In Planta Localization of Stilbenes within Picea abies Phloem^,

The axial parenchyma cells of Norway spruce phloem accumulate stilbenes, the amount of which varies with changes in cell type and cell volume from the inner to the outer phloem.

Abstract

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.

The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutive (i.e. continuously produced) or inducible (i.e. activated as a response to insect or pathogen attack; Krokene, 2015). Many defense traits exist in both forms (Franceschi et al., 2005). For example, axial phloem parenchyma cells (or polyphenolic parenchyma) are critical in conifer bark defense. These cells regularly form in Pinaceae during annual phloem formation (Franceschi et al., 1998, 2000; Krekling et al., 2000; Jyske et al., 2015) but also are produced on invasion (Franceschi et al., 2005; Krokene, 2015). In Norway spruce (Picea abies) phloem, axial parenchyma forms distinctive, continuous tangential sheets across conducting (i.e. noncollapsed) and nonconducting (i.e. collapsed) tissue.

Pioneering studies using microscopy with different dye agents and autofluorescence showed that the large vacuole is a special feature of the axial phloem parenchyma that contains phenolic substances (i.e. phenolic bodies; Franceschi et al., 1998). Microscopic imaging techniques also showed that polyphenolic content is highly dynamic (Franceschi et al., 1998, 2000, 2005) and changes seasonally (Krekling et al., 2000). Within the last 5 years, progress in laser microdissection (LMD) has facilitated the sampling of individual tissues and cells, providing information about the exact chemical composition of phenolic content. Li et al. (2012) used LMD to show that the axial parenchyma is the main site of phenolic accumulation in spruce bark, including that of stilbene compounds.

Stilbenes are secondary metabolites that are composed of two phenol moieties linked by a C₂ bridge. These compounds are derived from the phenylpropanoid pathway, in which the last steps of biosynthesis are catalyzed by stilbene synthase (Chong et al., 2009). There is increasing interest in these antioxidant, antibacterial, and antiinflammatory compounds for use in healthy human diets, therapeutic approaches, and as protective agents in materials sciences (Shibutani et al., 2004; Metsämuuronen and Siren, 2014; Reinisalo et al., 2015; Hedenström et al., 2016; Sirerol et al., 2016). The tetrahydroxystilbene glucosides trans-astringin (3,3ʹ,4ʹ,5-tetrahydroxystilbene 3-O-β-d-glucoside) and trans-isorhapontin (3,4ʹ,5-trihydroxy-3ʹ-methoxystilbene 3-O-β-d-glucoside) are the most abundant constitutive stilbene compounds of Norway spruce, while the trihydroxystilbene glucoside trans-piceid (resveratrol 3-O-β-glucoside) and stilbene aglycons (i.e. without the sugar moiety) are less abundant. Stilbene synthesis in spruce probably proceeds through the formation of resveratrol (i.e. aglycon of piceid) followed by further modifications (i.e. hydroxylation, O-methylation, and O-glycosylation) to yield tetrahydroxystilbene glucosides (Hammerbacher et al., 2011). Stilbenes are assumed to provide protection against a wide variety of environmental stressors (Franceschi et al., 2005; Witzell and Martin, 2008; Chong et al., 2009). Stilbenes appear to contribute to antifungal defense in spruce (Hammerbacher et al., 2011, 2013). The fungal inoculation of spruce bark with the blue-stain fungus Endoconidiophora polonica (previously named Ceratocystis polonica; de Beer et al., 2014) causes astringin levels to decrease, in parallel with increasing dimeric stilbene glucoside levels in the LMD-isolated axial phloem parenchyma (Li et al., 2012) or increasing levels of corresponding aglycons in bulk tissue (Viiri et al., 2001). During the annual formation of phloem in Norway spruce, the accumulation of stilbene glucosides inside the newest, LMD-isolated phloem ring is preceded by the formation and cellular development of a new band of axial parenchyma (Jyske et al., 2015). These observations strongly indicate that the inducible and constitutive stilbene compounds of spruce phloem are both stored and synthesized in the axial parenchyma.

New mass spectrometry imaging techniques provide significant improvements in the mapping of plant metabolites (Briggs and Seah, 1993; Vickerman and Briggs, 2001; Burrell et al., 2007; Cha et al., 2008; Lee et al., 2012; Bjarnholt et al., 2014; Aoki et al., 2016). To elucidate the synthesis, distribution, and metabolism of secondary plant metabolites, it is essential to gather positional information about them in a living state, as pretreatment of specimens, such as drying, may change the distribution and concentration features of soluble chemicals (Metzner et al., 2008; Li et al., 2012; Kuroda et al., 2013). In this study, we used a unique system of time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle (cryo-TOF-SIMS/SEM) to study the localization and accumulation patterns of stilbenes within cells and tissues of phloem. This system has been developed to study chemical distributions at high-spatial resolution (1 µm) directly from the surfaces of plant specimens in a frozen-hydrated state (i.e. in planta) representing living tissues (Kuroda et al., 2013; Aoki et al., 2016). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) directly detects organic and inorganic compounds on the specimen surface over a broad mass-to-charge ratio (m/z) range by mass spectrometry with high chemical sensitivity. Specimen surface morphology is visualized by the detection of total secondary ion content. The quality of cellular integrity may be further observed by scanning electron microscopy connected with a cryo-shuttle (cryo-SEM) imaging of the frozen surface of the same specimen. The cryo-TOF-SIMS/SEM system has still rarely been applied to the analysis of plant physiology (Metzner et al., 2008, 2010; Iijima et al., 2011; Kuroda et al., 2013; Aoki et al., 2016).

Mass spectrometer imaging techniques consist of an ionizer and a mass analyzer. In the TOF-SIMS system, secondary ion mass spectrometry is used as an ionizer and time-of-flight as a mass analyzer. In another mainstream imaging mass spectrometry technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization is used as ionizer. Compared with TOF-SIMS, MALDI-MS is more quantitative and has high-M_r acceptance, but the resolution of MALDI-MS is not high enough for cell-level detection (Aoki et al., 2016). Instead, the spatial resolution of TOF-SIMS is superior to focus on cell functions. The disadvantage of TOF-SIMS is that the ionization and fragmentation phenomenon may be affected by the matrix effect, causing some degree of uncertainty. However, when time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle (cryo-TOF-SIMS) is used in combination with quantitative gas chromatography, it is very powerful to study the positional and temporal distributions of metabolites within living plants.

To complement TOF-SIMS analysis, we applied quantitative chemical microanalysis methods to study the amounts of stilbene glucosides and to correlate those with the amounts of total extractives, monosaccharides and disaccharides, and water across phloem and bark. The methods include tangential cryo-sectioning of tissues and their chemical microanalysis by gas chromatography with flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).

