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Resveratrol accumulation and resveratrol synthase gene expression in response to abiotic stresses and hormones in peanut plants
Plant Science 164 (2003) 103 /109 www.elsevier.com/locate/plantsci
Resveratrol accumulation and resveratrol synthase gene expression in response to abiotic stresses and hormones in peanut plants Ill-Min Chung a, Myoung Ryoul Park b, Jae Chul Chun c,d, Song Joong Yun b,d,* a
Research Team of Friendly Environmental Low Input Natural Herbicide New Material Study, Konkuk University, Seoul 143-701, South Korea b Faculty of Biological Resources Sciences, Chonbuk National University, Chonju 561-756, South Korea c Faculty of Biotechnology, Chonbuk National University, Chonju 561-756, South Korea d Institute of Agricultural Science and Technology, Chonbuk National University, Chonju 561-756, South Korea Received 31 July 2002; received in revised form 12 September 2002; accepted 30 September 2002
Abstract The peanut is one of the limited number of plant species that synthesize resveratrol, which is both a phytoalexin with antifungal activity and a phytochemical associated with reduced cancer risk and reduced cardiovascular disease. We investigated resveratrol content and resveratrol synthase gene expression in response to various stresses and hormones in order to understand the mode of resveratrol synthesis in peanut plants. Resveratrol was present in substantial amounts (1.2 /2.6 mg/g FW) in leaves, roots and shells, but very little (0.05 /0.06 mg/g FW) was found in developing seeds and seed coats of field-grown peanuts. Accumulation of resveratrol in leaves increased over 200-fold in response to UV light, over 20-fold in response to paraquat, and between two- and ninefold in response to wounding, H2O2, salicylic acid (SA), jasmonic acid and ethephon, 24 h after treatment. No accumulation of resveratrol was induced by abscisic acid. Changes in resveratrol content were correlated with levels of RS mRNA, indicating a transcriptional control of resveratrol synthase activity. The results suggest that resveratrol synthesis is induced by biotic and abiotic factors through the regulation of RS transcription, and that stress hormones such as SA and ethylene are involved in the RS gene expression in peanut. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethylene; Jasmonic acid; Resveratrol; Resveratrol synthase; Salicylic acid; Yeast extract
1. Introduction Resveratrol, 3,5,4?-trihydroxystilbene, is synthesized by the catalysis of resveratrol synthases (RS; EC 2.3.1.95) using one molecule of coumaroyl-CoA and three molecules of malonyl-CoA. It has antifungal activity and its synthesis is induced in response to pathogen infections; thus, it is a member of the natural phytoalexins group [1]. For this reason, resveratrol has attracted attention as a plant defense agent against fungal infections. Transgenic monocot and dicot plants producing resveratrol by the overexpression of RS genes have shown increased resistance against various fungal infections [2,3].
* Corresponding author. Tel.: /82-63-270-2508; fax: /82-270-2640 E-mail address: [email protected] (S.J. Yun).
Resveratrol has been found in the hypocotyls and germinating seeds [4] of the peanut under conditions of fungal infection and wounding. Recently, however, resveratrol has been found in uninfected peanut seeds [5]. The finding that peanut seeds are a source of resveratrol focuses attention on the role of resveratrol as a phytochemical with human health benefits. Resveratrol has been associated with reduced human pathological processes such as atherosclerosis [6] and carcinogenesis [7]. Regulation of resveratrol synthesis in the peanut has been investigated in the simple cell culture system rather than in the intact plant system. Resveratrol synthesis is induced by pathogen infections, fungal elicitors, and UV light in peanut and grapevine suspension cells [8]. Because resveratrol is synthesized by a one-step reaction of RS, RS gene expression plays a regulatory role in resveratrol synthesis. A transient and parallel increase in RS mRNA and RS activity in response to fungal
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elicitors and UV irradiation in the cultured peanut cells [9] indicates a possible transcriptional control of RS gene expression and resveratrol synthesis. As in the cell cultures, resveratrol synthesis is induced in peanut hypocotyls by UV light and wounding [4,8]. However, little information is available on the accumulation of resveratrol and RS gene expression in peanut plant tissues of agronomic importance. It is well understood that signal molecules such as salicylic acid (SA), jasmonic acid (JA) and ethylene play critical roles in plant responses to various biotic and abiotic stresses [10]. SA is responsible for the induction of genes involved in the systemic acquired resistance (SAR) response [11], and JA and ethylene activate certain genes involved in the SA-independent response [12]. Although it has been demonstrated that RS gene expression and resveratrol synthesis are induced by biotic and abiotic factors, little information is available on the role of signal molecules in the regulation of RS gene expression and resveratrol synthesis in the peanut. Therefore, we investigated the accumulation of resveratrol and RS gene expression in response to stresses and hormones in peanut plant tissues. The results indicate that the expression levels of RS genes modulate resveratrol synthesis in peanut plants, and that stress hormones such as SA, JA, and ethylene are involved in resveratrol synthesis.
