Hydrogen Peroxide Induces Vessel Occlusions and Stimulates Sesquiterpenes Accumulation in Stems of Aquilaria Sinensis

Plant Growth Regul DOI 10.1007/s10725-013-9838-z

ORIGINAL PAPER

Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis

Zheng Zhang • Xingli Zhang • Yun Yang • Jian-he Wei • Hui Meng • Zhi-hui Gao • Yan-hong Xu

Received: 20 March 2013 / Accepted: 21 June 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Agarwood is highly valuable resinous and fra- wounding signal in A. sinensis that leads to the induction of grant heartwood, produced principally from tropical tree vessel occlusions formation and sesquiterpene biosynthe- species in the genus Aquilaria, which is used widely in sis, and thus H2O2 might play a vital role in agarwood countries of the Middle East, Southeast Asia and Japan. formation in pruned stems of A. sinensis. Generally, healthy trees will not produce agarwood, but wounding of the tree initiates the production of agarwood. Keywords Agarwood Á Aquilaria sinensis Á Hydrogen In this study, the pruning of actively growing saplings of peroxide Á Sesquiterpene Á Vessel occlusions Aquilaria sinensis resulted in hydrogen peroxide (H2O2) burst, which was followed by formation of vessel occlusions and sesquiterpene biosynthesis in the pruned stems. Introduction

