Tree Physiology Advance Access published November 26, 2014

Tree Physiology 00, 1–8 doi:10.1093/treephys/tpu090

Research paper

Tocochromanols in wood: a potential new tool for dendrometabolomics Eva Fleta-Soriano1, Beatriz Fernández-Marín1,2,5, José Miguel Olano3, Fátima Míguez1, Jon Molinero1, Jesús Julio Camarero4 and José Ignacio García-Plazaola1

Received April 2, 2014; accepted September 28, 2014; handling Editor Marilyn Ball

Tocochromanols are the most abundant lipid-soluble antioxidants in plants. Among them, α-tocopherol (α-Toc) shows a particularly high sensitivity to environmental stressors and its content is used as a stress biomarker even in non-photosynthetic tissues. Nevertheless, the presence of tocochromanols has not been described yet in the xylem of woody plants, even when their functions regarding cell membrane protection and the transport of photoassimilates may be crucial in this tissue and despite its potential utility in dendrometabolomics. Considering all these, we aimed to determine the presence and distribution of tocochromanols in the xylem of woody plants, to examine their responsiveness to high temperature and to evaluate their potential as environmental bioindicators. The analysis of 29 phyllogenetically diverse species showed that α-Toc is the most abundant and frequent tocochromanol in the xylem and is ubiquitously present in all the studied species, with a concentration ranging from 0.5 to 39.3 μg g−1 of dry weight. α-Tocopherol appeared to be mainly located in the parenchyma rays and was found in both the sapwood and the heartwood, suggesting that it is present even in dead parenchyma cells. The levels of α-Toc in the xylem did not change in response to locally induced xylem heating, but responded positively to the 3-year moving average of annual precipitation. The present findings suggest that α-Toc may be linked to changes in climatic stress. This should enhance further research on the environmental controls of α-Toc variation in the xylem as a first step towards a deeper understanding of dendrometabolomics. Keywords: heartwood, sapwood, tocopherol, tree ring, xylem.

Introduction Xylem is a complex tissue made up of conductive elements and parenchyma cells. It has a broad range of key functions in plants, including the transport of water and solutes, physical support and storage of reserves (Tyree and Zimmermann 2002, Olano et al. 2013). Historically, in the field of dendrochronology, most studies have focused on the anatomical properties of conductive elements, as these are associated with environmental factors such as climate (Martinez-Cabrera et al. 2009). In the last two decades, however, studies on xylem

parenchyma (Olano et al. 2013) and the dynamics of metabolites accumulated in woody stems (McDowell et al. 2008) have revealed their relevance for gaining an understanding of plant xylem physiology and have become a promising new sub-field for dendrochronological studies: dendrometabolomics. Even so, at this time, dendrometabolomic studies are restricted to a limited number of metabolites/molecules such as non-structural carbohydrates and nitrogen compounds (Palacio et al. 2007, McDowell et al. 2008). In contrast, many other metabolites with crucial roles in xylem parenchyma and with a potential for

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1Departament of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), Apdo 644, E-48080 Bilbao, Spain; 2Institute of Botany and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Sternwartestraße 15, Innsbruck A-6020, Austria; 3Departamento de Ciencias Agroforestales, EU de Ingenierías Agrarias, Universidad de Valladolid, Los Pajaritos s/n, E-42004 Soria, Spain; 4Instituto Pirenaico de Ecología (IPE-CSIC), Avda Montañana 1005, E-50192 Zaragoza, Spain; 5Corresponding author ([email protected])

