Similarly, a strong 126 % increase in total cysteine was measured within the infected bark, while the percentage of cystine remained unchanged (Fig. 7; Supplemental Fig. 2h). The total ascorbate levels and the percentage of dehydroascorbate were lowered (Fig. 8; Supplemental Fig. 2i).
Ascorbate–glutathione system On 18th April 2011 (3 dpi), C. polonica-inoculated bark samples exhibited significantly more oxidised glutathione pool accompanied by higher glutathione reductase activity (Figs. 5a, 6). The concentration of total glutathione was comparable to the controls (Fig. 5b; Supplemental Fig. 2f, g). The total cysteine dropped slightly below the levels of the uninfected samples and the cysteine redox state shifted towards more oxidised value when the samples were infected by C. polonica (Fig. 7; Supplemental Fig. 2h). Similar to glutathione, the tASC concentration remained at the control levels, whereas the infected bark exhibited a slightly more reduced ascorbate pool (Fig. 8; Supplemental Fig. 2i).
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Trees Fig. 5 (a) Glutathione disulfide (% of total) and (b) concentrations of total glutathione in phloem tissue of control trees and Ceratocystis polonica-infected trees during the period of sampling. Each value represents the median and median deviations (MD) of seven replicate samples. Significant differences were indicated by different letters (a– f). Decision rule P \ 0.05 was regarded as significant
Fig. 6 Glutathione reductase activity (munits/g DW) in phloem tissue of control trees and Ceratocystis polonicainfected trees during the period of sampling. Each value represents the median and median deviations (MD) of seven replicate samples. Significant differences were indicated by different letters (a, b). Decision rule P \ 0.05 was regarded as significant
On 15th July 2011, the glutathione concentration had increased significantly by almost 100 % within the infected bark in comparison to the controls, although the concentration levels of both the infected and uninfected groups were lower than in June (Fig. 5b; Supplemental Fig. 1f). The increase in glutathione had been accompanied by significantly increased glutathione reductase activity and restored glutathione redox balance (Figs. 5a, 6;
Supplemental Fig. 1 g). Total cysteine and the percentage of cystine had risen by 88 and 35 %, respectively, upon C. polonica infection (Fig. 7; Supplemental Fig. 1 h). Likewise, the total ascorbate contents and the percentage of dehydroascorbate had remained below the control levels (Fig. 8; Supplemental Fig. 1i). On 9th September 2011, the total glutathione levels were equal to those of the controls and a restored
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Trees Fig. 7 (a) Cystine (% of total) and (b) concentrations of total cysteine in phloem tissue of control trees and Ceratocystis polonica-infected trees during the period of sampling. Each value represents the median and median deviations (MD) of seven replicate samples. Significant differences were indicated by different letters (a– d). Decision rule P \ 0.05 was regarded as significant
Fig. 8 (a) Dehydroascorbate (% of total) and (b) concentrations of total ascorbate in phloem tissue of control trees and Ceratocystis polonicainfected trees during the period of sampling. Each value represents the median and median deviations (MD) of seven replicate samples. Significant differences were indicated by different letters (a– e). Decision rule P \ 0.05 was regarded as significant
glutathione redox balance was detectable within the C. polonica-infected bark, reflecting a new steady-state acclimation stage (Fig. 5; Supplemental Fig. 2f). The glutathione reductase activity was significantly higher within the infected samples but remained more or less at the same
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level as on the previous sampling date (Fig. 6; Supplemental Fig. 1 g). The total cysteine was higher within the infected samples but the trend was insignificant (Fig. 7b; Supplemental Fig. 1 h). A 25 % increase in total ascorbate was measured, whereas the percentage of
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dehydroascorbate was comparable to the controls (Fig. 8; Supplemental Fig. 2i).
