Distribution of resveratrol and stilbene synthase in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon) and the effect of UV-C on its accumulation

Plant Physiology and Biochemistry 48 (2010) 142e152

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Research article

Distribution of resveratrol and stilbene synthase in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon) and the effect of UV-C on its accumulation Wei Wang a, b,1, Ke Tang a,1, Hao-Ru Yang a,1, Peng-Fei Wen c, Ping Zhang d, Hui-Ling Wang a, Wei-Dong Huang a, * a

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Science, Beijing 100093, China College of Horticulture, ShanXi Agricultural University, Taigu, ShanXi 030801, China d Institute of Horticulture, XinJiang Academy of Agricultural Sciences, Urumuqi 830000, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 May 2009 Accepted 1 December 2009 Available online 17 December 2009

Current research indicated that the resveratrol was mainly accumulated in the skin of grape berry, however, little is yet known about the distribution of resveratrol, as well as the regulation mechanism at protein level and the localization of stilbene synthase (malonyl-CoA:4-coumaroyl-CoA malonyltransferase; EC 2.3.1.95; STS), a key enzyme of resveratrol biosynthesis, in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon). Resveratrol, whose constitutive level ranged from 0.2 mg kg
1 FW to 16.5 mg kg
1 FW, could be detected in stem, axillary bud, shoot tip, petiole, root and leaf of grape plants. Among them, stem phloems presented the most abundant of resveratrol, and the leaves presented the lowest. Interestingly, the level of STS mRNA and protein were highest in grape leaves. And the analysis of immunohistochemical showed the tissue-specific distribution of STS in different organs, presenting the similar results compared with the amount of protein. And the subcellular localization revealed that the cell wall in different tissues processed the most golden particles representing STS. Subjecting to UV-C irradiation, resveratrol and STS were both intensely stimulated in grape leaves, with the similar response pattern. Results above indicated that distribution of resveratrol and STS in grape was organ-specific and tissue-specific. And the accumulation of resveratrol induced by UV-C was regulated by transcriptional and translational level of STS. Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Grape plant Immunolocalization Resveratrol Stilbene synthase

1. Introduction Resveratrol, 3, 5, 40 -trihydroxystilbene, is a naturally occurring polyphenol produced by plants as a self-defense agent, i.e. phytoalexin, which has antifungal activity [1,2]. It has been found in some plants such as grapevine [3,4], pine [5] and peanut [1]. A body of reports has demonstrated that resveratrol was associated with reduced coronary heart disease mortality and atherosclerosis [6], inhibited low density lipoprotein oxidation [7], and carcinogenesis [8,9]. Due to the dual significances in both plant protection and

Abbreviations: BSA, bovine albumin; CTAB, cetyltrimethylammonium bromide; EDTA, ethylene diamine tetraacetic acid; PAL, phenylalanine ammonia lyase; PBS, phosphate-buffered saline; PMSF, phenylmenthylsulfonyl fluoride; RT-PCR, reverse transcription-polymerase chain reaction; SDS, sodium dodecyl sulfate; STS, stilbene synthase; Tris, tris (hydroxymethyl) e amino methane; UV, ultra violet. * Corresponding author. Tel./fax: þ86 10 6273 7553. E-mail address: [email protected] (W.-D. Huang). 1 These authors contribute equally to this work. 0981-9428/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.12.002

human health, the content and distribution of the resveratrol in grape and wine had attracted more and more attentions over the past several years, particularly in berries used for winemaking. Most of the previous researches focused on the synthesis in response to biotic or abiotic stress [10,11,12], concentration in the grape and/or wine [13,14] and the transcriptional regulation of STS gene [1,15]. Grapevine and wine is an important dietary source of resveratrol [16]. In grapevines (Vitis vinifera), resveratrol was synthesized in response to biotic and abiotic stress, such as fungal infection [1], ultra violet light exposure [17,18], ozone stress [15], anoxic treatment [4], and wounding [1]. In wines, the resveratrol content was depended largely on the wine type, grape variety [16,17,18,19], enology practices [20]. Since the resveratrol was mainly accumulated in the grape berry skin and seeds [4,21], much attention had been paid on the changes of resveratrol during grape berry development and vinification process. To our knowledge, there was no report on the distribution of resveratrol in grapevine.