2. Materials and methods 2.1. Chemicals and enzymes Chemicals and enzymes were purchased from Sigma (USA) and Roche (Germany), respectively. 2.2. Plant materials Peanut (Arachis hypogaea L. var. Jinpoong) plants were grown in a glasshouse and in the experimental field at Chonbuk National University, South Korea. Light intensity in a clear day during the growing period was as high as 1600 and 1000 mE/(m2/s), respectively, in the field and in a glasshouse. Plants grown in the field were used to determine resveratrol content and RS gene expression in the tissues under field conditions. Leaves and developing pods were collected separately from the plants grown in the field 40 days after flowering (DAF), and pods 15/20 mm in length were dissected into the shell, seed coat, and seed with a sterile surgical blade and forceps. The plants were grown aseptically in vitro by germinating surface-sterilized seeds on half-strength Murashige and Skoog (MS) medium under a 16 h light/8 h dark cycle for 4 weeks at 25 8C. Light intensity in the incubator was about 40 mE/(m2/s), 10 cm above the self. Roots and leaves of sterile plants were used for
induction experiments. Plants were also grown in a glasshouse as aseptically as possible for 8 weeks and their leaves were used for induction experiments. All tissue samples were frozen in liquid nitrogen and kept at /80 8C until needed. 2.3. Induction conditions Trifoliate leaves from healthy plants grown in a glasshouse were used for the induction experiments and were collected by cutting the uppermost part of the petioles with a razor blade. Leaves with petioles were immersed in the following sterile solutions for 0, 3, 12, and 24 h: H2O, 50 mM NaHPO4 (pH 6.9), 0.1 mM paraquat, 5 mM H2O2, 0.7 mM JA, 10 mM SA, 5 mM ethephon, or 100 mM abscisic acid (ABA) [13,14]. For the wounding treatment, the trifoliate leaves were punched with fine pins and floated on the sterile water. Leaves were kept for the first 12 h in the light with intensity of about 100 mE/(m2/s) and for the following 12 h in the dark at 25 8C. For the UV treatment, plants were irradiated under UV lamps at 1.35 mE/(m2/s) for 2 h and returned to the dark glasshouse. The irradiated leaves were collected 0, 3, and 12 h after irradiation. Leaves and roots of sterile plants that had been grown in vitro for 4 weeks were used for induction experiments. Plants grown in vitro were placed under dark conditions for 48 h prior to yeast extract treatments. For the elicitor treatment, roots of intact plants were incubated in halfstrength MS medium containing 25 mg/ml yeast extract and placed in the dark for 6 h. All tissue samples were frozen in liquid nitrogen and stored at /80 8C until needed. 2.4. Resveratrol analysis Resveratrol was analyzed using a modification of the method of Fettig and Hess [3]. Samples were protected from light during the analysis process and the free form of resveratrol was extracted by agitating the frozen tissue powder in methanol for 16 h at room temperature. Resveratrol was separated on a mBondapak C18 column (3.9 /300 mm, Waters, USA) with water /acetonitrile gradient elution at a flow rate of 1.0 ml/min and detected with a fluorescence detector (Waters 474, Milford, USA) set at 330 nm for excitation and 374 nm for emission. Samples from three replications were analyzed. 2.5. Northern blot analyses Northern blot analyses were carried out with total RNA prepared from sample tissues as described [15]. Northern blots were repeated at least three times and results from representative blots were presented. Twenty micrograms of total RNA was denatured,
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separated on a 1.2% formaldehyde/agarose gel and transferred onto a nylon membrane (Hybond-N , Amersham, UK). The membrane was prehybridized for 2 h at 50 8C in a DIG Easy Hyb solution (Roche, Germany), and incubated for 16 h at 50 8C after adding the DIG-labeled RS cDNA clone, pPRS3C [15], in the prehybridization solution. After hybridization, the membrane was washed to a final stringency of 0.5 / SSC and 0.1% SDS at 68 8C for 30 min. RNA on the membrane was detected using the DIG chemiluminescent detection system (Roche, Germany) as described [15].
3. Results
3.1. Resveratrol content and RS gene expression in fieldgrown peanut plants Regulation of resveratrol synthesis has been investigated in peanut suspension cells, but rarely in plant tissues. With regard to the beneficial roles of resveratrol for plant and human health, it is of interest to understand the mode of resveratrol synthesis regulation in peanut tissues of agronomic importance. To obtain the initial information for further investigation, resveratrol was analyzed in the tissues of healthy plants grown in the field up to 40 DAF. Free resveratrol contents in leaves, pods, and roots were 2.05, 1.34, and 1.19 mg/g FW, respectively (Fig. 1A). Peanut pods contain seeds inside the shell and the seeds are covered with the seed coat. To specifically locate resveratrol in pods, the pods at mid-maturity were dissected into the shell, seed coat, and seed. Resveratrol was present at 2.60, 0.06, and 0.05 mg/g FW in the shell, developing seed, and seed coat, respectively (Fig. 1A). Because resveratrol is synthesized by the catalysis of RS, RS mRNA levels were examined in the same plant tissues. RS-specific mRNA, about 1.4 kb in size, was detected at high levels in the roots and pods, but below the detection levels in the leaves (Fig. 1B). RS mRNA was present at high levels in the shell, but below the detection levels in the developing seed and seed coat, indicating that most of the RS mRNA detected in the pods was from the shell (Fig. 1B). Resveratrol content in mature peanut seeds ranges from 0.03 to 0.14 mg/g seed and is lower than that found in the seed coats [5]. Our results for resveratrol content in developing peanut seeds are consistent with a recent report on resveratrol in peanut seeds [5]. However, little information is available on the RS gene expression in tissues of field-grown peanut plants.
Fig. 1. Resveratrol (A) and RS mRNA (B) in the tissues of peanut plants grown in the field. (A) Resveratrol content was determined by high-performance liquid chromatography (HPLC) in leaves (LF), whole pods (WH), and roots (RT). Whole pods (WH) were dissected into the shell (SH), seed coat (SC), and seed (SE). (B) Expression of RS mRNA in peanut tissues as indicated in (A). The size of the RS mRNA detected was about 1.4 kb. Twenty micrograms of total RNA was resolved in a 1.0% (w/v) agarose/formaldehyde gel, transferred to a nylon membrane, then hybridized with a DIG-labeled pPRS3C insert. Ethidium bromide-stained rRNA bands as an indicator of equal loading (rRNA).