Discussion Sap flow and temperature measurements In the present paper, temporal analyses were performed regarding antioxidative responses in the phloem tissues and sap flow measurements of Norway spruce trees infected by C. polonica at low densities. The data from our sap flow measurements showed altered sap flow maxima and time shifts on the sap flow curves for the C. polonica-infected Norway spruce trees compared to the controls. Given that all the inoculated trees resisted low-density inoculations with C. polonica, our results are in agreement with observations that during most of the growing season the trees that are resistant to inoculations with C. polonica have higher sap flow than the more susceptible trees (Kirisits and Offenthaler 2002). In contrast, the heavily infected trees experienced a 50 % reduction in sap flow within 15–25 days after mass inoculations, leading to tree deaths (Cermak and Kucera 1990; Kirisits and Offenthaler 2002). Phenolic compounds Among the antioxidants, phenolics represent a more important multi-tiered system of the inducible defence strategy of spruce bark with spatial and temporal components. The spatial component is determined by the positions of the concentric rings of the PP cells from the periderm surface to the cambial zone analogous to concentric defences of castles. The temporal component of the system consists of the dynamic production of phenolics and is triggered by both the intensity of attack and weather conditions (Krekling et al. 2004; Franceschi et al. 2000, 2005; Urbanek Krajnc 2009). Phenolic composition in the phloem was previously used to predict Norway spruce resistance to both bark beetles and their associated fungi (Brignolas et al. 1998; Bonello et al. 2006; Witzell and Martin 2008; Urbanek Krajnc 2009). Evaluation of the impact of C. polonica infection on the phenolic status of a plant varies considerably among different authors (Brignolas et al. 1995, 1998; Evensen et al. 2000; Viiri et al. 2001; Zeneli et al. 2006). Trends are difficult to set, as different inoculation procedures, times of inoculations, degrees of infection, physiological status of the trees, different sampling times and especially different Norway spruce clones and weather conditions, must be taken into account when analysing the results of different experiments.
In the presented experiment, each of analysed phenolic compounds (catechin, astringin, isorhapontin, taxifolin) was interpreted according to its own characteristic time course, characterised by different degrees of accumulation/ degradation in comparison to the control and with certain time shifts in response. The initial C. polonica infection was characterised by a significant increase in isorhapontin and taxifolin concentrations and a significant decline in catechin content 3 days after C. polonica infection. As reported in literature, the results of the initial phenolic response to C. polonica inoculation could be quite different. Brignolas et al. (1998) studied the phenolic defence reaction 6 and 12 days post-inoculation with C. polonica in different spruce clones at different sampling locations from the inoculation side. Generally, the induced reactions 6 and 12 days after C. polonica inoculation were characterised by a significant increase in catechin levels but astringin, isorhapontin and taxifolin did not change. In the experiment performed by Hammerbacher et al. (2013), astringin and isorhapontin values increased slightly 8 days post-inoculation with one C. polonica isolate, whereas infection with other isolates decreased stilbenes immediately. In the presented experiment, a different phenolic shift was detected 3 weeks after inoculation. Increases in total phenolics, catechin, astringin, isorhapontin and taxifolin were monitored in comparison to controls but for individual phenolics the trend was insignificant. These results are in accordance with Evensen et al. (2000), which performed a similar designed C. polonica inoculation experiment with, however, 50 inoc./m2, and obtained on average higher concentrations of catechin and stilbenes 3 weeks after inoculation with C. polonica. Analysing each single phenolic compound, on later sampling dates, we observed that within certain time shifts the phenolics follow the ecophysiological concept and basically fit with the temporal sequence of changes in total phenolic concentrations after the moderate bark beetle attack reported in our earlier experiment on the spruce/bark beetle pathosystem (Urbanek Krajnc 2009; Urbanek Krajnc et al. 2011). The initial decline in catechin was followed by an increase in catechin concentration until September, although through the season the levels eased until the last sampling date. Taxifolin accumulated within the infected bark at significantly higher levels until June, later the concentrations dropped toward the control levels. After a slight accumulation of astringin in response to C. polonica infection in April and May, the concentrations dropped below the control levels at later sampling dates. A dramatic initial increase in isorhapontin in April soon ceased, later isorhapontin concentrations of infected samples diminished in comparison to the controls. This time course of changes in stilbene (astringin, isorhapontin) values was also in agreement with Hammerbacher et al. (2013). These authors
