Postharvest Biology and Technology 79 (2013) 18

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Postharvest Biology and Technology

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Arginase induction by heat treatment contributes to amelioration of chilling injury and activation of antioxidant enzymes in tomato fruit
Xinhua Zhang a,c , Lin Shen c , Fujun Li a, , Demei Meng c , Jiping Sheng b,c,
a b c

School of Agricultural and Food Engineering, Shandong University of Technology, Zibo 255049, Shandong, Peoples Republic of China School of Agricultural Economics and Rural Development, Renmin University of China, Beijing 100872, Peoples Republic of China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, Peoples Republic of China

a r t i c l e

i n f o

a b s t r a c t
Treatment of tomato (Solanum lycopersicum L. cv. Messina) fruit with hot air (HA) at 38 C enhanced the transcript levels of LeARG1 and LeARG2, the two genes encoding arginase, and arginase activity. The strongest induction of LeARG1 and LeARG2 transcripts was observed after fruit treated with 38 C HA for 12 h, which also effectively alleviated chilling injury (CI) of tomato fruit, manifested as decreased CI index, electrolyte leakage and malondialdehyde content during cold storage. To investigate the potential role of arginase in HA-induced chilling tolerance, fruit were treated with HA, or arginase inhibitor N hydroxy-nor-l-arginine (nor-NOHA) combined with HA and then stored at 2 C for up to 28 d. The results showed that HA-induced arginase activity was strongly inhibited by pretreatment with nor-NOHA and the reduction of CI by HA was nearly abolished by the arginase inhibitor. In addition, HA treatment increased activities of superoxide dismutase, catalase and ascorbate peroxidase, inhibited peroxidase activities, and promoted the accumulation of arginine, proline and putrescine. These effects were partially counteracted by nor-NOHA except that arginine and putrescine accumulation was unaffected. Our results indicate that arginase induction may be partly involved in HA-induced chilling tolerance in tomato fruit, possibly by a mechanism involving activation of antioxidant enzymes and an increase in proline levels. 2013 Elsevier B.V. All rights reserved.

Article history: Received 19 September 2012 Accepted 21 December 2012 Keywords: Arginase Chilling injury Hot air Tomato fruit Proline Antioxidant enzyme

1. Introduction Arginine is one of the most metabolically versatile amino acids in living cells. It plays multiple roles not only as a building block of proteins, but also as a precursor for the synthesis of polyamines (putrescine, spermidine, spermine), proline and nitric oxide (NO) (Morris, 2007; Jubault et al., 2008). Arginine can be catabolized through the action of three different enzymes: arginase, arginine decarboxylase (ADC) and nitric oxide synthase (NOS) (Morris, 2007). Arginase catalyzes the hydrolysis of arginine to ornithine and urea. In mammals, this reaction has been mostly studied as a key nal step in the urea cycle, which allows potentially toxic ammonium to be detoxied into urea (Morris, 2009). In addition to this detoxication function, arginase is involved in several important subsequent metabolic pathways due to its generation of ornithine which is a precursor for the synthesis of putrescine and proline via ornithine decarboxylase (ODC) and ornithine

Corresponding author. Tel.: +86 533 2786820; fax: +86 533 2786820. Corresponding author at: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, Peoples Republic of China. Tel.: +86 10 62737620; fax: +86 10 62736474. E-mail addresses: lifujun@sdut.edu.cn (F. Li), pingshen@cau.edu.cn (J. Sheng). 0925-5214/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.12.019

aminotransferase (OAT), respectively (Morris, 2009). Putrescine is the key substrate for the biosynthesis of both spermidine and spermine. Increased arginase expression has been shown to stimulate the production of polyamines or proline that is responsible for tumor immune escape, wound healing and axonal regeneration following injury (Morris, 2009; Munder, 2009). In contrast to our understanding of arginase regulation in animals, the physiological roles of arginase have been described in plants mostly for its involvement in nitrogen remobilization processes from protein degradation, especially during seed germination (King and Gifford, 1997; Goldraij and Polacco, 2000). More recently, arginase was also reported to be involved in stress responses. Enhanced expression of arginase upon stress imposition has been reported (Chen et al., 2004; Jubault et al., 2008; Brauc et al., 2011), and abrogation of clubroot-induced arginase activity in Arabidopsis by mutation of the arginase-encoding gene ARGAH2 resulted in enhanced gall size in infected tissue (Gravot et al., 2012). Browneld et al. (2008) also found that a loss of ARGAH2 function impacted on normal induction of At2g14610, a pathogenesis response family member, following methyl jasmonate treatment. In addition, overexpression of arginase in tomato was shown to increase resistance to phytophageous insects (Chen et al., 2005). In our previous study, the enhanced arginase activity and expressions of both LeARG1 and LeARG2 were also observed in mature

