Comparative studies of bark structure, lacquer yield and urushiol content of cultivated Toxicodendron vernicifluum varieties

Abstract

The bark structure and major chemical composition of raw lacquer in four cultivated varieties of the lacquer tree, Toxicodendron vernicifluum (Stokes) F. A. Barkley, were characterized using the paraffin section technique and high-performance liquid chromatography, respectively. The four varieties had roughly similar bark structures; however, they differed in bark thickness, raw lacquer yields and urushiol homologue content. Laticiferous canal diameter and bark thickness were positively correlated with raw lacquer yields and triene-urushiol content. Our data suggest that bark structure, raw lacquer yield and urushiol homologue content are useful indicators for distinguishing among these four varieties.

Keywords:

• Toxicodendron vernicifluum (Stokes) F. A. Barkley
• lacquer tree
• anatomy
• raw lacquer
• lacquer hydroxybenzene

Introduction

Lacquer trees, Toxicodendron vernicifluum (Stokes) F. A. Barkley (also known as sumac), are widely distributed in East and South East Asia. Some species include the Chinese–Japanese lacquer tree (T. vernicifluum), Japanese wax tree (Toxicodendron succedaneum), poison oak (Toxicodendron toxicarium and Toxicodendron diversilobum), poison sumac (Toxicodendron vernix), cashew nut (Anacardium occidentale) and poison ivy (Toxicodendron radicans and Toxicodendron rydbergii). These species belong to the family Anacardiaceae and have been introduced into parts of Europe, Australia and New Zealand, where they have caused allergic contact dermatitis (Rademaker & Dunffill 1995; Derraik 2007). Raw lacquer (also known as oriental lacquer) is the sap obtained by tapping lacquer trees (Niimura et al. 1996; Niimura & Miyakoshi 2006). It has long been used as a coating and painting material in China, Japan, Thailand, Vietnam and the Korean Peninsula because of its excellent characteristics, such as water resistance, anti-oxidation and corrosion resistance (Webb 2000; Zhang et al. 2007). A total of c. 100 lacquer tree varieties exist and most of them have been cultivated from the Chinese–Japanese lacquer tree (Wei et al. 2010), a species mainly found in East Asia (Nie et al. 2009).

During past decades, most studies on T. vernicifluum have focused on the morphogenesis and development of laticiferous canals (Zhao & Hu 1985, 1990) and the chemical composition of raw lacquer (Hatada et al. 1994; Hong et al. 1999). Laticiferous canals are mainly present in the phloem of lacquer trees (Zhao & Hu 1985), wherein raw lacquer is mostly synthesized and stored during their mature stage. Furthermore, the peripheral tissues are involved in synthesizing the precursors for raw lacquer (Zhao & Hu 1990). In the tree stem, most mature laticiferous canals are found in the non-functional phloem area. Raw lacquer mainly consists of urushiol, lacquer polysaccharides, lacquer enzymes and water. Among these, urushiol is the major effective component and is responsible for the utility of raw lacquer (Hatada et al. 1994). Thus, its content has been considered as an indicator of raw lacquer quality. However, to the best of our knowledge, no report has been made available on the association of bark structure with raw lacquer yield and urushiol homologue content (or other chemical components).

This study specifically aims to: (1) examine the correlation between the bark structure of the lacquer tree, raw lacquer yield and its major chemical components; and (2) provide references for varietal identification and cultivation, as well as the improvement of raw lacquer collecting technique.

Materials and methods

Sampling

Four cultivated varieties of T. vernicifluum were selected from their major production area in Shaanxi Province (China), i.e. Dahongpao, Gaobachi, Huoyanzi and Qieketou; all sampled trees were planted in 1994. In August 2009, 3 cm×3 cm bark specimens were collected from the main trunk (c. 1.5 m above the ground) of each variety. Five bark specimens from each variety were collected from five trees, respectively, and fixed with FAA fixation solution (90 mL 70% ethanol, 5 mL glacial acetic acid and 5 mL formalin). All voucher specimens (No: 200908001-004, Collector: Caiqin Liu) were stored in the Herbarium of the College of Life Sciences, Northwest University (Xi'an, China).

