Astringin and isorhapontin distribution in Sitka spruce trees

0031-9422/91 $3.00+0.00 0 1991PergamonPressplc

Vol. 30,No. 7, pp.2183-2189, 1991 Phytochemistry, Printedin GreatBritain.

ASTRINGIN AND ISORHAPONTIN DISTRIBUTION SITKA SPRUCE TREES CLAUDIA D. TOSCANO UNDERWOOD

IN

and RAYMOND B. PEARCE

Oxford Forestry Institute, Department of Plant Sciences, University of Oxford, South’Parks Road, Oxford OX1 3RB, U.K. (Received in revised form 14 December 1990)

Key Word Index-Picea astringin; isorhapontin.

sitchenti; Pinaceae; Sitka spruce; distribution, antifungal compounds; stilbene glucosides;

Abstract-High levels of the stilbene glucosides astringin and isorhapontin, previously identified as major constitutive antifungal compounds in Sitka spruce (Piceu sitchensis), were present in mature bark tissues. Levels in needles, sapwood and feeder roots were relatively low. Significant amounts of the aglycones astringenin and isorhapontigenin were not found in healthy spruce tissues. In the bark the stilbenes were present at high levels in certain parenchyma cells: the relative proportions of the two glucosides, as well as absolute amounts varied from the cambium to the pcriderm.

INTRODUCTION

The stilbene glucosides astringin (5,3’,4’-trihydroxystilbene-3-/I-D-glucoside) and isorhapontin (S$-dihydroxy3’-methoxystilbene-3-B_D-glucoside) (included in rhaponticin sensu la@), have been identified as major constitutive antifungal compounds in the bark of Sitka spruce [Picea sitckensis (Bong.) Carr.] [l]. For such constitutive compounds to be effective in defence, they must be present in the appropriate tissues at concentrations high enough to be biologically active. A knowledge of the distribution of these stilbene glucosides within the plant is thus necessary for understanding their role in its protection. Also, this information is a prerequisite for optimizing sampling procedures for use in studies of these compounds. Although there have been a number of studies of the distribution of monoterpenes in conifer species [eg. 231 there has been relatively little comparable work on the distribution of polyphenols in general, and stilbenes in particular. Some data on the distribution of these compounds in Sitka spruce trees has been published [4, 51, showing that mature bark tissues contain very high levels of stilbenes. In this paper we report a more detailed investigation of the distribution of astringin and isorhapontin in juvenile and mature Sitka spruce trees in both winter and summer.

tended to complex with trace metal ions in the solvent [cf. 63, resulting in unreliable quantification. Crude methanol extracts of spruce tissues could be assayed directly; detection of stilbenes by their UV absorbance at 325 nm eliminated interference from other compounds present in

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ASTRINGIN

Id,*-

lSOl?HAFONTlN ASTRINGENIN

RESULTS AND DIScUSSION

HPLC assay for

stilbenes

The HPLC analysis of methanol extracts of spruce bark tissues gave good resolution of the stilbene glucosides astringin and isorhapontin, and of their respective aglycones astringenin and isorhapontigenin (Fig. 1). These had R,s of ca 7.7, 11.3, 12.4 and 16.5 min respectively. Peak area calibration curves were linear for these compounds. Incorporation of EDTA at a minimum of 10 mgll’ in the aqueous solvent proved essential for reliable HPLC of astringin: in its absence the astringin

Fig. 1. HPLC chromatogram of an enxymic hydrolysate of astringin and isorhapontin (partially purified from a spruce bark extract Cl]), less than 30 min after addition of fl-n-glucoside glucohydrolase, when both stilbene glucosides and their aglycones are present.

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C. D.

TOSCANO UNDERWOODand R. B. PEARCE

the extracts (particularly from needles). This technique minimized the opportunities for the degradation of these rather labile compounds during assay by reducing the number of handling operations involved and by using a fully end-capped reverse phase silica HPLC column. Local distribution

ofstilbenes

within spruce bark

The variation in astringin and isorhapontin content in 15 mm bark plugs taken from the buttress roots of three mature Sitka spruce trees in Bagley Wood, Oxfordshire, U.K. was greater between trees than between replicated plugs from a single tree (P < 0.005) Detailed studies of the genotypic variation in stilbene glucoside levels in Sitka spruce are reported elsewhere [7]). The variation in stilbene levels was greater within trees than within bark plugs quartered prior to extraction (P < 0.001; P < 0.05 for astringin and isorhapontin, respectively). Absolute levels and relative proportions of astringin and isorhapontin in bark tissue varied according to their radial position (P
Fig. 2. Radial distribution of stilhene glucosides in the root bark of a 62-year-old Sitka spruce tree from Bagley Wood, Oxfordshire. Mean stilhene glucoside contents were obtained from six replicates of serial tangential 200 pm sections. Vertical bars represent s.e.m.

