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Changes in sugar metabolism caused by exogenous oxalic acid related to chilling tolerance of apricot fruit
Postharvest Biology and Technology 114 (2016) 10–16
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Changes in sugar metabolism caused by exogenous oxalic acid related to chilling tolerance of apricot fruit Zhen Wang, Jiankang Cao, Weibo Jiang* College of Food Science and Nutritional Engineering, China Agricultural University, PO Box 111, 17 Qinghuadonglu Road, Beijing 100083, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 August 2015 Received in revised form 23 November 2015 Accepted 26 November 2015 Available online xxx
Enhancement of chilling tolerance has been observed in fruits treated with oxalic acid (OA). To learn how OA may play a role in modulating chilling injury in apricot, the fruit were treated with OA (5 mM) and stored at 2 1 C for five weeks. OA treatment significantly inhibited chilling injury, electrolyte leakage and accumulation of hydrogen peroxide and malondialdehyde in apricot fruit. Sorbitol, sucrose, fructose and glucose were the major soluble sugars in the fruit. Contents of glucose and fructose were enhanced, meanwhile, the levels of sucrose and sorbitole were decreased by OA treatment during storage. OA treatment enhanced activities of the enzymes related to increasing glucose and fructose, and suppressed sucrose synthase synthesis activity which could explain the lower content of sucrose. The activities of sucrose synthase cleavage function, acid invertase, NAD+-sorbitol dehydrogenase and sorbitol oxidase were significantly enhanced by OA. Meanwhile, OA-treated apricot fruit showed lower sucrose synthase synthesis activity. The degradation of sorbitol in apricot fruit was accelerated by OA treatment. Our results indicate that higher chilling tolerance of OA treated fruit was associated with higher content of reducing sugars (glucose and fructose). ã 2015 Elsevier B.V. All rights reserved.
Keywords: Oxalic acid Sugar metabolism Glucose Fructose Chilling injury Apricot fruit
1. Introduction Apricots are popular around the world as a result of their high nutritional value and aroma (Solis-Solis et al., 2007). Various phenolic compounds including caffeic acid, ferulic acid, catechin, epicatechin, p-coumaric acid have been detected in apricot fruit (Sochor et al., 2010). Apricot fruit is usually stored at low temperatures to extend shelf life and maintain quality. During storage at low temperature, apricot fruit show some physiological disorders because apricot fruit is sensitive to low temperatures. Internal browning is one major symptom of chilling injury (CI) in apricot fruit (Saba et al., 2012). Recently, the metabolism of soluble sugars of fruit during the postharvest storage attracted the interest of numerous scientists (Borsani et al., 2009; Sun et al., 2011). The reason is that the profiles and concentrations of soluble sugars not only contribute to the organoleptic quality of fruit (Borsani et al., 2009), but also affect the chilling tolerance of fruit during cold storage (Agopian et al., 2011; Wang et al., 2013; Cao et al., 2013). In mandarin fruit, the
* Corresponding author. E-mail address: [email protected] (W. Jiang). http://dx.doi.org/10.1016/j.postharvbio.2015.11.015 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
level of sucrose is considered to enhance chilling tolerance (Holland et al., 2002). However, a higher level of fructose and glucose is believed to enhance the chilling tolerance of loquat fruit (Shao et al., 2013; Cao et al., 2013). Oxalic acid (OA), a natural organic acid, has been reported to play an important role in systemic resistance and response to environment (Zheng et al., 2012; Jin et al., 2014; Liang et al., 2009). Prestorage application with OA enhanced the antioxidant capacities of pomegranate (Sayyari et al., 2010) and inhibited the decay of mango (Zheng et al., 2012). Moreover, the application of OA enhanced the chilling tolerance of litchi, peach and plum fruit during the postharvest storage under low temperatures (Zheng and Tian, 2006; Jin et al., 2014; Wu et al., 2011). Some work has been reported on the effect of OA on the chilling tolerance of various fruits (Jin et al., 2014; Wu et al., 2011; Zheng and Tian., 2006). The effect of OA on the chilling tolerance of various fruits has been explained by various mechanisms (Jin et al., 2014; Wu et al., 2011; Zheng and Tian., 2006). To the best of our knowledge, there is no literature focus on the changes of soluble sugar content and metabolism of sugar in fruits caused by OA treatment. Then no work focuses on illuminating the relationship of changes of soluble sugar content caused by OA treatment and higher chilling tolerance of OA-treated fruit during