Subsequently, raw lacquer was collected from each tree using the traditional manual tapping method. First, a ‘V’ shape or sidelong kerf was tapped into the bark of the lacquer tree trunk using a knife. The ‘V’-shape kerf was c. 10 cm long with an angle of c. 60° (Fig. 1A), whereas the sidelong kerf has a length of c. 5 cm (Fig. 1B). ‘V’-shaped kerfs were tapped on the main trunk, whereas sidelong kerfs were tapped on the branch of sample trees bacause the diameter of the branch was not enough for ‘V’-shaped kerfs. The numbers of kerfs were same on each lacquer tree, and five trees of each variety were tapped in this study. Second, a shell (Fig. 1C) or folded leaf (Fig. 1D) was inserted into the bottom of the cut in a concave-up orientation to collect the raw lacquer that flowed from the wounded lacquer tree bark.

Figure 1  Manual tapping method for raw lacquer. A, Manual tapping method and ‘V’-shaped kerf. B, Sidelong kerf. C, Raw lacquer collecting in a shell (SH). D, Raw lacquer collecting in a folded leaf (FL).

Histological observation

Bark specimens were fixed with FAA fixation solution for 7 days, and then softened for 60 days in softener (50 mL 70% ethanol, 50 mL glycerol). The tissues were then sequentially dehydrated at room temperature in 70, 85, 95 and 100% ethanol (120 min each step), vitrified with a gradient from 100% ethanol to 100% xylene, infiltrated and embedded in paraffin. The sections (10 µm) were obtained from a microtome (Leica RM2135, Germany) and subsequently mounted on microscope slides. The mounted sections were double-stained with Safranin O and Fast Green FeF. The stained tissues were observed and photographed under a light microscope (Leica DC300F, Germany). The laticiferous canal diameter (µm) was measured using the Motic Images Plus 2.0 software. The summation of canals in unit area (mm²) was counted and the data were analysed with Statistica 2.0 software.

Raw lacquer yield of different cultivated varieties

Raw lacquer was collected using the traditional manual tapping method. The raw lacquer stopped flowing c. 20 h after tapping because the laticiferous canals become plugged. Under such conditions, the kerf was cut in the same way on each side to allow the raw lacquer to flow again. This process can be repeated 25 times on each kerf for raw lacquer collection during the tapping season (June to September). The raw lacquer collected for each variety during the tapping season was recorded as the annual yield.

Urushiol homologue content in raw lacquer

Raw lacquer (1.00 g) was initially suspended in 40 mL of methanol to extract urushiol. The suspension aliquot was chromatographed using a silica gel column (15 cm long and 1.5 cm in diameter). The eluent was ethyl acetate and petroleum ether (60:40, v/v). The eluate was collected using a fraction collector, and then concentrated under the reducing pressure condition. The residue was suspended with methanol to a final volume of 50 mL. Finally, the solution was diluted 25 times with methyl alcohol and filtered using 0.22 µm millipore filter. The resuspension was used as the sample for the subsequent analysis.

Resuspension aliquots (10 µL) were injected into the column (Diamonsil C₁₈, 5 µm, 4.6 mm×250 mm) of the HPLC system (SHIMADZU, LC-10ATvp, Japan), and the column temperature was maintained at 35 °C with detection at 225 nm. The HPLC mobile phase consisted of methanol (83%) and water (17%) with a flow rate of 0.6 mL min⁻¹ to isolate and collect urushiol homologue contents in raw lacquer.

Results

Comparison of secondary phloem structure among cultivated varieties of T. vernicifluum

The bark specimens of four cultivated varieties exhibited similar secondary phloem and periderm structures (Fig. 2). The secondary phloem can be divided into functional and non-functional parts. The functional phloem was produced in the same year, whereas the non-functional phloem found outside the functional phloem was formed in previous years. The secondary phloem comprises sieve tubes, parenchyma cells, stone cell clusters, laticiferous canals and phloem rays (Fig. 2).