Treatment of the pruned stems with scavenger of H2O2 (ascorbate, AsA) greatly reduced the amount of H2O2 Agarwood is a highly valuable resinous and fragrant released, the number of vessel occlusions, and the amount heartwood produced principally from tropical tree species of sesquiterpenes produced. In addition, exogenous H2O2 in the genus Aquilaria (Thymelaeaceae) (Zhang et al. also induced A. sinensis plants to form vessel occlusions 2012). The wealth of names for this dark and heavy wood and produce sesquiterpenes as pruning treatment. The (the Chinese name literally means ‘incense from the wood results indicated that H2O2 may be an important post- that sinks’) reflects its widespread and varied use over thousands of years (Nakashima et al. 2005). Agarwood is used as incense for religious ceremonies by Buddhists, Zheng Zhang and Xingli Zhang have contributed equally to this work. Hindus and Muslims, in perfumes notably in the Middle East, and as medicinal components in traditional Chinese, Z. Zhang Á X. Zhang Á J. Wei (&) Á Z. Gao Á Y. Xu National Engineering Laboratory for Breeding of Endangered Southeast Asian, Indian and Middle Eastern medicines Medicinal Materials, Institute of Medicinal Plant Development, (Chinese Pharmacopoeia Commission 2010). Aquilaria Chinese Academy of Medical Sciences and Peking Union sinensis (Lour.) gilg is the sole and legal plant resource in Medical College, Beijing 100093, People’s Republic of China China for agarwood production (Uddin et al. 2008), which e-mail: [email protected]; [email protected] is chiefly distributed and widely cultivated in the Hainan Z. Zhang Á Y. Yang Á J. Wei Á H. Meng and Guangdong provinces (Chen et al. 2012). Hainan Provincial Key Laboratory of Resources Conservation It is well known that mechanical wounding and infec- and Development of Southern Medicine, Hainan Branch of the tious diseases initiate the production of agarwood (Okudera Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences and Peking Union Medical College, and Ito 2009; Kumeta and Ito 2010). In response to Wanning 571533, People’s Republic of China mechanical wounding of A. sinensis, vascular occlusions are produced in the conduit lumen, which are often thought X. Zhang to be involved in wound sealing and reducing the risk of School of Food and Biological Engineering, Shandong Polytechnic University, Jinan 250353, pathogen intrusion. After wounding or fungal pathogen People’s Republic of China infection, phytoalexins such as phenolic compounds and 123 Plant Growth Regul sesquiterpenoids accumulate in the perivascular paren- before being exposed to air. 1.1-cm-long samples were chyma cells, and are released into the infected vessel lumen collected from the apical end of each treated stem and a to format vascular occlusions for restricting the vascular section of stem 1 mm long at the apical end of the cut stem ingress of the fungus (Zhang et al. 2010). Reactive oxygen was discarded, then five 2-mm-thick stem samples were species (ROS), which include superoxide, hydrogen per- collected, which represented the positions 2, 4, 6, 8, and oxide (H2O2) and hydroxyl radical, have been proposed to 10 mm from the cut surface. Samples were rapidly col- play a role in wound signaling (Ross et al. 2006; Angelini lected, immersed in liquid nitrogen and stored at -80 °C et al. 2008; Mittler et al. 2011). The H2O2 generated in for measurement of endogenous H2O2 at 0, 1, 2, 6, 12, and response to wounding can not only be used directly as 24 h after cutting. Other samples were fixed in formalin- antimicrobial agents but also acts as both local and dif- acetic acid-alcohol (FAA) for assessment of vascular fusible signal molecules for inducing formation of defense occlusion development at 0, 5, 10, 15, 20, 25 and 30 days. structures, such as callose (Luna et al. 2011), vessel Thirty-day-samples were collected and immersed in liquid occlusions (Lorrain et al. 2004). H2O2 also mediates the nitrogen and stored at -80 °C for HS–SPME–GC–MS elicitor-induced accumulation of secondary metabolites, analysis. For statistical analysis, data were calculated based such as capsidiol in tobacco (Perrone et al. 2003), isofl- on combined averages from five individual saplings avonoid glyceollin in soybean (Degousee et al. 1994; Guo (n = 5). et al. 1998), and p-coumaroyloctopamine in potato tubers Five saplings were treated with 1 mM H2O2 solution and (Matsuda et al. 2001). five received sterile ddH2O (control). A very small hole deep Our preliminary research showed that exogenous into the xylem was drilled in the stem. An infusion needle application of 1 mM H2O2 to the stem surface resulted in was inserted in the hole, and the H2O2 solution or ddH2O was the accumulation of vessel occlusions and sesquiterpenes, drawn into the sapling’s xylem cells by transpiration pull. It but little is known about the role of H2O2 in the formation required about 1 h for 20 mL of solution to be drawn into the of vessel occlusions and biosynthesis of sesquiterpenes. In stem. 2-mm-thick stem samples were collected at a height of the present study, to test the role of H2O2 in agarwood 10 cm above the hole. Samples were fixed in FAA for formation, stems of A. sinensis were pruned. We tested the assessment of vascular occlusion development at 0, 5, 10, 15, response to application of the scavenger of H2O2 (ascor- 20, 25 and 30 days. Thirty-day-samples were collected and bate, AsA) to the wounded stems. Our results indicated that immersed in liquid nitrogen and stored at -80 °C for HS–