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the postharvest storage. The objective of this study was to determine changes in sugar content and metabolism caused by exogenous oxalic acid related to chilling tolerance of apricot fruit. 2. Materials and methods 2.1. Materials and treatments Apricot fruit (Prunus armeniaca L. cv. Diaogan) of commercial maturity were obtained from local market in Beijing, PR China. The fruit were packaged in fibreboard cartons, transferred to our laboratory (Beijing, PR China) within 5 h via truck. During the transportation, the fruit was kept at low temperature (4–8 C). Fruit sorted by hand (uniform size and color, without physical damage) were used in the experiment. Apricots were dipped in 5 mM OA solution for 10 min at room temperature. Control fruit were treated with distilled water. The fruit were then air-dried at room temperature for about 30 min. For each treatment, forty apricot fruit were placed into a plastic boxes (600 100 70 mm), and then stored at 2 1 C. The relative humidity (RH) was 85–90%. Each treatment contained 360 apricots. About 60 fruits were collected in each sampling time, and 18 of the collected fruits were used for the biochemical analysis, and 18 of the fruits used for measuring chilling injury (CI) and electrolyte leakage (EL). The measurement of CI was done in three replicates (per replicate consisted of six fruits). For the measurement of EL, flesh tissues from six fruits were used in one measurement. For the biochemical analysis, the flesh from sampling fruits were mixed, immediately frozen in liquid nitrogen and then stored at 80 C. The flesh from 6 sampling fruits were used as one repeat (3 repeats for each biochemical measurement). 2.2. Determination of internal browning index (IBI), electrolyte leakage (EL), content of hydrogen peroxide (H2O2) and malondialdehyde (MDA) The symptom of chilling injury was expressed as internal browning index (IBI). IBI was determined according to previous method with slight modification (Saba et al., 2012). The severity of browning was visually assessed and classified as 5 grades: 0 = none; 1 = slightly (browning area < 5%); 2 = moderate (browning area 5–25%); 3 = moderate severe (browning area 25–50%); 4 = severe (browning area > 50%). The results were calculated as P follows: IBI = ( IBI level) (the number of fruits at this IBI level)/ (4 total number of fruits) 100%. A procedure previously reported was modified to determine the EL (Jin et al., 2014). For the determination of EL, ten flesh disks from fruits (the total weight of flesh was kept about 3 g) were collected with a 3-mm diameter cork borer, rinsed in 25 mL double distilled water for 30 min and was employed to measure the initial conductivity by one DJS-1C conductivity meter (Shanghai Analytical Instrument Co., Shanghai, China). Then the disks were placed at 20 C for 24 h, incubated in a boiling water for 30 min and then used to determine total electrolyte conductivity. The EL was expressed as relative conductivity: (the initial conductivity/the final conductivity) 100%. The content of MDA was determined according to method described by Jin et al. (2014) with some modification. Flesh tissue (2 g) was homogenized on ice with 3 mL trichloroacetic acid (100 g L 1) and then centrifuged at 12,000 g for 20 min at 4 C. A 0.5 mL supernatant was mixed with 2.5 mL thiobarbituric acid and then incubated in boiling water for 20 min. The reaction mixture was centrifuged at 12,000 g for 7 min at 4 C and absorbance of the supernatant was recorded at 450 nm, 532 nm, 600 nm respectively. MDA content in the supernatant was and
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calculated as follow: c (mM) = 6.45 (OD532–OD600)–0.56 OD450. The MDA content of flesh were expressed as nmol g 1 fresh weight. The content of H2O2 was assayed using the method previously described with slight modification (Patterson et al., 1984). The modification was that we used titanium tetrachloride-hydrochloric acid instead of titanium sulphate. Flesh tissue (2 g) was homogenized with 4 mL acetone (pre-chilled at 20 C) and then centrifuged at 12,000 g for 20 min at 4 C. A 1.0 mL supernatant with 0.1 mL titanium tetrachloride–hydrochloric acid and 0.2 mL ammonia (25%, v/v) was centrifuged at 12,000 g for 15 min at 4 C. The precipitation was collected and sufficiently suspended in 3 mL H2SO4 (10%, v/v), centrifuged at 12,000 g for 10 min at 4 C. Absorbance of the supernatant was recorded at 410 nm and the H2O2 content was expressed as nmol g 1 fresh weight. 