Tree Physiology Advance Access originally published online on December 5, 2008
Tree Physiology 2009 29(1):19-25; doi:10.1093/treephys/tpn006
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Wounding response in xylem of Scots pine seedlings shows wide genetic variation and connection with the constitutive defence of heartwood
1 Finnish Forest Research Institute, Punkaharju Research Unit, FI-58450 Punkaharju, Finland
2 Corresponding author (anni.harju{at}metla.fi)
3 Finnish Forest Research Institute, Vantaa Research Unit, P.O. Box 18, FI-01301 Vantaa, Finland
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Abstract |
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In this greenhouse experiment, 3-year-old Scots pine (Pinus sylvestris L.) seedlings were wounded by drilling holes through the stem. In the xylem next to the wound, the concentration of resin acids (RAC) increased, and the production of extractives typical for heartwood (stilbenes) and knotwood (stilbenes and lignans) of mature trees was induced. The induced stilbenes were pinosylvin (PS) and pinosylvin monomethyl ether (PSM), and the lignans nortrachelogenin (NTG) and matairesinol (MR). There was positive phenotypic correlation between concentrations of the different extractives. Except for the RAC, the extractive concentrations showed no correlation with the size of the seedlings. The treated seedlings belonged to half-sib families, which enabled the estimation of the genetic parameters for the response variables. The proportion of heritable variation (heritability, h2) in the concentration of PS, NTG and MR varied between 0.71 and 1.03, whereas for PSM and RAC the heritability was lower (0.35 and 0.31). Genetic correlation was significant between PS and PSM (r = 0.55, P = 0.018), and between NTG and MR (r = 0.50, P = 0.033). Heritabilities were also estimated on the basis of the regression of the offspring on their mothers $${h}_{\mathrm{OP}}^{2}$$
. These estimates were assessed for the concentration of PS, PSM and RAC in the wound response area of the seedlings and correspondingly in the heartwood of their mothers. The heritability was highest for the concentration of PS $$({h}_{\mathrm{OP}}^{2}=0.31)$$
. The findings of this study support the suggestion that the wounding of Scots pine seedlings may facilitate the development of an early testing method for breeding heartwood durability.
Keywords: early testing, genetic parameters, pinosylvin, Pinus sylvestris L., resistance
Received June 14, 2008; Accepted September 8, 2008
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Introduction |
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The living tissues in the trunk of Scots pine (Pinus sylvestris L.), namely the sapwood, phloem and the cambium located between them, are constitutively protected on the outside by the bark and on the inside by the heartwood (Franceschi et al. 2005). However, these preformed passive barriers are not always enough to protect the living tissues, and attack by damaging agents such as insects and their fungal associates triggers additional defensive responses. The defensive response of wood tissue in the form of induced production of secondary compounds and changes in wood anatomy has been widely studied. A large number of studies have been carried out on the reactions of phloem of various conifer species to attack by bark beetles and associated fungi (e.g., Lieutier et al. 1989, Nebeker et al. 1993, Lieutier et al. 1996, Bois and Lieutier 1997, Nagy et al. 2005, Raffa et al. 2005).
Even the mere mechanical wounding of Scots pine phloem (Bois and Lieutier 1997) and sapwood (Nilsson et al. 2002) induces the production of chemical defenses, such as phenolic compounds. After wounding, pinosylvin (PS), its pinosylvin monomethyl ether (PSM), pinocembrin and taxifolin appear in the phloem of trees classified as resistant (Bois and Lieutier 1997). The discoloured sapwood induced by mechanical wounding had a similar high performance liquid chromatogram as the natural heartwood (Nilsson et al. 2002). After wounding of bark and cambium, a ‘wound heartwood’ with PS and PSM was formed in the sapwood of Scots pine, which is a site where they do not normally occur (Lyr 1967).
Scots pine seedlings have been shown to produce stilbenes in the various types of tissue in response to different kinds of stress factor, such as UV light (Schoeppner and Kindl 1979, Gehlert et al. 1990, Zinser et al. 1998, Zinser et al. 2000), ozone (Rosemann et al. 1991, Zinser et al. 1998, Chiron et al. 2000, Zinser et al. 2000) and infection by fungi (Gehlert et al. 1990, Bonello et al. 1993, Johansson et al. 2004). The active defence mechanisms of the xylem against external stresses such as wounding have generally been ignored in these studies.