To combine the chemical information with phloem morphology, the cellular and subcellular features of the axial phloem parenchyma were analyzed by three-dimensional (3D) synchrotron radiation microtomography (µCT). µCT is a prominent tool that has gained popularity for 3D analysis of xylem structure and physiology (Brodersen, 2013; Cochard et al., 2015), but only recently has it been applied to the 3D analysis of phloem (Jyske et al., 2015). This method offers advantages over traditional light microscopic approaches, as high-throughput data at the submicrometer level can be produced from significantly larger tissue volumes. The data allow for representative volumetric analysis of cellular distributions along with 3D visualization of subcellular features.

In this study, we used a novel combination of cutting-edge techniques to analyze in parallel (1) in planta cellular localization and accumulation of stilbene glucosides across phloem and bark by semiquantitative cryo-TOF-SIMS/SEM; (2) tissue-level quantitative amounts of stilbene glucosides, total extractives, and monosaccharides and disaccharides across phloem and bark by tangential cryo-sectioning and GC-FID and GC-MS; (3) 3D cell abundance distributions across phloem and bark by µCT; and (4) variation in water content across phloem and bark (Fig. 1).

Figure 1.

Schematic presentation of the specimen structure and preparation for different analyses. Sample blocks were taken from living tree stem (A) or stem discs (B) at 1.3 m on the stem. The blocks (C) containing outer bark (periderm), phloem, cambium, and part of the outermost xylem ring (D; transverse view of phloem and bark) were further divided into subblocks (1–3; C and E). Subblocks 1 and 2 were quick frozen, and subblock 3 was fixed chemically. Subblock 1 was used for the direct chemical mapping of stilbenes across the phloem from the cambium to the outer bark (i.e. semiquantitative analysis of stilbene localization and accumulation across transverse and radial surfaces [purple] of the tissue block by TOF-SIMS; E-1). To obtain quantitative data on the amounts of stilbenes, other extractives, and carbohydrates across phloem and bark, tangential cryo-sections (250 or 450 µm each; cut slices illustrated with purple in E-2) were cut across subblock 2 and directed for chemical microanalysis by GC-FID (E-2). Subblock 3 was divided into four to six zones, and from each zone, small cuboids (illustrated with purple in E-3) were cut and directed for morphological analysis of phloem by phase-contrast µCT (E-3). Water content across the phloem and bark was analyzed from separate fresh blocks, which were further cut tangentially into thin sections. Black arrows indicate the radial direction from the cambium toward the outer bark. Purple areas show the analyzed locations of each subblock (E). Note that schematic drawings are not to scale.

RESULTS

Extractives, Carbohydrates, and Water within Phloem

The variation of stilbene glucoside content across phloem and bark was studied by quantitative chemical microanalysis (GC-FID and GC-MS) of tangentially cut cryo-sections (as demonstrated in Fig. 1). Total amounts of extractives, monosaccharides and disaccharides, and water content also were analyzed across phloem and bark in order to study how their radial quantitative patterns correlate with those of stilbene glucosides and changes in phloem morphology.

Total extractive content increased from the cambium outward, while total soluble sugar and water contents followed the opposite trend (Fig. 2). In the cambial zone and noncollapsed phloem, Suc represented over 80% of total soluble sugars, but it decreased to approximately 50% with increasing distance from the cambium (Fig. 2). Concurrently, the ratio of hexoses (Glc + Fru) increased from approximately 10% in the innermost phloem to approximately 50% in the collapsed outer phloem. The decreasing radial trend in water content was similar to that of Suc.

Figure 2.

Structure of Norway spruce phloem and the distribution of extractives, soluble sugars, and water across the phloem. A, Cryo-SEM image of frozen-hydrated tissue of older tree FO. A resin droplet is indicated with the arrow. C, Cambium; NColP, noncollapsed phloem; ColP, collapsed phloem; P, periderm. Bar = 500 µm. B, Distribution of the total amount of extractives, soluble sugars (i.e. monosaccharides and disaccharides), and water (g g⁻¹ phloem dry weight [DW]) across the phloem. C, Distribution of the ratio of Suc (circles; black line) and hexoses (i.e. Glc + Fru; squares; dark gray line) to the total soluble sugars, and the relative water content (RWC; diamonds; light gray line). Each data point in B and C represents a tangential section (250–450 µm thick) derived from a representative phloem zone. Data of older (FO) and younger (FY) trees are combined. Relative water content was calculated as the amount of water (g) in fresh tissue to the amount of water (g) in the same tissue after water saturation.

The quantitative amount of stilbene glucosides increased from the inner phloem to mid phloem and then decreased to the outermost phloem (Fig. 3). The free stilbene aglycon units were undetectable. Of the stilbene glucoside compounds, astringin had the highest content, while that of isorhapontin and especially piceid was lower. Unlike astringin and piceid, the maximum of the isorhapontin curve was in the innermost noncollapsed and outermost collapsed phloem of an older tree and in the noncollapsed and innermost collapsed phloem of a younger tree (Fig. 3). The maximal amount of stilbenes in the older tree was 38% higher than that in the younger tree.

Figure 3.

Radial changes of stilbene glucoside amounts analyzed by GC-FID across Norway spruce phloem of an older (FO; A) and a younger (FY; B) tree. Total, Total amount of stilbene glucosides; Ast, astringin; Isorh, isorhapontin; Pic, piceid. Each data point represents a tangential cryo-section (250–450 µm thick) representative of a different phloem zone. C, Cambium; NColP, noncollapsed phloem; ColP, collapsed phloem; P, periderm. Due to the low amount of piceid, its radial patterns are shown as inlets. DW, Dry weight.

TOF-SIMS Spectrum of Phloem

To reveal the localization of stilbene glucosides at the cellular and tissue levels across phloem and bark in planta, frozen-hydrated tissue blocks (Fig. 1) were analyzed by the semiquantitative cryo-TOF-SIMS/SEM system. A typical positive TOF-SIMS spectrum of Norway spruce phloem with characteristic ion peaks of stilbenes is shown in Figure 4. The ion peaks of stilbenes in positive and negative ion modes were verified by comparing them with the peaks from authentic stilbene compounds either under the frozen-hydrated condition (Table I) or as dry (Supplemental Fig. S1). The positive ion mode produced repeatable results; thus, it was selected for the analysis. The peaks that were detected from both the authentic compounds and the phloem specimens were selected as estimated peaks representing the stilbenes with the following m/z values: 229 for resveratrol (i.e. aglycon of piceid), 245 for piceatannol (i.e. aglycon of astringin), 259 for isorhapontigenin (i.e. aglycon of isorhapontin), 391 for piceid, 406 for astringin, and 420 for isorhapontin. Higher ion intensities were detected for the aglycons of stilbene glucosides at m/z 229, 245, and 259 than for the corresponding glucosides at m/z 391, 406, and 420. In TOF-SIMS analysis, it is a frequent phenomenon that fragment ions have higher intensity than that of molecular ions (Aoki et al., 2016). Here, the quantitative chemical analysis by GC-FID and GC-MS clearly showed the absence of free aglycons within the phloem. Thus, the peaks at m/z 229, 245, and 259 were selected as the estimates for stilbene glucosides when depicting their semiquantitative localization and accumulation within the phloem.

