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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, People’s Republic of China School of Agricultural Economics and Rural Development, Renmin University of China, Beijing 100872, People’s Republic of China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s 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 ﬁnal step in the urea cycle, which allows potentially toxic ammonium to be detoxiﬁed into urea (Morris, 2009). In addition to this detoxiﬁcation 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, People’s Republic of China. Tel.: +86 10 62737620; fax: +86 10 62736474. E-mail addresses: email@example.com (F. Li), firstname.lastname@example.org (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). Brownﬁeld 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
Absorbance of the reaction product of MDA with 3 mL of 0. 3. Nucleotide sequences of gene-speciﬁc primers used for quantitative real-time PCR were as described previously (Zhang et al. 3 = pitting covering <50% but >25% of surface and 4 = pitting covering >50% of surface. little information is available about the potential role of arginase in fruit chilling tolerance induced by heat treatment. the threshold cycle (Ct) value was normalized to Ubi3 and set relative to 0 h samples according to the 2− Ct method. 2007). Shang et al.. 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 = pitting covering <25% but >5% of surface.1 M mannitol under constant shaking. 0. 2007. Any remaining genomic DNA was removed by digestion with DNase I (DNA-free.. Six disks (3 mm thick) of mesocarp tissue were cut with a cork borer from the equator of the fruit. After that. 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-inﬁltrated under low pressure (−35KPa) for 0. were evaluated via qRT-PCR using the SYBR Green I MasterMix (Toyobo.. Osaka.6 ± 0. The weight of per fruit about 41. Materials and methods 2. 6. Zhang et al. 2012). For the combination nor-NOHA + HA treatment.. 2002). 2. After treatment. Arginase could provide a precursor for the biosynthesis of putrescine or proline by hydrolyzing arginine. Arginase assays Frozen fruit tissue (1 g) was ground and homogenized in 5 mL of 100 mM Tris–HCl (pH 7. 2010).. CI. 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. Furthermore.6% thiobarbituric acid and 1 mL of the extract at 532 nm was recorded and corrected for nonspeciﬁc absorbance at 600 nm. He et al. The severity of symptoms was evaluated visually according to the following four-stage scale: 0 = no pitting. The amount of MDA was calculated using an extinction coefﬁcient of 155 mM−1 cm−1 and expressed as mol g−1 fresh weight (FW).2 cm. Fluorescence signal was measured at the end of each annealing step. LeARG1 and LeARG2. 2007. 12 and 24 h using trizol reagent as described previously (Zhang et al. Before assays.. To address this question. To check the annealing speciﬁcity of each oligonucleotide. 2012. and the supernatants were used for the enzyme assays. Uniform sized fruit. MDA was measured using the thiobarbituric acid method as described previously (Ding et al. Heat shock treatment. To determine relative gene expression for each sample. malondialdehyde (MDA). is effective in reducing CI in a number of postharvest horticultural products (Mirdehghan et al. Messina) fruit were harvested at the mature green stage. Japan) on a Chromo4 real time PCR Detection System (Bio-Rad.3. / Postharvest Biology and Technology 79 (2013) 1–8 green tomato fruit in response to low temperature. Luengwilai et al. Transcript levels of the two genes encoding arginase. enzymes. 