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of enzymes, and will generate toxic products such as ethanol (McManmon and Crawford 1971, Pfanz and Heber 1989). Furthermore, the hypoxic conditions in plants induce oxidative stress (for a review, see Blokhina et al. 2003) and lipids, proteins and nucleic acids are the main cellular components susceptible to damage. Particularly, hydrogen peroxide (H2O2) is produced in plant tissues under these conditions (Blokhina et al. 2001, Garnczarska and Bednarski 2004, Vergara et al. 2012). In addition, the oxygen concentration within the stem varies strongly following daily and annual cycles (Pfanz et al. 2002), potentially leading to transient increases in H2O2 (i.e., the oxygen concentration in the outer sapwood decreases during the growing season reaching the minimum in summer and recovers again to values similar to those of air during the autumn (Eklund 1990)). Under these conditions it is reasonable to hypothesize that antioxidants, like tocochromanols, would be present in the xylem of woody plants playing an important role in counteracting the deleterious effects of anoxia or hypoxia on the living xylem (parenchyma, cambium) cells. All these things considered, in this work we investigate the presence of tocochromanols in the xylem tissues of woody plants. Specifically we aimed to: (i) check for the presence and distribution of tocochromanols among different taxonomic groups; (ii) evaluate their response to environmental stimuli, in particular to locally induced heating; and (iii) evaluate the potential of tocochromanols from wood as environmental ­bioindicators.

Materials and methods Sample collection In this study, the tocochromanol content was analysed in the secondary xylem of 29 woody species, mainly trees. Samples were collected in the field using a Pressler increment borer (inner diameter 5 mm) at 1.3 m above ground, during the winter and early spring of 2013 (for details about sampling locations and study sites, see Table S1 available as Supplementary Data at Tree Physiology Online). The phloem and cambium were removed in the field immediately after core extraction. To avoid the alteration of the tocochromanol content, we followed the method described in Esteban et al. (2009) and samples were introduced in a hermetically closed chamber with silica gel (relative humidity <5%, temperature 20 °C) and dried for 7 days before analytical measurements.

Experimental design Five different experiments were performed as follows. Experiment I: species screening  To determine the presence of tocochromanols in wood, 25 species from 22 different families were studied. Four to five trees were sampled for each species. From each sample, the first 1–2 cm of the core

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dendrochronological applications remain unexplored as yet. In this sense, antioxidants may represent a particularly interesting group of molecules due to their crucial role in cell integrity maintenance and due to their sensitivity in response to environmental factors. Tocochromanols are the most abundant lipophilic antioxidants in plants and changes in their content can reflect responses of tissues to environmental stressors (MèneSaffrané and DellaPenna 2010). This family of compounds includes tocopherols (with a saturated polyprenyl chain) and tocotrienols (with an unsaturated chain) with four different homologues each (α-, β-, γ- and δ-) (DellaPenna and Pogson 2006). Tocochromanols are exclusively synthesized by photosynthetic organisms in the plastids, where they are usually located and accumulated. It has been generally accepted that tocochromanols, particularly α-tocopherol (α-Toc), play key roles in membrane preservation as lipophilic antioxidants, acting as quenchers of reactive oxygen species (ROS), and as extremely efficient scavengers of singlet oxygen and lipid peroxyl radicals (Méne-Saffrané and DellaPenna 2010). Variations in α-Toc content have been used as an ageing signal in photosynthetic tissues (Hormaetxe et al. 2005) and as a stress biomarker (Sattler et al. 2004). Tocopherol content is very responsive to heat stress, increasing under high temperatures (García-Plazaola et al. 2008, Pinto-Marijuan et al. 2013), and the exogenous application of tocopherol improves the heat tolerance of crops (Sanjeev et al. 2013). Until now, tocopherols have been found ubiquitously in the seeds, leaves, roots, fruits, tubers, hypocotyls and cotyledons of photosynthetic organisms (Horvath et al. 2006). Tocotrienols, on the other hand, have only been found in the seeds and fruits of some angiosperm groups (Falk and Munné-Bosch 2010). To the best of our knowledge, the presence of tocochromanols in the xylem of woody plants has yet to be described, despite the fact that their synthesis is thought to take place in all plant tissues and that they presumably drive important functions in the living cells of stems. Low oxygen diffusion through the bark compared with the respiratory activity of the parenchyma cells produces a suboptimal oxygen concentration for the metabolism of living xylem cells (Pfanz et al. 2002), despite the fact that sapwood cells are still functional under those hypoxic conditions (Spicer and Holbrook 2005). Some species compensate for such a low oxygen concentration with the presence of photosynthetic cells below the peridermis (Aschan et al. 2001, Filippou et al. 2007), which release oxygen in the rays and in the pith or surrounding the pith. As an example of the extent of photosynthetic activity of wood, in young stems, the re-fixation of CO2 can compensate for up to 90% of the potential respiratory carbon loss (Pfanz et al. 2002). Depending on the species and on the conditions, however, fermentation is also possible (Pfanz et al. 2002) and this will alter the cellular pH affecting the activity

Tocopherol as a new tool for dendrosciences 3 (containing heartwood, sapwood or mixture) was used for the analysis.