H2O2 is an important post-wounding signal in A. sinensis SPME–GC–MS analysis. and that H2O2 production leads to the accumulation of The samples were immediately frozen in N2 and then vessel occlusions and sesquiterpenes, and thus may play a ground to a powder with a pestle and mortar. Analyses vital role in agarwood formation in pruned stems of were carried out using a H2O2 assay kit (Beyotime Bio- 2? 3? A. sinensis. technology, Haimen, China). H2O2 oxidized Fe to Fe , and then Fe3? reacted with xylenol orange leading to a colorimetric reaction that was measured with a spectrom- Materials eter. Briefly, test tubes containing 50 lL supernatants and 100 lL test solutions were placed at room temperature for Three-year-old A. sinensis saplings were grown in a 20 min and measured immediately with a spectrometer at a greenhouse at the Hainan Branch of the Institute of wavelength of 560 nm. The concentration of H2O2 released Medicinal Plant Development in Xinglong County, Wan- was calculated from a standard concentration curve. ning City, Hainan Province, China, and identified by Prof. The structure of vessel occlusions was evaluated as Jian-He Wei. The saplings were grown under day/night previously described (Sun et al. 2008). Briefly, for temperatures of 30 ± 2/25 ± 3 °C, respectively. The sap- assessment of vessel occlusion structure, one segment was lings were approximately 1.5 m in height, with a stem used for examination with a scanning electron microscope diameter of 2.0 ± 0.2 cm at a height of 10 cm above the (JSM-6510LV, JEOL, Tokyo, Japan). For vessel occlusions ground. numbers, freehand transverse sections were cut from each sample at 2, 4, 6, 8, and 10 mm from the cut end, mounted in water with a coverslip, and observed by light microscopy Methods (BX51, Olympus, Tokyo, Japan). In each section, five areas were randomly selected for analysis. In each area, 40–50 Pruning was imposed by cutting through stems at about vessels were examined for vessel occlusion development. 10 cm above the ground. Stems were cut in air, water or a For 30-days stem samples pruned in air, the apical 1 mm degassed solution of 0.1 mM AsA, respectively. And the section of the cut stem was discarded, then five 2-mm-thick cut ends remained in the inhibitor solution or water for 2 h stem samples were collected, which represented the 123 Plant Growth Regul positions 2, 4, 6, 8, and 10 mm from the cut surface. The (A) 60 samples were immediately frozen in N2 and then ground to 2 mm a powder with a pestle and mortar. For 30-days samples 4 mm 45 6 mm pruned in AsA or water and transfused with H2O2 or water, FW] 8 mm -1 the apical 5 mm section of the cut stem was discarded, then 10 mm 2-mm-thick stem samples were collected at a depth of 30 [nmolg

6 mm from the cut surface. The samples were immediately 2 O frozen in N2 and then ground to a powder with a pestle and 2 15 mortar. Accurately weighed 0.5 g powdered stem samples for GC–MS analysis. Vapor from the ﬂask was extracted 0 using a solid phase microextraction (SPME) ﬁber (100 lm 012 61224 polydimethylsiloxane, Supelco, Bellefonte, PA, USA) at (B) 15 60 °C for 1 h in accordance with the manufacturer’s pro- tocol (Okudera and Ito 2009), and injected into a GC–MS 12 2 mm (Varian 450 GC directly coupled to a Varian 300 mass 4 mm FW]6 mm H spectrometer). A 30 m 9 0.25 mm diameter HP1 -1 9 (0.25 lm) fused silica GC column was used, with the oven 8 mm 6 10 mm

temperature initially held at 60 °C for 2 min, then [nmolg

-1 2 increased to 230 °C at the rate of 4 °C min . The carrier O 2 gas was helium. Identiﬁcations of sesquiterpene com- H 3 pounds were based on an inhouse library of reference mass 0 spectra built up from standard compounds and oils of 0 1 2 6 12 24 known composition (Chen et al. 2011). (C) 60 Statistical analysis 2 mm 4 mm 45

Data were calculated based on combined averages from FW] 6 mm ﬁve individual saplings (n = 5). The signiﬁcance of dif- -1 8 mm ferences among data sets was evaluated with Duncan’s 30 10 mm [nmolg multiple range test. 2 O 2 15 H

Results and discussion 0 01261224 Hours after pruning (h) To investigate the function of H2O2 in wound-induced agarwood formation, the stem of actively growing saplings Fig. 1 Temporal changes in H2O2 concentration in xylem tissue of of A. sinensis were pruned. The production of H2O2 by wounded stems at different depths from the cut surface with and stems pruned in air was measured from 0 to 24 h post- without AsA. The H2O2 concentration was measured at 2, 4, 6, 8, wounding and at different depths from the wound surface. and 10 mm from the cut surface of stems pruned in air (a), AsA (b), and water (c). Error bars represent ±S.E, n = 5 Production of H2O2 increased in a biphasic manner with peaks at about 1 and 6 h after pruning and an intervening decline to the initial level at 2 h (Fig. 1a). The second peak In response to pruning, occlusions were observed in the was about double that of the ﬁrst peak and 30-times higher vessel lumens of the secondary xylem. Continued secretion than the initial concentration. By 24 h after pruning, the of vessel occlusions sealed some vessel lumens as early as