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green tomato fruit in response to low temperature, which was proved to increase the chilling tolerance of fruit (Zhang et al., 2010). Chilling injury (CI) is an economically important postharvest problem that reduces the overall quality and marketability of many tropical and subtropical horticultural products. Heat shock treatment, a physical treatment free from chemical residues, is effective in reducing CI in a number of postharvest horticultural products (Mirdehghan et al., 2007; He et al., 2012; Luengwilai et al., 2012). Arginase could provide a precursor for the biosynthesis of putrescine or proline by hydrolyzing arginine. Evidence has shown that the production of polyamines or proline can serve as an adaptive mechanism to chilling stress in higher plants (Mirdehghan et al., 2007; Zhang et al., 2011; Shang et al., 2011). Furthermore, in spite of the evidence that the increased polyamines with temperature were not necessarily related to chilling protection (Gonzlez-Aguilar et al., 1997, 2000b), it has been shown that the reduction in CI by heat treatment may be through a mechanism that involved elevation of polyamines levels in several chilling-sensitive fruit such as pepper (Gonzlez-Aguilar et al., 2000a), peach (Cao et al., 2010), and pomegranate (Mirdehghan et al., 2007). In view of the possible roles of polyamines and proline in plant tolerance to chilling stress, together with the fact that arginase appears to be involved in stress resistance mechanism in higher plants, we hypothesis that arginase may also be involved in heat-induced chilling resistance in postharvest fruit. However, little information is available about the potential role of arginase in fruit chilling tolerance induced by heat treatment. To address this question, tomato fruit were used, and research was focused on investigating the potential role of arginase in the induction of heat-induced chilling resistance. We examined the effects of heat treatment with or without pretreatment with exogenous N -hydroxy-nor-l-arginine (nor-NOHA, an ideal inhibitor to study the role of arginase) on arginase activity or gene expression, CI, lipid peroxidation, antioxidant enzyme activities, polyamines and arginine-related amino acid levels in tomato fruit during low temperature storage.

development of CI was measured after these fruit were transferred to 20 C for an additional 3 d. 2.2. Quantitative real-time PCR (qRT-PCR) assays Total RNAs were extracted from fruit treated with HA for 0, 3, 6, 12 and 24 h using trizol reagent as described previously (Zhang et al., 2010). Any remaining genomic DNA was removed by digestion with DNase I (DNA-free; Ambion) according to the manufacturers protocol. The cDNA was obtained as described by Zhao et al. (2009). Transcript levels of the two genes encoding arginase, LeARG1 and LeARG2, were evaluated via qRT-PCR using the SYBR Green I MasterMix (Toyobo, Osaka, Japan) on a Chromo4 real time PCR Detection System (Bio-Rad, Hercules, USA). The Ubi3 encoding ubiquitin was used as the reference gene. Nucleotide sequences of gene-specic primers used for quantitative real-time PCR were as described previously (Zhang et al., 2011). The following amplication protocols were used: 95 C for 2 min, followed by 40 cycles of 15 s at 95 C and 20 s at 60 C. To check the annealing specicity of each oligonucleotide, melting curve analysis (5594 C) was carried out at the end of amplication. Fluorescence signal was measured at the end of each annealing step. All experiments were run in triplicate with different cDNAs synthesized from three biological replicates. To determine relative gene expression for each sample, the threshold cycle (Ct) value was normalized to Ubi3 and set relative to 0 h samples according to the 2 Ct method. 2.3. CI index evaluation CI symptoms of tomato fruit were manifested as surface pitting (Ding et al., 2002). The severity of symptoms was evaluated visually according to the following four-stage scale: 0 = no pitting; 1 = pitting covering <5% of the fruit surface; 2 = pitting covering <25% but >5% of surface; 3 = pitting covering <50% but >25% of surface and 4 = pitting covering >50% of surface. The CI index was calculated using the following formula: CI index = (CI level) (number of fruit at the CI level) total number of fruit .