Experiment III: heartwood and sapwood tocopherol ­content  To check for the presence of tocochromanols in heartwood, seven species were chosen, in which the sapwood–heartwood boundary is defined with a clear visual distinction (colour): Crataegus monogyna Jacq., Fraxinus excelsior L., Laurus nobilis L., Platanus × hispanica Mill., Prunus dulcis Mill., Tilia platyphillos Scop. and Ulmus pumila L. Species with a high frequency of non-distinguishable heartwood, as is the case for Populus or Betula (Johansson and Hjelm 2013), were avoided. Stems were sampled deep enough with increment borers to reach the darker coloured heartwood. From each core, heartwood and sapwood were separately ground and analysed. Experiment IV: the responsiveness of the tocopherol content to locally induced steam heating  To study whether the concentration of tocochromanols responds to local environmental factors such as warming, the trunks of two angiosperms (Populus tremula L. and Quercus pyrenaica Willd) and one gymnosperm (Pinus sylvestris L.) species were heated in the field for 5 months (from January to May 2013). The trees were located at the Monte Valonsadero site (latitude 41°49′N, longitude 2°32′W), near the city of Soria, at an elevation of 1020 m above sea level, growing under a Mediterranean climate and continental conditions (mean annual temperature 10.6 °C, mean temperatures of the coldest and warmest months 2.9 and 20 °C, respectively, and annual precipitation of 502 mm). The heaters (20–25 cm wide) covered the entire perimeter of the main trunk at a height of 1.3 m. The temperatures in the outer bark and near the heating tape were monitored using several thermometers. Heaters were fixed at a constant temperature of ∼25 °C with a thermostat placed in every tree (to illustrate how air and xylem temperatures differed throughout the experiment, in the

Experiment V: assessment of tocopherol as a potential dendroclimatic marker  To study the potential use of tocochromanols as dendroenvironmental indicators, Populus deltoides W. Bartram was chosen due to the wide rings usually exhibited by this species. Radial wood samples were extracted with increment borers from 10 different trees living at the Soria site using a Pressler increment borer. These cores were fractionated in 40 individual annual rings (from 1973 to 2012). After crossdating the rings visually, samples from the same year were pooled together and their content in α-Toc was measured for each ring age. This approach allows an accurate quantification of the α-Toc (its limited concentration in the xylem would have compromised an accurate quantification in a tree-by-tree and ring-by-ring yearly analysis) and favours, at the same time, the evaluation of tocopherol changes in response to the environment at the population level. The year-to-year changes in α-Toc content in the youngest 28 rings (from 1985 to 2012) were related to annual or seasonal climatic variables obtained from the nearby Soria meteorological station (mean temperature, total precipitation). Data from rings beyond 1985 were excluded from the analysis due to the ageing-related decrease in the tocopherol content (data not shown).

Tocochromanol analyses Dry samples were ground with a vibratory grinder (Model MM 301, Fischer Bioblock Scientific, Madrid, Spain) until they were homogenized to dust. Approximately 30 mg of each sample were then extracted in 1 ml of pure heptane (Romil, Barcelona, Spain) and centrifuged at 16,100g for 20 min. The supernatants were filtered with 0.2 μm PTFE filters (Teknokroma, Barcelona, Spain). Tocochromanols were analysed by high-performance liquid chromatography (HPLC) following a modified method based on Bagci et al. (2004a, 2004b). Briefly, 15 μl of each sample were injected into the HPLC system. Tocochromanols × 4.6 mm, were separated in a Supercosil LC-Diol (250  5 μm of pore) column (Sigma-Aldrich, Bellefonte, USA). The system was operated with an effluent of heptane/tertiarybutyl methyl ether (97.5 : 2.5 v/v) at 1 ml min−1. Detection was performed with a fluorescence detector Waters 474 at 295 nm λ excitation and 325 nm λ emission. Pure standards of α-, β-, γ- and δ-tocopherols (Toc) and α-, β-, γ- and δ-tocotrienols (T3) (Sigma-Aldrich, Munich, Germany) were

