TISSUE LOCALIZATION OF THE CONDENSED TANNINS IN THE LEAVES OF THE BLACK CHERRY, PRUNUS SEROTINA EHRH.

Aleksandra Halarewicz
Department of Botany and Plant Ecology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

Phenolic compounds found in the black cherry, Prunus serotina Ehrh., seem to enrich this plant's biochemical defense. The aim of the study was to analyze the tannin content in this invasive tree species. The condensed tannin content in the leaves of P. serotina was determined using vanillin method and it was demonstrated that it changed seasonally, reaching the highest values in July. The results of the histological study, performed in order to observe the tissue localization of the secondary metabolites, suggest that tannin compounds are accumulated mainly in the vacuoles of the upper epidermis and the palisade parenchyma. The observation of the main leaf veins also reveals frequent presence of tannin cells.

Key words: condensed tannins, black cherry, Prunus serotina, leaf anatomy.

INTRODUCTION

Introduced alien plants, once released from under the pressure of their natural enemies: pathogens, herbivores or other competitor plants, spread easily over their new range [10, 21]. The mechanisms responsible for the interactions between the newcomers and their new enemies are still poorly understood [11].

The most recent research results suggest that the invasive character of the introduced plants is associated with their more potent biochemical defence compared to the species representing the native flora (novel weapons hypothesis) [4, 5, 18].

Black cherry, Prunus serotina Ehrh., is an example of the North American plant which owes its successful march through the European forest phytocenoses to, inter alia, the low number of enemy species, and in particular - of the soil pathogens [15]. It seems that the most essential line of this plant's biochemical defence is its ability to synthesise cyanogenic glucosides: prunasin and amygdalin, much the same way the other Prunus spp. do [1, 17, 19]. The presence of these metabolites in the plant tissues makes it rarely infested by generalist phytophages [9] as it is only the specialist phytophages that are equipped with biochemical mechanisms to inhibit cyanogenesis in plant or to detoxicate the cyanogenic glucosides contained in their diet [6, 7].

There is only few research articles discussing phenolics in the black cherry. A register of flavonoids is included in the work of Olszewska [13]. Flavonoids from extracts of wood and bark has been shown to have an inhibitory effect on polyphagous pests [14]. Furthermore, attention has been paid in the recent years to the considerably enhanced content of the condensed tannins in the leaves of P. serotina, compared to that in the native Prunus padus and other understory shrub species [22]. Also, the knowledge of the histological localization of condensed tannins might provide clues about their physiological function in plant [12].

The aim of the present work was to determine the content and tissue localization of the condensed tannins in the leaves of Prunus serotina.

MATERIAL AND METHODS

The study was carried out in 2010 within the Wołów Forest Division (Lower Silesia). Leaf samples were collected from nine selected trees of Prunus serotina (Ehrh.) in fresh mixed deciduous forest (51°18' N, 16°35' E). One branch in a shaded portion of each tree, northerly directed and situated ca 120 cm above ground level was selected and marked. About 30 leaves were sampled per tree at the beginning of June, July and August, each time from the same, marked branch. Five leaf blades from each tree were used to prepare the anatomical slides. The other material was immediately frozen and stored at -20°C until the time of chemical analysis.

The content of condensed tannins, after extraction from lyophilized leaves with methanol, was determined by vanillin method [2]. Commercial sample of (+)-catechin hydrate (Aldrich) was used as the standard. The measurements were carried out in 3 replicates for each sample of the plant material. Results were expressed as mg (+)-catechin per 1 g of the leaf dry weight.

In order to prepare the anatomical slides, samples measuring 3 × 5 mm were removed from the interveinal zone and veins of the midblade region of each leaf. For light microscopic detection of tannins, samples were fixed at room temperature in a mixture containing 5 % formalin and 10 % ferrous sulphate for 24 h [8]. Next, the samples were fixed in glutaraldehyde 2.5 % with paraformaldehyde 4%, in a phosphate buffer at pH 7.2, for 20 h at 5 °C. Then they were rised twice, each time for 10 min in the same buffer. The samples were further postfixed in OsO₄ 2 %, in the same buffer for 2 h and later dehydrated in ethanol series and embedded in an epoxy resin. Tannins stain brownish-orange. For light microscopic observations vertical 3-µm thick sections were cut on ultramicrotome and examined with microscope Axioscop 2 plus (Zeiss). From each one of the leaves 3 cross-sections were examined. For each cross-section the number of tannin-containing cells was recorded per 50 cells of consecutive leaf layers. In this way, the percentage of the tannin-containing cells in the analysed tissues could be determined.