H2O2 concentration was again at the initial level where it 15 days after wounding (Fig. 2a). Vascular occlusions remained with a slight diurnal oscillation. The peak in were absent in vessels of unpruned controls (data not

H2O2 concentration 6 h after pruning decreased with dis- shown). Following wounding, the percentage of vessels tance from the wounded surface, ranging from about with occlusions (PVO) per square millimeter generally 45 nmol g-1 FW at 2 mm to about 5 nmol g-1 FW at increased until 30 days after pruning in all sections 10 mm at 6 h after pruning (Fig. 1a). These results showed (Fig. 2b). PVO increased at a higher rate at 4, 6, and 8 mm that wounded A. sinensis stems had two bursts of H2O2 compared to 2 and 10 mm. The increase in PVO at 2 and production, which was similar to ryegrass and Arabidopsis 10 mm does not seem particularly different before or after (Le Deunff et al. 2004; Song et al. 2006). 15 days. The PVO at 30 days after pruning showed a 123 Plant Growth Regul

3 Our result was consistent with fact that H O plays a 2 mm (A) (B) 2 2 2.5 4 mm dual role in plants: at high concentrations, it leads to PCD 6 mm 8 mm or even necrosis at high levels (Gill and Tuteja 2010). Our 2 10 mm result showed that there were less vessel occlusions and 1.5 sesquiterpenes at surface 0–4 mm or deeper C10 mm, PVO [%] PVO 1 where too high or low concentrations of H2O2 resulted the 0.5 parenchyma cells necrosis or have no or little change, separately. While in middle at about 4–8 mm, there were 0 0 5 10 15 20 25 30 much more vessel occlusions and sesquiterpenes, where the Days after pruning (d) medial concentration could induce plant defense response, Fig. 2 Pruning-induced vessel occlusions and temporal changes in such as sesquiterpene cyclase gene expression and vessel percentage of vessel occlusions at different depths from the cut occlusions formation. surface. SEM micrographs of vessel occlusions (arrows) at 15 days The pattern of H2O2 production from the pruned end of after pruning (a), Temporal changes in PVO at different depths from stems treated with AsA differed dramatically from that of the cut surface (b). PVO, the percentage number of vessels with occlusions per square millimeter. Error bars represent ±S.E, n = 5 the controls. The treatment completely eliminated the first peak in H2O2 production, and the second increase was normal distribution against distance from the wound sur- greatly reduced (Fig. 1b). Thus, wounding-induced H2O2 face (Fig. 2b). production was greatly suppressed in the presence of AsA. The volatiles emitted from the control and at different However, there were no obvious differences H2O2 depths from the cut surface in pruned samples were production between the stems pruned in air and water investigated using headspace solid-phase microextraction (Fig. 1a, c). (HS–SPME). The GC–MS analysis showed that the vola- The temporal changes in vessels occlusion development tiles of pruned samples contained different components to were markedly affected by treatment of the stems with those of the control samples. The volatiles of pruned stems AsA. In control stems pruned in water (in which the cut end contained different sorts and contents of sesquiterpenes. remained in the water only for the first 2 h), the number of The relative abundance of sesquiterpenes was highest at vessel occlusions increased rapidly from 0 to 15 days after 6 mm, and decreased sharply above and below this depth pruning, with the PVO reaching 1.65 %. Thereafter, the (Table 1). Except for a-humulene and humulane-1,6-dien- PVO increased slowly to 2.32 % by 30 days after pruning, 3-ol, the other six sesquiterpenes detected were precursor which had no significant with treatment in air. However, in compounds or constituents of wild agarwood (Naef 2011). stems treated with AsA, the progress of vessel occlusion However, these sesquiterpenes were not detected in the development was slower and PVO was only 0.37 % and to control. These results showed that wound-induced H2O2 0.61 % at 15 and 30 days respectively (Fig. 3). These data production was accompanied by the formation of vascular indicate that suppression of H2O2 production greatly occlusions and accumulation of sesquiterpenes in pruned reduced and delayed wound-induced development of vessel stems of A. sinensis. occlusion.