2. Materials and methods 2.4. Electrolyte leakage and MDA content assays 2.1. Fruit and treatments Tomato (Solanum lycopersicum L. cv. Messina) fruit were harvested at the mature green stage. The weight of per fruit about 41.2 2.1 g in average and the mean diameter of fruit was 3.6 0.2 cm. Uniform sized fruit, free of blemishes or defects, were selected and randomly divided into three lots and subjected to following treatments: one lot of 270 fruit was dipped in 30 M arginase inhibitor nor-NOHA in a 50 L stainless steel vacuum container and vacuum-inltrated under low pressure (35KPa) for 0.5 min (12); thereafter, fruit were kept under ambient air pressure for 2 min and then air-dried. The remainder (930 fruit) were rst treated with water under the same conditions, and then 450 of these fruit were incubated at 20 C for 12 h serving as the control lot; and another 480-fruit lot was incubated with 38 C hot air (HA) for 12 h in an incubator provided with heating and humidifying systems. For the combination nor-NOHA + HA treatment, fruit pretreated with nor-NOHA were also incubated at 38 C for 12 h. In each lot, fruit were randomly divided into three replicates. After treatment, fruit were stored at 2 1 C with a relative humidity of 8090% for up to 28 d. The mesocarp from 10 fruit per replicate was collected at different hours or days for genes, enzymes, electrolyte leakage, malondialdehyde (MDA), polyamines and arginine-related amino acid analysis. Another sample of 15 fruit was removed after 3 or 4 weeks of storage at 2 1 C and the Electrolyte leakage rate was measured as described by Zhao et al. (2009) with minor modications. Six disks (3 mm thick) of mesocarp tissue were cut with a cork borer from the equator of the fruit. Conductivity was measured after 2 h of incubation in 25 mL of 0.1 M mannitol under constant shaking. After that, the solutions were boiled for 10 min and cooled to room temperature and the total conductivity was measured. The rate of electrolyte leakage was expressed as a percentage of the total: (initial/total) 100. MDA was measured using the thiobarbituric acid method as described previously (Ding et al., 2007). Absorbance of the reaction product of MDA with 3 mL of 0.6% thiobarbituric acid and 1 mL of the extract at 532 nm was recorded and corrected for nonspecic absorbance at 600 nm. The amount of MDA was calculated using an extinction coefcient of 155 mM1 cm1 and expressed as mol g1 fresh weight (FW). 2.5. Arginase assays Frozen fruit tissue (1 g) was ground and homogenized in 5 mL of 100 mM TrisHCl (pH 7.5) containing 1% (v/v) -mercaptoethanol, 0.1 mM phenylmethylsulfonyl uoride (PMSF) and 0.5% (w/v) polyvinyl polypyrrolidone (PVP). The homogenates were centrifuged at 12,000 g for 15 min at 4 C, and the supernatants were used for the enzyme assays. Before assays, the enzyme extract

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was activated with 1 mM MnCl2 at 37 C for 15 min. The reaction mixture contained 20 L of the activated arginase, 420 L of 120 mM glycinNaOH (pH 9.6), 40 L of 1.25 M arginine (pH 9.6) and 20 L of 50 mM MnCl2 . Reactions were carried out at 37 C for 20 min and stopped by adding 0.5 mL of 15% (v/v) perchloric acid. Urea released in the medium was determined spectrophotometrically as described previously (Chen et al., 2004). Absorbance was read at 515 nm and standard urea solutions were used for calibration. Arginase activity was expressed as nmol urea produced min1 mg1 protein. 2.6. Antioxidant enzyme assays Frozen tissue (1 g) was homogenized in 5 mL of 50 mM sodium phosphate buffer (pH 7.0), containing 0.1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ascorbic acid and 1% PVP in an ice bath. The homogenate was centrifuged at 12,000 g for 15 min at 4 C. The supernatant was used for assays of the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The SOD activity was determined by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium chloride (NBT) using the method of Rao et al. (1996). One unit of SOD activity was dened as the amount of enzyme that caused 50% inhibition of NBT reduction. CAT activity was assayed by monitoring the disappearance of H2 O2 according to the method of Chance and Maehly (1955). One unit of CAT activity was dened as the amount of enzyme that decomposed 1 mol H2 O2 min1 at 30 C. POD activity was determined in a reaction mixture containing 50 mM sodium acetate (pH 5.0), 0.1% H2 O2 , 0.1% guaiacol and 0.2 mL of crude enzyme extract (Chance and Maehly, 1955). One unit of POD activity was dened as the amount of enzyme that caused an increase in absorbance of 0.01 at 470 nm in 1 min. APX activity was determined by the method of Nakano and Asada (1981). One unit of APX activity was dened as the amount of enzyme that oxidized 1 mol ascorbate min1 at 30 C. Specic activities of all enzymes were expressed as units (U) mg1 protein. 2.7. Protein concentration Protein concentration in the enzymatic extracts was quantied according to the method of Bradford (1976) with bovine serum albumin as standard. 2.8. Arginine-related amino acids assays Free amino acids were extracted according to the method of Micallef and Shelp (1989) with minor modications. Samples were ground with liquid nitrogen, and the powder was suspended in 1 mL g1 of a 3% (w/v) solution of 5-sulfosalicylic acid. Following centrifuged for 10 min at 12,000 g, the supernatant was adjusted to pH 7 with 4 M NaOH to prepare for HPLC analysis. Samples were derivatized with 1-uoro-2, 4-dinitrobenzene (FDNB) as described previously (Wang et al., 2000), with minor modications. Approximately 0.5 mL of sample extracts were mixed with 0.5 mL of NaHCO3 solution (0.5 M, pH 9.0), followed by thorough mixing with 0.5 mL of 1% (v/v) FDNB in pure acetonitrile. The derivatization was complete in the dark for 60 min at 60 C. After cooling to room temperature, it was added to 3 mL of 50 mM KH2 PHO4 solution and vortexed for several seconds. After ltration through a 0.45 m membrane lter, 20 L of derivatized samples were injected onto a 4.6 mm 250 mm Zorbax ODS column and analyzed by reverse-phase HPLC using an Agilent 1200 series (Agilent, USA). Amino acids were eluted with 25 mM sodium acetate