tion. The increase in stilbene concentration towards the outside of the bark may reflect tissue volume changes. Studies with Picea mariana [8] showed that sieve cell volume decreased gradually from the cambium towards the outer dead phloem, as a result of the collapse of these elements. In contrast the volume of longitudinal parenchyma. shown in the present study to contain the stilbenes, increased. In view of the general similarity in bark structure in spruce species [9], this probably applies also in Sitka spruce. However, the change in proportions of astringin and isorhapontin in the different bark tissues provides evidence that metabolic processes must operate also. Storage of the stilbene glucosides in the parenchyma cells is likely to be vacuolar. This location is suggested by

Fig. 3. Transverse section of bark tissues stained with diazotized o-tolidine, showing the distribution of stilbenecontaining cells in the phloem tissues.

Stilbenes in Sitka spruce the staining responses observed. Indeed the deposition of phenolic compounds in the vacuoles of cortical parenchyma cells has been reported in many species of woody and non-woody angiosperms [lO-123. Similar studies in gymnosperms are more limited, although tannins were detected in the vacuoles of callus and suspension culture cells of Pseudotsuga menziesii, Pinus taeda and Picea glauca [13].

On the basis of these results it was concluded that the replicated bark plug sampling procedure used in this study for measuring the stilbenes in secondary bark tissues provided an adequate measure of these compounds at the site sampled. Other studies have shown that levels of the two compounds in trees are genotypically determined [7]: the residual variation e.g. that found in this study within quartered bark plugs, may be attributable to structural inhomogenities, such as resin ducts, in the small bark samples extracted. Stilbene distribution in Sitka spruce trees

Determinations of astringin and isorhapontin levels in the various tissues of a five-year-old Sitka spruce tree are given in Table 1. Astringin and isorhapontin levels, which were low in needles, did not differ significantly with needle age or position. In bark tissues, however, levels of both compounds increased significantly with age, both in branches in the crown (P
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sampled in spring or late summer showed broadly similar patterns of stilbene distribution in the crown and root tissues (Tables 2 and 3). This was essentially the same as that seen in young trees, with very high levels of stilbene glucosides in mature bark, lower levels in juvenile bark, and low levels in the needles and sapwood. No evidence for free stilbene aglycones was obtained for any healthy tissues. The distribution of stilbenes and their levels in the various tissues were similar in 62-year-old trees from Bagley Wood, Oxfordshire. The differences in overall stilbene glucoside content, and relative proportions of astringin and isorhapontin between the individual trees examined can be attributed to the high between-genotype variation in levels of these compounds found in Sitka spruce [7]. The results parallel those of Forrest [4], although he also reported the constitutive presence of the aglycones astringenin and isorhapontigenin in spruce tissues. This apparent difference may be the result of the different sample preparation and assay methods used in the two studies. In the present investigation samples were extracted with pure methanol, and crude extracts assayed by a single step HPLC method. In the earlier work the extracting solvent had a high water content (50% aq. MeOH and 25% aq. MeOH), which could have permitted some activity of endogenous plant glucosidases after cellular compartmentalization had been broken down, thus releasing the stilbcne aglycones. Also, stilbenes in the earlier work were measured after TLC purification on silica gel, which can itself cause their breakdown [ 141. Some evidence was obtained from these studies for seasonal variations in the distribution of stilbenes in the stem bark of mature trees. In trees felled in spring

Table 1. Stilbene contents of tissues in a five-year-old Sitka spruce tree Stilbe.necontent (mgg-’ fr. wt) Site

Tissue

Tissue age (yr)

Astringin

Isorhapontin

Stem*

Bark

Fibrous roots.7 Root tips? Thickened, translocating rootst Upper crown1

All All Bark

5 4 3 2 1 up to ca 1 juvenile 24

21.11 12.67 7.83 6.84 3.20 1.57 0.56 26.97

11.17 9.35 7.84 7.52 3.20 0.15 0.009 3.00

Bark

Needles Lower crowns

Bark Needles

2 1 current 1 current 2 1 current 1 current

*Mean of two replicates. tSingle measurement based on three pooled samples. $Mean of three replicates.