Table 1 Chemical composition and relative amounts of sesquiterpenes from pruned and intact stem samples after 30 days treatment No. Compounds Peaks areas (9109) Wounding CK 2 mm 4 mm 6 mm 8 mm 10 mm

1 a-Caryophyllene –b –b 0.32 ± 0.04a –b –b –b 2 a-Humulene 2.08 ± 0.32c 3.52 ± 0.48b 6.59 ± 1.24a 4.53 ± 0.89b 2.17 ± 0.43c –d 3 4,11-Selinadiene –c 2.23 ± 0.31b 3.19 ± 0.52a –c –c –c 4 b-Selinene 1.01 ± 0.09b 0.51 ± 0.12c 1.60 ± 0.20a 1.03 ± 0.13b –d –d 5 d-Guaiene – 0.34 ± 0.07c 1.18 ± 0.23a 0.66 ± 0.12b –d –d 6 c-Eudesmol 1.06 ± 0.15c 2.26 ± 0.36b 3.54 ± 0.45a 2.61 ± 0.35b 0.96 ± 0.18c –d 7 Agarospirol –b –b 0.24 ± 0.04a –b –b –b 8 Humulane-1,6-dien-3-ol –b 0.96 ± 0.22a –b –b –b –b 93-a,4-b-Dihydroxy-1,5,7-a-(H),6-b-(H)-guai- –b –b 0.30 ± 0.08a –b –b –b 10(15),11(13)-dien-6,12-olide Values followed by a different superscript letter are signiﬁcantly different according to Duncan’s multiple range test (P \ 0.05)

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6.00 genes (Ahuja et al. 2012). For example, in response to Pruning in air Pruning in wa ter fungal infection, rapid occlusion of xylem vessels by 5.00 Pruning in AsA Transfusion hydrogen peroxide tyloses followed by synthesis of fungitoxic terpenoid Transfusion water 4.00 aldehydes was observed in resistant cotton (Zhang et al. 3.00 1993; Daayf et al. 1997). To explain the mechanism of PVO [%] PVO 2.00 agarwood production, we hypothesized that a wound-acti- vated defense reaction results in agarwood formation 1.00 (Zhang et al. 2010). 0.00 In order to verify the role of wound signals in agarwood 0 5 10 15 20 25 30 DAYS AFTER PRUNING [d] formation, H2O2 were tested. Exogenous application of H2O2 resulted in accumulation of vessel occlusions and Fig. 3 Temporal changes in number of vessel occlusions at 6 mm sesquiterpenes. The number of vessels occlusions in the from the cut surface in stems pruned in air, AsA or water, and in stem induced by H2O2 was higher than that induced by stems transfused with 1 mM H2O2 or water. PVO, the percentage number of vessels with occlusions per square millimeter. Error bars pruning (Figs. 2, 3). Consistent with the scanning electron represent ±S.E, n = 5 microscopy results, exogenous H2O2 resulted in a rapid increase in PVO from 0 to 30 days after treatment, which As suppression of H2O2 production greatly reduced is almost double that observed in pruned stems at 6 mm wound-induced development of vessel occlusion, the kind from the cut surface 30 days after pruning (Fig. 3). The and quantity of the volatiles pruned in AsA decreased GC–MS analysis showed that the volatiles of H2O2-treated signiﬁcantly comparing with that in water and air. The samples comprised similar components to those of pruned