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 (González-Aguilar et al. the enzyme extract . electrolyte leakage. in spite of the evidence that the increased polyamines with temperature were not necessarily related to chilling protection (González-Aguilar et al.. 2000b). thereafter. a physical treatment free from chemical residues. free of blemishes or defects. development of CI was measured after these fruit were transferred to 20 ◦ C for an additional 3 d. Conductivity was measured after 2 h of incubation in 25 mL of 0. USA). In each lot. 2. The following ampliﬁcation protocols were used: 95 ◦ C for 2 min. Electrolyte leakage and MDA content assays 2.000 × g for 15 min at 4 ◦ C.. and pomegranate (Mirdehghan et al. 2. fruit were randomly divided into three replicates. fruit were stored at 2 ± 1 ◦ C with a relative humidity of 80–90% for up to 28 d. polyamines and arginine-related amino acid levels in tomato fruit during low temperature storage. Quantitative real-time PCR (qRT-PCR) assays Total RNAs were extracted from fruit treated with HA for 0. followed by 40 cycles of 15 s at 95 ◦ C and 20 s at 60 ◦ C.1 g in average and the mean diameter of fruit was 3. Fruit and treatments Tomato (Solanum lycopersicum L. Chilling injury (CI) is an economically important postharvest problem that reduces the overall quality and marketability of many tropical and subtropical horticultural products. fruit pretreated with nor-NOHA were also incubated at 38 ◦ C for 12 h. In view of the possible roles of polyamines and proline in plant tolerance to chilling stress. CI index evaluation CI symptoms of tomato fruit were manifested as surface pitting (Ding et al. together with the fact that arginase appears to be involved in stress resistance mechanism in higher plants. Ambion) according to the manufacturer’s protocol. 2010). However. and then 450 of these fruit were incubated at 20 ◦ C for 12 h serving as the control lot. The mesocarp from 10 fruit per replicate was collected at different hours or days for genes. 1997.. We examined the effects of heat treatment with or without pretreatment with exogenous N -hydroxy-nor-l-arginine (nor-NOHA. polyamines and arginine-related amino acid analysis. Hercules. Zhang et al. 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. The Ubi3 encoding ubiquitin was used as the reference gene.4.2.. melting curve analysis (55–94 ◦ C) was carried out at the end of ampliﬁcation..1. which was proved to increase the chilling tolerance of fruit (Zhang et al. cv. antioxidant enzyme activities. fruit were kept under ambient air pressure for 2 min and then air-dried. All experiments were run in triplicate with different cDNAs synthesized from three biological replicates.5 min (12). tomato fruit were used. 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. 2011). The rate of electrolyte leakage was expressed as a percentage of the total: (initial/total) × 100. an ideal inhibitor to study the role of arginase) on arginase activity or gene expression.. 2010). The remainder (930 fruit) were ﬁrst treated with water under the same conditions.2 X.. The homogenates were centrifuged at 12. 2011). 2007).1 mM phenylmethylsulfonyl ﬂuoride (PMSF) and 0. The cDNA was obtained as described by Zhao et al. 2. we hypothesis that arginase may also be involved in heat-induced chilling resistance in postharvest fruit. (2009).5) containing 1% (v/v) -mercaptoethanol.. 1 = pitting covering <5% of the fruit surface. and research was focused on investigating the potential role of arginase in the induction of heat-induced chilling resistance.5% (w/v) polyvinyl polypyrrolidone (PVP). peach (Cao et al. the solutions were boiled for 10 min and cooled to room temperature and the total conductivity was measured.2 ± 2. lipid peroxidation.5.. 2000a). (2009) with minor modiﬁcations. 2011.