Active defence mechanisms do not exist in Scots pine heartwood. However, the accumulated phenolic compounds, namely PS and PSM, as well as resin acids (RAC), increase the resistance of heartwood against wood-decaying fungi. High correlation has been reported between the concentration of phenolic compounds and mass loss in laboratory decay tests (Venäläinen et al. 2004, Heijari et al. 2005, Harju and Venäläinen 2006, Karppanen et al. 2007, Leinonen et al. 2008), and a number of studies have demonstrated that these compounds retard the growth of fungi in plate tests (e.g., Rennerfelt 1943, 1945, Rennerfelt and Nacht 1955, Loman 1970, Seppänen et al. 2004).
In this study, mechanical wounding was used to induce the production of typical heartwood compounds in the xylem of 3-year-old seedlings of Scots pine. We were especially interested in quantifying the variation among the seedlings and in the proportion of the heritable variation in response to the wounding. Also phenotypic and genetic correlations between the concentration of extractives and the seedling size were of interest. If mechanical wounding of the seedlings induces a reaction related to the extractive composition of their mother’s heartwood, then wounding could be used in developing an early testing method in the breeding of durable Scots pine heartwood.
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Materials and methods |
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Experiment
The seeds were collected in October 2000 from 18 unrelated trees, the heartwood of which had been studied earlier (Harju et al. 2001, 2002, 2003, Tiitta et al. 2003, Venäläinen et al. 2004). Seeds from each known mother-tree comprise a half-sib family, in which the fathers originate from an unknown stochastic pollen pool. The seeds were sown mother-tree-wise into plastic trays containing 5 x 9 cells in May 2001 to produce 18 half-sib progenies. The cells were filled with fertilized peat. The seedlings were grown in a greenhouse at the Punkaharju Research Unit (61°48' N and 29°19' E) of the Finnish Forest Research Institute. After two growing seasons, each seedling was allocated a random number, and transplanted into single pots (12 x 12 x 12 cm) at the end of March 2003. The pots were arranged on two greenhouse tables according to the randomly selected number, resulting in a completely randomized growing position for each seedling. The seedlings were grown for one more year. During the 3-year growth period, a sufficient level of irrigation, a low level of fertilization and no artificial illumination were applied. Aphids were controlled with pesticide (0.3% malation).
The mechanical wounding experiment had two factors: the treatment with two levels (wounded seedlings and unwounded controls) and the half-sib family with 18 levels. The experimental design was a split-plot design with single-tree-plots. The greenhouse tables were divided into 10 blocks, each consisting of two sub-blocks, giving a total of 20 sub-blocks. Each sub-block contained one seedling from each of the 18 half-sib families in a random order. Within the 10 blocks, the wounding treatment was randomly assigned to one of the two sub-blocks. As a result of this procedure, a total of 10 seedlings from each half-sib family were wounded, and 10 seedlings were left as controls. The seedlings were wounded on 20–21 April 2004, when elongation of the buds of the seedlings had just started. About 10 holes were drilled through the stem with a 2.5 mm drill at intervals of about 2 cm along the 2002 internode (2-year-old segment of the stem). The mean length of the wounded internode was 18.8 cm (SD = 2.4 cm). The seedlings were grown in the greenhouse for about 3 months (until the 17th of August) and then the wounded internodes were cut apart. After removing the bark and phloem, the internodes were frozen at –20 °C. Similar sections were cut from the control seedlings.
To prepare a sample for chemical analysis, each stem section was split longitudinally into two halves to expose the wounded area. After the examination under UV light (wavelength 313 nm, Figure 1), we decided to take a longitudinal 5-mm-section from above and below each wound hole as a sample. The pieces were transferred to a paper bag and stored at –20 °C. Later, these stem-wise samples were dried at 60 °C for 48 h, milled into powder with an analytical mill, sealed in small glass bottles and stored at –20 °C. Each milled section contained UV fluorescent xylem with induced compounds and a nonfluorescent xylem grown during summer after wounding (Figure 1). Because the volume ratio of the UV fluorescent and nonfluorescent xylem was dependent on the stem radius, a coefficient was calculated to adjust for the diameter-dependent dilution of the milled sample in the chemical analyses.