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In grape, resveratrol is synthesized by the catalysis of stilbene synthase (malonyl-CoA:4-coumaroyl-CoA malonyltransferase; EC 2.3.1.95; STS) using one molecule of coumaroyl-CoA and three molecules of malonyl-CoA [1,3,12]. The STS gene has been cloned in many plants including grapevine, and the regulation mechanism at transcriptional level, especially under the biotic and abiotic stress, has been extensively studied [22]. Although there has been a substantial amount of research on the STS, the regulation mechanism at transcriptional and translational level in different grape tissues still remains obscure. We have previously described that the full-length gene encoding STS has been cloned from wine grape berries (Cabernet Sauvignon) and a polyclonal antibody against grape STS has been prepared [23]. Here, we reported the distribution of resveratrol and expression of STS gene at transcriptional and translational level, as well as the localization of STS in different tissues of grape plants.

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Additionally, we studied the changes of resveratrol and STS mRNA and protein, and the relationship among them, in response to UV-C irradiation in grape leaves. The results obtained will provide some information for elucidating the synthesis and transport of resveratrol in grape plant, and increase our insight in regulation mechanism of plant secondary metabolism. 2. Results 2.1. Constitutive level of endogenous resveratrol and STS in different grape plant organs Constitutive level of endogenous resveratrol and STS in different grape plant organs were investigated (Figs. 1 and 2). The distribution of resveratrol in the grape plants was organ-specific (Fig. 2A). As shown in Fig. 2, the content of resveratrol in different organs of

Fig. 1. Diagrammatic representation of grape plant to show the experimental sampling site.

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grape plants ranged from 0.2 mg kg
1 FW to 16.5 mg kg
1 FW, and there was a significant difference among the organs. The maximum content of resveratrol was presented in the stem phloems, followed by axillary buds, roots, stem xylems, shoot tips and petioles; and the minimum was found in leaves. Fig. 2B showed the amount of STS protein in different organs of grape plants. A 43 kDa peptide was specifically detected with an anti-grape STS polyclonal antibody on SDS-PAGE gel of crude protein extract. The intensity of immune signal was significantly different in diverse organs, which indicated that the distribution of STS protein also depended on different organs. The most abundance of STS protein was obtained in leaves, which was about 2-fold abundance compared with other organs except shoot tip. These results illustrated that the STS protein was mainly assembled in leaves. The expression of STS gene in different grape organs was analyzed by RT-PCR (Fig. 2C) Actin1, which was expected to show a constitutive expression pattern, was used as the control to standardize the expression of the STS gene. As shown in Fig. 2C, there was significant difference on the STS gene transcription level in different organs. The high level of STS mRNA was found in leaves, roots and petioles (Fig. 2C), and the maximum was observed in leaves. The low level of STS mRNA was detected in other organs, and the minimum among them was in axillary buds. 2.2. Immunohistochemical localization of STS in grape plants The cell and tissue localization of STS in grape plants were investigated by the standard immunolocalization techniques and the results were shown in Fig. 3, and the blue-green signal reflected the distribution of STS in the grape plants. As shown in Fig. 3, STS presented in all kinds of grape organs, including leaf, leaf bud, tendril, stem and root. In grape leaf (Fig. 3A), strong STS signal was detected in palisade and spongy tissue in mesophyll cells and vascular bundle, especially in vein phloem. In the leaf bud (Fig. 3B), the STS signal presented in growth point, leaf primordium, young leaf and leaf bud primordium. Whereas, more blue-green signal was visualized in the growth point and leaf primordium. In grape stem (Fig. 3C), the STS mainly localized in the interfascicular cambium and primary phloem. However, only little STS signal was detected in the vascular bundle of grape tendril and no obvious STS signal was founded in other tissues (Fig. 3D). In grape root, the STS were mainly localized in the endoderm and primary phloem (Fig. 3E). In order to verify the reliability of the immunolocalization technique, two controls were carried out. In the first control we substituted normal rabbit serum for the primary STS antibody (Fig. 3F), and in the second one we omitted the primary STS antibody (Fig. 3G). All the other procedures proceeded as usual. There was very little STS signal detected in these sections, which indicated that the immunohistochemical localization technique was reliable and the antibody was highly specific. 2.3. Subcellular localization of STS in grape plant Fig. 2. Distribution level of resveratrol and stilbene synthase in various organs of grape plants. A, Resveratrol content in various organs of grape plant. As shown in Fig. 1, the whole grape plant was dissected into shoot tip (Sh T), axillary bud (Ax B), petiole, leaf, stem phloem (Stem P), stem xylem (Stem X), and root. B, Western blot analysis of STS in different organs of grape plant. For each lane, 20 mg protein was loaded. In all cases leaves from 10 seedlings were pooled for each protein extraction to obtain an average protein pattern. Proteins were probed with immune serum against grape STS (diluted in 1:1000). Three independent experiments were carried out. Bars represent standard error (SEs) and the bands in the nitrocellulose membrane are the result of one of the experiments. C, RT-PCR analysis of STS mRNA in different organs of grape plant. Amplification of grape Actin1 cDNA was used as control to standardize the expression of the STS gene. Bars represent the SEs of three samples.