2. remained unchanged at 20 ◦ C for 24 h.5 mL of sample extracts were mixed with 0. 2. which was detected rapidly within 3 h of HA treatment. However. POD activity was determined in a reaction mixture containing 50 mM sodium acetate (pH 5.5 M. Data analysis Data were analyzed using one-way analysis of variance (ANOVA) with SPSS 16. peroxidase (POD) and ascorbate peroxidase (APX). Approximately 0.05 was considered as signiﬁcant. The polyamines was detected using a UV detector at 254 nm and quantiﬁed using the benzoylated standard curves. 92%A. APX activity was determined by the method of Nakano and Asada (1981). The data were expressed as means ± standard error (SE).8. / Postharvest Biology and Technology 79 (2013) 1–8 3 was activated with 1 mM MnCl2 at 37 ◦ C for 15 min. peaked at the 12 h time point.25 M arginine (pH 9. 80% A.6). Polyamines in the supernatant was benzoylated. Speciﬁc activities of all enzymes were expressed as units (U) mg−1 protein. One unit of POD activity was deﬁned as the amount of enzyme that caused an increase in absorbance of 0. Absorbance was read at 515 nm and standard urea solutions were used for calibration.01 at 470 nm in 1 min. 2. 1955). 2). Samples were derivatized with 1-ﬂuoro-2.45 m pore ﬁlters before HPLC analysis. 4. Reactions were carried out at 37 ◦ C for 20 min and stopped by adding 0.5 mL of 1% (v/v) FDNB in pure acetonitrile. 1C). 0. CAT activity was assayed by monitoring the disappearance of H2 O2 according to the method of Chance and Maehly (1955). containing 1% (v/v) dimethyl formamide and pure acetonitrile at a ﬂow rate of 1 mL min−1 according to the following gradient: initial..5 mL of 15% (v/v) perchloric acid. Derivatized amino acids were detected at 360 nm using a UV detector. Protein concentration Protein concentration in the enzymatic extracts was quantiﬁed according to the method of Bradford (1976) with bovine serum albumin as standard.0).0). 92% A. 2000). HA-induced expression of LeARG genes was accompanied by an increase in arginase activity during heat treatment and the recovery period.6) and 20 L of 50 mM MnCl2 ..2 mL of crude enzyme extract (Chance and Maehly.1 mM ethylene diamine tetraacetic acid (EDTA). when compared with the control (Fig. 92% A and 50 min. 40 min. 60% A. Arginine-related amino acids assays Free amino acids were extracted according to the method of Micallef and Shelp (1989) with minor modiﬁcations. 11 min. Heat-treated fruit shared the same trend as control fruit. 2. is relatively stable in fruit tissue.6 mm × 250 mm Zorbax ODS column and analyzed by reverse-phase HPLC using an Agilent 1200 series (Agilent.7. then extracted with 2 mL of chilled diethyl ether. as previously described (Zhang et al. 6 min. 2004). Samples were ground with liquid nitrogen. Although there was a decline in the transcription of LeARG1 and LeARG2 after 24 h of HA treatment. The homogenate was centrifuged at 12.0 statistical software. once synthesized. The expression pattern of the two genes was very similar. the supernatant was adjusted to pH 7 with 4 M NaOH to prepare for HPLC analysis. 30 min. Changes of arginase gene expression and activity in response to HA treatment The expression of the two genes encoding arginase in tomato fruit. pH 6. Urea released in the medium was determined spectrophotometrically as described previously (Chen et al. The reaction mixture contained 20 L of the activated arginase. LeARG1 and LeARG2. 3. 5 m particle diameter.0). 88% A. 20 L of derivatized samples were injected onto a 4.1% H2 O2 . 2. (1996). Following 1 d of exposure to HA. and then decreased gradually (Fig.6 mm × 250 mm) and eluted with 64% methanol at a ﬂow rate of 1 mL min−1 . 40 L of 1. Amino acids were identiﬁed by cochromatography of individual standards and quantiﬁed by comparison of individual external calibration curves. 92% A. 1 mM ascorbic acid and 1% PVP in an ice bath. containing 0.1. pH 9. reaching a peak at day 7. followed by thorough mixing with 0. One unit of CAT activity was deﬁned as the amount of enzyme that decomposed 1 mol H2 O2 min−1 at 30 ◦ C. exposure of fruit to 38 ◦ C for 12 h before cold storage caused an enhanced arginase activity at all the time points when . Arginase activity was expressed as nmol urea produced min−1 mg−1 protein.6. The ether phase was dried under a stream of warm air and the residue was dissolved in 300 L of methanol (HPLC grade) and ﬁltered through 0. 