Postharvest Biology and Technology 40 (2006) 6472

Effect of salicylic acid on phenylpropanoids and phenylalanine ammonia-lyase in harvested grape berries
Jian-Ye Chen a,1,2 , Peng-Fei Wen a,b,1 , Wei-Fu Kong a , Qiu-Hong Pan a , Ji-Cheng Zhan a , Jing-Ming Li a , Si-Bao Wan a , Wei-Dong Huang a,
a

Center for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, Peoples Republic of China b College of Horticulture, ShanXi Agricultural University, ShanXi Taigu 030801, Peoples Republic of China Received 5 May 2005; accepted 19 December 2005

Abstract Current research indicates that both salicylic acid (SA), a likely signal in the response of plants to stress, and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), a key enzyme in phenylpropanoid metabolism, perform defense-related functions within plants. However, very little is yet know about the role SA might play in regulating PAL expression and phenylpropanoid biosynthesis. The present experiment was performed using in vivo inltration of 150 M salicylic acid into entire postharvest grape berries (Vitis vinifera L. cv. Cabernet Sauvignon). The results indicated that SA activated PAL by enhancing the accumulation of PAL mRNA, as well as enhancing the synthesis of a new PAL protein and enzyme activity. Further, the activation was found to be time course-dependent. A signicant accumulation of phenylpropanoids was also observed in the SA-treated berries. However, the induction of PAL expression and the accumulation of phenylpropanoids could be blocked by pretreatment with the protein synthesis inhibitor cycloheximide, mRNA transcription inhibitor actinomycin D, and PAL inhibitor 2-amino-2-indanophonic acid (AIP), respectively. These results further indicated that SA could induce PAL mRNA accumulation and as a result, enhance PAL protein amounts and activity as well as enhancing the accumulation of phenylpropanoids such as phenolic acids. 2006 Elsevier B.V. All rights reserved.
Keywords: Grape berries; Salicylic acid; Post-harvest; Phenylpropanoids; Phenylalanine ammonia-lyase

1. Introduction Phenolic compounds are a group of important secondary metabolites in grape berries and play an essential role in determining grape berry quality as well as wine characteristics such as color, avor, astringency, and bitterness (Chamkha et al., 2003). They are also involved in various biochemical and pharmacological activities, displaying antimicrobial, anticarcinogenic, and antioxidant properties (Frankel et al., 1995). Because phenolic compounds have also been shown

Corresponding author. Tel.: +86 10 62737024; fax: +86 10 62737553. E-mail address: huanggwd@263.net (W.-D. Huang). 1 Authors contributed equally to this work. 2 Present address: Guangdong Key Lab for Post-Harvest Science, College of Horticulture, South China Agricultural University, Guangzhou 510642, Peoples Republic of China. 0925-5214/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.12.017

to be involved in plant resistance to a range of biotic and abiotic stresses (Dixon and Paiva, 1995; Solecka and Kacperska, 2003), their biosynthesis and regulation in fruit has attracted close attention. The phenylpropanoid pathway is an important pathway in secondary plant metabolism, and produces a variety of phenolics with structural and defense-related functions including lignins, phenolic acids, stilbenes, and avonoids. Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is a crucial enzyme in phenylpropanoid metabolism, catalyzing the formation of trans-cinnamic acid via the l-deamination of phenylalanine. It is induced by various biotic (infection by viruses, bacteria, fungi, etc.) and abiotic (low and high temperatures, UV-B light, wounding, etc.) stresses, which result in the accumulation of such phenylpropanoids as phenolic acids and avonoids (Solecka and Kacperska, 2003; Sgarbi et al., 2003). However, application of the PAL inhibitor, 2-amino-

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

65

2-indanophonic acid (AIP), can decrease PAL activity. As a result, the phenolic contents are markedly decreased and resistance to stress is weakened (Janas et al., 2002; Solecka and Kacperska, 2003). This nding suggests that PAL may modulate the resistance to stress by regulating the biosynthesis of phenolic compounds. Salicylic acid (SA) and methyl-SA (MeSA) are considered to be plant signal molecules that play a key role in plant growth, development, and defense responses as well as functioning in the induction of systemic acquired resistance (SAR). These signal molecules are involved in some signal transduction systems, which induce particular enzymes catalyzing biosynthetic reactions to form defense compounds such as phenolics, alkaloids or pathogenesis-related (PR) proteins (Hahlbrock and Scheel, 1989; Ding et al., 2002). Exogenous application of SA or MeSA may also induce the expression of many defense genes in tobacco (Fraissinet-Tachet et al., 1998), tomatoes (Ding et al., 2002), and parsley (Thulke and Conrath, 1998). In parsley, the pretreatment of parsley cells with SA enhanced elicitation of the phenylpropanoid phenylalanine ammonia-lyase and 4-coumarate:CoA ligase genes (Thulke and Conrath, 1998). Past rov a et al. (2004) reported that application of SA induced the accumulation of phenylpropanoids such as coumarin metabolites. However, the relationship among SA, PAL, and phenolic compounds in biosynthesis is far from understood. Here, we report that SA acts on PAL at all levels of mRNA transcription, translation, and post-translation to activate the enzyme and consequently, enhances the accumulation of phenylpropanoids such as phenolic acids in harvested grape berries.