3.2. Accumulation of resveratrol and RS mRNA in response to elicitor Resveratrol and RS mRNA showed tissue-specific distribution in peanut plants grown in the field. To investigate the tissue-specific expression of resveratrol synthesis, we examined the effects of various stresses and hormones on resveratrol synthesis in peanut plants. First, we examined the effect of elicitor on resveratrol synthesis in sterile peanut leaves and roots based on the previous report on the elicitor effect of yeast extract in peanut suspension cells [9] and plant tissues [15]. Sterile leaves and roots contained trace amounts (0.15 mg/g FW) of resveratrol. Yeast extract induced an accumulation of resveratrol in the leaves and roots. Resveratrol accumulated up to 1.2 and 2.8 mg/g FW in leaves and roots, respectively, after treatment with yeast extract for 6 h, which corresponded to 8- and 19-fold increases relative to untreated control tissues (Fig. 2A). Levels of RS mRNA also increased in leaves and roots in the yeast extract treatment (Fig. 2B), and these increases were correlated with the increase in resveratrol contents in the tissues. RS transcription is induced by a yeast extract similar to the elicitor prepared from Phytophthora megasperma f. sp. glycinea in peanut suspension cells [9] and peanut roots [15]. Our result indicates that yeast extract induces
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RS mRNA was detected in the leaves treated with water or 50 mM sodium phosphate buffer for up to 24 h. However, RS mRNA increased in response to other treatments, generally in a time-dependent manner. The induction kinetics, however, was also quite different between the treatments. It took less than 3 h for the induction of detectable amount of RS transcripts by UV light and paraquat, but over 12 h by wounding and H2O2 (Fig. 3B). The increase in RS mRNA was correlated with resveratrol accumulation, indicating transcriptional control of RS gene expression. Induction of resveratrol and RS mRNA synthesis in response to UV light has been observed in peanut suspension cultures [8,9]. The induction of resveratrol and RS mRNA by paraquat, the generator of the superoxide radical, and H2O2 is consistent with the observation of RS mRNA induction by ozone in grapevine leaves [16]. Fig. 2. Accumulation of resveratrol (A) and RS mRNA (B) in peanut leaves and roots in response to yeast extract. (A) Resveratrol content was determined by HPLC in the leaf (LF) and root (RT) tissues treated for 6 h with yeast extract (YE). CT, control. (B) Accumulation of RS mRNA in peanut leaves and roots treated with YE as indicated in (A). The size of the RS mRNA detected was about 1.4 kb. Twenty micrograms of total RNA was resolved in a 1.0% (w/v) agarose/ formaldehyde gel, transferred to a nylon membrane, then hybridized with a DIG-labeled pPRS3C insert. Ethidium bromide-stained rRNA bands as an indicator of equal loading (rRNA).
resveratrol accumulation via transcription of RS mRNA in peanut plants. 3.3. Accumulation of resveratrol and RS mRNA in response to abiotic stresses Since resveratrol synthesis was induced in peanut plants by elicitor, we further examined the effect of abiotic stresses and reactive oxygen species (ROS) on resveratrol synthesis with the leaves of peanut plants grown as aseptically as possible in a glasshouse. No, or little, resveratrol accumulated in the leaves treated with water or 50 mM sodium phosphate buffer for up to 24 h, and a small amount (0.25 mg/g FW) was detected in leaves treated with water for 24 h. However, resveratrol increased in the leaves treated with various stresses 2- to over 200-fold relative to the amount detected in the leaves treated with water. Resveratrol increased up to 225-fold 12 h after the UV treatment for 2 h, and also increased 23-, 2-, and 2-fold by the paraquat, H2O2, and wounding treatments for 24 h, respectively. Accumulation kinetics was different between the treatments, but in general the accumulation increased with the period of treatment up to 24 h (Fig. 3A). Accumulation of RS mRNA in response to the abiotic stresses was similar to that of resveratrol. No, or little,
3.4. Accumulation of resveratrol in response to stress hormones The induction of resveratrol synthesis by abiotic stresses and ROS suggested that stress hormones might be involved in resveratrol synthesis in peanut plants. Therefore, we investigated effects of stress hormones on resveratrol synthesis in peanut leaves. Resveratrol accumulated in response to SA, JA, and ethylene in a time-dependent manner up to 24 h. The effect of the hormones on resveratrol accumulation was quite different: it increased up to three-, two-, and eightfold in response to SA, JA, and ethylene, respectively. However, no resveratrol accumulated in response to ABA in peanut leaves (Fig. 3A). RS mRNA also increased in response to SA, JA, and ethylene at least 12 h after treatment. RS mRNA also increased in a time-dependent manner in response to SA and ethylene, and the increase reached its maximum 24 h after treatment. In contrast, RS mRNA was not induced by ABA (Fig. 3B). The levels of RS mRNA increased in response to the hormones correlated with the amounts of resveratrol accumulated, indicating transcriptional control of RS gene expression.