3.67 1.74 0.24 0.028 0.037 6.51 1.92 0.17 0.028 0.028

3.67 1.70 0.36 0.009 0.017 6.23 1.35 0.21 0.035 0.017

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C. D. TOSCANOUNDERWOOD and R. B. PEARCE Table 2. Stilbene contents of root and crown tissues in a 35-year-old Sitka spruce tree from Hundred Acres Wood, Amersham, felled in April Stilbene content (mgg-’

fr. wt)*

Site

Tissue

Tissue age (yr)

Astringin

Isorhapontin

Fibrous roots Secondary (buttress) roots

All Bark

up to ca 1 Mature

4.69 47.65

0.36 12.26

Wood Bark Wood Bark

Mature Mature Mature 4 2 1 4 2 1 4 4 2 1 4 2 1 4

0.25 59.57 0.14 17.40 8.54 5.01 0.21 0.15 0.10 0.91 10.32 4.81 3.28 0.094 0.13 0.12 0.40

0.069 26.82 0.087 7.22 3.58 2.78 0.061 0.052 0.096 0.061 4.55 2.60 2.26 0.044 0.052 0.035 0.096

Root collar Upper crown

Needles

Lower crown

Wood Bark

Needles

wood

*Values represent a single measurement from three pooled samples. Table 3. Stilbene contents of root and crown tissues in a 35-year-old Sitka spruce tree from Hundred Acres Wood, Amersham, felled in September Stilbene content (mg g- ’ ft. wt)* Site

Tissue

Tissue age (yr)

Astringin

Isorhapontin

Fibrous roots Secondary (buttress) roots Root collar

All Bark

up to ca 1 Mature

4.39 30.32

0.43 10.48

Wood Bark Wood Bark

Mature Mature Mature 3 1 current 3 1 current 3 3 1 current 3 1

0.27 30.40 0.52 13.03 5.56 1.80 0.15 0.037 0.047 0.037 9.32 2.06 1.27 0.037 0.047 0.047 0.051

4.15 11.09 0.24 3.92 3.96 3.27 0.061 0.035 0.000 0.009 5.06 1.61 0.94 0.026 0.035 0.017 0.10

Upper crown

Needles

Lower crown

Wood Bark

Needles

Wood

3

*Values represent a single measurement from three pooled samples.

astringin levels decreased with height, but no comparable height related changes in isorhapontin were evident (Fig. 4), although, in the tree figured, the isorhapontin levels were very high in the lower 2 m of the stem. For astringin the data fitted a quadratic regression model (P < 0.001).

In the bark of trees felled in late summer, astringin levels reached a maximum at a height of 3 m or more above ground level, levelling off and ultimately declining towards the top of the tree. The data fitted a quadratic regression model (P
Stilbenes in Sitka spruce

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Fig. 4. Astringin and isorhapontin contents of Sitka spruce stem bark along the height of a tree from Hundred Acres Wood, Amersham, determined in April.

Fig. 5. Astringin and isorhapontin contents of Sitka spruce sapwood along the height of a tree from Hundred Acres Wood, Amersham, determined in April.

with height (tree from Hundred Acres Wood) or remained essentially constant (tree from Bagley Wood). Data from more trees, sampled at additional times throughout the year would be required to clarify the trends observed. Most studies of seasonal variations in secondary metabolites in conifers have been concerned with terpenes. Evidence for seasonal variations in the volatile oil of young leaves of several Picea species has been obtained [lS-181, although no evidence for similar changes in the main stem oleoresin was obtained in Sitka spruce [S]. Such seasonal changes as may occur in stilbene glucosides are no greater than the between tree variation observed [7], and are of a magnitude unlikely to influence the disease resistance of these tissues (Toscano Underwood and Pearce, unpublished data). Stilbene glucoside levels in the sapwood of Sitka spruce trees were much lower than in the bark. In the trees from Hundred Acres Wood a decrease with height in the levels of both astringin and isorhapontin could be identified as a significant trend in the trees examined in spring (Fig 5) and in late summer. Significant height-related changes in either compound were not evident in the sapwood of trees from Bagley Wood. The high levels of astringin and isorhapontin, the principal constitutive antifungal compounds in Sitka spruce [l] in the older bark of trees may confer on these tissues enhanced resistance to potential pathogens. Disease damage to juvenile tissues, which comprise the outer part of the crown and root system is less serious than destruction of the essentially irreplaceable main stem or, to a lesser extent, major roots and branches. The integrity