buffer (solvent A), pH 6.4, containing 1% (v/v) dimethyl formamide and pure acetonitrile at a ow rate of 1 mL min1 according to the following gradient: initial, 92% A; 6 min, 92% A; 11 min, 88% A; 20 min, 80% A; 30 min, 70% A; 35 min, 50% A; 40 min, 60% A; 45 min, 92% A and 50 min, 92%A. Derivatized amino acids were detected at 360 nm using a UV detector. Amino acids were identied by cochromatography of individual standards and quantied by comparison of individual external calibration curves. Proline content was determined by the ninhydrin method, as described by Zhao et al. (2009). Results were expressed as mol g1 FW. 2.9. Free polyamine determination Frozen tissue was extracted in 4 volumes of 5% (w/v) cold perchloric acid and centrifuged at 12,000 g for 30 min at 4 C. Polyamines in the supernatant was benzoylated, as previously described (Zhang et al., 2011), then extracted with 2 mL of chilled diethyl ether. The ether phase was dried under a stream of warm air and the residue was dissolved in 300 L of methanol (HPLC grade) and ltered through 0.45 m pore lters before HPLC analysis. Derivatized samples (20 L) were injected onto a reverse phase C18 column (Zorbax ODS, 5 m particle diameter, 4.6 mm 250 mm) and eluted with 64% methanol at a ow rate of 1 mL min1 . The polyamines was detected using a UV detector at 254 nm and quantied using the benzoylated standard curves. Results were expressed as nmol g1 FW. 2.10. Data analysis Data were analyzed using one-way analysis of variance (ANOVA) with SPSS 16.0 statistical software. Signicant differences were performed by Duncans multiple comparison procedure. A probability of P < 0.05 was considered as signicant. All experiments were conducted in a completely randomized design with three replicates for each treatment. The data were expressed as means standard error (SE). 3. Results 3.1. Changes of arginase gene expression and activity in response to HA treatment The expression of the two genes encoding arginase in tomato fruit, LeARG1 and LeARG2, remained unchanged at 20 C for 24 h, while treatment of fruit with HA at 38 C resulted in strong induction of LeARG1 and LeARG2 (Fig. 1A and B). The expression pattern of the two genes was very similar, which was detected rapidly within 3 h of HA treatment, peaked at the 12 h time point, and declined thereafter. HA-induced expression of LeARG genes was accompanied by an increase in arginase activity during heat treatment and the recovery period, when compared with the control (Fig. 1C). Following 1 d of exposure to HA, arginase activity reached a maximum with a value 91% higher than that of control (Fig. 1C). Although there was a decline in the transcription of LeARG1 and LeARG2 after 24 h of HA treatment, arginase activity remained elevated thereafter. This observation indicates that the enzyme, once synthesized, is relatively stable in fruit tissue. 3.2. Heat-induced arginase activity is inhibited by nor-NOHA during cold storage Arginase activity in control fruit increased in response to cold stress, reaching a peak at day 7, and then decreased gradually (Fig. 2). Heat-treated fruit shared the same trend as control fruit. However, exposure of fruit to 38 C for 12 h before cold storage caused an enhanced arginase activity at all the time points when