4. Discussion The beneficial roles of resveratrol in human health, as well as in plant health, focus much attention on the synthesis of resveratrol in peanut plants. Therefore, it is of interest to understand resveratrol synthesis in the peanut tissues of agronomic importance. In peanut plants grown in the field to mid-maturity, resveratrol and RS mRNA were relatively abundant in the roots and shells, but not in seed coats and seeds, indicating tissue-specific regulation of resveratrol synthesis in pea-
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Fig. 3. Accumulation of resveratrol (A) and RS mRNA (B) in peanut leaves in response to abiotic stresses and hormones. (A) Resveratrol content was determined by HPLC in the leaves treated with water, 50 mM sodium phosphate (pH 7.0; SP), 0.1 mM paraquat (PQ), 5 mM H2O2 and wound (WD), 5 mM ethylene, 10 mM SA, 0.7 mM JA, and 10 mM ABA for 0, 3, 12 and 24 h. UV light (UV) was treated for 2 h and resveratrol was determined 0, 3 and 12 h after treatment (S.D.: standard deviation). (B) Accumulation of RS mRNA in peanut leaves treated as indicated in (A). The size of the RS mRNA detected was about 1.4 kb. Twenty micrograms of total RNA was resolved in a 1.0% (w/v) agarose/formaldehyde gel, transferred to a nylon membrane, then hybridized with a DIG-labeled pPRS3C insert. Ethidium bromide-stained rRNA bands as an indicator of equal loading (rRNA).
nut plants. In leaves of field-grown peanut plants, however, RS mRNA was very low, even though a substantial amount of resveratrol was present in the tissue. This may be due to the resveratrol accumulated prior to the previous stimuli in the leaves in which RS was not expressed at the time of investigation. Evidence on resveratrol synthesis indicates that RS gene expression plays an important role in resveratrol accumulation in plant tissues [2,3]. Our results also show a correlation between resveratrol and RS mRNA accumulation, indicating that resveratrol synthesis is regulated through the transcriptional control of RS genes. Previous studies also indicate that RS gene expression is induced by several biotic and abiotic stresses. RS
genes are induced by fungal infection, elicitor, and UV treatments in peanut cultured cells [9] and in plant tissues [15]. There is a high possibility of wounding and microbial infection in roots and shells during growth in soils colonized with various soil fungi. Roots undergo wounding during the penetration of young lateral roots through the cortex and epidermis of the main roots [17]. Infection of peanut shells by soil fungi such as Aspergillus flavus takes place in the soil [18]. Therefore, it is possible that accumulation of resveratrol in the roots and shells was induced by factors such as wounding and fungal infections. Our results also support the view that resveratrol is accumulated by elicitor and abiotic stresses such as wounding and UV light through the expression of RS genes in peanut leaves and roots.
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Furthermore, RS mRNA expression was induced in peanut leaves in response to SA, JA, and ethephon. SA, JA, and ethylene have been shown to be involved in the activation of various plant defense responses following pathogen attack. SA plays a central role in the establishment of SAR and the elaboration of local defense responses against pathogen attack [19]. On the other hand, JA and ethylene play an important role in the induction of nonspecific disease resistance through signaling pathways distinct from the classic SAR response pathway regulated by SA [10]. In addition to its role in plant pathogenesis, SA, JA, and ethylene play a major role in plant responses to abiotic stresses such as UV-B and ozone [20], and wounding [21]. Therefore, induction of RS expression by SA, JA, and ethylene raises the possibility that these molecules share a common signaling pathway in the regulation of RS gene expression in the peanut. Induction of RS mRNA by paraquat is interesting because paraquat causes the generation of ROS such as superoxide and hydrogen peroxide [22]. ROS has been shown to stimulate the accumulation of SA [23] and ethylene [24], and is also required for the establishment of SAR to avirulent pathogens in plants [25]. Wounding, UV-B and ozone also lead to the generation of ROS [26,27]. Induction of RS mRNA by UV and paraquat, together with its induction by H2O2, suggests that ROS play a role as an intermediate in the signaling pathways [28] for the expression of peanut RS genes. It is noteworthy that accumulation of resveratrol and RS transcripts was faster and higher in the UV and paraquat treatments. This observation is supported by the ability of UV and paraquat to generate superoxide and hydrogen peroxide from multiple sources [22,27], and induction of SA and ethylene by ROS [23,24]. Our results indicate that the tissue-specific distribution of resveratrol in the field-grown peanut is due to the differential expression of RS genes induced by biotic and abiotic stresses. In addition, stress hormones such as SA, JA, and ethylene, and ROS mediate the transduction of signals triggered by biotic and abiotic stresses, which finally result in the expression of RS genes for the synthesis of resveratrol. Therefore, tissues such as roots and shells, which are vulnerable to abiotic and biotic stresses, should be more likely to produce resveratrol than would protected tissues such as seeds and seed coats. However, developmental regulation of RS gene expression in the seed and seed coat cannot be excluded without further dissection studies with pods at different levels of maturity. However, our results suggest that levels of RS expression in seeds and seed coats may be lower than those in other tissues such as roots and shells. In peanut cells, most of the resveratrol is used as an intermediate for the insoluble materials deposited in the cell wall [29]. Peanut shells play multiple roles, including the structural protection of seeds from injury and
infections. Lignocellulosic materials found in high amounts in the shell are the major chemical components for the structural roles of the tissue [30]. Results from this study also indicate that the shell contains conjugated resveratrol at about half the amount of the free form (data not shown). However, most of the resveratrol is present as a free form in other peanut tissues. Therefore, the synthesis of resveratrol by the expression of RS in peanut tissues could provide resistance to pathogen infection through the direct antifungal effect of resveratrol and the reinforcement of shells by the synthesis of cell wall materials with resveratrol as an intermediate.