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Chemical analysis
The analysis of stilbenes, RAC and lignans in the seedlings was carried out in accordance with the procedure for stilbenes described in Karppanen et al. (2007). Quantifications were made using an internal standard and response corrections with pure PS and PSM (Arbonova) for PS and PSM, and with 75% abietic acid (Fluka 00010) for RAC. The lignans were identified using MS library (matairesinol, MR) and literature (nortrachelogenin, NTG) (Ekman et al. 2002), and they were quantified with an internal standard (diethylstilbesterol).
The sampling procedure and the chemical analysis of the heartwood of the mothers have been described in detail in Harju et al. (2002) and in Venäläinen et al. (2004).
Statistical analysis
On the individual seedling level, the measured phenotypic value (P) of a trait (e.g., concentration of a chemical compound) is assumed to be the sum of the additive genetic effect (A) and of the independent environmental effect (E); E also includes the remaining genetic effects that are independent of A. Thus, P = A + E. Furthermore, the phenotypic variance at the population level is assumed to be composed of genetic and environmental components, 
. The narrow-sense heritability is estimated using 
.
For the experiment, we constructed a model and estimated the narrow-sense heritabilities on an individual seedling basis, assuming that all random effects in the studied properties were pairwise independent. Variance components were estimated with the restricted maximum likelihood (REML) technique using the MIXED procedure of the Statistical Package for Social Sciences (SPSS) system. As the control seedlings appeared to contain none or only small amounts of the studied extractives, the experimental design for the induced seedlings was regarded as a randomized block design with single-tree-plots:
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The block effect (
b) was regarded as fixed, and the family effect (bf) as random. The random effects were assumed to be normally distributed with the expectation zero and the variance 
, and 
. The individual-tree heritability for the half-sib progenies was estimated using the formula 
, assuming true half-sibs and unrelated parents. This may be an overestimate due to the possible existence of full-sibs (see Squillace 1974).
The MIXED procedure of the SPSS Inc. (2006) system also provided approximate standard deviations for the estimated variance components. The standard deviation for the heritability estimate was calculated using 
. The coefficient of additive genetic variation (CVA) was calculated by dividing 
by the overall mean value of the trait.
Pearson’s coefficients of correlation were estimated as phenotypic correlations for the data consisting of all individual seedlings. The correlation of the family mean values (Spearman’s rank order correlation) was estimated to approximate the genetic correlation between the studied characteristics.
We applied the regression of offspring on their mothers 
to estimate the heritability (Falconer 1981, pp 134–147) of the concentration of PS, PSM, their sum and the concentration of RAC in the UV fluorescent xylem of the offspring and in the heartwood of the mothers. Heritability was also estimated by a correlation approach using the coefficient of genetic prediction between offspring and parents (CGPOP) (Baradat 1976).
In each chemical variable there were some family-wise outliers (Sokal and Rohlf 1995, pp 406–407), and these were not included in the estimation of the parameters. Exclusion of the outliers reduced the differences between the families.
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Results |
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The drill-induced wound reaction in the xylem of the pine seedlings was clearly visible under UV light as an area of light-blue fluorescence above and below each hole (Figure 1). Chemical analysis of the methanol extract of the xylem next to the wound showed that the seedlings had synthesized the stilbenes, PS and PSM (Table 1). Moreover, the seedlings were also found to have synthesized two lignans, NTG and MR. The same chemical analyses were carried out on 54 non-wounded control seedlings that were grown in three blocks. The control seedlings contained no detectable amount of stilbenes or lignans.
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The main components of the methanol extract of the xylem next to the wound were RAC (Table 1) that were also found in small amounts in the control seedlings (mean = 3.36 mg g–1, SD = 1.25). However, the concentration of RAC after the wounding was significantly higher, on an average 21-fold, than that in the control seedlings (P < 0.001 in Mann–Whitney test).
The phenotypic correlations of the studied extractives at the individual seedling level are presented in Table 2. The coefficients of correlation between the summed concentration of stilbenes, lignans and RAC, as well as between the individual compounds (PS, PSM, NTG and MR) are given. All the correlations were significant except for the correlation between the concentrations of PS and MR. The magnitude of the correlation coefficients between the groups of extractives was 0.3–0.5. The correlation coefficients between the extractives and the two size variables of the seedlings, diameter and height, are presented in Table 2. Except for the concentration of RAC, the production of extractives was independent of the seedling size.