The data were analysed using Kruskall-Wallis ANOVA in Statistica 9.0 package (StatSoft), at p<0.05.

RESULTS AND DISCUSSION

The smallest content of condensed tannins was found in the leaves of Prunus serotina at the beginning of June, when it amounted to the average of 29.13 mg (+)-catechin per 1g of the lyophilized leaves (Tab.). At the beginning of July the nearly 3-fold increase in the concentration of the condensed tannins was recorded, up to the average of 114.94 mg per 1g of the leaves. In August, the content of the examined metabolites decreased to the average level of 94.08 mg per 1g of the leaves. The difference in tannin concentrations between June and the other two sampling dates was statistically significant. Therefore the obtained data conform to the information on the seasonal accumulation of phenolic compounds in plants, which has been also observed in the leaves of other tree species [16]. Furthermore, in case of P. serotina, the relatively high content of condensed tannins [22] suggests the presence of the robust constitutive (preformed) defence and may be a premise to confirm the novel weapons hypothesis for this alien plant. However, as much as the role of induced phenolics in the resistance of trees to pathogens is evident, the effect of constitutive phenolics still remains unclear [20].

Differences in tissue localization of the tannin compounds in the leaves of P. serotina were determined using light microscopy. Cells containing tannins were practically absent from samples collected in June (Tab., Fig.1.), whereas in the leaves from July and August tannins were mostly located in the upper epidermis, visible there as small clusters (Fig. 2.). Tannins were absent from the cells of the lower epidermis.

Figure 1. Cross-section through the leaf of Prunus serotina, June 2010. Epidermis and mesophyll cells without droplet of tannins. Bar 30 µm.

Figure 2. Cross-section through the leaf of Prunus serotina, July 2010. The arrows indicate cells filled with tannins. Bar 30 µm.

In the palisade mesophyll the investigated secondary metabolites were observed sporadically and only in a number of single cells. However, in spongy mesophyll cells, tannins cluster together forming droplets in the vacuoles. Cells containing tannins occur side by side with those that are apparently tannin-free. It therefore indicates that only specialized cells in the mesophyll tissue show the ability to accumulate tannins. Compared to that, in the leaves of beech tree (Fagus sylvatica) tannins are accumulated in the upper epidermis and in the palisade mesophyll and at a later stage they are retranslocated to the wall of the epidermal cells [3].

In the vascular bundle of P. serotina leaves, cells with single large vacuole filled with tannins were noticed too (Fig. 3.). Under such conditions cell cytoplasm degenerates and the organelles may disappear [3]. The presence of tannin cells in xylem and phloem tissue suggests that probably this tannin compounds are actively involved in transport. Mosjidis et al. [12] conclude that phenolics, including condensed tannins, my play an active role in some of the physiological processes or they could represent a form in which plant stores its excess photosynthates.

Table 3. Cross-section through the main vein of the leaf blade, July 2010. Droplets of tannins in cells stained brownish-orange. Bar 20 µm.

CONCLUSIONS

1. Young leaves of Prunus serotina have low concentration of condensed tannins. During their maturation a rapid increase in the concentration of these polyphenols is observed.
2. Tannin compounds are mostly deposited in the upper epidermal cells.
3. The largest associations of tannin droplets in tissues of P. serotina were found in the specialized cells of spongy parenchyma and in the vascular bundles.

Acknowledgements

The author would like to thank dr Anna Sokół-Łętowska and dr Alicja Kucharska, both from the Department of Fruit, Vegetable and Cereals Technology in Wrocław University of Environmental and Life Sciences, for their performing the biochemical determination of (+)-catechin content in the leaves.

REFERENCES

Accepted for print: 16.12.2011

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