Figure 4.

A, Positive TOF-SIMS spectrum of frozen-hydrated Norway spruce phloem. The spectrum was obtained from the transverse surface at the middle region of collapsed secondary phloem of tree FO. Arrows indicate the peaks generated from glucosides (m/z 391, 406, and 420 for piceid, astringin, and isorhapontin, respectively) and their aglycons (m/z 229, 245, and 259 for resveratrol, piceatannol, and isorhapontigenin, respectively). B, The molecular structure of trans-stilbenoids. Piceatannol, R1 = OH, R2 = H; resveratrol, R1 = OH, R2 = OCH₃; isorhapontigenin, R1 = OGlc, R2 = OH; astringin, R1 = OGlc, R2 = H; piceid, R1 = OGlc, R2 = OCH3; isorhapontin, OGlc = 3-O-β-d-glucoside.

Table I. Positive (+) and negative (−) ion peaks representative of stilbene glucosides and their aglycons as detected by TOF-SIMS from authentic stilbene standards in frozen-hydrated and freeze-dried conditions (Standards).

Corresponding positive ion peaks that were detected predominantly from Norway spruce phloem are highlighted in brackets. Positive and negative ion peaks (estimated to represent stilbenes) were obtained from the freeze-dried extracts of Norway spruce phloem (Bark). No peaks were representative of stilbene compounds at m/z ≥ 200 in phloem specimens that had had their extracts chemically removed (i.e. extracted and dried specimens).

Compound	Frozen-Hydrated		Freeze-Dried
	+	−	+	−
Standards
Resveratrol	228, [229]	227	[228], 229	227, 228
Piceatannol	244, [245]	243	244, [245]	243
Isorhapontigenin	258, [259]	257	258, [259]	257
Piceid	228, [229], 391	227	[228], 229, 390	227, 389
Astringin	429, [406]a	243, 405	244, [245], 406	243, 405
Isorhapontin	258, [259], [420], 421	257, 419	258, [259], 420	258, 419
Bark
Phloem extracts			228, 229, 244, 245, 258, 259, 406, 408, 420, 421, 422	243, 405, 406
Extracted phloem tissue	–	–	Nob	Nob

^aVery low peak counts were observed from the standard.

^bNonobservable amounts of stilbene peaks at m/z ≥ 200 when the phloem specimen analyzed by TOF-SIMS were subjected to chemical extraction and then reanalyzed by TOF-SIMS. In contrast, some stilbene-related peaks were detected in the extracts of the phloem (i.e., freeze-dried extracts).

Mapping of Stilbenes within Phloem

In order to detect the localization and accumulation patterns of stilbenes across phloem and bark, we detected the peaks at m/z 229, 245, and 259 from the transverse surface (Fig. 1) of a frozen-hydrated phloem specimen of the older tree (FO) by obtaining and joining 13 images from the innermost to the outermost phloem and periderm (Fig. 5). The total ion images (Fig. 5) show the sum of all ion intensities in each pixel, clearly depicting phloem morphology (compare Figs. 1 and 2). All stilbenes were clearly detected in the axial phloem parenchyma that formed distinctive tangential layers. The localization of all the stilbenes at m/z 229, 245, and 259 was similar; however, peak intensity varied between the compounds.

Figure 5.

Positive TOF-SIMS images of the transverse surface of Norway spruce (FO) phloem from the cambium (at left) to the outermost bark (at right). Images of m/z 229 (A), 245 (B), and 259 (C) represent trans-piceid (A), trans-astringin (B), and trans-isorhapontin (C), respectively, and total ion images (D) represent the morphology of the sample surface. The color of the pixels corresponds to the ion intensity of the compounds on the specimen surface. Specifically, stilbenoids were absent in dark areas, showed midrange concentrations in red areas, and had the highest concentration in yellow areas. C, Cambium; NColP, noncollapsed phloem; ColP, collapsed phloem; P, periderm. Bars = 100 µm.

To analyze radial changes in semiquantitative stilbene content, we computed relative ion intensities at m/z 229 and 391, 245 and 406, and 259 and 420 per mille of total ion counts within each consecutive image from the cambium to the outermost periderm (this normalization was needed as total ion counts varied slightly among images; Fig. 6). The relative ion intensities of astringin derivatives (m/z 245 and 406) were highest in the mid phloem, whereas the relative ion intensities of isorhapontin derivatives (m/z 259 and 420) were highest in the innermost and outermost phloem. These results well corresponded with the tissue-level quantitative amounts of stilbene glucosides as detected by gas chromatography (Fig. 3). The ratio of stilbene glucosides to their aglycons decreased slightly from the innermost phloem to the periderm (Fig. 6).

Figure 6.

A, Radial changes in relative ion intensities of trans-stilbenoids obtained by TOF-SIMS spectra from the transverse surface of frozen-hydrated Norway spruce (FO) phloem. The relative ion intensities of m/z values representing derivatives of piceid (229 and 391), astringin (245 and 406), and isorhapontin (259 and 420) were calculated per mille of total ion counts for each consecutive TOF-SIMS image shown in Figure 5. B, Ratio of peak counts of the sum of all stilbene glucosides to that of their aglycons across the phloem. Error bars show sd between different compounds.

The semiquantitative cellular accumulation of stilbenes was compared between axial parenchyma and intermediate phloem tissue (i.e. comprising ray parenchyma and collapsed and noncollapsed sieve cells) by observing stilbene peaks at m/z 229, 245, and 259 from the radial surface (Fig. 1) of the frozen-hydrated specimen FO (Supplemental Figs. S2–S4). Phloem and periderm were divided into six zones from the cambium to the outermost periderm. Then, we obtained TOF-SIMS images from each zone. From the images at m/z 229, 245, and 259, the regions of interest (ROIs) were selected to represent the axial parenchyma and intermediate areas (for details, see Supplemental Fig. S3). In the ROIs of the axial parenchyma, the relative ion intensities were significantly higher than those in the intermediate areas, which only had ion intensities at the background level (P < 0.05 for m/z 245 and 259 and P < 0.01 for m/z 229; Supplemental Figs. S2 and S4). Stilbenes also were present in the living cells produced by cork cambium (Fig. 5; Supplemental Fig. S2).

Differences between Frozen-Hydrated and Freeze-Dried Specimens

To analyze the effect of specimen water-status on TOF-SIMS analysis, we compared frozen-hydrated and freeze-dried phloem tissue and authentic stilbene compounds in dry and frozen-hydrated conditions (Fig. 7; Table II). Compared with the frozen-hydrated condition (Fig. 7, B and C), freeze-drying of the specimen (Fig. 7, F and G) decreased the ion intensities of stilbene glucoside peaks (e.g. m/z 406 for astringin; Fig. 7, B and F) relatively more than those of stilbene aglycons (e.g. m/z 245 for piceatannol; Fig. 7, C and G). The result was statistically significant for astringin and isorhapontin derivatives within phloem and for the authentic piceid compound (Table II).