of these, in the region of the cambium at least, is required for the continuing survival of all distal parts of the tree. The distribution of stilbenes in Sitka spruce contrasts with that known in pines. In the latter the major stilbenes, pinosylvin and pinosylvin 3-methyl ether, are normally present as aglycones. These are found in the xylem, either as a heartwood constituent Cl93 or induced as an apparently phytoalexin-like compound in reaction zones at the margin of wood decaying infections [20]. These compounds occur also in the needles [21, Pearce, R. B, unpublished data], but appear generally absent or are present only at very low levels in pine bark [Pearce, R. B, unpublished data]. Preliminary data [Toscano Underwood and Pearce, unpublished data] suggest that high stilbene levels are not induced in reaction zones in Sitka spruce, although this requires more rigorous confirmation. It is noteworthy that pines appear to be susceptible to more pathogens of the bark and cambial tissues than are spruces [see 22,231. Also, the root and butt rot pathogen Heterobasidion annosum is prone to attack the cambial and bark tissues in pines, causing tree death, whilst in spruces the wood may be extensively decayed without damage to the vital cambium [23]. Although the high levels of antifungal stilbenes in spruce bark have not been directly related to this apparently high general resistance, their involvement is an attractive possibility.

decreased

EXPERIMENTAL

Plant material. All Sitka spruce [Picea sitckansis (Bong.) Carr.] trees used in this study were of Queen Charlotte Island pro-

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C. D. TOSCANOUNDERWOOD and R. B. PEARCE

venance. Juvenile trees, 4-6-years-old, had been purchased as 2year-old plants (Woodland Improvement Nurseries, Huntley, Glou~stershire, U.K.) and grown on in large plastic pots (ca 20 cm) in open frames. 35-year-old trees, growing at Hundred Acres Wood, Amersham, Buckinghamshire, U.K. and 62-yearold trees from Bagley Wood, Oxfordshire, U.K. provided the mature plants used in this study. Sampling and extraction procedures. Bark samples from the main stem of juvenile plants were removed using small tubular stainless steel punches, 5 mm i.d. 1 mm wall thickness, cn 70 mm long, sharpened at one end. Other samples (roots, needles, bark from small branches) were cut from random locations on the plant, and the requisite tissues dissected out, Samples of bark and sapwood from mature trees were obtained using 15 mm i.d. stainless steel extractors, as previously described [i]. Samples for assay development and for examination of the local distribution of stilbenes within bark tissues were obtained from random sites on the lowest 1 m of the trunk, or from excavated buttress roots at distances of up to 1 m from the root collar. Trees for stilbene distribution studies were felled, and the bark and sapwood was sampled at intervals of 1 m from the root collar to the top of the tree. Three replicate samples were taken at each height. Samples of needles, and branch shoots were taken from three random positions in the upper and lower parts of the crown. Root samples were obtained from excavations at three random positions near the base of the sampled tree. Tissue samples (0.05-2.0 g) from juvenile trees were extracted immediately in MeOH (IO-20 ml; 4”; 24 hr, in the dark). Material from mature Sitka spruce trees was stored for not more than 24 hr at 4” before storage at -20” or extraction. All extractions were completed within 5 days of sampling The three replicate samples obtained for all sites on mature trees were pooled for extraction. Needles, fine roots and stripped bark from young shoots (0.05-2 g) were cut up finely and extracted in MeOH (20 ml) as above. Bark and sapwood plugs were extruded from the extractors; wood was separated from bark at the cambium, and the dead rhytidome tissue discarded. The living bark and sapwood samples were cut up and extracted as above. For studies of the local distribution of stilbenes in bark tissues, sampie plugs were quartered transversely and axially and the resultant pieces extracted separately. To examine the radial distribution of the stilbenes in bark, cambial and rhytidome tissues were manually removed from three replicate bark plugs, the remainder of which were cut into four tangential sections, 2OOpm thick, using a sliding microtome fitted with a freezing attachment. Corresponding tissues were pooled and extracted as described. The resultant MeOH extracts from al1 Sitka spruce tissues were stored (-20”, in the dark) until required for assay. Measurement ufstiibenes. Extracts were further diluted with MeOH as necessary before assay. Aliquots (20 4) were separated by reverse phase HPLC (Novapak Cl8 Radial-Pak cartridge column 10 cm x 8 mm i.d.) using a solvent mixt. of H,O, containing 10 mg I- 1EDTA (Solvent A) and MeOH (Solvent B). An elution gradient was used with an initial solvent ratio of 3: 1 (A:B), linearly changing to 13:7 after 3 min, then to 3:7 in a concave curve {Waters 600 gradient controller, curve 8) at 15 min. This increased linearly to 100% B at 17 min. and was mamtained at this composition for a further 5 min before returning to the initial solvent composition (via 100% A) over a total cycle (including equilibration at the initial conditions) of 40 min. A Row rate of 1.5 mlmin-’ was used, and detection was by absorbance at 325 nm. A Cl8 precolumn catndge (P-Bondapak) was used to protect the column. Stilbenes were quantilied on a peak area basis against an external, purified isorhapontin