Acknowledgements This work was supported by Korea Science and Engineering Foundation under Project No. R01-1999000-00172.
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Resveratrol accumulation and resveratrol synthase gene expression in response to abiotic stresses and hormones in peanut plants Ill-Min Chung a, Myoung Ryoul Park b, Jae Chul Chun c,d, Song Joong Yun b,d,* a
Research Team of Friendly Environmental Low Input Natural Herbicide New Material Study, Konkuk University, Seoul 143-701, South Korea b Faculty of Biological Resources Sciences, Chonbuk National University, Chonju 561-756, South Korea c Faculty of Biotechnology, Chonbuk National University, Chonju 561-756, South Korea d Institute of Agricultural Science and Technology, Chonbuk National University, Chonju 561-756, South Korea Received 31 July 2002; received in revised form 12 September 2002; accepted 30 September 2002
Abstract The peanut is one of the limited number of plant species that synthesize resveratrol, which is both a phytoalexin with antifungal activity and a phytochemical associated with reduced cancer risk and reduced cardiovascular disease. We investigated resveratrol content and resveratrol synthase gene expression in response to various stresses and hormones in order to understand the mode of resveratrol synthesis in peanut plants. Resveratrol was present in substantial amounts (1.2 /2.6 mg/g FW) in leaves, roots and shells, but very little (0.05 /0.06 mg/g FW) was found in developing seeds and seed coats of field-grown peanuts. Accumulation of resveratrol in leaves increased over 200-fold in response to UV light, over 20-fold in response to paraquat, and between two- and ninefold in response to wounding, H2O2, salicylic acid (SA), jasmonic acid and ethephon, 24 h after treatment. No accumulation of resveratrol was induced by abscisic acid. Changes in resveratrol content were correlated with levels of RS mRNA, indicating a transcriptional control of resveratrol synthase activity. The results suggest that resveratrol synthesis is induced by biotic and abiotic factors through the regulation of RS transcription, and that stress hormones such as SA and ethylene are involved in the RS gene expression in peanut. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethylene; Jasmonic acid; Resveratrol; Resveratrol synthase; Salicylic acid; Yeast extract
1. Introduction Resveratrol, 3,5,4?-trihydroxystilbene, is synthesized by the catalysis of resveratrol synthases (RS; EC 2.3.1.95) using one molecule of coumaroyl-CoA and three molecules of malonyl-CoA. It has antifungal activity and its synthesis is induced in response to pathogen infections; thus, it is a member of the natural phytoalexins group [1]. For this reason, resveratrol has attracted attention as a plant defense agent against fungal infections. Transgenic monocot and dicot plants producing resveratrol by the overexpression of RS genes have shown increased resistance against various fungal infections [2,3].
* Corresponding author. Tel.: /82-63-270-2508; fax: /82-270-2640 E-mail address: [email protected] (S.J. Yun).
Resveratrol has been found in the hypocotyls and germinating seeds [4] of the peanut under conditions of fungal infection and wounding. Recently, however, resveratrol has been found in uninfected peanut seeds [5]. The finding that peanut seeds are a source of resveratrol focuses attention on the role of resveratrol as a phytochemical with human health benefits. Resveratrol has been associated with reduced human pathological processes such as atherosclerosis [6] and carcinogenesis [7]. Regulation of resveratrol synthesis in the peanut has been investigated in the simple cell culture system rather than in the intact plant system. Resveratrol synthesis is induced by pathogen infections, fungal elicitors, and UV light in peanut and grapevine suspension cells [8]. Because resveratrol is synthesized by a one-step reaction of RS, RS gene expression plays a regulatory role in resveratrol synthesis. A transient and parallel increase in RS mRNA and RS activity in response to fungal
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elicitors and UV irradiation in the cultured peanut cells [9] indicates a possible transcriptional control of RS gene expression and resveratrol synthesis. As in the cell cultures, resveratrol synthesis is induced in peanut hypocotyls by UV light and wounding [4,8]. However, little information is available on the accumulation of resveratrol and RS gene expression in peanut plant tissues of agronomic importance. It is well understood that signal molecules such as salicylic acid (SA), jasmonic acid (JA) and ethylene play critical roles in plant responses to various biotic and abiotic stresses [10]. SA is responsible for the induction of genes involved in the systemic acquired resistance (SAR) response [11], and JA and ethylene activate certain genes involved in the SA-independent response [12]. Although it has been demonstrated that RS gene expression and resveratrol synthesis are induced by biotic and abiotic factors, little information is available on the role of signal molecules in the regulation of RS gene expression and resveratrol synthesis in the peanut. Therefore, we investigated the accumulation of resveratrol and RS gene expression in response to stresses and hormones in peanut plant tissues. The results indicate that the expression levels of RS genes modulate resveratrol synthesis in peanut plants, and that stress hormones such as SA, JA, and ethylene are involved in resveratrol synthesis.