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Experiment II: tissular location of tocopherol within the ­sapwood  Sapwood is considered to be the most metabolically active part of the xylem, with conductive elements that are still functional and with a higher density of live parenchyma cells. We checked whether the location of tocopherol was restricted to this more metabolically active tissue (sapwood) and, specifically, whether the tocopherol is accumulated in the parenchyma rays (with a high density of parenchyma cells). To characterize the location of tocochromanols within the different xylematic tissues (parenchyma rays vs vessels), five stem discs, each from a different tree of Quercus ilex L., were cut with a saw. This species was chosen due to the abundance of conspicuous parenchyma rays in its xylem. From these discs, two kinds of samples were visually separated under a stereomicroscope and collected with a gouge: tissue enriched in parenchyma rays and tissue devoid of visible parenchyma rays.

first week of March 2013, the air temperature was 2.20 °C, the mean xylem temperature of non-heated stems averaged across all trees was 4.65 °C and of heated trees 19.79 °C). Data were recorded and saved every 15 min. Two heated individuals per species were heated whereas two non-heated individuals of a similar diameter (30–50 cm) of the same species and growing nearby the same site were selected as controls. This allowed us to calculate a simple one-way analysis of variance (ANOVA) for the three species. Samples were collected at the end of May.

4  Fleta-Soriano et al. used for the identification and quantification of tocochromanols. Concentrations are expressed as μg g−1 of dry weight (DW).

Data analyses

Results and discussion Presence of tocochromanols in the xylem of woody plants In the present survey, it is shown for the first time that tocochromanols are present in the xylem, expanding the list of plant organs and structures in which the presence of tocochromanols has been documented. In addition, tocochromanols were found in all the taxa analysed, demonstrating their ubiquitous presence among woody plants (Table 1). Among tocochromanols, α-Toc was found in all the species while tocotrienols and plastochromanol-8 exhibited a minor presence and a scattered distribution among some unrelated taxa (Table S2 available as Supplementary Data at Tree Physiology Online). Despite its presence in all the species analysed, the α-Toc concentration in the xylem showed a substantial variability

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Family

Taxon

Gymnosperms Cupressaceae Cupressus arizonica Greene Juniperus thurifera L. Pinaceae Pinus sylvestris L. Angiosperms Lauraceae Laurus nobilis L. Magnoliaceae Magnolia grandiflora L. Buxaceae Buxus sempervirens L. Platanaceae Platanus × hispanica Mill. Santalaceae Viscum album L. Myrtaceae Eucalyptus globulus Labill. Betulaceae Betula alba Ehrh. Fagaceae Fagus sylvatica L. Juglandaceae Juglans regia L. Moraceae Ficus carica L. Rosaceae Prunus dulcis Mill. Crataegus monogyna Jacq. Salicaceae Populus nigra L. Populus alba L. Ulmaceae Ulmus pumila L. Cistaceae Cistus laurifolius L. Sapindaceae Acer monspessulanum L. Malvaceae Tilia platyphyllos Scop. Ericaceae Arbutus unedo L. Oleaceae Fraxinus excelsior L. Araliaceae Hedera helix L. Adoxaceae Viburnum tinus L.