Figure 2  Lenticular characteristics of bark structure among diverse varieties. A, Dahongpao. B, Gaobachi. C, Huoyanzi. D, Qieketou. SC, Stone cell; FP, functional phloem; C, cambium; PR, phloem ray; PE, periderm. Scale bar=250 µm.

The secondary phloem structures showed similar arrangements in the four varieties (Figs 3A to 3D). Sieve tubes, companion cells and parenchyma cells were disposed as strips wherein the components were arranged alternately. The parenchyma cells had one or two layers of sieve tubes at regular intervals for every one to three layers; this arrangement was typical in the functional phloem. In the non-functional phloem, sieve tubes were crushed by parenchyma cells, where the strip-arrangement in the non-functional phloem was atypical. Laticiferous canals were distributed between the phloem rays in a radial direction. The laticiferous canals were encircled by secretory cells enclosed by two or three layers of sheath cells (Fig. 3E). Stone cells were present only in the non-functional phloem, where several stone cells assembled as clusters (Fig. 3F).

Figure 3  Lenticular characteristics of secondary phloem structure among diverse varieties and the structure of laticiferous canals and stone cells. A, Secondary phloem structure of Dahongpao. B, Gaobachi. C, Huoyanzi. D, Qieketou. E, Structure of laticiferous canal. F, Structure of stone cell cluster. SE, secretory cell; SH, sheath cell; PR, phloem ray; SC, stone cell. Scale=100 µm (A–D), 25 µm (E), 80 µm (F).

The quantity of laticiferous canals per unit area (mm²) showed significant differences among the cultivated varieties (Table 1). The laticiferous canal diameters were higher in the Dahongpao variety than in the other three varieties. The Dahongpao variety exhibited a thicker bark, suggesting the occurrence of large interspaces for raw lacquer production and excretion.

Table 1  Comparison of the structure of secondary phloem among four cultivated varieties of T. vernicifluum.

	Variety
Index	Dahongpao	Gaobachi	Huoyanzi	Qieketou
Average diameter of laticiferous canals (µm)	259.7^a±3.99	152.6^b±2.65	111.2^b±1.95	102.4^b±1.30
Quantity of laticiferous canals (10 mm^−2)	28.3^ab±0.498	32.2^a±2.307	27.1^ab±0.711	25.4^b±2.108
Average thickness of bark (cm)	1.16^a±0.075	0.80^b±0.062	0.50^c±0.071	0.44^c±0.035
Phloem ray lines (mm^−2)	1–4.41	1–4.03	1–3.22	1–3.61
Quantity of phloem rays (mm^−2)	4.44^a±0.178	4.58^a±0.293	4.44^a±0.151	4.98^a±0.246
Note: Results are given as mean±standard error. Different superscript letters in the same row are used to indicate significant differences, p<0.05.

Annual yield of raw lacquer

The annual raw lacquer yields for the four T. vernicifluum varieties were estimated and ranked from high to low as follows: Dahongpao=Gaobachi > Huoyanzi > Qieketou (Table 2).

Table 2  Annual yield of raw lacquer in the four cultivated T. vernicifluum varieties.

	Variety
Index	Dahongpao	Gaobachi	Huoyanzi	Qieketou
Average yield of one time tapping (g)	8.95^a±0.54	8.76^a±0.43	3.44^b±0.32	1.90^c±0.33
Average annual yield (g)	223.79^a±4.75	219.04^a±5.34	85.89^b±3.18	47.38^c±3.20
Note: Results are given as mean±standard error. Different superscript letterss in the same row are used to indicate significant differences, p<0.05. Five trees of each variety were used in the experiment.

Comparison of urushiol homologue content among different varieties

Seven urushiol homologue components were detected from the samples of the four varieties. Five urushiol homologue chemical compositions were identified using HPLC (Kim et al., 2003). The triene-urushiol content was the highest among all varieties (88.3–93.4%), followed by monoene-urushiol (5.0–5.8%). The lowest homologue was diene-urushiol (0.5–0.9%). Five identified urushiol homologues comprised the major proportion (94.8–98.0%) of the total urushiol content. The minor proportion of unidentified urushiols consisted of only 2.0–5.2% of the total content (Table 3).