2. Materials and methods 2.1. Chemicals and enzymes Chemicals and enzymes were purchased from Sigma (USA) and Roche (Germany), respectively. 2.2. Plant materials Peanut (Arachis hypogaea L. var. Jinpoong) plants were grown in a glasshouse and in the experimental field at Chonbuk National University, South Korea. Light intensity in a clear day during the growing period was as high as 1600 and 1000 mE/(m2/s), respectively, in the field and in a glasshouse. Plants grown in the field were used to determine resveratrol content and RS gene expression in the tissues under field conditions. Leaves and developing pods were collected separately from the plants grown in the field 40 days after flowering (DAF), and pods 15/20 mm in length were dissected into the shell, seed coat, and seed with a sterile surgical blade and forceps. The plants were grown aseptically in vitro by germinating surface-sterilized seeds on half-strength Murashige and Skoog (MS) medium under a 16 h light/8 h dark cycle for 4 weeks at 25 8C. Light intensity in the incubator was about 40 mE/(m2/s), 10 cm above the self. Roots and leaves of sterile plants were used for
induction experiments. Plants were also grown in a glasshouse as aseptically as possible for 8 weeks and their leaves were used for induction experiments. All tissue samples were frozen in liquid nitrogen and kept at /80 8C until needed. 2.3. Induction conditions Trifoliate leaves from healthy plants grown in a glasshouse were used for the induction experiments and were collected by cutting the uppermost part of the petioles with a razor blade. Leaves with petioles were immersed in the following sterile solutions for 0, 3, 12, and 24 h: H2O, 50 mM NaHPO4 (pH 6.9), 0.1 mM paraquat, 5 mM H2O2, 0.7 mM JA, 10 mM SA, 5 mM ethephon, or 100 mM abscisic acid (ABA) [13,14]. For the wounding treatment, the trifoliate leaves were punched with fine pins and floated on the sterile water. Leaves were kept for the first 12 h in the light with intensity of about 100 mE/(m2/s) and for the following 12 h in the dark at 25 8C. For the UV treatment, plants were irradiated under UV lamps at 1.35 mE/(m2/s) for 2 h and returned to the dark glasshouse. The irradiated leaves were collected 0, 3, and 12 h after irradiation. Leaves and roots of sterile plants that had been grown in vitro for 4 weeks were used for induction experiments. Plants grown in vitro were placed under dark conditions for 48 h prior to yeast extract treatments. For the elicitor treatment, roots of intact plants were incubated in halfstrength MS medium containing 25 mg/ml yeast extract and placed in the dark for 6 h. All tissue samples were frozen in liquid nitrogen and stored at /80 8C until needed. 2.4. Resveratrol analysis Resveratrol was analyzed using a modification of the method of Fettig and Hess [3]. Samples were protected from light during the analysis process and the free form of resveratrol was extracted by agitating the frozen tissue powder in methanol for 16 h at room temperature. Resveratrol was separated on a mBondapak C18 column (3.9 /300 mm, Waters, USA) with water /acetonitrile gradient elution at a flow rate of 1.0 ml/min and detected with a fluorescence detector (Waters 474, Milford, USA) set at 330 nm for excitation and 374 nm for emission. Samples from three replications were analyzed. 2.5. Northern blot analyses Northern blot analyses were carried out with total RNA prepared from sample tissues as described [15]. Northern blots were repeated at least three times and results from representative blots were presented. Twenty micrograms of total RNA was denatured,
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separated on a 1.2% formaldehyde/agarose gel and transferred onto a nylon membrane (Hybond-N , Amersham, UK). The membrane was prehybridized for 2 h at 50 8C in a DIG Easy Hyb solution (Roche, Germany), and incubated for 16 h at 50 8C after adding the DIG-labeled RS cDNA clone, pPRS3C [15], in the prehybridization solution. After hybridization, the membrane was washed to a final stringency of 0.5 / SSC and 0.1% SDS at 68 8C for 30 min. RNA on the membrane was detected using the DIG chemiluminescent detection system (Roche, Germany) as described [15].
3. Results
3.1. Resveratrol content and RS gene expression in fieldgrown peanut plants Regulation of resveratrol synthesis has been investigated in peanut suspension cells, but rarely in plant tissues. With regard to the beneficial roles of resveratrol for plant and human health, it is of interest to understand the mode of resveratrol synthesis regulation in peanut tissues of agronomic importance. To obtain the initial information for further investigation, resveratrol was analyzed in the tissues of healthy plants grown in the field up to 40 DAF. Free resveratrol contents in leaves, pods, and roots were 2.05, 1.34, and 1.19 mg/g FW, respectively (Fig. 1A). Peanut pods contain seeds inside the shell and the seeds are covered with the seed coat. To specifically locate resveratrol in pods, the pods at mid-maturity were dissected into the shell, seed coat, and seed. Resveratrol was present at 2.60, 0.06, and 0.05 mg/g FW in the shell, developing seed, and seed coat, respectively (Fig. 1A). Because resveratrol is synthesized by the catalysis of RS, RS mRNA levels were examined in the same plant tissues. RS-specific mRNA, about 1.4 kb in size, was detected at high levels in the roots and pods, but below the detection levels in the leaves (Fig. 1B). RS mRNA was present at high levels in the shell, but below the detection levels in the developing seed and seed coat, indicating that most of the RS mRNA detected in the pods was from the shell (Fig. 1B). Resveratrol content in mature peanut seeds ranges from 0.03 to 0.14 mg/g seed and is lower than that found in the seed coats [5]. Our results for resveratrol content in developing peanut seeds are consistent with a recent report on resveratrol in peanut seeds [5]. However, little information is available on the RS gene expression in tissues of field-grown peanut plants.
Fig. 1. Resveratrol (A) and RS mRNA (B) in the tissues of peanut plants grown in the field. (A) Resveratrol content was determined by high-performance liquid chromatography (HPLC) in leaves (LF), whole pods (WH), and roots (RT). Whole pods (WH) were dissected into the shell (SH), seed coat (SC), and seed (SE). (B) Expression of RS mRNA in peanut tissues as indicated in (A). The size of the RS mRNA detected was about 1.4 kb. Twenty micrograms of total RNA was resolved in a 1.0% (w/v) agarose/formaldehyde gel, transferred to a nylon membrane, then hybridized with a DIG-labeled pPRS3C insert. Ethidium bromide-stained rRNA bands as an indicator of equal loading (rRNA).