70% A.5 mL of NaHCO3 solution (0.000 × g for 30 min at 4 ◦ C. 4-dinitrobenzene (FDNB) as described previously (Wang et al. One unit of APX activity was deﬁned as the amount of enzyme that oxidized 1 mol ascorbate min−1 at 30 ◦ C. Amino acids were eluted with 25 mM sodium acetate buffer (solvent A). 45 min. 2. with minor modiﬁcations. Following centrifuged for 10 min at 12.X. it was added to 3 mL of 50 mM KH2 PHO4 solution and vortexed for several seconds. Free polyamine determination Frozen tissue was extracted in 4 volumes of 5% (w/v) cold perchloric acid and centrifuged at 12. and declined thereafter. Zhang et al. 420 L of 120 mM glycin–NaOH (pH 9. Antioxidant enzyme assays Frozen tissue (1 g) was homogenized in 5 mL of 50 mM sodium phosphate buffer (pH 7. and the powder was suspended in 1 mL g−1 of a 3% (w/v) solution of 5-sulfosalicylic acid. arginase activity reached a maximum with a value 91% higher than that of control (Fig. arginase activity remained elevated thereafter.000 × g for 15 min at 4 ◦ C. One unit of SOD activity was deﬁned as the amount of enzyme that caused 50% inhibition of NBT reduction. Heat-induced arginase activity is inhibited by nor-NOHA during cold storage Arginase activity in control fruit increased in response to cold stress.9. 0. 2011). Results were expressed as nmol g−1 FW. Results 3. catalase (CAT).10. 1A and B). 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. USA). After ﬁltration through a 0. 35 min. 3. Proline content was determined by the ninhydrin method. (2009). Derivatized samples (20 L) were injected onto a reverse phase C18 column (Zorbax ODS. All experiments were conducted in a completely randomized design with three replicates for each treatment. Signiﬁcant differences were performed by Duncan’s multiple comparison procedure. as described by Zhao et al.4.45 m membrane ﬁlter. 1C). 50% A. The derivatization was complete in the dark for 60 min at 60 ◦ C. This observation indicates that the enzyme. Results were expressed as mol g−1 FW.000 × g. A probability of P < 0. while treatment of fruit with HA at 38 ◦ C resulted in strong induction of LeARG1 and LeARG2 (Fig.. 20 min. After cooling to room temperature. The supernatant was used for assays of the activity of superoxide dismutase (SOD).1% guaiacol and 0.
control. a highly speciﬁc inhibitor of arginase. However. normalized to Ubi3 gene and set relative to control samples from 0 h according to the 2− Ct method. Different letters indicate signiﬁcant differences at P < 0. The presence of nor-NOHA in heat-treated fruit had almost no effect on the content of arginine. 3. Although norNOHA signiﬁcantly inhibited the heat-induced increase in proline Fig. 3B) and APX (Fig.05 by Duncan’s multiple range test. there were no signiﬁcant differences in CI index and MDA content between controls and (norNOHA + HA)-treated fruit. CAT (Fig. However. during most of the storage time. 4B).4. day 0). 3. Moreover. whereas nor-NOHA + HA treatment markedly decreased its content in fruit during the entire storage periods. / Postharvest Biology and Technology 79 (2013) 1–8 21 Arginase activity (nmol·min-1·mg -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. the proline content was increased immediately after exposure to HA for 12 h by 67. CAT.3% relative to the control (Fig. CAT and APX were partially inhibited by exposing fruit to nor-NHOHA before HA treatment. However. APX and POD considered to play an important role in cellular defense against oxidative stress caused by chilling injury (Sevillano et al. electrolyte leakage and MDA content in HA-treated fruit as compared to the . In contrast. 3C) activities compared with the controls during most of the storage period. 3. although the electrolyte leakage in (norNOHA + HA)-treated fruit was lower than that in control. 3). the highest POD activity (Fig. in heat-treated fruit. HA treatment had little effect on ornithine content relative to the controls. exogenous addition of norNOHA. arginine content in control fruit remained mostly unchanged throughout the storage periods. 