berries were immersed in the equilibrium buffer for 30 min. Then, 1 kg of equilibrated grape berries were immediately immersed in a solution containing an equilibrium buffer with 150 M SA, 120 M AIP (kindly provided by Dr. Jerzy Zo n), 150 M SA +120 M AIP, 150 M SA +100 M cycloheximide, and 150 M SA +10 g/l actinomycin D, respectively. Berries in the equilibrium buffer with equal amount of the distilled water were taken as the control. A pressure of about 0.1 MPa for 30 min was used for the SA inltration following a pilot experiment that indicated this pressure did not cause the berry any harm. After reversing the pressure, the berries were kept in the media for another 30 min prior to removal and placement in moist chambers at 25 C. The berries were sampled, respectively, at 0, 8, 16, 24, 32, 40, and 48 h after treatment and before they were frozen in liquid nitrogen and stored at 80 C. Each treatment contained three independent replicates. 2.3. Determination of total phenolics Total phenolics were determined spectrophotometrically using FolinCiocalteus reagent as described by Singleton and Rossi (1965) and Bonilla et al. (2003). Briey, 4 g fresh berries (the seed discarded) were ground in liquid nitrogen. A sample was then extracted in 2% HCl in methanol for 24 h in the dark and at room temperature. After centrifugation at 12,000 g for 20 min at 4 C, the supernatant was diluted with the same extract solvent at a suitable concentration for assaying total phenolics. Two hundred microliters of diluted extraction was introduced into a 5.0 ml test tube. One millilitres of FolinCiocalteu reagent and 0.8 ml carbonate (7.5%) were then added and the contents mixed and allowed to stand for 30 min. Absorption at 765 nm was measured in a Shimadzu UVvis spectrophotometer (Shimadzu UV-1601). Total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of sample using a standard curve generated with 50, 100, 150, 200, 250, 300, 350, 400, and 500 mg/l of gallic acid. 2.4. Extraction and determination of phenolic acid contents Phenolic acids were extracted as described by Hakkinen et al. (1998). Briey, 10 g of fresh berries (seed discarded) were grounded in liquid nitrogen. A sample was then rinsed with 30 ml of extraction solution (70% methanol +1.2 M HCl +80 mg ascorbic acid) into a 100 ml round-bottom bottle, carefully mixed, and then sonicated for 2 min. The remaining air in the bottle was replaced by nitrogen gas. The mixture was shaken in 35 C water bath in the dark. After 16 h, the extract was allowed to cool and was centrifuged at 12,000 g for 20 min. The supernatant was diluted to 50 ml and a 30 ml portion of the supernatant evaporated to dryness using a rotary evaporator and a 35 C water bath. The residue was dissolved in 1.5 ml of methanol (HPLC grade). Before analysis, the samples were ltered through a 0.45 m lter for organic

2. Materials and methods 2.1. Plant material Grape berries (Vitis vinifera L. cv. Cabernet Sauvignon) were harvested from a vineyard in the suburbs of Beijing. The freshly harvested berries were selected on the basis of similar size and maturity, without physical injuries or infections. After washing with distilled water, the berries were pre-cooled and air-dried. All the chemicals were purchased from Sigma (St. Louis, MO 63178, USA) unless otherwise noted. 2.2. In vivo inltration of SA and the inhibitors into intact grape berries The experiment was performed according to Beruter and Studer (1995) with some modications. The equilibrium buffer contained 50 mM MesTris [Mes = 2-(N-morpholino)ethane sulfonic acid; and Tris = tris(hydroxymethyl)-amino methane] (pH 5.5), 1 mM ethylenediamine tetra-acetic acid (EDTA), 5 mM ascorbic acid, 5 mM CaCl2 , 1 mM MgCl2 , and 200 mM mannitol. First, the grape