The subcellular localization of STS in grape plants was performed using immunogold electron-microscope technique. In the grape plant root, STS visualized by gold particles were primarily present in cell wall, cytoplasm and vacuole (Fig. 4BeD). Moreover, there was no obvious immuno-signal in other cellular compartments (data was not shown). In stem, both cell wall and cytoplasm (Fig. 5B and C) were largely labeled by gold particles, while few particles were observed in the chloroplast (Fig. 5B). In grape plant leaf, gold particles representing STS were visualized in cell wall

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Fig. 3. Immunohistochemical distribution of stilbene synthase in different organs of grape plant. A, Tissue localization of STS in grape leaf (mesophyll, vein). UE, upper epidermis; LE, lower epidermis; VB, vascular bundle; X, xylem; PPh, primary phloem; PT, palisade tissue; Sc, sclerenchyma; ST, spongy tissue. B, Immunohistochemical distribution of STS in grape leaf bud. GP, growth point; LP, leaf primordium; YL, young leaf; LBP, leaf bud primordium; PRC, primary cambium. C, Immunohistochemical localization of STS in grape stalk. PPh, primary phloem; PX, protoxylem; SX, secondary xylem; PiR, pith ray; Fa, fascicular cambium. D, Immunohistochemical localization of STS in grapevine tendril. EP, epidermis; Co, cortex; VB, vascular bundle; Pi, pith; PiR, pith ray. E, Tissue localization of STS in grapevine root. EP, epidermis; Co, cortex; En, endoderm; PPh, primary phloem; PX, protoxylem; Vc, vascular cylinder. F, The primary STS antibody was substituted with the normal serum of rabbit. No substantial immuno-signal was detected. G, Without the primary STS antibody. No substantial immuno-signal was detected.

(Fig. 6A) and nucleus (Fig. 6D), while few gold particles were visualized in cytoplasm and chloroplast (Fig. 6B and C). Furthermore, no gold particles were detected when the pre-immune serum was used and the polyclonal antiserum against STS was

omitted during the immuno-labeling, which suggested that the immunogold electron-microscopy localizations identified in the experiment were both specific and reliable (Figs. 4E and F, 5D and E, 6E and F).

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Fig. 4. Immunogold electron-microscope localization of stilbene synthase in the root of grape plant A, Ultrastructure of grape root cells. BeD, Immunogold localization of STS in the root cells of grape plants. Gold particles mainly resided in the cell wall and cytoplasm, while some particles labeled in vacuole (50,000). EeF, The antiserum omission and preimmune serum control of the STS immunogold localization, respectively. No substantial immuno-signal was detected. Abbreviations: CW, cell wall; Cyt, cytoplasm; V, vacuole.

2.4. Change of resveratrol and STS in response to UV-C of grape plant leaves In order to investigate the involvement of resveratrol accumulation, STS gene expression and STS protein accumulation, change of them were studied in grape plant leaves after UV-C irradiation. As shown in Fig. 7A, resveratrol could be induced intensely by UV-C light in grape plant leaves. After UV-C irradiation, content of endogenous resveratrol started to increase at 8 h and reached its peak value at 16 h and 24 h, whereas, it declined afterwards and finally reached values similar to the control level. In this two time points of peaks, the resveratrol content had no significant differences, which were 196.43 and 180.93 folds relative to the control, respectively. Similarly, accumulation of STS protein in grape plant leaves also occurred at 16 h and 24 h, respectively, with no significant difference (Fig. 7B). It was notable that the first peak of STS accumulation disappeared immediately, while the latter one maintained at a relatively high level after decreasing a little.