3D) was observed in control. severe CI symptoms were observed on treated and untreated tomato fruit (Table 1). and the lowest was recorded in HA-treated fruit. As expected. 2. followed by nor-NOHA + HA.. Changes in the content of arginine-related amino acids and polyamines As shown in Fig. 4C. HA treatment maintained signiﬁcantly higher SOD (Fig. 2009) were determined for mesocarp tissue of tomato fruit subjected to various treatments (Fig. and maintained a signiﬁcantly higher level than controls during subsequent storage up to 28 d. However. the HA-induced activities of SOD. In contrast.4% higher compared with the controls at the end of storage.5. before HA treatment signiﬁcantly inhibited heat-induced arginase activity.4 X. its content increased immediately after HA treatment (Fig. Gene expression and activity of arginase in tomato fruit at different time intervals during or after hot air (HA) treatment. Zhang et al. proline content also increased with storage time in all samples. Effect of hot air (HA) treatment on arginase activity in tomato fruit during storage at 2 ◦ C with or without pretreatment by nor-NOHA. the inductive effects of HA on chilling tolerance were almost prevented by treatment fruit with nor-NOHA before heating. Similar to ornithine. Changes in activities of antioxidant enzymes The activities of SOD. Thus. and no signiﬁcant peak in arginase activity was found during the entire cold storage period. Especially after 4 weeks at 2 ◦ C. 1. as observed by the lower value of CI index. Heating the fruit for 12 h at 38 ◦ C before storing them at 2 ◦ C reduced the development of CI symptoms. Data are expressed as mean ± SE of triplicate assays. Data are expressed as mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. compared with the control. The expression levels of LeARG1and LeARG2 encoding arginase were evaluated by quantitative real-time PCR.3. which was relatively lower than that of control fruit. 4A. 3A). Expression of LeARG1 (A) and LeARG2 (B) and arginase activity (C). 4A. Changes in chilling injury After 3 or 4 weeks at 2 ◦ C. ornithine content in control and treated fruit increased in response to chilling stress during storage at 2 ◦ C (Fig. proline content in HA-treated fruit was also higher than that in controls during the subsequent cold storage period and remained 23. day 0).
23d 39. Storage time 21 d Treatment Control HA Nor-NOHA + HA Control HA Nor-NOHA + HA CI index 2. 2009).13a 1. Zhang et al.62 ± 0.76 ± 1. 2012). and there were little effects of HA treatment on the content of them expect on the earlier 3 d (Fig. when lower levels of putrescine were detected. In addition. dysfunction of the cell membrane at low temperature is considered to be the primary molecular event.40 ± 3. Effect of hot air (HA) treatment on SOD (A). Electrolyte leakage and MDA content were measured immediately after transferring the fruit from 2 ◦ C to 20 ◦ C. CI index was measured after transferring the fruit from 2 ◦ C to 20 ◦ C for 3 d. fruit treated with nor-NOHA + HA showed a signiﬁcantly higher CI index. which is frequently related to an increase in membrane permeability and lipid peroxidation (Sevillano et al.. 3.79 ± 3. but also effectively reduced CI symptom in tomato fruit (Table 1).98 ± 0.71 ± 0.79b 12.16b 3. electrolyte leakage and MDA content.37c 11. content. 2008.10c 2. Vertical bars represent the standard errors of the means.22 ± 0. 2009.15a 30. Gravot et al. we found that HA treatment at 38 ◦ C for 12 h not only resulted in strong induction of LeARGs. To evaluate the participation of arginase in HAinduced chilling tolerance. the nor-NOHA + HA treatment also showed no signiﬁcant difference with controls during the whole storage periods.42a 9. Zhang et al. spermidine and spermine contents ﬂuctuated during the storage periods.53 ± 0. Data are accompanied by standard errors of the means. 4D–F). Putrescine was the predominant polyamines. followed by spermidine and spermine in tomato fruit (Fig.71b 55. Although research on plant arginase has mainly focused on its role in nitrogen remobilization processes from protein degradation. At the end of cold storage. The putrescine content increased in response to chilling stress during the storage periods. 