66

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

solvents (Acrodisc LC13 PVDF lter, Gelman/Pall Life Sciences, MI) to remove any suspended material. Qualitative and quantitative analysis was performed by reverse phase HPLC (Guillen et al., 1996). The HPLC apparatus consisted of a Shimadzu (Japan) LC-6A series pumping system, SIL-6A automatic injector furnished with a 50-l loop, SPD-6AV UVvis detector, and C-R6A chromatography data station software. Separation was carried out using a Merck LiChrospher 100RP-18e (250 mm 4.0 mm i.d., 5 m) column joined with a Merck RP-18 (10 mm 4.0 mm) guard column at 30 C. The volume injected was 10 l. A constant ow rate of 1.0 ml/min was used with two solvents: solvent A, 10% methanol2% acetic acid in water; solvent B, 90% methanol2% acetic acid in water. All solvents used were of HPLC grade. For the elution program, the following proportions of solvent B were used: 025 min, 015% B; 2545 min, 1550% B; 4553 min, 500% B. The chromatograms were monitored at 280 nm. The system precolumn column was washed with 100% mobile phase B and equilibrated with 100% mobile phase A before the next sample injection. Identity of phenolic acids was conrmed by co-chromatrography on HPLC with authentic standards. 2.5. Assay for PAL activity PAL activity was measured as previously described by Solecka and Kacperska (2003) with some modications. Briey, 4 g of berries were homogenized with mortar and pestle in 12 ml of extraction buffer (50 mM TrisHCl buffer, pH 8.9, 15 mM -mercaptoethanol, 5 mM EDTA, 5 mM ascorbic acid, 10 M Leupeptin, 1 mM PMSF, 0.15%, w/v, PVP). The homogenate was ltrated through four layers of cheesecloth and centrifuged at 12,000 g for 20 min at 4 C. The supernatant was used as a source of crude enzyme for assaying PAL activity. The reaction mixture (3 ml) contained 16 mM l-phenylalanine, 50 mM TrisHCl buffer (pH 8.9), 3.6 mM NaCl, and 0.5 ml the crude enzyme. Incubation was performed at 37 C for 1 h and the reaction was stopped by the addition of 500 l of 6 M HCl. The reaction mixture was then centrifuged for 10 min at 12,000 g, to pellet the denatured protein. The absorbance was measured at 290 nm before and after incubation. One unit of enzyme activity equals the amount of PAL that produced 1 mol of cinnamic acid in 1 h, and is expressed as mol cinnamic acid mg1 protein h1 . Protein was estimated according to Bradford (1976) using BSA as a standard. To determine whether the reaction was enzymatic, a sample extract was boiled and assayed. 2.6. Protein extraction and Western blot Total proteins were extracted according to Famiani et al. (2000) with modication. The extraction buffer consisted of 50 mM TrisHCl (pH 8.9), 2% (w/v) SDS, 5 mM ascorbic acid, 5 mM EDTA, 1 mM PMSF, 14 mM -mercaptoethanol, and 0.15% (w/v) PVP. The separation of the extracted proteins was performed by SDS-PAGE in a 10% polyacrylamide

gel as described by Laemmli (1970). The identical amount of protein (8 g) was loaded per lane. After electrophoresis, the proteins were electro-transferred to nitrocellulose (0.45 m, Amersham LIFE SCIENCE) using a transfer apparatus (Bio-Rad) as in Isla et al. (1998). For Western blot analysis, immunological detection of proteins on the NC membrane was carried out using a primary polyclonal PAL antibody (kindly provided by Dr. Somssich) in a 1/1000 dilution and alkaline phosphatase conjugated anti-rabbit IgG antibody from goat (Sigma; 1/500 dilution) as a secondary antibody at 25 C. Following that, 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 was terminated by double distilled water. The intensity of iummunobloting signal was determined by densitometer. 2.7. Isolation of total RNA Total RNA was isolated from the berry tissues using methods described in Cheng et al. (1993) with slight modications. All steps were performed at 4 C. One gram portion of berry tissue was ground in liquid nitrogen and transferred into a 2 ml washing buffer of 0.1 M Trisboracic acid (pH 7.4), 0.35 M sorbitol, 10% PEG6000 (w/v), 2% (v/v) -mercaptoethanol. After centrifugation at 12,000 g for 8 min, 2 ml of the extraction buffer containing 0.1 M Trisboracic acid (pH 7.4), 1.4 M NaCl, 0.02 M EDTA, and 2% cetyltrimethyl ammonium bromide (CTAB) was added and allowed to rest for 20 min at 55 C prior to adding 200 l 5 M potassium carbonate, 200 l ethanol, and 2 ml chloroform. After centrifugation at 12,000 g for 10 min, 1/3 volume 10 M LiCl and 0.8 volume isopropylalcohol were added before centrifugation at 15,000 g. The pellet was dried and then re-suspended in 0.5 ml diethylpyrocarbamate (DEPC)-treated water and 0.5 ml water-saturated phenol added. After centrifugation at 15,000 g for 15 min, 0.5 ml chloroform/isoamylalcohol was added before centrifugation at 15,000 g. Total RNA was then precipitated over night after addition of 1/3 volume 10 M LiCl. After centrifugation (15,000 g, 30 min), the pellet was washed in 75% ethanol and re-suspended in DEPCwater. RNA concentration was determined by absorbance at 260 nm, and purity was established with a 260/280 ratio. 2.8. RT-PCR analysis The mRNA expression patterns of PAL and Actin1 were examined using a quantitative reverse transcription polymerase chain reaction, using AMV reverse transcriptase (Promega, A3500). The amplication of Actin1 cDNA was used as an internal control. According to published sequences for grapes (GenBank accession nos. X75967 and AY680701), two pairs of oligonucleotide primers were designed to amplify the cDNA clones selected for expression analysis. Gene-specic primers for PAL (forward: 5 -GCCAATCCTGTCACCAAC3 ; reverse: 5 -CCAAACTGCCTTACCTT-3 ) and Actin1