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reported a slight increase in astringin and isorhapontin concentrations 8 days after inoculation with one C. polonica clone, later both stilbenes declined until 28 days postinoculation. Similarly, a number of previous studies had reported a decline in astringin and isorhapontin concentrations, unfortunately, in these studies the bark was collected for a shorter period (few dpi, maximum 2 months) after onset of experiment (Brignolas et al. 1998; Viiri et al. 2001; Li et al. 2012; Hammerbacher et al. 2013). These declines in stilbenes were considered very puzzling and contradictory to the results of the authors reporting elevated transcript levels of stilbene synthase genes and thus up-regulation of stilbene biosynthesis (Brignolas et al. 1995; Hammerbacher et al. 2013). It has been recently investigated that C. polonica metabolises astringin to openring lactones, aglycones, and dimers by fungal enzymes, the activities of which were induced when C. polonica was in a stilbene-rich environment (Hammerbacher et al. 2013). It is not surprising that fungi specialised for living in stilbene-rich plant material such as C. polonica have developed mechanisms to prevent the deleterious effects of these compounds. Stilbenes are known to inhibit fungal growth by interfering with microtubule assembly (Woods et al. 1995; Adrian et al. 1997), disrupting plasma membranes and uncoupling electron transport within fungal spores and germ tubes (Pont and Pezet 2008; Adrian and Jeandet 2012). Ring opening and lactonisation of stilbenes is therefore a logical early step of fungal catabolism and is considered to also be a successful detoxification strategy for other plant pathogens as it results in a more polar product, which diffuses more slowly into fungal cells and, therefore, shows decreased toxicity (Hammerbacher et al. 2013). Previously, within the same pathosystem, a temporal analysis was performed for chalcone synthase (CHS), a key enzyme involved in the syntheses of stilbenes and flavonoids (Nagy et al. 2004). The authors showed that PP cell activation, which involves enlargement and changes in polyphenolic content, is preceded by up-regulation of CHS. Transcript levels started to increase from day 3, peaked during days 10–16, and then declined toward day 37. The time course of transcript levels after C. polonica infection observed by Nagy et al. (2004) is in accordance with our time-course analysis of total phenolic metabolites, suggesting that defence responses to infection are transient and cease when the further accumulation of metabolites becomes no longer necessary. Ascorbate–glutathione system In addition to phenolics, the research priority of this paper was to determine the efficiency of the low molecular weight thiols (cysteine, glutathione) in Norway spruce
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against C. polonica infection, and their significance in ascorbate–glutathione cycle and phenolic metabolism. The initial reaction of Norway spruce to C. polonica infection 3 days after C. polonica inoculation resulted in a significantly more oxidised glutathione pool accompanied by higher glutathione reductase activity. The total cysteine dropped slightly below the levels of the uninfected samples and the cysteine redox state shifted towards a more oxidised value. Similarly, in one of our previous experiments, 2 weeks after the exposure of spruce trees to bark beetles slight but insignificant decreases were measured for total cysteine as well as total glutathione, which were accompanied by a slightly more oxidised glutathione redox state (Urbanek Krajnc 2009). At the beginning of May, 3 weeks after C. polonica inoculation, obvious accumulations of both total cysteine and total glutathione were detected within infected bark. As glutathione levels depend on the availability of cysteine (Zhao et al. 2008; Noctor 2006; Foyer and Noctor 2011), increased concentrations of cysteine found in C. polonicainfected bark might allow an enhanced rate of glutathione synthesis, thus resulting in the observed increase in glutathione content. Additionally, thiols accumulated to a significantly higher extent in both infected tissues and controls when compared to previous sampling dates. These increases can be explained as wound reactions induced by mechanical injury at the first sampling date, which then eased until June (Christiansen et al. 1999; Franceschi et al. 2005; Ralph et al. 2006; Urbanek Krajnc 2009). Until the middle of July, the infected bark was characterised by a gradual increase in total glutathione, reaching twofold higher levels in July when compared to the control. This gradual increase was accompanied by a significantly increasing glutathione reductase activity. The glutathione disulphide (% of total) was equal to controls indicating a restored glutathione redox balance. Furthermore, within the infected bark the total cysteine remained elevated over the whole sampling period. Based on our previous experiment (Urbanek Krajnc 2009), it can be concluded that the antioxidant shift within the C. polonica-infected bark indicated a successful defence reaction, which was characterised by a higher accumulation of thiols and a more reduced redox state. In September, the glutathione system within the C. polonica-infected bark had reached again a steady state, comparable to the controls. Glutathione reductase activity and total cysteine concentrations remained increased at a similar level to that on previous sampling dates. Glutathione is essential for the regeneration of ascorbate within the ascorbate–glutathione cycle (Tausz et al. 2003, 2004; Foyer and Noctor 2011). In addition to being the most abundant water-soluble antioxidant in plant cells (Smirnoff and Wheeler 2000), ascorbic acid is highly reactive against phenoxyl radicals generated by