α-Toc (μg g−1 DW) 1.4 ± 0.1 0.5 ± 0.3 10.5 ± 1.4 33.1 ± 6.4 3.7 ± 0.9 8.2 ± 2.6 8.4 ± 2.4 39.3 ± 7.5 1.2 ± 0.3 9.9 ± 2.4 5.4 ± 1.5 8.3 ± 0.7 4.0 ± 1.6 3.8 ± 0.9 10.0 ± 1.5 9.3 ± 2.0 11.9 ± 1.5 5.2 ± 1.5 26.0 ± 1.0 2.9 ± 0.6 8.8 ± 1.9 2.0 ± 0.7 1.6 ± 0.4 1.5 ± 0.2 3.5 ± 0.5

(ranging from 0.5 to 39.3 μg g−1 DW) across species and families (Table 1). The hemiparasitic plant species Viscum album L. showed the highest α-Toc concentration due to the abundance of photosynthetic tissues in stems (García-Plazaola and Flexas 2012). Overall, the α-Toc content in the xylem of the studied vascular plant species was low, within one to three orders of magnitude lower than in other organs such as leaves, seeds and flowers. For instance, in leaves, its content is one or two orders of magnitude higher than in wood, reaching concentrations of 1080–2162 μg g−1 DW in Spinacia oleracea L. (Lester and Hallman 2010, Lester and Makus 2010) or 431–1622 μg g−1 DW in Lactuca sativa L. (Lizarazo et al. 2010). As in the xylem, α-Toc is the main isoform found in photosynthetic tissues, while γ-Toc is the most abundant form in seeds of numerous plant species (Mène-Saffrané and DellaPenna 2010). Generally, the α-Toc content of seeds is usually lower than that in leaves, but even higher than in the xylem. For example, the α-Toc content in the seeds of Glycine max L. is 17–135 μg g−1 DW (Seguin et al. 2010) and in the seeds of Secale cereale (L.) M. BIEB. is 54–82 μg g−1 DW (Ryynänen et al. 2004). In other organs such as the flower buds of caper (Capparis spinosa L.), the reported concentration is 56 μg g−1 DW (Tlili et al. 2010). The content of α-Toc in different plant groups does not support any phylogenetic trend (Figure 1). Nevertheless, in spite of our modest sample size for each taxa, asterids seem

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To check for changes in α-Toc content either in response to local heating or in order to make a comparison between tissues (sapwood vs heartwood), one-way ANOVA was performed. Raw or log-transformed data were used. The homogeneity of variances was checked with Levene tests. The analysis of tocochromanols as dendroenvironmental indicators in response to annual precipitation was done in a two-step process. First, to remove ontogenetic trends in α-Toc contents, a three-­parameter exponential accumulation model was adjusted: y = a + b (1 − e−ct), where y is the concentration of α-Toc (μg g−1 DW), t is the cambial age of the tree ring (years), a is the initial α-Toc at t = 0 (μg g−1 DW), b is the increase in α-Toc (μg g−1 DW) and c is the accumulation rate (year−1). In this model, the sum of a plus b represents the maximum α-Toc concentration in a tree ring. Then, residuals (referred to as residual α-Toc hereafter) were obtained as the difference between the observed and predicted α-Toc concentrations and associated with the aforementioned annual precipitation at single- and multi-annual (2–6 years moving averages) scales by using linear regressions. The three-parameter exponential accumulation model described the observed pattern of tocopherol in the tree rings very well. Furthermore, all the model parameters were meaningful to explain the observed tocopherol pattern. The model was fitted to the data with a ‘least-squares algorithm’. Then, Student's t-test was used to check the significance of each parameter, an ANOVA was used to test whether the amount of variance in the data explained by the model was significant or not and, finally, the Shapiro test was performed to test whether the residuals follow a normal distribution. The shape of the tocopherol content curve suggests an exponential accumulation model and the statistical analyses validate its use.

Table 1.  Concentrations of α-Toc in the xylem of analyzed woody plant species. Data represent means ± SE (n = 4–5 replicates per species).

Tocopherol as a new tool for dendrosciences 5 Table 2. Differences of α-Toc content between xylematic tissues rich and poor in ray parenchyma in Quercus ilex. Data are means ± SE (n  = 5). Xylematic tissue

α-Toc (μg g−1 DW)

Rich in rays Poor in rays

22.3 ± 5.9 10.9 ± 1.7

to have lower α-Toc concentrations than the rest of groups, whereas basal angiosperms show higher α-Toc levels. These data indicate that the presence of α-Toc in the xylem represents a trait phylogenetically conserved across the different plant lineages.