2.3. Analysis of soluble sugar content Extraction and assays of soluble sugars were carried out with some modification according to a previous method (Shao et al., 2013). Five grams of frozen fresh from fruit samples were homogenized with 25 mL extraction solution on ice and kept still for 20 min. Special solution contained 3.2 g L 1 acetic acid and 2.4 g L 1 potassium ferrocyanide (dissolved with double distilled water). The mixture was centrifuged at 12,000 g for 20 min at 4 C. The supernatant was collected and diluted to 150 mL with double distilled water. Before the HPLC analysis, the resulting extract was filtered through a 0.22 mm membrane filter. A 10 mL sample was injected into an ultrafast liquid chromatography (UFLC). The UFLC contained HPLC (Agilent 1260, Agilent Corp, America., USA; column: Hedera, NH2; column temperature: 50 C) and an evaporative light-scattering detector (Agilent 1260, Agilent Corp, America). The mobile phase composition was acetonitrile and water (80:20). The total flow rate of mobile phase composition was 1 mL min 1. Individual sugars were identified by the retention of time and quantified based on standard curves of individual sugars. 2.4. Extraction and assays of activity of sucrose phosphate synthase (SPS), sucrose synthase synthesis activity (SS-synthesis), sucrose synthase cleavage activity (SS-cleavage), acid invertase (AI) and neutral invertase (NI) For the assay of relative activity of SPS, SS-synthesis, SScleavage, AI and NI, the extraction process was modified from a previous method (Shao et al., 2013). Flesh tissues (3 g) were homogenized on ice with 0.5 g crosslinking polyvingypyrrolidone (PVPP) and 6 mL buffer containing 50 mM HEPES–NaOH (pH 8.5), 10 mM MgCl2, 2.5 mM DTT, 0.1% TritonX-100 (v:v) and 10 mM vitamin C, 10 mM b-mercaptoethanol, 20% (v:v) glycerol, 10 mg mL 1 leupeptin, 10 mg mL 1 chymostatin. The homogenate was centrifuged at 12,000 g for 30 min at 4 C. A cold pre-equilibrated Sephadex G-25 column was used to desalt the supernatant. These crude extracts were used for the measurement of activity of enzymes. For the assay of activity of SPS, the reaction system consisted of HEPES–NaOH buffer (50 mM, pH 7.5; containing 15 mM MgCl2), 25 mM fructose 6-phosphate, 25 mM glucose 6-phosphate, 25 mM UDP-glucose and crude enzyme extract. The reaction system was incubated for 30 min at 37 C. At last, 0.1 mL 30% (w:v) KOH was added and then the mixture was incubated in boiling water for 5 min to terminate the reaction. The content of sucrose produced by this reaction was determined using the anthrone assay (van Handel, 1963). SS-synthesis was assayed as SPS but with 60 mM fructose instead of fructose-6-phosphate, and in the absence of glucose-6phosphate. The content of sucrose was determined using the
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anthrone assay (van Handel, 1963). The activities of SPS and SSsynthesis were expressed as katal kg 1 protein. For the assay of activity of SS-cleavage, the reaction system consisted of acetic acid-sodium acetate buffer (100 mM, pH 5.5; containing 5 mM NaF), 100 mM sucrose and 20 mM UDP and enzyme extract. The reaction system was incubated for 30 min at 25 C and then terminated by transferring to a water bath (80 C) for 10 min. The content of glucose produced by this reaction was determined using the DNS method (Miller, 1959). Activity of AI was analyzed by a previously described method (Cao et al., 2013). The reaction mixture contained 100 mM sucrose, 100 mM acetic acid-sodium acetate (pH 5.0) and enzyme solution. The reaction mixture was incubated at 37 C for 1 h, and then terminated by incubating in boiling water for 5 min. 1 mL 3, 5dinitrosalicylic acid (DNS) was added to the mixture to determine the content of glucose. The amount of reducing sugar produced was calculated based on a standard glucose curve. The determent of activity of NI was similar to AI. The only difference was that the pH of 100 mM potassium phosphate buffer was 7.5. The activities of SS-cleavage, AI and NI were expressed as katal kg 1 protein. The quantity of protein was determined using a previous method (Bradford, 1976). 2.5. Extraction and assays of activity NAD+-dependent sorbitol dehydrogenase (NAD+-SDH) and sorbitol oxidase (SOX) For the assay of relative activity of NAD+-SDH and SOX, the extraction process was modified from a previous method (Sun et al., 2011). For NAD+-SDH, flesh tissue (2 g) was homogenized on ice with 0.5 g PVPP. And then the mixture was extracted with 6 mL buffer containing 0.1 M Tris–HCl (pH 9.0), 20 mM b-mercaptoethanol, 8% (v/v) glycerol, 0.1% (v/v) Tween 20. For SOX, flesh tissue (2 g) was homogenized on ice with 0.5 g PVPP and 6 mL of 0.1 M HEPES–NaOH buffer (pH 7.5). The buffer contained 10 mM dithiothreitol (DTT), 3 mM acetate, 8% (v/v) glycerol, 0.1% (v/v) Tween 20. The homogenate was centrifuged at 12,000 g for 30 min at 4 C. The supernatant was desalted with Sephadex G-25 column. Activity of NAD+-SDH was analyzed by a previously described method (Cao et al., 2013). The reaction mixture