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peroxidases during oxidative stress (Kawano and Muto 2000). In May, 3 weeks after C. polonica infection, the accumulation of thiols was accompanied by a significant degradation of total ascorbate within the C. polonicainfected bark until July, followed by a significant accumulation in September. However, when comparing the total ascorbate contents in the C. polonica-infected samples on five occasions between the 18th April and 9th September 2011 the concentrations increased continuously from the spring onwards to September and this trend was significant. These events reflected an increased pressure on the ascorbate–glutathione cycle. DHA is re-converted to ascorbic acid via an ascorbate– glutathione pathway, which uses reduced glutathione as an electron donor for regenerating ascorbate from its oxidised form (Foyer and Noctor 2011). It has recently been shown that flavonoids and hydroxycinnamic acids are capable of scavenging H2O2 by acting as electron donors for guaiacol peroxidases (Sakihama et al. 2002). Under conditions of severe stress, the scavenging capacity of the plastids may be exceeded when plastid ascorbate pools become oxidised. It has been proposed that phenolics, particularly polyphenols as opposed to monophenols, act as antioxidants to support the primary ascorbate-dependent detoxification system as a back-up defence mechanism of vascular plants (Sakihama et al. 2002). When phenolics act as antioxidants, either by enzymatic or direct radical scavenging mechanisms, they are univalently oxidised to their respective phenoxyl radicals. Phenoxyl radicals can be reduced to their parent compounds by non-enzymatic reactions with ascorbate. It has also been shown that phenoxyl radicals can be reduced to parent phenolics by monodehydroascorbate reductase, an enzyme of the ascorbate–glutathione cycle that catalyses the reduction of monodehydroascorbate radical, the univalent oxidised product of ascorbate, to regenerate the parent compound ascorbate. In regard to our experiment, this interaction explains the shift of the ascorbate redox state towards a more reduced value that was observed immediately after C. polonica infection, as ascorbate is needed for reducing the phenoxyl radicals formed during pathogen defence. It prevents the pro-oxidant activity of phenoxyl radicals and consequently cellular DNA degradation under prolonged microbial attack (Sakihama et al. 2002). These multi-tiered interactions within the antioxidant defence system explain the observed degradation in the ascorbate 3 weeks after C. polonica infection and later the continuous increases in total ascorbate levels towards September. Therefore, the ascorbate– glutathione cycle must be recognised as a crucial part of bark defence strategies with respect to bark phenolic metabolisms.
Conclusion In the presented in vivo experiment, changes in the antioxidative system of Norway spruce during low-density C. polonica infection have been pointed out as a dynamic process with temporal and spatial components. In the earliest stage of pathogen infection, the SAR responses could be seen to rapidly increase the concentrations of antioxidant compounds involved in defence against C. polonica infection, with except of catechin and total cysteine, which were lowered 3 days after inoculation. An initial reaction was followed by increased cysteine and glutathione concentrations, increased glutathione reductase activity, and a more reduced redox state of those molecules. In view of the stress-response concept of the glutathione system (Tausz et al. 2004), higher concentrations of thiols would confer better antioxidative protection and would be considered an acclimation. Similarly, total phenolics, catechin and taxifolin concentrations increased continuously in response to low-density C. polonica inoculation, whereas the astringin and isorhapontin contents had diminished within the infected bark from June onwards. These events were accompanied by a significant degradation of the total ascorbate. A new steady state of the ascorbate system was achieved in September, when total ascorbate contents raised significantly above the control levels. The time-course analysis of the individual antioxidant molecules enabled us to establish a relationship between the phenolics and the ascorbate–glutathione system in response to low-density C. polonica infection. Changes in the phenolic contents reflect an increased pressure on the ascorbate–glutathione system as the system is needed for reducing the phenoxyl radicals formed during the pathogen defence. Based on the ascorbate–glutathione concept of Foyer and Noctor (2011), phenolics within the concentric layers of PP cells can be viewed as a wheel of the bark defence mechanism, driven by the ascorbate–glutathione system, as a central part or redox hub of the wheel that integrates metabolic information and environmental stimuli to tone defence responses against C. polonica infection. The implementation of the underlying results into generalised models of SAR concept would not only improve the physiological and biochemical understanding of Norway spruce defence against bark beetles and associated fungi but could also allow the predictions of antioxidative defence responses in other conifer pathosystems. Author contribution statement M.N., N.K. and A.U.K., designed the experiments. N.K., N.Z., A.U.K., F.M., and M.N., performed the field experiments (inoculation of test trees, sampling procedure and sap flow measurements). M.N., N.Z., M.F. carried out the analyzes of single phenolic compounds at National Institute of Chemistry. M.F. and A.U.K. carried out the analyzes of thiols, ascorbate and glutathione reductase at Faculty of Agriculture and Life Sciences. A.U.K., M.N., N.K., M.L. and R.K. prepared the manuscript.
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Trees Acknowledgments We thank Peter Kramer from Maribor University Agriculture Centre and Ciril Zupan from the Slovenian Forest Service for permission and support during the experimental work. We gratefully acknowledge support and technical assistance from Vilma Sˇusˇtar, Janez Gorensˇek, Anja Ivanusˇ and Andreja Sˇober. Conflict of interest of interests.
The authors declare that they have no conflict
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