3.2. Accumulation of resveratrol and RS mRNA in response to elicitor Resveratrol and RS mRNA showed tissue-specific distribution in peanut plants grown in the field. To investigate the tissue-specific expression of resveratrol synthesis, we examined the effects of various stresses and hormones on resveratrol synthesis in peanut plants. First, we examined the effect of elicitor on resveratrol synthesis in sterile peanut leaves and roots based on the previous report on the elicitor effect of yeast extract in peanut suspension cells [9] and plant tissues [15]. Sterile leaves and roots contained trace amounts (0.15 mg/g FW) of resveratrol. Yeast extract induced an accumulation of resveratrol in the leaves and roots. Resveratrol accumulated up to 1.2 and 2.8 mg/g FW in leaves and roots, respectively, after treatment with yeast extract for 6 h, which corresponded to 8- and 19-fold increases relative to untreated control tissues (Fig. 2A). Levels of RS mRNA also increased in leaves and roots in the yeast extract treatment (Fig. 2B), and these increases were correlated with the increase in resveratrol contents in the tissues. RS transcription is induced by a yeast extract similar to the elicitor prepared from Phytophthora megasperma f. sp. glycinea in peanut suspension cells [9] and peanut roots [15]. Our result indicates that yeast extract induces
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RS mRNA was detected in the leaves treated with water or 50 mM sodium phosphate buffer for up to 24 h. However, RS mRNA increased in response to other treatments, generally in a time-dependent manner. The induction kinetics, however, was also quite different between the treatments. It took less than 3 h for the induction of detectable amount of RS transcripts by UV light and paraquat, but over 12 h by wounding and H2O2 (Fig. 3B). The increase in RS mRNA was correlated with resveratrol accumulation, indicating transcriptional control of RS gene expression. Induction of resveratrol and RS mRNA synthesis in response to UV light has been observed in peanut suspension cultures [8,9]. The induction of resveratrol and RS mRNA by paraquat, the generator of the superoxide radical, and H2O2 is consistent with the observation of RS mRNA induction by ozone in grapevine leaves [16]. Fig. 2. Accumulation of resveratrol (A) and RS mRNA (B) in peanut leaves and roots in response to yeast extract. (A) Resveratrol content was determined by HPLC in the leaf (LF) and root (RT) tissues treated for 6 h with yeast extract (YE). CT, control. (B) Accumulation of RS mRNA in peanut leaves and roots treated with YE as indicated in (A). The size of the RS mRNA detected was about 1.4 kb. Twenty micrograms of total RNA was resolved in a 1.0% (w/v) agarose/ formaldehyde gel, transferred to a nylon membrane, then hybridized with a DIG-labeled pPRS3C insert. Ethidium bromide-stained rRNA bands as an indicator of equal loading (rRNA).
resveratrol accumulation via transcription of RS mRNA in peanut plants. 3.3. Accumulation of resveratrol and RS mRNA in response to abiotic stresses Since resveratrol synthesis was induced in peanut plants by elicitor, we further examined the effect of abiotic stresses and reactive oxygen species (ROS) on resveratrol synthesis with the leaves of peanut plants grown as aseptically as possible in a glasshouse. No, or little, resveratrol accumulated in the leaves treated with water or 50 mM sodium phosphate buffer for up to 24 h, and a small amount (0.25 mg/g FW) was detected in leaves treated with water for 24 h. However, resveratrol increased in the leaves treated with various stresses 2- to over 200-fold relative to the amount detected in the leaves treated with water. Resveratrol increased up to 225-fold 12 h after the UV treatment for 2 h, and also increased 23-, 2-, and 2-fold by the paraquat, H2O2, and wounding treatments for 24 h, respectively. Accumulation kinetics was different between the treatments, but in general the accumulation increased with the period of treatment up to 24 h (Fig. 3A). Accumulation of RS mRNA in response to the abiotic stresses was similar to that of resveratrol. No, or little,
3.4. Accumulation of resveratrol in response to stress hormones The induction of resveratrol synthesis by abiotic stresses and ROS suggested that stress hormones might be involved in resveratrol synthesis in peanut plants. Therefore, we investigated effects of stress hormones on resveratrol synthesis in peanut leaves. Resveratrol accumulated in response to SA, JA, and ethylene in a time-dependent manner up to 24 h. The effect of the hormones on resveratrol accumulation was quite different: it increased up to three-, two-, and eightfold in response to SA, JA, and ethylene, respectively. However, no resveratrol accumulated in response to ABA in peanut leaves (Fig. 3A). RS mRNA also increased in response to SA, JA, and ethylene at least 12 h after treatment. RS mRNA also increased in a time-dependent manner in response to SA and ethylene, and the increase reached its maximum 24 h after treatment. In contrast, RS mRNA was not induced by ABA (Fig. 3B). The levels of RS mRNA increased in response to the hormones correlated with the amounts of resveratrol accumulated, indicating transcriptional control of RS gene expression.