In addition, STS mRNA level in grape plant leaves was investigated by RT-PCR to elucidate the mechanism of accumulation of resveratrol in response to UV-C irradiation (Fig. 7C). Similar to accumulation of STS protein in response to UV-C light, STS mRNA also responded to UV-C irradiation in a biphasic manner, with two peaks of accumulation at 16 h and 24 h, respectively. The results shown here indicated that resveratrol and gene and protein of STS in response to UV-C light in a similar manner in grape plant leaves. 3. Discussion 3.1. Distinct distribution of resveratrol and STS in normal developmental grape plants So far, resveratrol has been founded in at least 72 plant species distributed among 31 genera and 12 families [33], and a number of these are components of the human diet, such as grapes, wine, cranberries peanuts and chocolate. Grape and its products, especially wine, are the most important human dietary containing

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Fig. 5. Immunogold electron-microscope localization of stilbene synthase in the stem of grape plant. A, Ultrastructure of grape stem cells. BeC, Immunogold localization of STS in the stem cells of grape. Gold particles mainly resided in the cell wall and cytoplasm, while some particles labeled in chloroplast (50,000). DeE, The antiserum omission and preimmune serum control of the STS immunogold localization, respectively. No substantial immuno-signal was detected. Abbreviations: CW, cell wall; Chl, chloroplast; Cyt, cytoplasm.

resveratrol. Therefore, it is of interest to understand resveratrol synthesis and accumulation in grape tissues. In peanut, the resveratrol were mainly accumulated in the shell of pods, the roots and leaves [1]. However, results obtained from this research showed that the resveratrol were mainly accumulated in the phloem of stem, axillary bud and root (Fig. 2A) in grape plant. This discrepancy implied that there existed different mechanism of resveratrol accumulation in different species. Besides, it was observed that the maximum accumulation of resveratrol in grape plant occurred in the phloem of stem. Together with that the phloem tissue was considered to function in long distance transport of signaling molecules and pathogens and for nutrient reallocation [34], it suggested that the resveratrol may be transported among the different tissues/organs in grape plants subjected to stresses. This was also supported by the visualized STS in phloem tissues of leaf vein, stem and root (Fig. 3A, C and E). Nevertheless, because little was known about the cellular and molecular biological mechanisms of STS action in grape plants, it was still difficult to propose the physiological significance of such distributions that were different with previous reports. Many reports indicated that the synthesis and accumulation of resveratrol in plant tissue were regulated at both transcriptional [1,35] and translational levels [12]. In order to evaluate the involvement between STS gene and resveratrol, the transcriptional and translational level of STS gene and endogenous level of resveratrol in different grape organs were investigated under normal developmental condition (Fig. 2B and C). The results indicated that the maximum amount of STS mRNA and protein were obtained in leaves. However, compared with other tissues/ organs, there was no obvious occurrence of resveratrol in grape leaves. In general, the leaves are considered to be the main organ to apperceive the changes of environment. And the changes of environment, such as ozone exposure [15], UV irradiation [12,19]

and fungal infection [36], induced the STS gene expression and the accumulation of protein and resveratrol. Hence, the difference between endogenous resveratrol and STS in grape leaves under normal developmental condition may be relative to its physiology function against stresses. Thus, defense pattern may be speculated as below: resveratrol maintained at a relative low level in plant under normal developmental process, but was newly synthesized with abundance of its conserved STS as soon as subjecting to stresses to be abundant enough to induce plant defense response, then reduced extremely the degree of damage against stresses. The relationship between histochemical and/or subcellular localization and physiological function was the highlight of enzymology research. In previous studies, using the grape berry as materials, the STS protein was found mainly to distribute in the skin tissues. The particular localization of STS on the skin tissues was considered to be related to its role in synthesizing defense compounds [37]. The STS protein was also detected on the cell wall [12,37] and chloroplast, even in the endoplasmic [12] of skin tissue of grape berry at subcellular level. In our study, however, the STS protein appeared in all kind of organs of grape plants, including leaves, root, bud, tendril, and stem (Fig. 3); Moreover, it was shown that not only in the cell wall and chloroplast (Figs. 4e6) which was in agreement with the distribution in grape berry under UV irradiation reported by Pan et al. (2008) [12], but also in cytoplasm (Figs. 4e6) and vacuole (Fig. 4), even in the nucleus (Fig. 6D) existed the STS protein. It was worthy to investigate the obvious difference on the subcellular localization of STS in different organs, which indicated that different regulation mechanism might exist in different organs. To our knowledge, it was the first time that localization of STS in the whole grape plant. However, it is difficult to propose the physiological significance of such distribution that was different to previous studies.