Figure 2.  Content of α-Toc in the sapwood and heartwood of seven different species (see species and sampling locations in Table S1 available as Supplementary Data at Tree Physiology Online). Bars are means ± SE (n ≥ 5). Asterisks denote significant differences between the two kinds of wood in L. nobilis (P < 0.001).

Location of α-Toc in woody tissues To study the exact location of α-Toc within the xylem (parenchyma cells vs vessels), we compared wood samples either enriched or poor in parenchyma rays. Samples with a higher content of rays also had a higher concentration of α-Toc than samples poor in rays (Table 2). These data suggest that most, if not all, of the α-Toc pool in the xylem is mainly located in the parenchyma rays mostly formed by living cells. To check whether α-Toc is also present in heartwood, sapwood and heartwood were compared in several species assuming that the different wood colours correspond to morphological sapwood and heartwood, respectively. Tocopherol was present in both sapwood and heartwood, in all the analysed species (Figure 2). Comparable concentrations were found in both types of wood in all the species with the exception of L. nobilis in which sapwood contained significantly higher α-Toc concentrations (33.1 ± 6.4 vs 14.0 ± 4.1 μg g−1 DW in the heartwood, P < 0.001). Taking into account the fact that heartwood is a dead tissue characterized by the absence of living parenchyma cells (Spicer and Holbrook 2007), these data indicate that α-Toc is preserved in the xylem even after parenchyma cell death. Even though this might seem counterintuitive, the toughness of tocopherol in dead plant tissues has been proved, as it has been found even in 380-million-year old Cretaceous sediments (Melendez et al. 2013).

Tocopherol as an environmental chronoindicator Since the content of α-Toc in plants is highly responsive to environmental conditions (Munne-Bosch 2005), we investigated

Figure 3.  Artificial local heating did not alter the xylematic content of α-Toc in three different tree species (Pinus sylvestris, Populus tremula and Quercus pyrenaica). No statistical differences were found between control and heated treatments for any of the species at α ≤ 0.05. Bars are means ± SE (n = 2).

the effect of the locally induced heating of outer sapwood on α-Toc in three coexisting species (Figure 3). No significant differences were found between heated and non-heated trees, indicating that α-Toc levels do not directly correspond to a local environmental increase in temperature in the vicinity of the tissue. Once the effect of local temperature had been discarded, the influence of climatic conditions on the levels of α-Toc was investigated in Populus trees through an analysis of their annual rings. An age-dependent cumulative pattern of α-Toc was clearly observed during the youngest 10 rings of Populus trees, when the parenchyma is fully active (Figure 4a). This pattern has also been described in other organs and seems

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Figure 1. Phylogenetic tree of the analysed taxa. The width of each branch is directly proportional to their α-Toc concentration. To prevent figure congestion, data from Viscaceae (core eudicots) are not represented in the figure.

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to be widespread in leaves (Molina-Torres and Martinez 1991, Tramontano et al. 1992, Hormaetxe et al. 2005, Lizarazo et al. 2010). As shown in leaves, the basal α-Toc levels increase with age and over this basal pattern, peaks and valleys in response to stress events and optimal conditions are

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Figure 4.  (a) α-Tocopherol concentrations in Populus deltoides (n = 10) growth rings adjusted to a three-parameter exponential accumulation model. (b) Residual α-Toc and the 3-year moving average of annual rainfall (normalized anomalies calculated for the common period 1985–2009). (c) Linear regression between the residual α-Toc and the 3-year moving average of annual rainfall.