GC–MS analysis showed that the volatiles of stems pruned samples. The volatiles of H2O2-treated stems r yield the in water had similar components to stems in air. Both these highest peak area for 8 sesquiterpenes except for d-guaiene volatiles comprised 8 sesquiterpenes compounds and peak than in the pruned stems at 6 mm from the cut surface area for each sesquiterpene was not statistically deferent. (Table 2). H2O2 solution was injected into the xylem part However, the volatiles of stems pruned in ascorbate solu- of Aquilaria trees through transfusion sets invented by our tion had signiﬁcantly different components from that in lab (Liu et al. 2013). Due to water transportation, H2O2 are water and air. There only two sesquiterpenes, a-humulene transported to the whole body of the tree, thus forming an and 4,11-selinadiene identiﬁed in stems pruned in ascor- overall wound in the tree, and as a result, a large number of bate solution which had lower peak area than treatment in vessel occlusions and sesquiterpenes accumulated in the air and water (Table 2). Aquilaria trees in a short period of time (30 days). Vessel It is well known that wounding or microbial infection occlusions and sesquiterpenes were not detected in water- induces the biosynthesis of structural barriers (including transfused samples, which is identical to that of unpruned cell wall thickening, callose deposition, and vascular stems (Fig. 3; Table 2). occlusions), production of soluble inhibitory intermediates The present study demonstrated that both exogenous or phytoalexins, and induced expression of defense-related H2O2 and pruning induce vessel occlusion formation and

Table 2 Chemical composition and relative amount of sesquiterpenes from stems pruned in air, AsA or water, and stems transfused with H2O2 or water after 30 days treatment No. Compounds Relative amount (%)

Wound in air Wound in Wound in H2O2- Water- AsA water transfusion transfusion

1 a-Caryophyllene 0.32 ± 0.04b –c 0.33 ± 0.05b 2.44 ± 0.35a –c 2 a-Humulene 6.59 ± 1.24b 1.97 ± 0.30c 5.38 ± 0.92b 11.67 ± 1.11a –d 3 4,11-Selinadiene 3.19 ± 0.52a 1.50 ± 0.29b 3.34 ± 0.47a 3.29 ± 0.36a –c 4 b-Selinene 1.60 ± 0.20b 0.21 ± 0.41c 1.96 ± 0.23b 5.95 ± 0.77a –d 5 d-Guaiene 1.18 ± 0.23b –c 1.62 ± 0.28b 4.34 ± 0.62a –c 6 c-Eudesmol 3.54 ± 0.45b –c 2.90 ± 0.51b 5.32 ± 0.78a –c 7 Agarospirol 0.24 ± 0.04b –c 0.13 ± 0.02b 1.77 ± 0.32a –c 8 Humulane-1,6-dien-3-ol –b –b –b 0.27 ± 0.03a –b 93-a,4-b-Dihydroxy-1,5,7-a-(H),6-b-(H)-guai- 0.30 ± 0.08b –c 0.20 ± 0.03b 2.88 ± 0.49a –c 10(15),11(13)-dien-6,12-olide Values with a different letter are signiﬁcantly different according to Duncan’s multiple range test (P \ 0.05)

123 Plant Growth Regul sesquiterpene biosynthesis in stems of A. sinensis. The Chinese Pharmacopoeia Commission (2010) Pharmacopoeia of the results indicated that H O might regulate vascular occlu- People’s Republic of China (Vol I). China Medical Science 2 2 Press, Beijing sion formation and sesquiterpene biosynthesis in pruned Daayf F, Nicole M, Boher B, Pando A, Geiger JP (1997) Early stems of A. sinensis. It is hypothesized that pruning caused vascular defense reactions of cotton roots infected with a the production of a wounding signal (H2O2), and H2O2 defoliating mutant strain of Verticillium dahlia. 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