2000). By contrast. Brauc et al. 2005.6 ± 1. a product CAT activity (U·g-1 protein) SOD activity (U·g-1 protein) 110 88 66 44 22 A 20 16 12 8 4 0 54 0 45 36 27 18 9 0 0 B APX activity (U·g-1 protein) 120 90 60 30 0 0 7 14 21 Days of storage at 2°C 28 POD activity (U·g-1 protein) 0 180 0 C 150 Control HA nor-NOHA+HA 7 14 21 Days of storage at 2°C 28 D 7 14 21 Days of storage at 2°C 28 7 14 21 Days of storage at 2°C 28 Fig.30b MDA content ( mol g−1 FW) 13.85 ± 0. 1).54 ± 0. similar to HA treatment. arginase induction was reported to be part of a defense mechanism against biotic and abiotic stress (Chen et al.0% higher than that in controls..87 ± 0. Discussion Recently. especially during seed germination (King and Gifford. / Postharvest Biology and Technology 79 (2013) 1–8 5 Table 1 Effect of hot air (HA) treatment on chilling injury (CI) index. arginase in mammals has garnered considerable attention due to its potential role in a very wide range of physiological and pathophysiological conditions (Morris. which led to the increased arginase activity (Fig.03 ± 0.X. For spermidine and spermine contents. CI index directly reﬂected development degree of CI symptoms and was widely used in study of chilled postharvest fruit for judging cold tolerance. fruit were incubated with the arginase inhibitor nor-NOHA before the imposition of HA. There were little effect of nor-NOHA treatment on heat-induced putrescine accumulation during the chilling stress periods except on days 3 and 7. In our present study. Munder. as well as increased electrolyte leakage and MDA content (Table 1).15c 48. 2010. Brownﬁeld et al. .28a 2. Compared with HA-treated fruit. levels in fruit treated with nor-NOHA + HA were still higher than those in controls at most sampling times. 1997.58 ± 0. 4E and F).05 by Duncan’s multiple range tests.10a Electrolyte leakage (%) 49. 2011.45b 13.32 ± 2.39 ± 0. APX (C) and POD (D) activities in tomato fruit during storage at 2 ◦ C with or without pretreatment by nor-NOHA. 4D). Therefore.31 ± 4. 4. and HA treatment enhanced the accumulation compared with the control fruit throughout the cold storage period (Fig.23 ± 0. electrolyte leakage and MDA content in tomato fruit during storage at 2 ◦ C with or without nor-NOHA pretreatment.. Goldraij and Polacco.. CAT (B). 2009).03a 28 d Means in a column followed by a different letter for the same storage period differ signiﬁcantly at P < 0. Data are expressed as mean ± SE of triplicate assays.. the content of putrescine in HA-treated fruit was 15.03a 36.17b 3.87a 8..
Some reports indicated a requirement of 2–3 d at 38 ◦ C to ameliorate CI in tomato fruit (Lurie and Klein. conﬂicting results concerning the relationship between CI severity and POD activity have been reported (Saﬁzadeh et al.6 X.14 0. Effect of hot air (HA) treatment on arginine (A). Jin et al. CAT and POD. treatment with HA signiﬁcantly reduced the CI symptoms and increased the activities of SOD. 2007. 2008. HA treatment decreased POD activity and increased the CAT and APX activity.. 2008). the activities of SOD. CAT and APX (Saﬁzadeh et al. 1991.. 2).. Our results and these prior reports suggest that the balance between SOD. 2009). SOD catalyzes the dismutation of the superoxide free radicals to O2 and H2 O2 (Sevillano et al. ornithine (B).. 2009). 2009). of lipid peroxidation. 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. 2007. 2007).6 0.3 Spermidine (nmol g -1 FW) Spermidine c (nmol g-1 FW) Ornithine ( mol g -1 FW) Proline ( mol g-1 FW) 0.07 0.. APX and POD are the main enzymes responsible for H2 O2 scavenging.6 0. The positive effects of heat treatments on the chilling injury and antioxidant system in tomato fruit were also reported in several other studies.1 1500 1200 900 1. 2008.. In the present work. 2007. POD activity in fruit treated with nor-NOHA + HA was higher than that in HA-treat fruit (Fig.2 0. suggesting that HA altered the activities of these enzymes at least in part through the induction of arginase. which not only scavenged hydrogen peroxide. In our study. which are very important for scavenging active oxygen species (AOS) to protect cell membranes. CAT. CAT. HA treatment had a negative effect on POD activity compared to the control fruit. As expected. APX. In addition. APX and POD activity is critical to cell survival during cold storage. 