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

67

(forward: 5 -GATTCTGGTGATGGTGTGAGT-3 ; reverse: 5 -GACAATTTCCCTTAGCAG-3 ) were used in RT-PCR with expected sizes of PCR products at 376 and 168 bp. For RT-PCR, rst-strand cDNA was synthesized from 1 g of total RNA in a volume of 20 l containing 20 mM TrisHCl, pH8.3, 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 dT (15) for 45 min at 42 C. One microliter of the rst-strand solution was used for PCR reaction in a total volume of 50 l with 20 mM TrisHCl, pH 8.3, 100 mM KCl, 2.5 mM dNTP, 5 units of Taq DNA polymerase (TaKaRa), 5 mM MgCl2 , 10 pmol of each genespecic amplication primer. Reverse transcription was performed at 42 C for 45 min, followed by PCR amplication for 26 cycles of denaturation at 94 C for 30 s (10 min for the rst cycle), annealing at 50 C for 30 s, and extension at 72 C for 1 min, with a nal extension at 72 C for 10 min, in a GeneAmp PTC-100 cycler. 2.9. Electrophoresis and blot The amplied DNA samples were separated on an agarose gel, transferred to a HybondTM -N+ nylon membrane, and hybridized with radiolabeled cDNA clone as probes (Random Primed DNA Labeling Kit, TaKaRa). Membranes were prehybridized at 65 C in 0.5 M Na2 HPO4 (pH 7.2), 1% bovine serine albumin, 7% sodium dodecyl sulfate (SDS), and 1 mM EDTA. Hybridization was performed overnight at 65 C in the same buffer. Membranes were washed twice in 2 SSC/0.1% SDS for 15 min and exposed to Kodak lm. 2.10. Statistics All experiments were repeated and analyzed at least three times. Means and standard errors were calculated from pooled data. When present in the gure, the vertical line associated with each point or on the top of each bar represents the standard error. 3. Results 3.1. SA treatments induced phenolics accumulation Fig. 1 indicates that application of 150 M SA had an obvious effect on the accumulation of phenolics. Eight hours after SA treatment, total phenolic contents began to increase with the maximum appearing 24 h after treatment and then decreasing, although 48 h after treatment, the total phenolic contents were still higher than those of the control. AIP treatments markedly decreased total phenolic contents, reaching a minimum at 32 h at 62.4% of the control level. Additionally, the co-application of SA with AIP, cycloheximide, and actinomycin D produced total phenolic contents similar to those of the control, suggesting that the accumulation of phenolics by SA could be suppressed by AIP, cycloheximide, and actinomycin D.

Fig. 1. Effect of SA and AIP, cycloheximide, and actinomycin D inltration into intact grape berries on the contents of total phenolics. Vertical lines represent 1 standard error of the mean (n = 3).

3.2. Effect of SA on the accumulation of phenylpropanoids Eleven phenolic acids, including 6 hydroxybenzoic acid derivatives (gallic acid, protocatechuic acid, gentisic acid, phydroxybenzoic acid, vanillic acid, and syringic acid), and 5 hydroxycinnamic acid derivatives (chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) were analyzed by reverse phase HPLC. As shown in Figs. 2 and 3, the contents of four of the hydroxybenzoic acid derivatives (gallic acid, p-hydroxybenzoic acid, vanillic acid, and syringic acid, Fig. 2) and the ve hydroxycinnamic acid derivatives (Fig. 3) increased signicantly after postharvest application of 150 M SA, with maximum accumulations occurred at 24 or 32 h after treatment. After 32 h, the contents all decreased slightly, but still maintained values higher than the control. However, two of the hydroxybenzoic acid derivatives (protocatechuic and gentisic acid) were not affected by SA treatment or other treatments (data not shown). The AIP inhibitor treatment remarkably decreased the gallic, phydroxybenzoic, vanillic, syringic, chlorogenic, caffeic, pcoumaric, ferulic, and sinapic acid contents at 16 and 24 h after the AIP treatment. Moreover, the contents of berries cotreated with SA and AIP, cycloheximide, and actinomycin D were similar to those of the control, indicating that the AIP, cycloheximide, and actinomycin D inhibitors can eliminate the SA-induced accumulation of these four hydroxybenzoic acid derivatives and ve hydroxycinnamic acid derivatives. 3.3. Time course of PAL activation by SA Postharvest application of 150 M SA was shown to induce PAL activation after 8 h of treatment. A maximum activation was attained 16 h after SA treatment, with an