Table 3  Proportions of various urushiols of raw lacquer in different T. vernicifluum varieties.

Variety	T-urushiol (%)	D-urushiol (%)	M-urushiol (%)	Un-urushiol (%)
Dahongpao	92.87^a±0.10	0.48^b±0.10	5.63^a±0.13	1.01^b±0.06
Gaobaochi	93.36^a±0.07	0.60^b±0.09	4.99^b±0.08	1.05^b±0.02
Huoyanzi	90.61^b±0.09	0.88^a±0.13	5.64^a±0.03	2.86^b±0.04
Qieketou	88.31^b±0.12	0.71^a±0.11	5.78^a±0.14	5.23^a±0.11
Note: Results are given as mean±standard error. Different superscript letterss in the same row are used to indicate significant differences, p<0.05. Five trees of each variety were used in the experiment.

Table 3 shows the different proportions of the three known urushiol homologues among the four varieties. These varieties can be divided into two groups: Dahongpao and Gaobaochi with >91% triene-urushiol content, and Huoyanziand Qieketou with a lower content. The annual raw lacquer yield was also significantly higher in the first of these two groups (Table 2). The raw lacquer yield pattern was also different from that of the triene-urushiol content (Table 3).

The annual raw lacquer yield was positively correlated with triene-urushiol content; however, it was negatively correlated with the of the other urushiol homologue contents. In nature, the mixture of urushiol derivatives co-exists in the lacquer because urushiol polymerizes when catalysed by laccase, and finally forms an extremely hard and insoluble film (Niimura and Miyakoshi, 2006). The test of urushiol homologue contents indicated that triene-urushiol is the main chemical component of raw lacquer, thus, it is the basic ingredient of the raw lacquer that transformed from solid to film. The proportion of triene-urushiol that exceeded 90% should be considered as an index for the selection of lacquer tree variety. The correlation between triene-urushiol and other urushiols suggest that an inverse relationship existed between the synthesis and accumulation of urushiol homologue.

The correlation analysis of bark components and raw lacquer outputs is shown in Table 4. The annual raw lacquer yield was significantly and positively correlated with the diameter and quantity of laticiferous canals as well as bark thickness, with the correlation coefficients of 0.8036, 0.8086 and 0.8946, respectively. The data suggest that the high quantity and large diameters of laticiferous canals as well as the thicker barks may be a precondition for high lacquer yields. In addition, these traits can be used to distinguish cultivated lacquer tree varieties. The diameters of sieve tubes and number of stone cell clusters were also positively correlated with raw lacquer yield, exhibiting lower correlation coefficients. The study confirmed that the quantity of phloem rays correlated negatively with raw lacquer yield.

Table 4  Correlations among bark components, and annual yield of raw lacquer and T-urushiol content.

Bark components	Quantity of canals	Average diameter of canals	Quantity of phloem rays	Average thickness of bark
Annual yield	0.8086	0.8036	−0.5996	0.8946
T-urushiol content	0.8735	0.6917	−0.427	0.7995

Discussion

Varieties of cultivated plants are usually distinguished by their external features, harvest quality and yield (Rouphael & Colla, 2005). In previous studies, tree crown sizes, reproductive organ characteristics and the morphology of different organs have been considered as the index to differentiate lacquer tree varieties. Recently, amplified fragment length polymorphism molecular markers have also been used as novel indicators to identify highly yielding cultivated lacquer tree varieties (Wei et al. 2010). Yield and quality are considered the main economic characters for the raw lacquer production in T. vernicifluum. Previous studies have mainly focused on the economic characters for cultivated lacquer tree varieties (Yamauchi et al. 1982; Fu et al. 2007). These studies were carried out separately; thus, information on the yield–quality relationship has not yet been fully elucidated. Therefore, a comparative anatomical approach on bark structures with economic characters and phytochemistry was used in the present study to resolve this drawback.