4. Discussion The beneficial roles of resveratrol in human health, as well as in plant health, focus much attention on the synthesis of resveratrol in peanut plants. Therefore, it is of interest to understand resveratrol synthesis in the peanut tissues of agronomic importance. In peanut plants grown in the field to mid-maturity, resveratrol and RS mRNA were relatively abundant in the roots and shells, but not in seed coats and seeds, indicating tissue-specific regulation of resveratrol synthesis in pea-
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Fig. 3. Accumulation of resveratrol (A) and RS mRNA (B) in peanut leaves in response to abiotic stresses and hormones. (A) Resveratrol content was determined by HPLC in the leaves treated with water, 50 mM sodium phosphate (pH 7.0; SP), 0.1 mM paraquat (PQ), 5 mM H2O2 and wound (WD), 5 mM ethylene, 10 mM SA, 0.7 mM JA, and 10 mM ABA for 0, 3, 12 and 24 h. UV light (UV) was treated for 2 h and resveratrol was determined 0, 3 and 12 h after treatment (S.D.: standard deviation). (B) Accumulation of RS mRNA in peanut leaves treated as indicated in (A). The size of the RS mRNA detected was about 1.4 kb. Twenty micrograms of total RNA was resolved in a 1.0% (w/v) agarose/formaldehyde gel, transferred to a nylon membrane, then hybridized with a DIG-labeled pPRS3C insert. Ethidium bromide-stained rRNA bands as an indicator of equal loading (rRNA).
nut plants. In leaves of field-grown peanut plants, however, RS mRNA was very low, even though a substantial amount of resveratrol was present in the tissue. This may be due to the resveratrol accumulated prior to the previous stimuli in the leaves in which RS was not expressed at the time of investigation. Evidence on resveratrol synthesis indicates that RS gene expression plays an important role in resveratrol accumulation in plant tissues [2,3]. Our results also show a correlation between resveratrol and RS mRNA accumulation, indicating that resveratrol synthesis is regulated through the transcriptional control of RS genes. Previous studies also indicate that RS gene expression is induced by several biotic and abiotic stresses. RS
genes are induced by fungal infection, elicitor, and UV treatments in peanut cultured cells [9] and in plant tissues [15]. There is a high possibility of wounding and microbial infection in roots and shells during growth in soils colonized with various soil fungi. Roots undergo wounding during the penetration of young lateral roots through the cortex and epidermis of the main roots [17]. Infection of peanut shells by soil fungi such as Aspergillus flavus takes place in the soil [18]. Therefore, it is possible that accumulation of resveratrol in the roots and shells was induced by factors such as wounding and fungal infections. Our results also support the view that resveratrol is accumulated by elicitor and abiotic stresses such as wounding and UV light through the expression of RS genes in peanut leaves and roots.
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Furthermore, RS mRNA expression was induced in peanut leaves in response to SA, JA, and ethephon. SA, JA, and ethylene have been shown to be involved in the activation of various plant defense responses following pathogen attack. SA plays a central role in the establishment of SAR and the elaboration of local defense responses against pathogen attack [19]. On the other hand, JA and ethylene play an important role in the induction of nonspecific disease resistance through signaling pathways distinct from the classic SAR response pathway regulated by SA [10]. In addition to its role in plant pathogenesis, SA, JA, and ethylene play a major role in plant responses to abiotic stresses such as UV-B and ozone [20], and wounding [21]. Therefore, induction of RS expression by SA, JA, and ethylene raises the possibility that these molecules share a common signaling pathway in the regulation of RS gene expression in the peanut. Induction of RS mRNA by paraquat is interesting because paraquat causes the generation of ROS such as superoxide and hydrogen peroxide [22]. ROS has been shown to stimulate the accumulation of SA [23] and ethylene [24], and is also required for the establishment of SAR to avirulent pathogens in plants [25]. Wounding, UV-B and ozone also lead to the generation of ROS [26,27]. Induction of RS mRNA by UV and paraquat, together with its induction by H2O2, suggests that ROS play a role as an intermediate in the signaling pathways [28] for the expression of peanut RS genes. It is noteworthy that accumulation of resveratrol and RS transcripts was faster and higher in the UV and paraquat treatments. This observation is supported by the ability of UV and paraquat to generate superoxide and hydrogen peroxide from multiple sources [22,27], and induction of SA and ethylene by ROS [23,24]. Our results indicate that the tissue-specific distribution of resveratrol in the field-grown peanut is due to the differential expression of RS genes induced by biotic and abiotic stresses. In addition, stress hormones such as SA, JA, and ethylene, and ROS mediate the transduction of signals triggered by biotic and abiotic stresses, which finally result in the expression of RS genes for the synthesis of resveratrol. Therefore, tissues such as roots and shells, which are vulnerable to abiotic and biotic stresses, should be more likely to produce resveratrol than would protected tissues such as seeds and seed coats. However, developmental regulation of RS gene expression in the seed and seed coat cannot be excluded without further dissection studies with pods at different levels of maturity. However, our results suggest that levels of RS expression in seeds and seed coats may be lower than those in other tissues such as roots and shells. In peanut cells, most of the resveratrol is used as an intermediate for the insoluble materials deposited in the cell wall [29]. Peanut shells play multiple roles, including the structural protection of seeds from injury and
infections. Lignocellulosic materials found in high amounts in the shell are the major chemical components for the structural roles of the tissue [30]. Results from this study also indicate that the shell contains conjugated resveratrol at about half the amount of the free form (data not shown). However, most of the resveratrol is present as a free form in other peanut tissues. Therefore, the synthesis of resveratrol by the expression of RS in peanut tissues could provide resistance to pathogen infection through the direct antifungal effect of resveratrol and the reinforcement of shells by the synthesis of cell wall materials with resveratrol as an intermediate.
Acknowledgements This work was supported by Korea Science and Engineering Foundation under Project No. R01-1999000-00172.
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