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The narrow-sense heritabilities (h2) for the measured characteristics are presented in Table 3. The heritability of the chemical characteristics in the narrow sense was the highest for the concentration of MR. The heritability of PS and lignans was clearly higher than that of PSM and of the sum of RAC. The variables describing the seedling size also had high heritabilities. The CVA was the highest for the lignans and the lowest for the size variables.
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Most of the family-mean correlations between the studied characteristics (Tables 4 and 5) were nonsignificant. The only statistically significant correlations were between the concentration of PS and PSM, as well as between NTG and MR (Table 4). None of the chemical characteristics correlated with the growth rate of the seedlings (Table 5).
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Heritability
estimated from the regression of the offspring mean values (induced concentration of extractives in the xylem) on their mothers (concentration of extractives in the heartwood), and the CGP, suggests that there might be a connection between the constitutive and inducible synthesis of PS (Table 6).
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, and the coefficient of genetic prediction, CGPOP. |
Discussion |
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The lignans, NTG and MR, found in the xylem next to the wound, have not been reported previously in Scots pine seedlings or in the lesions of older trees. Typically they are present in the knotwood of Scots pine (Holmbom et al. 2003, Willför et al. 2003, Hovelstad et al. 2006). In herbaceous plants, several lignans have been reported to possess feeding regulating effects on herbivores (Bernard et al. 1995, Kubanek et al. 2001, Harmatha and Dinan 2003).
The two stilbenes, PS and PSM, found in the xylem next to the wound, are typical for Scots pine heartwood (Erdtman 1939) and for the knotwood of older trees (Willför et al. 2003, Hovelstad et al. 2006). They have been detected in the reaction zones of injured phloem of Scots pine (Lieutier et al. 1996, Bois and Lieutier 1997), but are found either in trace amounts or not in the healthy sapwood of older trees (Willför et al. 2003) or indoor-grown young seedlings (e.g., Chiron et al. 2000).
The most evident result of the mechanical wounding was the abundant resin flow that sealed the wound. The concentration of RAC measured in the UV fluorescent xylem was manifold compared to that in the sapwood of the control seedlings. In phenotypic level, seedling size seemed to affect the concentration of RAC in the xylem next to the wound.
The low phenotypic correlations between the concentrations of different extractives (stilbenes, lignans and RAC) indicate that there may be at least some divergence among the seedlings regarding the kind of extractives they produce under stress.
The proportion of heritable variation and the CVA ranged from moderate to high for the chemical characteristics that were studied. In Scots pine heartwood, the high estimates for heritability (Fries et al. 2000) indicated strong genetic control of the extractive concentration. In our study, the uniform greenhouse environment was favourable for revealing the heritable nature of the induction reaction.
The genetic correlations express to what extent the studied characteristics are defined by the same set of genes. The family mean correlations that we used to estimate the genetic correlations were highest between PS and PSM, as well as between MR and NTG. The correlations between the chemical and the size characteristics were not significant. In about 10-year-old loblolly pines (Pinus taeda L.), additive genetic correlations between the oleoresin yield and the growth traits were found to be positive and moderately high (Roberds et al. 2003).
The estimated offspring–parent heritability for PS suggests that there was a connection between the natural processes that provide passive constitutive defence in the heartwood and the induced production of secondary compounds that provide the active defences for the seedlings. The xylem of the seedling is the tissue that is transformed into heartwood at a later age. The extractive producing reactions in both cases may be related, e.g., to the desiccation of the tissues. According to Jorgensen (1961), Higuchi et al. (1967) and Lyr (1967), PSs are produced in slowly dying ray parenchyma cells.
Because of its natural decay resistance, Scots pine heartwood is an environmentally friendly alternative to impregnated wood. However, the resistance of heartwood to decay can be measured only on mature trees and thus an early testing method is needed for forest regeneration and tree breeding purposes. The findings of this study support the suggestion that the wounding of Scots pine seedlings may facilitate the development of an early testing method for breeding heartwood durability. Moreover, the estimated offspring–parent heritabilities indicate that a good ability of seedlings to defend themselves is coupled with the production of decay-resistant heartwood.
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Acknowledgements |
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The authors thank Heikki Kinnunen, Jouko Lehto, Hui Nan, Anna Naukkarinen, Kari Paakkunainen, Veli-Matti Suhonen, Jussi Tiainen and Liu Xinxin for practical assistance in all stages of this study, and John Derome for language edition. Prof. Katri Kärkkäinen and anonymous reviewers gave valuable comments on the manuscript.
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