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Fig. 6. Immunogold electron-microscope localization of stilbene synthase in the leaf of grape plant. AeD, Immunogold localization of STS in the leaf cells of grape. Gold particals resided in the cell wall, nucleus, while some particles labeled in the cytoplasm and chloroplast (50,000). EeF, The antiserum omission and pre-immune serum control of the STS immunogold localization, respectively. No substantial immuno-signal was detected. Abbreviations: Chl, chloroplast; CW, cell wall; M, mitochondrion; N, nucleus; P, plastid; V, vacuole.

3.2. Accumulation of resveratrol in response to UV-C was involved in its de novo biosynthesis in grape leaves In order to testify the hypothesis above, we studied the changes of resveratrol and STS mRNA and protein, and the relationship among them, in response to UV-C irradiation in grape leaves. Resveratrol content started to increase at 8 h after UV-C irradiation (Fig. 7A), but the amount of STS gene and protein didn't increase obviously (Fig. 7A and B), which indicated that grape plants might make use of its conserved STS firstly to synthesize

resveratrol rapidly, thereby they could respond to UV-C timely. Plants always reserve large number of defense-related genes and proteins which could be utilized directly to synthesize components for defense response promptly. Due to the omission of the process from transcription, translation to the activation of enzymes, plants could respond to environmental stress factors effectively and promptly, which made the plants damaged to the minimum extent. This may be an important manner to defense stresses in plants. Besides, it was noticed that accumulation of STS mRNA and protein induced by UV-C both presented two peaks, occurring at

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16 h and 24 h, respectively. And the pattern of STS mRNA and protein in response to UV-C was in consistent with that of endogenous resveratrol. Similar results had been reported in Pinot Noir, Chardonnay [38] and grape cell suspensions [39,40]. In addition, Liswidowati et al. (1991) and Wiese et al. (1994) [39,40] have suggested that the STS gene family may be divided into two groups: some are expressed early with rapid degradation of the mRNA produced, and others are expressed later and slowly activated, but provide more stable mRNA. They also stated that the occurrence of two peaks in STS mRNA accumulation was typical of induced grape cell cultures, indicating a modulated phytoalexin response that certainly enhances defense potential. All these results demonstrated that accumulation of resveratrol elicited by UV-C was modulated by both STS transcription and translation level, which may be involved in enhancing plant defense ability. In conclusion, distribution of resveratrol and STS in grape was organ-specific and tissue-specific, respectively, and endogenous resveratrol responded rapidly to UV-C irradiation in grape plants, and its endogenous level was regulated subsequently through the modulation of transcription and translation of STS. The change of endogenous level of resveratrol may be related to the defense response of seedlings induced by UV-C. Additionally, resveratrol represented the characteristic of a signal to some extent in response of UV-C irradiation: firstly, employing small molecular weight (FW 228.25); secondly, accumulating rapidly and disappearing immediately in response to UV-C irradiation (Fig. 7A); thirdly, may be mobile (indirect evidence obtained in Figs. 2 and 3). However, more direct and convincing evidence should be collected to testify whether resveratrol functions as a signal molecule in the defense response of plants. 4. Materials and methods 4.1. Plant materials One-year-old potted grapevines of V. vinifera L. cv. Cabernet Sauvignon plants were used throughout the experiments. The grape plants were pre-cultured in a controlled environment glass greenhouse at 25  C/18  C (day/night), 65% relative humidity. Plants with uniform vegetative growth were selected for the study, and the experiment was initiated when the plants with 8e10 functional leaves (Fig. 1). The different plant parts were cut as Fig. 1 showed and were either embedded for localization of stilbene or stored at
80  C after freezing in liquid N2 until further analysis. All chemicals were purchased from the Sigma Corporation (St. Louis, USA) unless otherwise noted. 4.2. UV-C irradiation