68

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

Fig. 2. Effect of SA and AIP, cycloheximide, and actinomycin D inltration into intact grape berries on the contents of the six hydroxybenzoic acid derivatives including gallic acid, protocatechuic acid, gentisic acid, p-hydroxybenzoic acid, vanillic acid, and syringic acid.

increase 1.39-fold more than that of the control. The activation effect maintained a comparable level for 32 h, when it began to decrease rapidly (Fig. 4). Fig. 4 also shows that application of AIP strongly inhibited PAL activity, with PAL activity inhibited by about 50% of the control at 24 h. Moreover, when SA was co-applicated with AIP, cycloheximide, and actinomycin D, PAL activity was similar to the control, indicating that AIP, cycloheximide, and actinomycin D could block the activation of PAL by SA. 3.4. SA induced the synthesis of new PAL proteins The results of the effects of SA on PAL protein amounts are shown in Fig. 5. A 67 kDa peptide was specically detected with an anti-parsley PAL polyclonal antibody on SDS-PAGE gel of the berry crude protein extract (raised against the parsley enzyme, 65 and 92 kDa polypeptides; Koopmann et al., 1999). Compared to the control without any inhibitor (Lanes 1 and 2 in Fig. 5) and SA treatments, the immunoblotting signal in the berries 16 and 24 h after SA treatment (Fig. 5, Lanes 7 and 8) was obviously stronger. After 32 h, the immunoblotting signal was weakened. Application of AIP strongly inhibited PAL activity (Fig. 4), but did not inhibit the amount of PAL protein (Lane 3 in Fig. 5). Moreover, co-application of SA with AIP (Lane 4 in Fig. 5), cycloheximide (Lane 5 in Fig. 5), and actidione D (Lane 6 in Fig. 5), respectively, still did not increase the PAL protein amounts, which were simi-

lar with the controls (Lanes 1 and 2 in Fig. 5). These results suggested that SA could activate PAL activity by inducing the synthesis of a new PAL protein in grape berries. 3.5. SA elevated PAL mRNA level To evaluate the effect of SA on the PAL mRNA level, the expression of PAL gene at different times after treatment was analyzed by RT-PCR. The amplied products were separated on a DNA gel and then detected with a radiolabeled genespecic probe (Fig. 6). Five different times after treatment (CK 8 h, SA 8 h, SA 16 h, SA 24 h, and SA 32 h) were selected for PAL mRNA level analysis. Actin1, which was expected to show a constitutive expression pattern, was used as the control. A rapid accumulation of PAL mRNA in response to SA was observed. The increase in the transcription began to appear 8 h after SA treatment, and attained a maximum at 16 and 24 h. At 32 h, the PAL mRNA level slightly decreased but was still higher than the control, indicating that the transcription of PAL gene was enhanced by SA. 4. Discussion Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) converts l-phenylalanine to trans-cinnamic acid, a precursor of various phenylpropanoids such as lignins, phenolic acids, coumarins, and avonoids (Schuster and Retey, 1995). PAL

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

69

Fig. 3. Effect of SA and AIP, cycloheximide, and actinomycin D inltration into intact grape berries on the contents of the ve hydroxycinnamic acid derivatives including chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid.

is considered to be a key enzyme between primary (shikimate pathway) and secondary (phenylpropanoid) metabolism (Dixon and Paiva, 1995), regulating the ux of phenylalanine to the biosynthesis of phenolic compounds. PAL activity could be induced by stresses such as chilling susceptibility (Leyva et al., 1995; Sanchez-Ballesta et al., 2000; Lafuente et al., 2003), wounding (Peiser et al., 1998; Hisaminato et al., 2001; Campos-Vargas et al., 2005), UV-B light (Teklemariam and Blake, 2004), ozone (Sgarbi et al., 2003), pathogeninvasion (Jones, 1984), and application of exogenous hormones inculding JA, SA, ABA, and ethylene (Hyodo et al., 1978; Campos-Vargas and Saltveit, 2002; Lafuente et al.,

2004). The present work shows that application of 150 mol/l exogenous SA noticeably increased PAL activity in postharvest grape berry, especially at 16 and 24 h after treatment (Fig. 4). These results are similar to those noted in the younger leaves of wounded lettuce (Campos-Vargas and Saltveit, 2002). 2-Amino-2-indanophosphonic acid (AIP) appeared to be a very effective competitive inhibitor of PAL (Zo n and Amrhein, 1992). Previous studies have shown that application of AIP inhibits PAL activity by 6090% (Solecka and Kacperska, 2003; Cvikrov a et al., 1999). In the present study, postharvest application of 120 mol/l AIP inhibited

70

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

Fig. 4. Effect of SA and AIP, cycloheximide, and actinomycin D inltration into intact grape berries on PAL activity. Vertical lines represent 1 standard error of the mean (n = 3).