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21

Arginase activity (nmolmin-1mg -1 protein)

18 15 12 9 6 3 0
0 7 14 21 28 Days of storage at 2 C Con trol HA nor-NOHA+HA

Fig. 2. Effect of hot air (HA) treatment on arginase activity in tomato fruit during storage at 2 C with or without pretreatment by nor-NOHA. Data are expressed as mean SE of triplicate assays. Vertical bars represent the standard errors of the means.

control. However, the inductive effects of HA on chilling tolerance were almost prevented by treatment fruit with nor-NOHA before heating. Especially after 4 weeks at 2 C, there were no signicant differences in CI index and MDA content between controls and (norNOHA + HA)-treated fruit, although the electrolyte leakage in (norNOHA + HA)-treated fruit was lower than that in control. 3.4. Changes in activities of antioxidant enzymes The activities of SOD, CAT, APX and POD considered to play an important role in cellular defense against oxidative stress caused by chilling injury (Sevillano et al., 2009) were determined for mesocarp tissue of tomato fruit subjected to various treatments (Fig. 3). HA treatment maintained signicantly higher SOD (Fig. 3A), CAT (Fig. 3B) and APX (Fig. 3C) activities compared with the controls during most of the storage period. However, the HA-induced activities of SOD, CAT and APX were partially inhibited by exposing fruit to nor-NHOHA before HA treatment. In contrast, during most of the storage time, the highest POD activity (Fig. 3D) was observed in control, followed by nor-NOHA + HA, and the lowest was recorded in HA-treated fruit. 3.5. Changes in the content of arginine-related amino acids and polyamines As shown in Fig. 4A, arginine content in control fruit remained mostly unchanged throughout the storage periods. However, in heat-treated fruit, its content increased immediately after HA treatment (Fig. 4A, day 0), and maintained a signicantly higher level than controls during subsequent storage up to 28 d. The presence of nor-NOHA in heat-treated fruit had almost no effect on the content of arginine. In contrast, ornithine content in control and treated fruit increased in response to chilling stress during storage at 2 C (Fig. 4B). Thus, HA treatment had little effect on ornithine content relative to the controls, whereas nor-NOHA + HA treatment markedly decreased its content in fruit during the entire storage periods. Similar to ornithine, proline content also increased with storage time in all samples. However, the proline content was increased immediately after exposure to HA for 12 h by 67.3% relative to the control (Fig. 4C, day 0). Moreover, proline content in HA-treated fruit was also higher than that in controls during the subsequent cold storage period and remained 23.4% higher compared with the controls at the end of storage. Although norNOHA signicantly inhibited the heat-induced increase in proline

Fig. 1. Gene expression and activity of arginase in tomato fruit at different time intervals during or after hot air (HA) treatment. Expression of LeARG1 (A) and LeARG2 (B) and arginase activity (C). The expression levels of LeARG1and LeARG2 encoding arginase were evaluated by quantitative real-time PCR, normalized to Ubi3 gene and set relative to control samples from 0 h according to the 2 Ct method. Data are expressed as mean SE of triplicate assays. Different letters indicate signicant differences at P < 0.05 by Duncans multiple range test.

compared with the control. As expected, exogenous addition of norNOHA, a highly specic inhibitor of arginase, before HA treatment signicantly inhibited heat-induced arginase activity, which was relatively lower than that of control fruit; and no signicant peak in arginase activity was found during the entire cold storage period. 3.3. Changes in chilling injury After 3 or 4 weeks at 2 C, severe CI symptoms were observed on treated and untreated tomato fruit (Table 1). Heating the fruit for 12 h at 38 C before storing them at 2 C reduced the development of CI symptoms, as observed by the lower value of CI index, electrolyte leakage and MDA content in HA-treated fruit as compared to the

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Table 1 Effect of hot air (HA) treatment on chilling injury (CI) index, electrolyte leakage and MDA content in tomato fruit during storage at 2 C with or without nor-NOHA pretreatment. CI index was measured after transferring the fruit from 2 C to 20 C for 3 d. Electrolyte leakage and MDA content were measured immediately after transferring the fruit from 2 C to 20 C. Storage time 21 d Treatment Control HA Nor-NOHA + HA Control HA Nor-NOHA + HA CI index 2.58 0.13a 1.53 0.10c 2.22 0.16b 3.62 0.28a 2.54 0.17b 3.23 0.10a Electrolyte leakage (%) 49.31 4.15a 30.85 0.23d 39.6 1.71b 55.40 3.03a 36.79 3.15c 48.32 2.30b MDA content ( mol g1 FW) 13.98 0.87a 8.03 0.37c 11.87 0.45b 13.39 0.42a 9.71 0.79b 12.76 1.03a