Fig. 7. Change of resveratrol and stilbene synthase in response to UV-C irradiation. A, Effects of UV-C treatment on resveratrol content in grape leaves. Values represent the mean of three independent measurements (n ¼ 3); bars represent the standard error (SE), each consisting of 10 seedlings. B, Western blot analysis of STS in response to UV-C irradiation in grape plant leaves. For each lane, 20 mg protein was loaded. In all cases leaves from 10 seedlings were pooled for each protein extraction to obtain an average protein pattern. Proteins were probed with immune serum against grape STS (diluted in 1:1000). Three independent experiments were carried out. Bars represent SEs and the bands in the nitrocellulose membrane are the result of one of the experiments. C, RT-PCR analysis of the expression of STS mRNA in UV-treated grape plants. Amplification of grape Actin cDNA was used as control to standardize the expression of the STS gene. Bars represent the SEs of three samples.

Grape plants were irradiated by UV-C (254 nm, Spectroline, Model ZQJ-254, output 300 mW/cm2) for 10 min at 15 cm distance, as well as negative controls consisting of non-irradiated plants. Following irradiation, both the UV-treated and the control plants were transferred into a Robert manual incubation (Model PRX350D) and then incubated in the dark at 25  C with relative humidity 80% for 60 h. At each incubation period of 0, 1, 4, 8, 16, 20, 24, 36, 48, 60 h, the inferior fourth leaf from top to base were collected, frozen in liquid nitrogen and stored at
80  C. Each treatment had at least three independent replicates, and each replicate consisted of ten plants. 4.3. Extraction and HPLC analysis of resveratrol/stilbene The stilbene in different grapevine parts was extracted according to the methods described by Sun et al. (2006) [24] with slight

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modification. In briefly, 4 g of grape tissue was firstly ground to fine powder under liquid nitrogen and then was extracted by 100 ml methanol for with agitation at room temperature under darkness for 48 h. After centrifugation at 10,000  g for 15 min, the supernatant was evaporated at less than 35  C to dryness and recovered by distilled water. The aqueous phenolic solution obtained was further purified by liquideliquid extraction. 20 ml of aqueous extract were extracted three times with 20 ml of ethyl acetate at room temperature under darkness. The organic phases were combined, evaporated to dryness at less than 35  C. The residue was recovered by 2 ml of 50% ethanol in water and stored at
20  C for further HPLC analysis. Qualitative and quantitative analysis was performed by reverse phase HPLC apparatus consisting of a WATERS(USA) 2695XE series pumping system, automatic injector furnished with a 50-ml loop, WATERS-2996 PDA detector and chromatography data station software. The separation of compounds was carried out using a Merck Li-Chrospher 100RP-18e column (250  4.0 mm ID, 5 mm) joined to a Merck RP-18 (10 mm  4.0 mm) guard column at 30  C. The injected volume was 20 mL. A constant flow rate of 1.0 mL/min was used with two solvents: solvent A, 1% acetic acid in water; and solvent B, acetonitrile. All the solvents used were of HPLC grade. For the elution program, the following proportions of solvent B were used: 0e20 min, 10% B; 20e36 min, 10e50% B; 36e40 min, 50e10% B. The chromatograms were monitored at 280 nm. The system precolumn column was washed with 100% mobile phase A before injection of the next sample. Identity of stilbene was confirmed by cochromatography on HPLC with authentic standards. 4.4. Protein extraction and gel blot hybridization Total proteins were extracted according to the method of Famiani et al. (2000) [25] with minor modification. Briefly 2 g sample was firstly ground to a fine powder under liquid nitrogen and then was rinsed with 6 mL extraction solution for 2 min. The extraction solution was consisted of 50 mM TriseHCl [Tris ¼ tris (hydroxymethyl)-amino methane] (pH 8.9), 2% SDS (sodium dodecyl sulfate), 5 mM ascorbic acid, 5 mM EDTA (ethylenediamine tetraacetic acid), 14 mM b-mercaptoethanol, 10 mM leupeptine, 1 mM PMSF (phenylmenthylsulfonyl fluoride) and 0.15% (w/v) PVP (polyvinyl pyrrolidone). The homogenate was filtered through 4 layers of cheesecloth and centrifuged at 12,000  g for 20 min. The supernatant was used as the total protein. Protein concentration was determined as described in Bradford (1976) [26] using bovine albumin (BSA) as the standard. The separation of total protein was performed using SDS-PAGE in 12% polyacrylamide gels as described by Laemmli (1970) [27]. The identical total proteins (8 mg/lane) were separation using Bio-Rad Mini electrophoresis system (Bio-Rad, Richmond California, USA). After electrophoresis, the proteins were electro-transferred to nitrocellulose (0.45 mm, Amersham LIFE SCIENCE) using a transfer apparatus (Bio-Rad) technique described by Isla et al. (1998) [28]. For protein gel blot analysis, immunological detection of proteins on the NC membrane was carried out using a primary polyclonal STS antibody (preparation by the author [23]) in a 1/1000 dilution at 25  C with an alkaline phosphatase conjugated anti-rabbit IgG antibody from a goat (1/500 dilution) as a secondary antibody. The membrane was stained with 10 mL of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) in the dark and the reaction terminated by addition of double distilled water. 4.5. Isolation of total RNA Total RNA was isolated from the berry using methods described in Wen et al. (2005) [29] with slight modifications. All steps were