4. we observed a signiﬁcant reduction in POD activity in HA-treated fruit.8 1. Data are expressed as mean ± SE of triplicate assays. A reduction in CI by treatments along with an increase in POD activity in horticultural crops under cold stresses has also been reported (Saﬁzadeh et al. Sevillano et al. McDonald et al.. but alleviated the production of phytotoxic free radicals liberated by POD. CAT.3 0.. 2009). Yahia et al. (2007) reported that hot air treatment at 38 ◦ C . Accompanying the alleviation of CI. However. putrescine (D).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 2°C 28 3 0 0 7 14 21 Days of storage at 2°C F 28 Fig. 2007. 3A–C). spermidine (E) and spermine (F) contents in tomato fruit during storage at 2 ◦ C with or without pretreatment by nor-NOHA.0 1. and APX during cold storage. is thought to be a major mechanism of resistance to chilling stress (Saﬁzadeh et al.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 0.9 0. the changes in the activities of these enzymes in HAtreated fruit were diminished when arginase activity was inhibited by nor-NOHA. Zhao et al. Imahori et al.0 0. / Postharvest Biology and Technology 79 (2013) 1–8 1. Previous studies have shown that the improvement of chilling tolerance in harvested horticultural crops is related to enhancement in activities of SOD.. which was conﬁrmed in our results. and APX in fruit treated with nor-NOHA + HA decreased compared with those treated with HA alone (Fig..21 0.2 0 0.. Zhang et al.. CAT. Imahori et al. However. have been used to evaluate chilling resistance of plant tissues (Imahori et al. 2009).. These free radicals are highly phytotoxic. Vertical bars represent the standard errors of the means. However. Zhao et al.. proline (C).5 Putrescine (nmol g -1 FW) Arginine ( mol g -1 FW) 2.28 0 0. but the mode of action of POD differs from CAT action in that POD liberates free radicals instead of oxygen (Saﬁzadeh et al. The coordinated action of antioxidant enzymes such as SOD. However. 3D). 1996).
. 2000b).M. 1976. Angenon. P5C. 1993). 2000a. but promoted putrescine accumulation (Fig. 2008). Claeys. The results of this study showed that HA treatment not only increased arginase activity but also signiﬁcantly promoted arginine content (Fig.. For the sake of clarity. Zhang et al. In the present study. However. plays an important role in heat-induced chilling tolerance.. 248–254.. associated with the increased arginase activity. Alternatively.. our results indicate that arginase may be involved in HA-induced chilling tolerance of tomato fruit partly through promotion of proline synthesis.. such as arginine biosynthesis. 31272215. levels of ornithine and proline markedly decreased in fruit treated with nor-NOHA + HA. and zucchini fruit (GonzálezAguilar et al. it would be interesting to determine if NOS. in our previous studies we found that the ODC pathway also contributed to chillinginduced putrescine accumulation in tomato fruit (Zhang et al. 2011) and loquat fruit (Cao et al. The biosynthetic pathways of polyamines in higher plants. 4C) and/or putrescine (Fig. Acknowledgments This study was supported by the National Basic Research Program of China (No. P5C reductase. These results indicated that other factors. have been thoroughly investigated (Groppa and Benavides. 2010). which uses arginine as the substrate to generate the signaling molecule NO. Treatment with nor-NOHA reduced the chilling tolerance induced by HA treatment. References Bradford. 4B). ornithine content remained unchanged in HA-treated compared with control fruit (Fig... 1993). suggesting that other arginine-regulating mechanisms may be induced by HA treatment.. which may be associated with the accumulation of proline (Fig.. 4D).. 4A). In conclusion. the gene expression or enzyme activity for OAT and ODC also needs to be further studied. however. the increased arginase activity coincided with putrescine accumulation in HA-treated fruit. the accumulation of proline in tomato fruit induced by HA treatment was. 2012). Zhang et al. 2007. Cao et al.. ornithine aminotransferase. 67. 39–45. These results suggest that inhibiting arginase activity promotes the metabolic ﬂux from arginine to putrescine via ADC.K. it has been reported that in plant responses to abiotic stress. However. 5) (Morris. 