standard. Astringin was estimated using a conversion factor based on the molar extinction coefficients of these two compounds, taken as 4.37 and 4.35 for astringin and isorhapontin, respectively. These represented the mean between mean published values [2426] and the values determined in an allied study Cl]. To calibrate the assay for the aglycones astringenin and isorhapontigenin a purified mixt. of the stilbene glucosides in Na citrate buffer pH 5.0 was hydrolysed with a /J-D-ghtcoside glucohydrolase (15 mgml-‘) (30 min, 37”). Freshly prepared hydrolysates were separated using the HPLC gradient system described. Stuc~st~cu~treatment. Statistical analysis of stilbene distribution data was by analysis of variance (ANOVAR). Staining for phenolic compounds. Stem lengths (cu 2 cm) from juvenile trees or bark plugs extracted from older trees were stained in bulk by immersion (with vacuum infiltration) in diazotized o-tolidine [20] for 24 hr, followed by a wash in H,O. Sections (20-3Opm) were cut from these stained bark ttssues using a sliding microtome, and were examined microscopically for the location of the red-brown insoluble deposits formed by reaction with phenolic compounds. Acknowledgements-The authors thank the Forestry Commission for making trees in the Hundred Acres Wood available for use in this research. They also thank Mr C. Wingad (Waters Chromatography) for his helpful suggestions regarding the HPLC methods used. C.D.T.W. acknowledges the receipt of a Brazilian Government (CAPES) scholarship. REFERENCES 1. Woodward, S. and Pearce, R. B. (1988) Physiol. Mol. Plant Pathol. 33, 127. 2. Roberts, D. R. (1970) Phytochemistry 9, 809. 3. Forrest, G. I. (1980) Can. J. For. Res. 10,452. 4. Forrest, G. I. (1975) Can. J. For. Res. 5, 26. 5. Forrest, G. I. (1975) Can. J. For. Rex 5, 38. 6. RiWreau-Gayon, P. (1972) Piant Phenotics. Oliver & Boyd, E~nburgh. 7. Toscano Underwood, C. D. and Pearce, R. B. (1991) Eur. J. For. Path. (submitted). 8. Brudermann, G. and Koran, 2. (1973) Can. J. Botany 51, 1649. 9. Chang, Y.-P. (1954) USDA Tech Bull No 1095. 86 pp. 10. Wardrop, A. B. and Cronshaw, J. (1962) Nature (Land.) 193, 90. 11. Mueller, W. C. and Beckman, C. H. (1976) Can J. Botany !I4, 2074. 12. Lynch, D. V. and Rivera, E. R, (1981) Bat. Gaz, 142,63. 13. Chafe, S. C. and Durzan, D. J. (1973) Pfunaa 113, 251. 14. Hart, J. H. (1981) clan. Rev. Phytopathol. 19,437. 15. Hrutfiord, B. F., Hopley, S. M. and Gara R. I. (1974) Phytochemistry 13, 2167. 16. Von Rudloff, E. (1972) Can. J. Botany 50, 1025. 17. Von Rudloff, E. (1975) Can. J. Botany 53, 2978. 18. Von Rudloff, E. (1975) Phytochemistry 14, 1695. 19. Erdtman, H., Frank, A. and Linsted, C. (1951) Swn. Pupperstidn. 54, 275. 20. Sham, L. (1967) Phytopat~~ogy S7, 1034.

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