performed at 4  C. The berry of 1 g was ground in liquid nitrogen and transferred into 2 ml washing buffer of 10 mM Triseboric acid (pH 7.4), 0.35 M sorbitol, 10% PEG6000 (w/v) and 2% b-mercaptoethanol (v/v). After centrifugation at 12,000  g for 8 min, the 2 ml extraction buffer containing 10 mM Triseboric acid (pH 7.4), 140 nM NaCl, 2 mM EDTA and 2% cetyltrimethylammonium bromide (CTAB) was added and rest incubated for 20 min at 55  C. Then 200 ml 5 M potassium carbonate, 200 ml ethanol and 2 ml chloroform were added. After centrifugation at 8000  g for 10 min, 1/3 volume 10 M LiCl and 0.8 volume isopropyl alcohol were added before centrifugation at 10,000  g. The pellet was dried and resuspended in 0.5 ml DEPC-water and 0.5 ml water-saturated phenol was added. After centrifugation at 15,000  g for 15 min, 0.5 ml chloroform/isoamyl alcohol was added before centrifugation at 15,000  g. Total RNA was then precipitated overnight after addition of 1/3 volume 10 M LiCl. After centrifugation at 15,000  g for 30 min, the pellet was washed in 75% ethanol and re-suspended in DEPC-water. RNA concentration was determined by absorbance at 260 nm, and purity was established with a 260/280 nm. 4.6. RT-PCR analysis The mRNA expression patterns of STS and Actin1 were examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), using AMV reverse transcriptase (Promega A3500). The amplification of Actin1 cDNA was used as an internal control. According to the published sequences for grapes (GenBank accession nos. EU156062 and AY680701), two pairs of oligonucleotide primers were designed to amplify the cDNA clones selected for expression analysis. Gene-specific primers for STS (forward: 50 -TCGAAACATCGGTTAGAAGAG-30 ; reverse: 50 -GACAATTTCCCGTTC AGCAGT-30 ) and Actin1 (forward: 50 -GATTCTGGTGATGGTGTGAGT-30 ; reverse: 50 -GACAATTTCCCGTTCAGCAGT-30 ) were used in RT-PCR. The expected sizes of the PCR products were 275 and 168 bp respectively. For RT-PCR, the first-stand cDNA was synthesized from 1 mg total RNA in a volume of 20 ml containing 20 mM TriseHCl, pH 8.3, 100 mM KCl, 2.5 mM dNTP, 20 units of RNase inhibitor, 5 mM MgCl2, 5 units of AMV reverse transcriptase, 2.5 pmol of Oligo dT15 for 45 min at 42  C. One microliter of the first-strand solution was used for PCR reaction in a total volume of 50 ml containing 20 mM TriseHCl, pH 8.3, 100 mM KCl, 2.5 mM dNTP, 5 mM MgCl2, 5 units of Taq DNA polymerase (TaKaRa), 10 pmol of each gene-specific amplification primer. Reverse transcription was performed at 42  C for 45 min, followed by PCR amplification for 26 cycles of denaturation at 94  C for 30 s (10 min for the first cycle), annealing at 50  C for 30 s, and extension at 72  C for 1 min, with a final extension at 72  C for 10 min, in a GeneAmp PTC-200 cycler. The amplified DNA samples were separated on 1% agarose TAE gel, and analyzed with Gene analysis software package (Gene Company). 4.7. Immunohistochemical localization of STS The immunohistochemical localization of STS was performed as the method described by Hou and Huang (2005) [30] with slight modifications. After the grape tissue was cut, they were post-fixed overnight in a solution containing 4% paraformaldehyde and 2.5% glutaraldehyde at 4  C, dehydrated with a graded ethanol series, embedded in paraffin, and sectioned into slices. The slides were spread with polylysine before the fixing of the sections. Dried sections were deparaffinized with xylene and hydrated in an ethanol-water series. After immersion in 10 mM phosphate-buffered saline (PBS, pH 7.0, containing 0.2 g/L KCl, 2.19 g/L Na2HPO4$12H2O, 0.482 g/L KH2PO4) for 5 min, slides were incubated in a blocking solution [BS, 10 mM PBS, 0.1% (v/v)Tween-20, 1.5% (w/v)