PAL activity by 50% at 24 h (Fig. 4), and implying that AIP is a powerful tool for use in understanding the biological function of PAL in different plant systems. Several reports have suggested that PAL activity is most often affected by promoting the transcription or translation of the PAL gene (Bolwell, 1992; Thulke and Conrath, 1998; Campos-Vargas et al., 2005) or its slow turnover (Crosby

and Vayda, 1991). In Romanine lettuce tissue, the wounding induced PAL activity by increasing the turnover of the induced PAL protein (Campos-Vargas et al., 2005). In the present study, a 67 kDa peptide was specically detected both in SA-treated and other treatments, with anti-parsley PAL polyclonal antibody on SDS-PAGE gel of the berry proteins. Compared with the controls, application of SA markedly increased 67 kDa peptide amounts, particularly at 16 and 32 h (Fig. 5), which is consistent with changes of PAL activity (Fig. 4). Although the time-dependence of SA activating PAL may be associated with the time course of SA accumulating in treated berries on the one hand, it probably has more to do with the time required for the enzymeencoding gene expression that was shown to be involved in this SA-induced effects. Furthermore, when application of SA, together with cycloheximide, a general protein synthesis inhibitor or mRNA transcription inhibitor actinomycin D, the PAL protein amount (Fig. 5), and the activity (Fig. 4) were similar to the control. These results indicate that PAL activation by SA should be attributed to the synthesis of new PAL protein and expression of the PAL gene in grape berries. Fraissinet-Tachet (1998) and Thulke and Conrath (1998) found that exogenous SA could induce an increase of PAL mRNA. The view was also conrmed in the present work. SA induced the accumulation of PAL mRNA, particularly at 16 and 24 h after SA application (Fig. 6), whereas there is no marked difference in the mRNA level of Actin1 at any phase

Fig. 5. Effect of SA and AIP, cycloheximide, and actinomycin D inltration into intact grape berries on the amounts of PAL protein. An identical amount of protein (8 g) was loaded per lane. Lanes 19: CK 0 h, CK 16 h, AIP 16 h, SA+ AIP 16 h, SA+ cycloheximide 16 h, SA+ actidione D 16 h, SA16 h, SA 24 h, SA 32 h.

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472

71

Fig. 6. Effect of SA on the transcripts of PAL gene in grape berries. M, 15: marker, CK 8 h, SA 8 h, SA 16 h, SA 24 h, and SA 32 h. Total RNA from grape berries of control (8 h) and SA treatments (SA 8 h, SA 16 h, SA 24 h, SA 32 h) were reverse-transcribed in the presence of an Oligo dT (15) primer. The PCR amplication was performed using PAL cDNA-spceic primers. As an internal control, Actin1 was amplied with PAL cDNA by adding Actin1-specic primers to the PCR amplication.

(Fig. 6). Therefore, we speculated that SA, at the transcriptional level, regulated PAL activity. As a consequence of the increased PAL activity, phenylpropanoid derivatives (such as phenolic acids, avonoids, anthocyanins, etc.) accumulated in the stress-affected tissues and were thought to protect plants against various biotic and certain abiotic stresss (Dixon and Paiva, 1995). Phenylpropanoids served a specic role in pathogen defense, antiherbivory, and abiotic resistance as well as structural components (Dixon et al., 2002). Solecka and Kacperska (2003) have shown an increase in PAL activity and accumulation of phenylpropanoids (especially hydroxycinnamic acids) in plants acclimated to cold. This phenomenon was also observed in the present study. As result of PAL activity increases by postharvest application of SA, total contents of nine phenolics including four hydroxybenzoic acid derivatives and ve hydroxycinnamic acid derivatives in grape berries were obviously increased (Figs. 2 and 3). These results indicated that SA could induce the accumulation of phenylpropanoids in grape berry by activating PAL activity. SA is considered one of the key endogenous signals involved in the activation of numerous plant defense responses. Application of SA could signicantly induce resistance against a variety of biotic and abiotic stresses (Yalpani et al., 1994; Dat et al., 1998; Kang et al., 2003). These properties subsequently were found to mimic the natural defense response when elevated endogenous SA levels were correlated with induced resistance to the invading pathogen (Malamy et al., 1990). PAL is an enzyme at the entry-point of the phenylpropanoid pathway. trans-Cinnamic acid, a product of PAL catalysis, is a substrate common to the biosynthesis of different classes of phenylpropanoid products. Due to the nature and defense-related function of these products, PAL activity, and the activation of PAL under stress condition have been considered a part of defense mechanism (Dixon and Paiva, 1995). The fact from the present work supports the view that development of acquired resistance mediated by SA may be attributed, at least partly, to the SA-