Figure 7.

Comparison of TOF-SIMS analysis between frozen-hydrated (A–D) and freeze-dried (E–H) older Norway spruce (FO) phloem. A and E show scanning electron microscopy images of the middle region of collapsed phloem tissue. Rays are highlighted in green and axial parenchyma in violet. Boxes in A and E indicate the same tissue areas as those of positive TOF-SIMS images of B to D and F to H, respectively. Compared with the frozen-hydrated condition (B and C), the ion intensities of the freeze-dried condition (F and G) were small either in m/z 406 (representative for astringin; B and F) or m/z 245 (representative for piceatannol; C and G). The distribution of m/z 406 and 245 is shown in violet (B, C, F, and G), and that of m/z 39 (representative for potassium) is shown in green. Total ion images (D and H) represent the general morphology of the tissue surface. Maximum ion counts (max) of each compound per pixel are shown in each image. Bars = 100 μm.

Table II. Percentage ratio of positive ion counts of stilbene glucosides to those of stilbene aglycons (Glu/Agl) in the authentic stilbene standards and specimens of Norway spruce phloem (middle of collapsed secondary phloem in tree JO) under frozen-hydrated and freeze-dried conditions.

To obtain the spectra of stilbene standards under frozen-hydrated conditions, their aqueous solutions were prepared with potassium chloride. Arithmetic means (and sd) for the standards are based on spectral data obtained from all of the standards of stilbene glucosides, while those for the phloem are based on spectra obtained from separate cell types (i.e., axial parenchyma, ray parenchyma, and intermediate areas composed mainly of collapsed sieve cells) isolated manually by the ROI tool in the data analysis software. P values in boldface are statistically significant (P < 0.05).

Specimen	Glu/Agl		F	P
	Frozen-Hydrated	Freeze-Dried
	%
Standards
Piceid	25 (7)	8 (6)	8.54	0.043
Astringin	40 (15)	15 (12)	5.16	0.086
Isorhapontin	52 (29)	15 (12)	4.29	0.107
Phloem
Piceid	22 (7)	50 (36)	1.87	0.243
Astringin	45 (10)	12 (11)	15.62	0.017
Isorhapontin	75 (24)	21 (16)	10.56	0.031

Volumetric Analysis of Phloem Cell Distribution

To obtain understanding of how the chemistry of phloem correlates with phloem morphological features, we carried out 3D visualization of the phloem and bark zones from the cambium outward (Figs. 1 and 8; Supplemental Fig. S5). We quantified the volumetric cellular distributions and classified axial parenchyma into subgroups based on their vacuolar contents.

Figure 8.

Typical µCT images of the phloem of the older Norway spruce tree (FO). Images show outer xylem (on left in A), cambium and noncollapsed phloem (A), inner (B), middle (C and D), and outer layers (E) of the collapsed phloem, and outermost phloem (F). The size of the cube is 730 × 730 × 730 µm³, and the imaged volume of the phloem is equal to the largest possible cylinder that fits inside the cube.

Most of the innermost phloem was composed of noncollapsed sieve cells, the proportion of which declined beyond this zone (Fig. 9; Supplemental Fig. S6). Concurrently, the volume ratio of rays and collapsed sieve cells increased with increasing distance from the cambium to the mid phloem. Subsequently, the ratio of these cells decreased toward the outermost phloem layers. In contrast, the volume ratio of the axial parenchyma increased with increasing distance from the cambium.

Figure 9.

The change of phloem cell volume ratios from the cambium (top) toward the outermost bark (bottom) in older Norway spruce (FO). A to F, Volume ratios of all cell types within the phloem. G to L, Volume ratios of axial parenchyma cell types to the total axial parenchyma. For details on the classification of cell types, see Figure 10 and Supplemental Figure S7.

The axial parenchyma cells were classified into seven different cell types based on their inner contents: specifically, cells with (1) smooth and light solid contents filling most of the intracellular space; (2) droplet-type contents; (3) densely packed contents in ring-type form next to the periphery of the intracellular space; (4) porous contents with small pore size; (5) porous contents with large pore size; (6) cells containing calcium oxalate crystals; and (7) empty parenchyma (Figs. 9 and 10; Supplemental Figs. S6 and S7). Of all axial parenchyma cells, the volume of solid-, droplet-, and ring-type cells (i.e. young type) was highest in the young inner half of the phloem (with an average of 63% compared with 22% in the older half). The volume of cells with small and large porous contents (i.e. old type) represented 10% to 17% of axial parenchyma in the inner phloem, decreased to a minimum in the mid phloem, and again clearly increased toward the outermost phloem, reaching values between 10% (younger tree) and 51% (older tree). The volume of empty parenchyma also clearly increased from the inner to the outer phloem. Ray volume was positively correlated with the volume of young type axial parenchyma (i.e. solid-, droplet-, and ring-type cells pooled), whereas a significant inverse relationship was obtained between ray volume and older type parenchyma (i.e. pooled small and large porous cells; Supplemental Fig. S8).

Figure 10.

3D renderings of different types of axial parenchyma cells within older Norway spruce (FO) phloem (left), with their intracellular features highlighted at right. Axial parenchyma cell types with their cellular contents are classified as solid (A), droplets (B), ring next to the vacuolar periphery (C), small porous (D), large porous (E), and calcium oxalate crystals (F). Bars = 50 µm.

Relationship among Phloem Properties

The quantitative chemical data and the data on morphological features of phloem were combined in the analysis to reveal interactions and the possible mechanisms explaining the patterns of stilbene localization and accumulation across phloem and bark.

Relative water content and the total amount of soluble sugars were positively correlated with ray volume and the volume of young type axial parenchyma, but they were negatively correlated with the volume of total axial parenchyma, empty parenchyma, and old type axial parenchyma with porous intracellular content (Supplemental Figs. S9 and S10). The total amount of stilbene glucosides was positively correlated with the total volume of axial parenchyma (Supplemental Fig. S10). To obtain an estimate for stilbene packing density (mg g⁻¹ dry weight mm⁻³) inside the cells, the content of stilbene glucosides (mg g⁻¹ dry weight) in each phloem zone analyzed by chemical microanalysis was divided by the estimated volume of stilbene-containing axial parenchyma (i.e. the sum of solid-, droplet-, ring-type, and porous cells) obtained with µCT at an equivalent distance from the cambium. The obtained stilbene packing density values were then plotted against the ratios (%) of young and old type axial parenchyma cells to the total volume of stilbene-containing axial parenchyma in each zone. Thus, we evaluated how the packing density of stilbenes inside the axial parenchyma was related to the abundance of different types of axial parenchyma. We found that, while there was a high ratio of young type cells (i.e. when the total volume of axial parenchyma was low), stilbenes were densely packed in the cells (Fig. 11). As the ratio of old type porous parenchyma increased (i.e. when the total volume of axial parenchyma increased), overall stilbene concentration in the cells decreased (Fig. 11).