contained 0.1 M Tris–HCl (pH 9.0), 1 mM NAD and crude enzyme extract. Before the start of reaction, the mixture was incubated at 25 C for 5 min. Then the mixture was added 300 mM sorbitol to start the reaction, and the change of absorbance was monitored at 340 nm during 5 min. The relative activity of NAD+-SDH was expressed as katal kg 1 protein. Activity of SOX was analyzed by a previously described method (Sun et al., 2011). The reaction mixture contained 100 mM acetic acid-sodium acetate (pH 4.5), crude enzyme extract and 300 mM sorbitol. The reaction mixture was incubated at 30 C for 30 min and then transferred to boiling water for 10 min to terminate the action. The content of glucose produced by this reaction was determined using the DNS method (Miller, 1959). The relative activity of SOX was expressed as katal kg 1 protein. The quantity of protein was determined using a previous method (Bradford, 1976). 2.6. Statistical analysis For each treatment, the determination was done in triplicate. Analyses of data were performed by one-way analysis of variance (ANONA) from SPSS 12.0 window. Significant differences were calculated by least significant difference (LSD) test and defined as P < 0.05.
3. Results 3.1. Effect of OA on internal browning index (IBI), electrolyte leakage (EL), content of hydrogen dioxide (H2O2) and malondialdehyde (MDA) For control fruit, IBI (the symptom of chilling injury in apricot fruit) was detected at 14th day of storage (Fig. 1A). OA treatment delayed the time that chilling occurred and induced the increase of chilling tolerance of apricot fruit (P < 0.05). EL (Fig. 1B) of apricot fruit (including control and OA treated fruit) showed a continuous increase during the storage and OA treated fruit showed lower EL than that of control fruit (P < 0.05). The content of H2O2 in all fruit gradually increased to the end of storage (Fig. 1C). Fruit treated with OA showed significantly lower content of H2O2 than control fruit (P < 0.05). The content of MDA in fruit showed an intense increase during the storage which was similar to the pattern of H2O2 in fruit (Fig. 1D). The accumulation of MDA in fruit was significantly inhibited by OA treatment (P < 0.05). 3.2. Effect of OA on content of soluble sugar Sucrose content in control fruit slightly increased during the early 21 d of storage and then decreased (Fig. 2A). During the early 21 d of storage, OA treatment suppressed the increase of sucrose content in fruit and then enhanced the decrease of sucrose content at the following days. As a consequence, the content of sucrose in fruit treated with OA was significantly lower than that in control fruit (P < 0.05). Content of sorbitol in control fruit continually decreased during the storage and OA treatment enhanced the degradation of sorbitol (Fig. 2B). OA treated fruit showed significantly lower content of sorbitol than control fruit (P < 0.05). No sorbitol was detected in fruit treated with OA at 21th day and the following day of storage. Levels of glucose in control fruit decreased during the early 14 days of storage and then increased (Fig. 2C). However, the levels of glucose in fruit treated with OA decreased during the early 7 d of storage and then increased. OA treated fruit showed significantly higher content of glucose than control fruit (P < 0.05). Content of fructose in control fruit slightly decreased during the early 7 d of storage, whereas the content of OA treated fruit continually increased during the storage (Fig. 2D). OA treated fruit showed profoundly higher content of fructose than control fruit (P < 0.05). 3.3. Effect of OA on sucrose synthase synthesis activity (SS-synthesis), sucrose phosphate synthase activity (SPS), sucrose synthase cleavage activity (SS-cleavage), acid invertase activity (AI) and neutral invertase activity (NI) As shown in Fig. 3, activity of sugar metabolism enzymes in apricot fruit could be altered by the application of OA treatment during the postharvest storage. Activity of SS-synthesis in control fruit was stimulated to increase at early storage and reached the peak after 21 d of storage. Activity of SS-synthesis in OA treated fruit was significantly (P < 0.05) inhibited and kept in a lower status than that in the control fruit (Fig. 3A). Activity of SPS in control fruit showed increase during the early 14 d of storage and afterwards decreased continually (Fig. 3B). Our work indicated that activity of SPS in fruit was significantly (P < 0.05) enhanced by OA treatment (P < 0.05). As to SS-cleavage, the maximum value of activity in control fruit was observed at 28th day of storage (Fig. 3C). For fruit treated with OA, higher activity of SS-cleavage was observed during the storage than that in control fruit. As to control fruit, the activity of NI continually increased (Fig. 3D). In our work, the activity of NI was intensified by OA treatment (P < 0.05). The increase of activity of AI in control fruit