1997. at least in part. peach (Shang et al. in addition to putrescine.C. The physiological effects of proline accumulation may result from alleviation of oxidative stress as a hydroxyl radical scavenger. G. These results indicate that the response to heat treatment depends on fruit size and/or cultivar. 5. either directly from ornithine by ODC or indirectly from arginine by ADC. . 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. osmotic regulation and prevention of protein degradation (Delauney and Verma. 2008).. ODC. Arginase catalyzes the conversion of arginine to ornithine. M. 1 -pyrroline-5-carboxylate. but had little effect on heat-induced putrescine content. In addition. Brownﬁeld. M. 2008). In contrast and as expected. Geuns.X. 2007. 2011). possibly by a mechanism bolstering the antioxidant system and by increasing proline levels. 2002. M. J. Analysis of Arabidopsis arginase gene transcription patterns indicates speciﬁc biological functions for recently diverged paralogs. 72. the possible role of arginase in polyamines synthesis is unclear. Brauc et al.. Biochem. 429–440.. the biosynthesis of putrescine generally depends on the ADC pathway (Groppa and Benavides. C. accumulation of putrescine has been reported in chilling-injured tomato. Furthermore. not all reactants and products are shown. 2011). whereas the putrescine content remained largely unaffected with the inhibition of arginase activity by norNOHA pretreatment. 1993). It has been proposed that proline accumulation can serve as an adaptive mechanism to chilling stress in higher plants (Delauney and Verma. this is the ﬁrst report that the reduction of CI in a postharvest horticultural product by heat treatment may be partly dependent on arginase induction.. Todd. Martínez-Téllez et al. 4D). 2009). In addition.. the potential role of polyamines in the chilling tolerance induced by HA should be shed light on in the future study. In the present investigation. 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. 2008. / Postharvest Biology and Technology 79 (2013) 1–8 7 Fig. 2000a. Zhang et al. OAT. 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. The induction of putrescine by heat-treatment associated with increased resistance to chilling has been previously reported in several CI-sensitive fruit (González-Aguilar et al.M.L.D. The increased proline content associated with increased resistance to chilling during cold storage is well-known in several horticultural crops. 14. Speciﬁcally. were required for HA-induced chilling tolerance in tomato fruit. Mirdehghan et al. for 24 h caused heat injury and resulted in negative effects on the antioxidant system of ‘Rhapsody’ tomato fruit. indicating that the metabolic ﬂux from arginine to proline was attenuated by inhibiting arginase activity. Biol.. Brauc. 2009). pepper. Höfte. P5CR.. ornithine decarboxylase. arginase may not be essential for the HAinduced putrescine accumulation in tomato fruit. our results indicate that arginase induction plays a part role in the chilling tolerance in tomato fruit induced by HA treatment. Schematic diagram of arginine hydrolysis by arginase. which in turn can serve as precursor for synthesis of proline and polyamines (Fig.. Plant Mol. 31201432. Overexpression of arginase in Arabidopsis thaliana inﬂuences defence responses against Botrytis cinerea. However. Moreover. HA treatment not only alleviated the CI. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.. However. transport or related events. 2013CB127106) and the National Natural Science Foundation of China (Nos. S. To our knowledge. De Vooght. there is also evidence to suggest that the increased polyamines by heat treatment were not necessarily related to chilling protection (González-Aguilar et al. E. 2008. such as tomato (Zhao et al. the possible role of arginase in polyamines synthesis has not yet been reported. D.. 2011).. 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. and 31101587). 2011). Plant Biol. M. There is evidence that the enhancement of polyamines accumulation is positively correlated with chilling tolerance in plants (Mirdehghan et al. Despite the fact that proline can also be synthesized from glutamate in plant (Delauney and Verma. 2011... Anal. Deyholos. Therefore.
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