Figure 11.

Relationship of the stilbene packing density to the ratio of young (A) or old (B) axial parenchyma cells. The young and old axial parenchyma cells are solid-, droplet-, and ring-type cells and cells with small and large porous contents, respectively. The stilbene-containing axial parenchyma cells are a total of young and old axial parenchyma cells that contain stilbenes. The packing density was calculated as the stilbene content in each studied phloem zone (mg g⁻¹ dry weight [DW] analyzed by GC-FID) divided by the absolute volume (mm⁻³) of stilbene-containing axial parenchyma cells within each zone. For details on cell types, see Figure 10 and Supplemental Figure S7.

DISCUSSION

This study presents novel in planta information about the localization and accumulation patterns of stilbene glucosides within the tissues and cells of Norway spruce phloem and bark. We used an innovative combination of cutting-edge techniques: semiquantitative chemical mapping at the cellular level by cryo-TOF-SIMS/SEM, tangential cryo-sectioning and quantitative chemical microanalysis at the tissue level by gas chromatography, and 3D analysis of phloem tissues and cells by µCT. Based on our study and the published literature, we propose a six-point concept to explain the radial accumulation patterns of stilbenes within Norway spruce phloem and discuss the points in the following sections. (1) Stilbenes synthesize and accumulate in the axial phloem parenchyma for several years. (2) Newly forming cork cambium and its living derivatives provide the outer layers of bark with a possible new defensive barrier, as stilbenes are localized in these cells in addition to the axial parenchyma. (3) Over time, the secondary changes of phloem and the deterioration of collapsed phloem structures reduce cell connectivity, causing a decline in the energetic and water resources of the axial parenchyma in the outer layers of the phloem and decelerating the synthesis of defensive substances. (4) Concurrently, the secondary oxidation and polymerization of stilbenes, and/or β-glycosidase activity, occurs, resulting in condensation of stilbenes with other substances, such as tannins, and/or stilbene glucoside-to-aglycon transformations. (5) Stilbenes of the axial parenchyma provide phloem with defenses; however, their effectiveness in older bark primarily depends on the preformed level and accumulation, because aging-dependent changes may prevent older axial parenchyma from inducing sufficient stilbene synthesis and/or metabolism in the case of major attack, such as by fungi. (6) The defensive efficacy of stilbenes is related to their preformed level, in terms of their quantity and composition inside cells (e.g. glucoside-to-aglycon ratio), and the ratio of methylated to nonmethylated stilbene compounds.

Stilbenes Synthesize and Accumulate in the Axial Phloem Parenchyma

Our study clearly showed the localization of stilbenes inside the axial phloem parenchyma cells. TOF-SIMS provided data of very high spatial resolution as compared with quantitative chemical microanalysis by gas chromatography. The amount of stilbenes in the intermediate spaces (i.e. composed of ray parenchyma and noncollapsed/collapsed sieve cells) was very low, because the detected ion intensities by TOF-SIMS corresponded to background-level intensities. Li et al. (2012) also showed that laser-microdissected axial parenchyma cells of Norway spruce contained noticeably higher levels of stilbene derivatives than their surrounding sieve cells. During the formation of a new phloem ring in Norway spruce, stilbene glucosides were found to accumulate in the newly formed and older phloem tissue only after the new band(s) of axial parenchyma form (Jyske et al., 2015). Cork cambium (phellogen) and its living derivatives also were found to accumulate stilbenes, providing another mechanism of protection for the outermost phloem.

The resolution of TOF-SIMS analysis did not allow for the detection of stilbenes at the level of separate cell organelles. However, our data support earlier findings about the vacuolar location of phenolic materials in the axial phloem parenchyma cells of Norway spruce (Franceschi et al., 1998, 2005; Krokene, 2015). In other plant species, such as grape (Vitis vinifera) leaves, stilbene autofluorescence has been localized in the epidermal cell vacuoles and intercellular space and/or vacuoles of the mesophyll, as analyzed by confocal microscopy coupled to microspectrofluorimetry (Bellow et al., 2012). In the cell suspension cultures of cv Cabernet Sauvignon, elicitation with ultraviolet C irradiation, methyl jasmonate, and salicylic acid have been shown to increase the stilbene production (trans-piceid, cis-piceid, trans-resveratrol, and ε-viniferin) inside the cells and extracellular trans-resveratrol accumulation in the culture medium (Xu et al., 2015).

The relative intensity data of TOF-SIMS (Fig. 6) showed similar radial trends of stilbene accumulation to the quantitative analysis by gas chromatography (Fig. 3). The maximum astringin amount appeared in the middle of the collapsed phloem, while that of isorhapontin was in the innermost phloem and next to the cork cambium in the older tree. Previous studies have detected higher isorhapontin content in the innermost phloem of Norway spruce (Jyske et al., 2014) and Sitka spruce (Picea sitchensis; Toscano Underwood and Pearce, 1991). The difference in the radial pattern between astringin and isorhapontin may be related to a higher requirement for protection from stress in the innermost and outermost phloem. The innermost phloem is vital for the axial translocation of photoassimilates (Pfautsch et al., 2015a, 2015b). In turn, the outermost phloem is a critical barrier against invaders and abiotic stress. These different functions may be reflected in the different chemical structures and spatial distribution of astringin and isorhapontin. Isorhapontin has an O-methylated group (Hammerbacher et al., 2011) and thus might be less reactive to oxidative enzymes than astringin or piceid (Shibutani et al., 2004).

We found that the composition and content of stilbenes varied depending on the water status of the specimen (i.e. between frozen-hydrated and freeze-dried phloem; Fig. 2; Table II). In TOF-SIMS, the ionization and fragmentation behavior is affected by the matrix effect. The glucoside-to-aglycon ratio (Fig. 6) showed a similar trend to the water content curves within phloem (Fig. 2). Thus, in consideration of the frozen-hydrated and freeze-dried TOF-SIMS results, it is clear that the frozen-hydrated state is important in order to detect the molecular ions of stilbene glucosides.

Secondary Chemical Changes of Stilbenes across Phloem

The radial pattern of stilbene accumulation across the phloem was correlated with subcellular changes in axial parenchyma and secondary chemical modifications of stilbenes. In the inner layers of phloem, axial parenchyma had solid-, droplet-, and ring-type contents. Franceschi et al. (1998) also found that young axial parenchyma cells of Norway spruce (recently formed cells in spring and summer) typically had ring-type and porous vacuolar contents and sometimes droplets. They proposed that the porous structures inside vacuoles are a mixture of dense phenolic substances and soluble vacuolar sap, as detected by transmission electron microscopy/scanning electron microscopy (Franceschi et al., 1998).