Our study revealed that raw lacquer tree varieties were similar in bark structure; however, they differed in laticiferous canal quantity per unit area, laticiferous canal diameter, bark thickness and other aspects. The differences were consistent among these varieties and may be used as an anatomic classification index for lacquer tree varieties, which were similar to the findings in Hevea brasiliensis (Gomez 1982). Latex vessels are rubber-synthesizing structures present in the bark of a rubber plant; the high-yield variety has larger quantity than the ordinary varieties (Hao et al. 1980). Systemic studies have shown that the quantity and diameter of latex vessels for several rubber plants had diverse yields. Furthermore, the results showed that the quantity and diameter of latex vessels of the high-yield varieties were higher than those in the middle and low-yield varieties; thus, the diameter of latex vessels exhibits a significant positively correlation coefficient with rubber yield (Ashplant 1928).

Commercial resin is tapped from pine trees using a method similar to that applied for harvesting raw lacquer. The oleoresin from pine trees is synthesized and stored in mature laticifers (Rodrigues et al. 2008). Laticifers are distributed within all the pine organs and oleoresin is produced in the secondary xylem. A previous study has shown that the number, diameter, depth and density of the inner and outer resin canal measurements differ significantly among pine species (Boucher et al. 2001). External features and anatomic indicators were combined to select pine tree varieties with high oleoresin yield and have proven to provide better results than the forest-type method (Z Wang & Huang 1993). Therefore, these characters likely function as an important index for distinguishing cultivated species using the combined biological and economic characters in T. vernicifluum variety classification.

Laticiferous canals are mainly formed and extended in the functional phloem; however, most mature laticiferous canals are present in the non-functional phloem (Q Wang & Hu 1979). The microstructure of laticiferous canals in the lacquer tree showed that raw lacquer is synthesized in the secretory cells surrounded by the canals. Raw lacquer is deposited in the lumen of laticiferous canals through the cell walls by plasmodesmata or diffusion penetration. Furthermore, the surrounding tissues of laticiferous canals are important in the synthesis of raw lacquer precursor substances (Zhao & Hu 1990). Differences in the quantity and diameter of laticiferous canals from the bark thickness of lacquer tree varieties can affect the ability to produce and store raw lacquer. Sieve tubes are the channels that transport organic compounds inside plants. These compounds may serve as precursors for raw lacquer components, and the average diameter of sieve tubes may affect the ability to produce or transport raw lacquer.

Our results revealed that the relationship between the bark structure and raw lacquer output of lacquer trees correlated positively in terms of the number of laticiferous canals, the average diameter of laticiferous canals and tubes, and the average bark thickness; the Dahongpao variety had the highest annual raw lacquer yield because it has the highest average diameter of laticiferous canals and average bark thickness. The quantity and average diameter of laticiferous canals and tubes, as well as average of bark thickness, are likely to be the key factors for determining raw lacquer yield. Thus, these characteristics should be considered as anatomical index for the selection of lacquer tree varieties.

The chemical compositions and proportions of raw lacquer are listed as follows: urushiol (60–70%), lacquer polysaccharides (5–7%), lacquer enzymes (<1.0%) and water (20–30%) (Hatada et al. 1994). Urushiol is a mixture of catechol derivatives and contains monoene-, diene- and triene-urushiol, with triene-urushiol as the major component (Zhang et al. 2007). Triene-urushiol in raw lacquer is the basic reagent used in its transformation from a solid to the basic skeleton of raw lacquer film. This transformation process can directly affect the performance of lacquer film burnish, adherence and toughness (Lu et al. 2004).

Laticiferous canals are specialized cells that contain latex (Fahn 1979) and have been found to take part in the biosynthesis of specialized metabolites in several plants, such as H. brasiliensis (van Beilen & Poirier 2007). In the present work, the structural support of phloem, laticiferous canals and raw lacquer contents were analysed. The results showed that these structural factors exhibited a relationship with the annual raw lacquer yields and urushiol content. Therefore, these findings should be considered for raw lacquer production and for the further investigation of lacquer tree varieties in support of appraisal and selection.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No.: 30870136). The authors would like thank Dr Yen-Kuang Ho and Dr Zeng-qiang Qian for their assistance in preparing the manuscript.