observed (Hormaetxe et al. 2005). By contrast, a decreasing pattern in α-Toc content was found in rings >28 years old (data not shown). In angiosperms, the ageing of parenchyma cells is associated with a decline in metabolic activity (respiration), even in the sapwood (Spicer and Holbrook 2007). Thus, the lower α-Toc content might be expected in decadent cells that are unable to renew the α-Toc pool due to their weakened metabolic vigour. This decadent metabolic activity, which will finally lead to death, together with the higher proportion of dead cells in the innermost xylem, may be the reason for the decrease in α-Toc concentration observed in our ring samples older than 28 years. To test the relationship between α-Toc concentration and climatic variables, this ontogenetic pattern was modelled and the residual α-Toc content was compared with annual and seasonal mean temperatures and total rainfall amounts, using either annual data or 2- to 6-year moving averages (data not shown). The best correlation was obtained between the residual α-Toc content and the 3-year moving average of annual rainfall (Figure 4b and c) of the youngest 28 rings. This suggests that the α-Toc content of the xylem is positively correlated with the fall in precipitation during the first 3 years of ring life. Our data reveal the usefulness of the α-Toc content as a bioindicator for at least the last 28 years of tree life in the studied species. We conclude that tocopherol loses its value as an indicator once the ageing-related decrease starts (∼28 years in our species and population), and this pattern may be similar in other woody plants. The ‘usefulness range’ (i.e., delimitated by the point at which the ageing-related decrease of α-Toc starts), however, is likely to be species dependent. Consequently, in future works, it should be studied specifically in different taxa. Toc is synthesized in plants to physically quench and chemically scavenge the ROS (Peñuelas and Munne-Bosch 2005). In the photosynthetic tissues of Mediterranean plants, ROS accumulation is enhanced during drought periods (MunneBosch and Alegre 2000). By contrast, in non-photosynthetic tissues (i.e., roots), the accumulation of ROS is favoured by peaks of high metabolic activity (respiration) following rain episodes in the case of Mediterranean plants (Carbone et al. 2011). Our results match this line of reasoning as an increase in the content of α-Toc during rainy periods was observed in the xylem of trees growing under a Mediterranean climate. Furthermore, in addition to its antioxidant function, α-Toc is also involved in the transport and storage of photoassimilates (Hofius et al. 2004), which would also be stimulated under favourable climatic conditions. This is especially consistent with the α-Toc location in the parenchyma rays, considering that these cells are fundamental for the transport and storage of photoassimilates in the wood (Gartner et al. 2000). In any case, with the necessary caution due to the novelty and exploratory nature of this research, the matching between α-Toc changes and annual rainfall together with the fact that

Tocopherol as a new tool for dendrosciences 7 it persists once the tissue is dead would enable the use of xylematic α-Toc as an environmental proxy of past stressing events. Up to now, most research on the wood anatomy in the xylem has focused mainly on hydraulic features and the xylem parenchyma has been neglected. However, the relevance of parenchyma rays and their related metabolic functions is gaining in importance. The metabolic studies of xylem parenchyma can provide information that is not necessarily linked to anatomical changes in the xylem such as seasonal or inter-annual variations in α-Toc. The findings presented here regarding the location of α-Toc in xylematic tissues of woody plants and its relationship to environmental factors may contribute to an understanding of xylem functions and set a new baseline for future works on dendrometabolomics.

Supplementary data for this article are available at Tree Physiology Online.

Acknowledgments The authors thank Gonzalo Juste for the laboratory treatment of samples, Raquel Esteban for her helpful comments and Brian Webster for English revision.

Conflict of interest B.F.-M. received two postdoctoral fellowships from the Research Vicerrectorate of the UPV/EHU and a Marie Curie IEF grant (328370 MELISSA) from the European FP7-PEOPLE programme. F.M. received a fellowship from the Basque Government.

Funding This research received funding from the following institutions: Spanish Ministry of Education and Science (BFU 2010-15021), Spanish Ministry of Economy and Competitivity (GL201234209, CGL2011-26654), ARAID foundation and the Basque Government (UPV/EHU-GV IT-624-13, IT-302-10).

References Aschan G, Wittmann C, Pfanz H (2001) Age-dependent bark photosynthesis of aspen twigs. Trees 15:431–437. Bagci E, Bruehl L, Özçelik H, Aitzetmuller K, Vural M, Sahim A (2004a) A study of the fatty acid and tocochromanol patterns of some Fabaceae (Leguminosae) plants from Turkey. Grasas Aceites 55:378–384. Bagci E, Bruehl L, Aizetmuller K, Altan Y (2004b) Fatty acid and tocochromanol patterns of some Turkish Boraginaceae—a chemotaxonomic approach. Nord J Bot 22:719–726.

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Supplementary data

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