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glycine, 5% (w/v) bovine serum albumin] for 45 min at room temperature, then rinsed in a regular salt rinse solution [RSR, 10 mM PBS, 0.1% (v/v) Tween-20, 0.8% (w/v) BSA, 0.88% (w/v) NaCl] for 5 min, and washed briefly with 10 mM PBS solution that was containing 0.8% (w/v) BSA (PB) to remove the Tween-20. A drop of 1 mg/mL primary STS antibody was added to each slide before covering the inner membrane with plastic gloves, then incubated overnight in a humidity chamber at 4  C. The slides were washed vigorously twice in a high-salt rinse solution [10 mM PBS, 0.1% (v/v) Tween-20, 0.1% (w/v) BSA, 2.9% (w/v) NaCl] followed by a 10 min wash with RSR and a brief rinse with 10 mM PBS. Then, 100 ml secondary antibody were added to the slides [1:100, (v/v) dilution of the antimouse IgG-alkaline phosphatase-conjugate (1 mg/L, Promega)] and incubated overnight in a humidity chamber at room temperature. After rinsing twice in RSR and once in water, the slides were developed for approximately 1 h by adding 200 ml of Western Blue stabilized substrate for alkaline phosphatase (Promega). When the blue-green color appeared on the sections, they were rinsed with water, dehydrated and mounted with a cover glass for photographing.

4.8. Subcellular localization of STS Tissue preparation was adapted from Wang and Huang (2003) [31], with small pieces (1 mm3) cut from grapevine different parts. The obtained pieces were rinsed and fixed overnight in a fixation solution containing 200 mM sodium cacodylate (pH 7.6), 4% (w/v) paraformaldehyde and 2% (w/v) glutaraldehyde at 4  C after a few minutes of vacuum infiltration to remove the air from the intercellular spaces. Tissues were then washed (4  10 min) in 0.1 M phosphate buffer (pH 7.6) at 4  C. After dehydration through an ethanol gradient series, the tissues were infiltrated with an LR White resin embedding medium and polymerized for 24 h under ultra violet light at
20  C. Ultrathin sections (60e80 nm) were cut with a diamond knife on an OM-U3 ultramicrotome, mounted on nickel thin-bar grids (100 mesh) and covered with 0.3% Formvar film. The immunostaining procedure followed that of Ruelland et al. (2003) [32]. The ultrathin sections were incubated with TBSTG buffer (100 mM TriseHCl, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 3% (w/v) BSA) for 30 min, then crossed directly with affinity-purified IgG to STS antibody (preparation by the author) in TBSTG. Following washing three times for 5 min each with TBSTG buffer, the sections were incubated for 1 h with a goat anti-rabbit IgG conjugated to 20 nm gold particles at 1:100 dilution in the TBSTG buffer at room temperature. The sections were rinsed consecutively with TBSTG and double distilled water, then stained with 2% uranyl acetate and 4% lead citrate. The ultrathin sections were examined with a JEM-100S electron microscope. In order to test the specificity of the immunogold localization two controls were employed. The first involved staining the sections with pre-immune rabbit serum instead of an antibody of STS. For the second control antibodies against STS were omitted. Neither of the treatments should lead to labeling the gold particles in the sections.

4.9. Statistics All treatments were repeated at least three times and all samples were analyzed three times. Means and standard errors were calculated from pooled data. In figures, the bar on each point represents the standard error.

151

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