induced PAL gene expression and activation. Nevertheless, plants most likely synthesize SA from trans-cinnamic acid, the product of PAL activity (Yalpani et al., 1993). So it is reasonable to enquire into the inter-regulation between SA and PAL in plant response to the stresses to nally induce the development of the resistance. This question may be a key to elucidate the mechanism of resistance induction mediated by SA, which is of interest for future study. In conclusion, the work presented here showed that postharvest application of SA could activate PAL by enhancing the accumulation of PAL mRNA, the synthesis of new PAL protein, the enzyme activity, and consequently enhances the accumulation of phenylpropanoids such as phenolic acids.

Acknowledgements This research was supported by grants nos. 30270918 and 30471192 from the National Natural Science Foundation of China and State 863 High Technology Program (no. 2003AA241170). We also greatly thank Dr. Imre E. Somssich (Max Planck Institute for Plant Breeding Research, K oLN, Germany) and Dr. Jerzy Zo n (Institute of Organic Chemistry, Biochemistry and Biotechnology, Technical University of Wroclaw, Wroclaw, Poland) for the generous gifts of parsley PAL polyclonal antibody and AIP, respectively.

References
Beruter, J., Studer, F.M.E., 1995. Comparison of sorbitol transport in excised tissue discs and cortex tissue of intact apple fruit. J. Plant Physiol. 146, 95102. Bolwell, G.P., 1992. A role for phosphorylation in the down regulation of phenylalanine ammonia-lyase in suspension cultured cells of French bean. Phytochemistry 31, 40814086. Bonilla, E.P., Akoh, C.C., Sellappan, S., Krewer, G., 2003. Phenolic content and antioxidant capacity of Muscadine grapes. J. Agric. Food Chem. 51, 54975503.