We found that, toward the outer phloem, the volume of axial parenchyma with porous vacuolar contents clearly increased. At the same time, the tissue stilbene content decreased and the stilbene packing density inside the cells declined. These findings indicate the occurrence of secondary biochemical changes of the compounds in the cells. Over time, stilbenes may react and become condensed with other compounds, such as tannins (Zhang and Gellerstedt, 2008; Kemppainen et al., 2014; Bianchi et al., 2015), which would explain their observed decline across the outer phloem. Stafford et al. (1988) also suggested that the secondary oxidation and polymerization of proanthocyanidins in the bark of Pseudotsuga menziesii led to a considerable loss of compounds from the middle to the outer bark. Similarly, the extractable flavanol content in Cryptomeria japonica and Pinus densiflora bark was found to decrease considerably from the inner to the outer layers, and at the same time, the molar mass of flavanols increased (Samejima and Yoshimoto, 1982).

TOF-SIMS analysis also indicated a possibility for a higher glucoside-to-aglycon ratio of stilbenes in the inner phloem than in the outer phloem. The aglycons have been proposed to accumulate in Norway spruce phloem due to (1) the activities of glucoside degrading β-glucosidase of the tree itself (Johansson and Stenlid, 1985) or (2) by attacking fungi (Woodward and Pearce, 1988). Glucosylated substrates and their degrading enzymes are localized separately in the plant cells, providing a two-component defense system that protects the cells from their own defense compounds (Morant et al., 2008). In the case of tissue disruption, such as the secondary structural changes in the older phloem and periderm, glucosides can make contact with their degrading enzymes, thus forming more toxic aglycons (Morant et al., 2008).

Do Aging-Dependent Changes in Phloem Lead to the Loss of Defense Potential of Axial Parenchyma?

A remarkable decrease in both water and soluble sugar contents from the cambium and noncollapsed to collapsed phloem correlated with the increasing volume of empty parenchyma, possibly indicating a decline in the hydraulic integrity of the collapsing tissues. At most, from one-third to one-half of the outer phloem volume was composed of empty parenchyma (Fig. 9; Supplemental Fig. S6). Our results are in agreement with Rosner et al. (2001), who found that the decreasing water content from the inner to the outer phloem in Norway spruce was related to aging-dependent structural changes (i.e. the collapse of sieve and Strasburger cells and an increase in phloem dry weight). We hypothesize that symplasmic connections among axial parenchyma (Krekling et al., 2000) and between ray and axial parenchyma diminish during phloem aging. Consequently, nutrition and water flow decelerate from the conducting to the outer phloem. Although sugar supplies in the outer phloem may not be completely depleted, it remains unclear whether the living axial parenchyma are able to use these supplies. The soluble sugars we detected in the outer phloem were possibly composed of both stored sugars in the living parenchyma and sugars compartmentalized within the collapsed tissues. This inference was based on the positive correlation between hexose content and the volume of collapsed sieve cells. As phloem age increases, the energy reservoirs of the axial parenchyma may decline (Krekling et al., 2000). Thus, the older parenchyma may lack the necessary machinery to induce sufficient synthesis or metabolism needed in the defense against massive fungal attacks (Franceschi et al., 1998). As a result, the defensive efficacy of stilbenes in the axial parenchyma is related to their preformed level, in terms of the quantity and composition inside the cells.

CONCLUSION

To our knowledge, this study is the first to present in planta information about how polyphenolic stilbenes are localized in the axial phloem parenchyma cells of Norway spruce phloem. Our observations highlight the importance of using frozen-hydrated samples to evaluate the positional and temporal distribution of chemicals within living plants, especially when studying their biosynthesis and metabolism at the tissue and cellular levels. The tetrahydroxylated stilbene glucosides astringin and isorhapontin are storage forms of constitutive stilbenes in Norway spruce phloem and are highly compartmentalized in axial phloem parenchyma cells. The decline in living cell connectivity between axial and ray parenchyma during tissue maturation and collapse from the cambium outward was assumed to reduce axial parenchyma vitality and stilbene synthesis in the outer phloem. Concurrently occurring secondary chemical modifications of stilbenes were seemingly associated with (1) the increased porosity of intracellular (i.e. vacuolar) contents of axial parenchyma and (2) decreasing stilbene amounts in the axial parenchyma and phloem tissue. Further studies are required to elucidate the symplasmic interconnections between axial and ray parenchyma. The results of this study demonstrate the power of using concurrent analyses of anatomical, chemical, and functional properties of phloem to understand its functions.

MATERIALS AND METHODS

Plant Material

Three Norway spruce (Picea abies) trees were selected for this study. The first was an older tree (approximately 40 years old, 41 cm in diameter, and 13 m in height) growing on the campus of the Forestry and Forest Products Research Institute, southeast of Honshu, Japan (Tsukuba; 36°01′N, 140°06′E, 33 m above sea level). The other two trees were of different ages, both of which grew in southern Finland (Loppi; 60°44′N, 24°30′E, 120 m above sea level; Jyske et al., 2014; Jyske and Hölttä, 2015).

From the older tree in Japan (JO), bark blocks (approximately 1.5 cm tangential × 4 cm longitudinal × 1.5 cm radial) containing the outermost bark, phloem, cambium, and outermost xylem rings were collected at breast height (1.3 m) in June 2013 (Fig. 1). The samples were immediately quick frozen with liquid Freon 22 (−160°C), which was cooled by using liquid nitrogen, and stored at –80°C.

Tree FY was 18 years old, 12 cm in diameter (at 1.3 m height on the stem), and 9.4 m in height. Tree FO was 37 years old, 29 cm in diameter, and 22.1 m in height. Both trees represented different clones of good growth and quality performance, and both originated from southern Finland. The trees were grown on fertile old agricultural soil. The trees were harvested in October 2011. Sample discs were sawn at breast height, and bark blocks (approximately 3 × 2 × 2 cm in longitudinal, radial, and tangential directions, including the whole bark, phloem, cambium, and a few xylem rings) were immediately cut from the southern sides of the discs (Fig. 1). A subsample of each block was rapidly frozen in dry ice (–78°C). The frozen samples were transported to the laboratory in dry ice and stored at –80°C. Another subsample of each block was fixed with 3% aqueous glutaraldehyde (in 0.1 m phosphate buffer, pH 7.4) and stored at 4°C until further processing.

TOF-SIMS Analysis of the Phloem and Bark

To map the distribution of stilbene compounds within the tissues and cells of the phloem and bark under water-hydrated and freeze-dried conditions, we prepared frozen-hydrated and freeze-dried sample blocks for TOF-SIMS measurements (Fig. 1).