28 d

Means in a column followed by a different letter for the same storage period differ signicantly at P < 0.05 by Duncans multiple range tests. Data are accompanied by standard errors of the means.

content, levels in fruit treated with nor-NOHA + HA were still higher than those in controls at most sampling times. Putrescine was the predominant polyamines, followed by spermidine and spermine in tomato fruit (Fig. 4DF). The putrescine content increased in response to chilling stress during the storage periods, and HA treatment enhanced the accumulation compared with the control fruit throughout the cold storage period (Fig. 4D). At the end of cold storage, the content of putrescine in HA-treated fruit was 15.0% higher than that in controls. By contrast, spermidine and spermine contents uctuated during the storage periods, and there were little effects of HA treatment on the content of them expect on the earlier 3 d (Fig. 4E and F). There were little effect of nor-NOHA treatment on heat-induced putrescine accumulation during the chilling stress periods except on days 3 and 7, when lower levels of putrescine were detected. For spermidine and spermine contents, similar to HA treatment, the nor-NOHA + HA treatment also showed no signicant difference with controls during the whole storage periods. 4. Discussion Recently, arginase in mammals has garnered considerable attention due to its potential role in a very wide range of

physiological and pathophysiological conditions (Morris, 2009; Munder, 2009). Although research on plant arginase has mainly focused on its role in nitrogen remobilization processes from protein degradation, especially during seed germination (King and Gifford, 1997; Goldraij and Polacco, 2000), arginase induction was reported to be part of a defense mechanism against biotic and abiotic stress (Chen et al., 2005; Browneld et al., 2008; Zhang et al., 2010; Brauc et al., 2011; Gravot et al., 2012). In our present study, we found that HA treatment at 38 C for 12 h not only resulted in strong induction of LeARGs, which led to the increased arginase activity (Fig. 1), but also effectively reduced CI symptom in tomato fruit (Table 1). To evaluate the participation of arginase in HAinduced chilling tolerance, fruit were incubated with the arginase inhibitor nor-NOHA before the imposition of HA. Compared with HA-treated fruit, fruit treated with nor-NOHA + HA showed a signicantly higher CI index, as well as increased electrolyte leakage and MDA content (Table 1). CI index directly reected development degree of CI symptoms and was widely used in study of chilled postharvest fruit for judging cold tolerance. In addition, dysfunction of the cell membrane at low temperature is considered to be the primary molecular event, which is frequently related to an increase in membrane permeability and lipid peroxidation (Sevillano et al., 2009). Therefore, electrolyte leakage and MDA content, a product

CAT activity (Ug-1 protein)

SOD activity (Ug-1 protein)

110 88 66 44 22

20 16 12 8 4 0 54 0 45 36 27 18 9 0 0

APX activity (Ug-1 protein)

120 90 60

30
0 0 7 14 21 Days of storage at 2C 28

POD activity (Ug-1 protein)

0 180 0 C 150

Control HA nor-NOHA+HA 7 14 21 Days of storage at 2C 28

7 14 21 Days of storage at 2C

28

7 14 21 Days of storage at 2C

28

Fig. 3. Effect of hot air (HA) treatment on SOD (A), CAT (B), APX (C) and POD (D) activities in tomato fruit during storage at 2 C with or without pretreatment by nor-NOHA. Data are expressed as mean SE of triplicate assays. Vertical bars represent the standard errors of the means.

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1.8
1.5

Putrescine (nmol g -1 FW)

Arginine ( mol g -1 FW)

2.1

1500 1200
900

1.2 0.9 0.6 0.3

0.0 1.2 0 0.9 7

Control HA nor-NOHA+HA
14 Time (d) 21

600 300 0 100 0 80 60

40

A
28

D
7 14 Time (d) 21 28

0.6 0.3

Spermidine (nmol g -1 FW) Spermidine c (nmol g-1 FW)

Ornithine ( mol g -1 FW)

Proline ( mol g-1 FW)