72

J.-Y. Chen et al. / Postharvest Biology and Technology 40 (2006) 6472 tion to the development of russet spotting caused by ethylene. Plant Physiol. 62, 3135. Isla, M.I., Vattuone, M.A., Sampietro, A.R., 1998. Essential group at the active site of Frapaeolum invertase. Phytochemistry 47, 11891193. Janas, K.M., Cvikrov a, M., Palagiewicz, A., Szafranska, K., Posmyk, M.M., 2002. Constitutive elevated accumulation of phenylpropanoids in soybean roots at low temperature. Plant Sci. 163, 369373. Jones, D.H., 1984. Phenylalanine ammonia-lyase: regulation of its induction, and role in plant development. Phytochemistry 23, 13491359. Kang, G.Z., Wang, Z.X., Sun, G.C., 2003. Participation of H2 O2 in enhancement of cold chilling by salicylic acid in banana seedings. Acta Bot. Sin. 45, 567573. Koopmann, E., Logemann, E., Hahlbrock, K., 1999. Regulation and functional expression of cinnamate 4-hydroxylase from Parsley. Plant Physiol. 119, 4955. Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680685. Lafuente, M.T., Sala, J.M., Zacarias, L., 2004. Active oxygen detoxifying enzymes and phenylalanine ammonia-lyase in the ethylene-induced chilling tolerance in citrus fruit. J. Agric. Food. Chem. 52, 36063611. Lafuente, M.T., Zacarias, L., Martinez-Telez, M.A., Sanchez-Ballesta, M.T., Granell, A., 2003. Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus. Postharvest Biol. Technol. 29, 308317. Leyva, A., Jarillo, J.A., Salinas, J., Martinez-Zapater, J.M., 1995. Low temperature induces the accumulation of phenylalanine ammonialyase and chalcone synthase mRNA of Arabidopsis thaliana in a light-dependent manner. Plant Physiol. 108, 3946. Malamy, J., Carr, J.P., Klessig, D.F., Raskin, I., 1990. Salicylic acid: a likely endogenous signal in the resistance response of tobacco viral infection. Science 250, 10021004. Past rov a, A., Repc ak, M., Elia sov a, A., 2004. Salicylic acid induces changes of coumarin metabolites in Matricaria chamomilla L. Plant Sci. 167, 819824. Peiser, G., Lopez-Galvez, G., Cantwell, M., Saltveit, M.E., 1998. Phenylalanine ammonia lyase inhibitors control browning of cut lettuce. Postharvest Biol. Technol. 14, 171177. Sanchez-Ballesta, M.T., Lafuente, M.T., Zacarias, L., Granell, A., 2000. Involvement of phenylalanine ammonia-lyase in the response of Fortune mandarin fruits to cold temperature. Physiol. Plantarum 108, 382389. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticult. 16, 144158. Schuster, B., Retey, J., 1995. The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine. Proc. Natl. Acad. Sci. U.S.A. 92, 84338437. Sgarbi, E., Fornasiero, R.B., Lins, A.P., Bonatti, P.M., 2003. Phenol metabolism is differentially affected by ozone in two cell lines from grape (Vitis vinifera L.) leaf. Plant Sci. 165, 951957. Solecka, D., Kacperska, A., 2003. Phenylpropanoid deciency affects the course of plant acclimation to cold. Physiol. Plantarum 119, 253262. Teklemariam, T.A., Blake, T.J., 2004. Phenylalanine ammonia-lyaseinduced freezing tolerance in jack pine (Pinus banksiana) seedlings treated with low, ambient levels of ultraviolet-B radiation. Physiol. Plantarum 122, 244253. Thulke, O., Conrath, U., 1998. Salicylic acid has a dual role in the activation of defence-related genes in parsley. Plant J. 14, 3542. Yalpani, N., Enyedi, A.J., Leon, J., Raskin, I., 1994. Ultroviolet light and ozone stimulate accumulation of salicylic acid pathogenesis-related proteins and virus resistance in tobacco. Planta 193, 372376. Yalpani, N., Le on, J., Lawton, M.A., Raskin, I., 1993. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiol. 103, 315321. Zo n, J., Amrhein, N., 1992. Inhibitors of phenylalanine ammonia-lyase: 2aminoindan-2-phosphonic acid and related compounds. Liebigs. Ann. Chem., 625628.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Bichem. 72, 248254. Campos-Vargas, R., Nonogaki, H., Suslow, T., Saltveit, M.E., 2005. Heat shock treatments delay the increase in wound-induced pheylalanine ammonia-lyase activity by altering its exptession, not its induction in Romaine lettuce (Lactuca sativa) tissue. Physiol. Plantarum 132, 8291. Campos-Vargas, R., Saltveit, M.E., 2002. Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettue. Physiol. Plantarum 114, 7384. Chamkha, M., Cathala, B., Cheynier, V., Douillard, R., 2003. Phenolic composition of champagnes from Chardonnay and Pinot Noir vintages. J. Agric. Food Chem. 51, 31793184. Cheng, S., Purgear, J., Cairney, J.A., 1993. Simple and efcient method for isolation RNA from pine trees. Plant Mol. Biol. Rep. 11, 113116. Crosby, J.S., Vayda, M.E., 1991. Stress-induced translational control in potato tubers may be mediated by polysome-associated proteins. Plant Cell 3, 10131023. Cvikrov a, M., Binarov a, P., Eder, J., V agner, M., Hrubcov a, M., Zon, J., Vach ackov a, I., 1999. Effect of inhibition of phenylalanine ammonialyase activity on growth of alfalfa cell suspension culture: alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins, and polyamines. Physiol. Plantarum 107, 329337. Dat, J.F., Lopez-Delgado, H., Foyer, C.H., Scott, I.M., 1998. Parallel changes in H2 O2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedings. Plant Physiol. 116, 13511357. Ding, C.K., Wang, C.Y., Gross, K.C., 2002. Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit. Planta 214, 895901. Dixon, R.A., Achnine, L., Kota, P., Liu, C.J., Srinivasa, M.S., Wang, L.J., 2002. The phenylpropanoid pathway and plant defencea genomics perspective. Mol. Plant Pathol. 3, 371390. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 10851097. Famiani, F., Walker, R.P., Tecsi, L., Chen, Z.H., Proietti, P., Leegood, R.C., 2000. An immunohistochemical study of the comparmentation of metabolism during the development of grape berries. J. Exp. Bot. 51, 675683. Frankel, E.N., Waterhouse, A.L., Teissedre, P.L., 1995. Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density lipoproteins. J. Agric. Food Chem. 43, 890894. Fraissinet-Tachet, L., Baltz, R., Chong, J., Kauffmann, S., Fritig, B., Saindrenan, P., 1998. Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid. FEBS Lett. 437, 319323. Guillen, D.A., Barroso, C.G., Perez, J.A., 1996. Selection of column and gradient for the separation of polyphenols in sherry wine by highperformance liquid chromatography incorporating internal standards. J. Chromatogr. A 724, 117124. Hahlbrock, K., Scheel, D., 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 347469. Hakkinen, S.H., Karenlampi, S., Heinonen, I.M., Mykkanen, H.M., Torronen, A.R., 1998. HPLC method for screening of avonoids and phenolic acids in berries. J. Sci. Food Agricult. 77, 543551. Hisaminato, H., Murata, M., Homma, S., 2001. Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis. Biosci. Biotechnol. Biochem. 65, 1016 1021. Hyodo, H., Kuroda, H., Yang, S.F., 1978. Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in rela-