For the analysis of frozen-hydrated phloem by cryo-TOF-SIMS, the frozen sample blocks were cut into small specimens (approximately 5 × 5 × 7 mm) on a shallow container filled with liquid nitrogen. Then, the specimens were freeze-attached to the sample holder of the cryo-TOF-SIMS device and transferred to the glove box using a plastic box filled with liquid nitrogen to prevent the water in the sample from melting. The specimen holder was fixed at the stage of a sliding microtome (approximately –30°C) inside the glove box (filled with nitrogen gas; temperature inside was approximately –20°C). The specimen surface (transverse or radial; Fig. 1) was then cut evenly using a sliding microtome (REM-710; Yamato Koki). Then, the specimen was placed in an introchamber of a cryo-vacuum shuttle connecting the glove box, cryo-TOF-SIMS, and cryo-SEM devices (for details, see Kuroda et al., 2013). For cryo-TOF-SIMS analysis, a special cryo-stage for the specimen was used (approximately –130°C) to prevent the ice from sublimating during the analysis.

To analyze the freeze-dried phloem, small frozen specimens (approximately 5 × 5 × 7 mm) were prepared, and their transverse surfaces was smoothly cut with a rotating cryo-microtome (Microm HM505 E; Thermo Fisher Scientific) at –30°C. The specimens were then freeze-dried for 72 h (FD-1000; Eyela), after which they were subjected to TOF-SIMS.

TOF-SIMS measurements were carried out using a TRIFT III spectrometer (ULVAC-PHI). The pressure in the TOF-SIMS specimen chamber was approximately 1 × 10⁻⁹ Pa. Positive spectra were obtained using 22-keV AU₁⁺ ions at a current of 5 to 7 nA. The measured surface areas were 200 × 200 µm² and 500 × 500 µm², with a sample surface primary ion dosage of less than 2.3 × 10⁹ and secondary ion counts greater than 2 × 10⁶.

To obtain a sequential image of the transverse surface across the phloem (of tree FO), overlapping images were taken and joined. For representative images in a radial direction, areas were selected and imaged to cover all of the zones of the phloem and bark from the cambium to the outermost bark. Each image (raw data) contained a full mass spectrum with a resolution of 256 × 256 pixels. The mass spectrum data for the estimated candidate peaks of stilbene compounds within phloem specimens were represented both as spectral data and images. The stilbene compounds within the phloem were identified by direct comparison of their peaks at an m/z value, with the peaks of authentic stilbene standards being analyzed by TOF-SIMS (Table I). We used commercial piceid (Polydatin), resveratrol, piceatannol, and isorhapontigenin (Sigma-Aldrich), and astringin and isorhapontin (Polyphenols Laboratories). Astringin and isorhapontin also were extracted and purified from the bark by preparative HPLC. Standard spectra of stilbenes under dried conditions were measured by TOF-SIMS (both spectral and image-mode data). To obtain the standard spectra of stilbene compounds under frozen-hydrated conditions, we prepared aqueous solutions with 0.1 m potassium chloride (0.4 mg mL⁻¹ aqueous potassium chloride). The frozen solutions were then measured with cryo-TOF-SIMS.

For the spatial analysis of stilbene distribution and accumulation across the phloem and bark, the peak intensities of the estimated stilbenes at m/z 229 and 391 (piceid derivatives), m/z 245 and 406 (astringin derivatives), and m/z 259 and 420 (isorhapontin derivatives) were detected and related to the total secondary ion counts (i.e. relative peak intensities) in each image and are presented as per mille of total ion counts. The ROI function of the WinCadence program (version 5.1.2.8; ULVAC-PHI) also was used to manually isolate specific tissue and cell regions within each image and to produce and compare mass spectral data between these ROIs.

Tangential Cryo-Sectioning and Quantitative Gas Chromatography Analysis of the Phloem

The quantitative amounts of stilbene glucosides and their aglycons, total extractives, and monosaccharides and disaccharides were analyzed with chemical microanalysis methods including GC-FID and GC-MS, for quantitative and qualitative analysis, respectively. For the analysis, another frozen subsample of the bark block was used (Fig. 1). The frozen blocks were cut into smaller subblocks (approximately 10 × 10 × 10 mm) on the bucket of dry ice and were freeze-attached on the stage of the rotary microtome (approximately –25°C). Serial tangential cryo-sections (250 or 450 µm each) were cut throughout the bark, from the outermost bark toward the cambial zone (Fig. 1). The cryo-sections (5–25 mg each) were placed in Eppendorf vials and immediately freeze-dried. The freeze-dried phloem tissues were weighed and extracted in a test tube three times with 3 mL of acetone:water (9:1, v/v) at room temperature in the dark (totaling 24 h). The test tube was periodically agitated. The clear extracts were transferred to another test tube with Pasteur pipettes, and the solvents from the cumulative extracts were evaporated in a water bath at 40°C with N₂ flow and carefully dried in a vacuum desiccator at 40°C for 1 h in the dark. The dried extractive content was determined gravimetrically, and 10 mL of dried acetone was added to each test tube to obtain a stock solution of the extractives. An aliquot of the stock solution was placed in separate test tubes to obtain about 1 mg of extractives, and 100 µL of 0.1 mg mL⁻¹ xylitol solution (in methanol) was added as the standard. To the same test tube, 2 mL of another standard solution containing 0.02 mg mL⁻¹ betulinol in MTBEL also was added. The solvents were then evaporated using N₂.

Silylation was completed at 70°C for 40 min with a 4:1:1 mixture of BSTFA:trimethyl chlorosilane:pyridine, and the derivatized sample was left overnight in the dark. The silylated samples were then analyzed by GC-FID using a Perkin-Elmer Clarus 500 gas chromatograph equipped with PH-1 (25 m × 0.2 mm; 0.11 μm film thickness) and PH-5 (25 m × 0.2 mm; 0.11 μm film thickness) capillary columns, with the temperature programming providing simultaneous analysis of monosaccharides/disaccharides and stilbene glucosides during one gas chromatography run: 100°C, 8-min hold, 2°C min⁻¹, 170°C, 12°C min⁻¹, 310°C, and 7-min hold. The temperature of the injector and detector were 250°C and 320°C, respectively. The sample injection volume was 1 μL, and the split ratio was 1:30. Monosaccharides and disaccharides were quantified against xylitol using correction factors that were determined by the separate analysis of authentic monosaccharides/disaccharides. Stilbene glucosides were quantified against betulinol without using a correction factor (i.e. correction factor 1).

The quantitative results were presented for each cryo-section as a function of a cumulative distance from the cambium (the middle point of each cryo-section in the center). To facilitate the comparative analysis of quantitative chemical results (stilbene glucosides, total extractives, and monosaccharides and disaccharides) with phloem structural properties (i.e. cell type volume ratios), as well as water content through the phloem, we computed the arithmetic means for two to three consecutive cryo-sections. Phloem tomography was completed for four (tree FY) to six (tree FO) zones across the phloem and bark.

Cryo-SEM Imaging of the Phloem

The transverse surfaces of frozen-hydrated (JO and FO) and freeze-dried (JO) specimens were observed by cryo-SEM and scanning electron microscopy systems (S4500; Hitachi) after cryo-TOF-SIMS and TOF-SIMS analyses. Secondary electron images were detected at an acceleration voltage of 1.5 kV.