0.0 0.28 0 0.21 0.14 0.07 0.00 0

B
7 14 21 Time (d) 28

20 0 15 0 12 9
6

E
7 14 Time (d) 21 28

C
7 14 21 Days of storage at 2C 28

3 0 0 7 14 21 Days of storage at 2C

F
28

Fig. 4. Effect of hot air (HA) treatment on arginine (A), ornithine (B), proline (C), putrescine (D), spermidine (E) and spermine (F) contents in tomato fruit during storage at 2 C with or without pretreatment by nor-NOHA. Data are expressed as mean SE of triplicate assays. Vertical bars represent the standard errors of the means.

of lipid peroxidation, have been used to evaluate chilling resistance of plant tissues (Imahori et al., 2008; Zhao et al., 2009). This study demonstrated an important role for arginase since the HAinduced CI tolerance in tomato fruit was strongly reduced with the inhibition of arginase activity by nor-NOHA pretreatment (Fig. 2). The coordinated action of antioxidant enzymes such as SOD, APX, CAT and POD, which are very important for scavenging active oxygen species (AOS) to protect cell membranes, is thought to be a major mechanism of resistance to chilling stress (Sazadeh et al., 2007; Imahori et al., 2008; Sevillano et al., 2009). In the present work, treatment with HA signicantly reduced the CI symptoms and increased the activities of SOD, CAT, and APX during cold storage. As expected, the activities of SOD, CAT, and APX in fruit treated with nor-NOHA + HA decreased compared with those treated with HA alone (Fig. 3AC). However, HA treatment had a negative effect on POD activity compared to the control fruit. In addition, POD activity in fruit treated with nor-NOHA + HA was higher than that in HA-treat fruit (Fig. 3D). Previous studies have shown that the improvement of chilling tolerance in harvested horticultural crops is related to enhancement in activities of SOD, CAT and APX (Sazadeh et al., 2007; Imahori et al., 2008), which was conrmed in our results. However, conicting results concerning the relationship between CI severity and POD activity have been reported (Sazadeh et al., 2007; Zhao et al., 2009). Accompanying

the alleviation of CI, we observed a signicant reduction in POD activity in HA-treated fruit. A reduction in CI by treatments along with an increase in POD activity in horticultural crops under cold stresses has also been reported (Sazadeh et al., 2007; Jin et al., 2009). Our results and these prior reports suggest that the balance between SOD, CAT, APX and POD activity is critical to cell survival during cold storage. SOD catalyzes the dismutation of the superoxide free radicals to O2 and H2 O2 (Sevillano et al., 2009). CAT, APX and POD are the main enzymes responsible for H2 O2 scavenging, but the mode of action of POD differs from CAT action in that POD liberates free radicals instead of oxygen (Sazadeh et al., 2007). These free radicals are highly phytotoxic. In our study, HA treatment decreased POD activity and increased the CAT and APX activity, which not only scavenged hydrogen peroxide, but alleviated the production of phytotoxic free radicals liberated by POD. However, the changes in the activities of these enzymes in HAtreated fruit were diminished when arginase activity was inhibited by nor-NOHA, suggesting that HA altered the activities of these enzymes at least in part through the induction of arginase. The positive effects of heat treatments on the chilling injury and antioxidant system in tomato fruit were also reported in several other studies. Some reports indicated a requirement of 23 d at 38 C to ameliorate CI in tomato fruit (Lurie and Klein, 1991; McDonald et al., 1996). However, Yahia et al. (2007) reported that hot air treatment at 38 C

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Fig. 5. Schematic diagram of arginine hydrolysis by arginase. For the sake of clarity, not all reactants and products are shown. ODC, ornithine decarboxylase; OAT, ornithine aminotransferase; P5C, 1 -pyrroline-5-carboxylate; P5CR, P5C reductase.