Synchrotron Phase-Contrast Imaging of the Phloem

We visualized secondary changes within the phloem tissue from the cambium to the outer bark and determined the distribution of different cell types across the phloem using phase-contrast µCT. The µCT analysis was performed on a chemically fixed subspecimen of the same block sample that was used for the chemical and TOF-SIMS analyses (Fig. 1). The fixed samples were serially cut into approximately 1- × 1- × 5-mm³ cuboids (Fig. 1) and were dehydrated in a graded series of ethanol, followed by critical point drying according to Leroux et al. (2009). The cuboids were mounted upright on carbon fiber rods, and microtomography scans were performed on beamline ID19 at the European Synchrotron Radiation Facility. The x-ray beam energy was 18 keV, and the sample-to-detector distance was 2 cm. Prior to reconstruction, Paganin phase retrieval with δ/β = 1,500 was used to enhance contrast in the transmission images. The voxel size (edge length of one cubical 3D image pixel) was 0.355 µm. Avizo (FEI) and VGStudioMAX (Volume Graphics) software were used for the segmentation of the 3D images and data visualization.

Quantitative Analysis of Cell Volumes within the Phloem

We determined the volumetric proportions of different cell types and tissue structures across the zones of the phloem and bark imaged by µCT. These structures included ray and axial parenchyma, noncollapsed and collapsed sieve elements, the cell wall, and other structures, such as cambial, xylem, and periderm cells. In addition to this general partitioning of different cell types, the phloem axial parenchyma cells were further separated into different types based on their cellular contents.

For the quantitative analysis, a program was written using Matlab software (Mathworks) to determine the relative volumes of different cell types interactively in a 3D tomography scan of the tissue. The Matlab function randomly selected voxels from the input data (from a total of 2,048 × 2,048 × 2,048 voxels per analyzed zone of phloem), displayed their surroundings in three cardinal slices, and prompted the user to identify the type of cell in the center of the images according to a predefined classification (Supplemental Fig. S5). The results presented here are based on a total of 1,000 randomly selected points per each radial zone of phloem and bark. To reduce subjective errors in the interpretation of cell types, the results were based on two individual interpretations.

Calculation of Stilbene Density within the Axial Parenchyma

We used quantitative chemical data and cellular distribution data to investigate how cellular changes across the phloem are linked with concurrent changes in stilbene content as a function of distance from the cambium. For each phloem block visualized by µCT, we determined the distance of the block from the cambium (i.e. for the inner and outer edges of each block). This spatial information was used to select data about stilbene content for the corresponding location (i.e. the results of stilbene content for tangential serial sections at the same distances from the cambium). Then, the arithmetic means for stilbene content (mg g⁻¹ dry weight) for each location (i.e. six locations in the older tree and four locations in the younger tree) were calculated. The assimilated information about the dimensions of these selected tangential sections was then used to calculate the combined total volume. Phloem volume (mm³) and cell volume ratio (%) data were then used to estimate the absolute volumes of different cell types in each location. An estimate for the packing density of stilbenes within axial parenchyma (mg g⁻¹ dry weight mm⁻³) was computed by dividing the stilbene content in each location (mg g⁻¹ dry weight) by the absolute volume of axial parenchyma that presumably contained stilbenes (i.e. solid, droplet, ring, small, and large porous axial parenchyma). This estimate was then compared against the relative volume of different types of axial parenchyma to evaluate how stilbene packaging density changes with changes in cell distribution across the phloem.

Analysis of Phloem Water Content

We analyzed the radial changes in phloem water content and relative water content from the cambium to the outermost bark by collecting small intact blocks (approximately 3 × 2 × 2 cm in longitudinal, radial, and tangential directions, including the bark, phloem, cambium, and a few xylem rings) from the trunk of tree JO at breast height. After collection, the blocks were placed into plastic tubes and were transported directly to the laboratory. Then, the blocks were tangentially cut into six to eight zones, and their fresh weights were measured. The subsamples for the water content analysis were then oven-dried at 102°C for 48 h, after which the dry weight was measured and the water content was determined (g g⁻¹ dry weight). For the relative water content measurements, other subsamples were measured to obtain the fresh weight and were then soaked in a water bath for 48 h under vacuum conditions. Subsequently, the water-saturated weight was measured, the samples were over-dried, and the relative water content was determined as the percentage ratio of water content in the fresh sample to the water content in the water-saturated sample.

Statistical Analysis

We analyzed the statistical significance of differences in stilbene content (i.e. secondary ion peak counts and the relative ion intensities of estimated stilbene compounds to total ion counts) between different cell types (i.e. axial parenchyma and their intermediate spaces, which were composed of sieve cells and ray parenchyma) and between frozen-hydrated and freeze-dried specimens by ANOVA (IBM SPSS Statistics 22; IBM) after first testing the assumption of homogeneity of variances. When equal variances could not be assumed, Welch tests were used for comparisons. The relationship of the radial patterns of phloem chemical properties with water content and cellular volume ratios was analyzed by fitting power law, in addition to linear and exponential functions, to the measured data.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Positive TOF-SIMS spectra of authentic stilbenes under dry conditions.

• Supplemental Figure S2. Positive TOF-SIMS images at the radial-longitudinal surface of older Norway spruce phloem.

• Supplemental Figure S3. ROIs were manually isolated from the TOF-SIMS images throughout the phloem and bark using total ion images and the images of the peaks at m/z 245.

• Supplemental Figure S4. Relative ion intensities of stilbene compounds at m/z 229 (piceid), 245 (astringin), and 259 (isorhapontin) detected by TOF-SIMS from the ROIs.

• Supplemental Figure S5. µCT images of the phloem layers from the xylem ring border, cambium, and noncollapsed phloem.

• Supplemental Figure S6. Phloem cell volume ratios of younger Norway spruce from the cambium toward the outermost bark.

• Supplemental Figure S7. 3D tomography scans of the phloem in three cardinal slices and the resulting 3D segmentation of the axial phloem parenchyma of Norway spruce.

• Supplemental Figure S8. Interrelationship between the cell type-specific volume ratios of the ray and axial parenchyma within the phloem of older and younger Norway spruce.

• Supplemental Figure S9. Relative water content and the amount of total soluble sugars within the phloem as a function of cell type-specific volume ratios of the rays and different types of axial parenchyma.

• Supplemental Figure S10. Total amounts of stilbene glucosides and soluble sugars as a function of the total cell volume of axial phloem parenchyma within phloem tissue in younger and older trees.

Glossary

LMD

laser microdissection

cryo-TOF-SIMS/SEM

time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle

TOF-SIMS

time-of-flight secondary ion mass spectrometry

cryo-SEM

scanning electron microscopy connected with a cryo-shuttle

MALDI-MS

matrix-assisted laser desorption/ionization mass spectrometry

cryo-TOF-SIMS

time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle

GC-FID

gas chromatography with flame-ionization detection

GC-MS

gas chromatography-mass spectrometry

3D

three-dimensional

ROI

region of interest