for 24 h caused heat injury and resulted in negative effects on the antioxidant system of Rhapsody tomato fruit. In our present work showed that 12 h at 38 C prior to 2 C storage eliminated CI symptom development in Messina tomatoes for up to 4 weeks without causing any heat injury. These results indicate that the response to heat treatment depends on fruit size and/or cultivar. Arginase catalyzes the conversion of arginine to ornithine, which in turn can serve as precursor for synthesis of proline and polyamines (Fig. 5) (Morris, 2009). The results of this study showed that HA treatment not only increased arginase activity but also signicantly promoted arginine content (Fig. 4A), suggesting that other arginine-regulating mechanisms may be induced by HA treatment, such as arginine biosynthesis, transport or related events. In addition, ornithine content remained unchanged in HA-treated compared with control fruit (Fig. 4B), which may be associated with the accumulation of proline (Fig. 4C) and/or putrescine (Fig. 4D). In contrast and as expected, levels of ornithine and proline markedly decreased in fruit treated with nor-NOHA + HA, indicating that the metabolic ux from arginine to proline was attenuated by inhibiting arginase activity. Despite the fact that proline can also be synthesized from glutamate in plant (Delauney and Verma, 1993), the accumulation of proline in tomato fruit induced by HA treatment was, at least in part, associated with the increased arginase activity. An increase in arginase activity or expression of ARGAH2 gene along with an increase in proline content in Arabidopsis has also been reported (Jubault et al., 2008; Brauc et al., 2011). It has been proposed that proline accumulation can serve as an adaptive mechanism to chilling stress in higher plants (Delauney and Verma, 1993). The physiological effects of proline accumulation may result from alleviation of oxidative stress as a hydroxyl radical scavenger, osmotic regulation and prevention of protein degradation (Delauney and Verma, 1993). The increased proline content associated with increased resistance to chilling during cold storage is well-known in several horticultural crops, such as tomato (Zhao et al., 2009), peach (Shang et al., 2011) and loquat fruit (Cao et al., 2012). Therefore, our results indicate that arginase may be involved in HA-induced chilling tolerance of tomato fruit partly through promotion of proline synthesis. Polyamines have been regarded as a new type of plant growth regulators that are purported to be involved in a broad-spectrum of physiological processes and stress responses (Groppa and Benavides, 2008). There is evidence that the enhancement of polyamines accumulation is positively correlated with chilling tolerance in plants (Mirdehghan et al., 2007; Zhang et al., 2011). Specically, accumulation of putrescine has been reported in

chilling-injured tomato, pepper, and zucchini fruit (GonzlezAguilar et al., 2000a; Martnez-Tllez et al., 2002; Zhang et al., 2011). The induction of putrescine by heat-treatment associated with increased resistance to chilling has been previously reported in several CI-sensitive fruit (Gonzlez-Aguilar et al., 2000a; Mirdehghan et al., 2007; Cao et al., 2010). However, there is also evidence to suggest that the increased polyamines by heat treatment were not necessarily related to chilling protection (Gonzlez-Aguilar et al., 1997, 2000b). In the present study, HA treatment not only alleviated the CI, but promoted putrescine accumulation (Fig. 4D). Treatment with nor-NOHA reduced the chilling tolerance induced by HA treatment, however, but had little effect on heat-induced putrescine content. These results indicated that other factors, in addition to putrescine, were required for HA-induced chilling tolerance in tomato fruit. Furthermore, the potential role of polyamines in the chilling tolerance induced by HA should be shed light on in the future study. The biosynthetic pathways of polyamines in higher plants, either directly from ornithine by ODC or indirectly from arginine by ADC, have been thoroughly investigated (Groppa and Benavides, 2008). However, the possible role of arginase in polyamines synthesis has not yet been reported. In the present investigation, the increased arginase activity coincided with putrescine accumulation in HA-treated fruit, whereas the putrescine content remained largely unaffected with the inhibition of arginase activity by norNOHA pretreatment. These results suggest that inhibiting arginase activity promotes the metabolic ux from arginine to putrescine via ADC. Alternatively, arginase may not be essential for the HAinduced putrescine accumulation in tomato fruit. In addition, it has been reported that in plant responses to abiotic stress, the biosynthesis of putrescine generally depends on the ADC pathway (Groppa and Benavides, 2008). However, in our previous studies we found that the ODC pathway also contributed to chillinginduced putrescine accumulation in tomato fruit (Zhang et al., 2011). Whether the ADC or ODC pathway is mostly responsible for putrescine synthesis in HA-treated fruit and the possible role of arginase in putrescine synthesis remain to be explored. In conclusion, our results indicate that arginase induction plays a part role in the chilling tolerance in tomato fruit induced by HA treatment, possibly by a mechanism bolstering the antioxidant system and by increasing proline levels. To our knowledge, this is the rst report that the reduction of CI in a postharvest horticultural product by heat treatment may be partly dependent on arginase induction. However, the possible role of arginase in polyamines synthesis is unclear; the gene expression or enzyme activity for OAT and ODC also needs to be further studied. Moreover, it would be interesting to determine if NOS, which uses arginine as the substrate to generate the signaling molecule NO, plays an important role in heat-induced chilling tolerance. Acknowledgments This study was supported by the National Basic Research Program of China (No. 2013CB127106) and the National Natural Science Foundation of China (Nos. 31201432, 31272215, and 31101587). References
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