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Fig. 1. Effects of OA on internal browning index (A), electrolyte leakage (B), and contents of H2O2 (C), MDA (D) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
Fig. 2. Effects of OA on contents of sucrose (A), sorbitol (B), glucose (C), fructose (D) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
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Fig. 3. Effects of OA on activity of SS-synthesis (A), SPS (B), SS-cleavage (C), NI (D) and AI (E) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
did not stop until 28th day of storage (Fig. 3E). OA treatment profoundly enhanced the activity of AI (P < 0.05). 3.4. Effect of OA on the activity of sorbitol oxidase (SOX) and NAD+sorbitol dehydrogenase (NAD+-SDH) For control fruit, activity of SOX increased slightly and reached the peak after 14 d of storage (Fig. 4A). As to OA treated fruit, activity of SOX was significantly enhanced and kept in a higher status than that in the control fruit throughout the storage (P < 0.05). Activity of NAD+-SDH increased during the early 14 d of storage and then decreased (Fig. 4B). Activity of NAD+-SDH was significantly enhanced by OA treatment (P < 0.05). 4. Discussion Our present study showed that treatment with OA could reduce chilling injury of the apricot fruit during storage at 2 C for 5 weeks. Similar effects of OA on chilling tolerance have also been observed
in peach, litchi and plum fruits (Jin et al., 2014; Zheng and Tian et al., 2006; Wu et al., 2011). Damage of membrane system is a primary event of chilling injury in plant, which will lead to the loss of cellular compartmentalization and increase of electrolyte leakage and lipid peroxidation (Wongsheree et al., 2009; Cao et al., 2011). Hence, membrane permeability is usually used to assess of plant cell membrane stability. It is suggested that maintaining membrane stability can enhance chilling tolerance of fruit stored a chilling temperature (Zhang and Tian, 2009). Our study on the apricot fruit showed that the OA-treatment could prevent increase of electrolyte leakage from the flesh cells, as well as suppressing accumulations of MDA and H2O2. H2O2 can lead to peroxidation and breakdown of unsaturated fatty acids in membrane lipids because of its relatively long half-life, which will do harm to the membrane system (Quan et al., 2008). MDA, one product of lipid peroxidation, reflects the peroxidation of lipids and affects membrane structure, disturbing normal physiological metabolism (Zhang and Tian, 2009).
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Fig. 4. Effects of OA on activity of SOX (A) and SDH (B) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
Our result showed that sorbitol, sucrose, fructose and glucose were the major soluble sugars in apricot fruit of cultivar ‘Diaogan’. A previous work has reported that sucrose, fructose and glucose are the major sugars in apricot fruit of cultivar ‘Shannong Kaixin’ and other nine cultivars of apricot, but no ‘Diaogan’ (Chen et al., 2006). The total sugar content in fruit of cultivar ‘Diaogan’ used in our experiment was more than 160 g kg 1, while the highest content of total sugar was less than 90 g kg 1 in fruit the cultivars in the previous study (Chen et al., 2006). Several the other studies on species of Rosaceae, such as peach, pear, apple and loquat, have shown that sucrose, fructose, sorbitol, glucose are major soluble sugars of these fruits (Sun et al., 2011; Yamaki, 2010; Zhang et al., 2010; Cao et al., 2013). An earlier study reported that grape fruit with a higher content of reducing sugars (including glucose and fructose) showed a higher chilling tolerance (Purvis and Grierson., 1982). Nowadays, it is widely believed that soluble sugars could be associated with chilling tolerance of plants at low temperature. There is a controversy on whether sucrose or reducing sugar (glucose and fructose) are responsible for the beneficial effect of sugar on the chilling tolerance of plant (Holland et al., 2002; Shao et al., 2013). It has been suggested that heat treatment prevented the decline of sucrose content and enhanced the chilling tolerance of mandarin fruit (Holland et al., 2002). However, our work indicated that the correlation between sucrose content and relative electrolyte leakage was positive and not significant (r = 0.27713, P > 0.05). Moreover, lower sucrose content accompanied with higher relative electrolyte leakage and chilling injury has been reported in other work (Shao et al., 2013; Lara et al., 2009). A study on loquat fruit storage at 1 C for 20 days showed that the cultivar having higher sucrose and glucose contents and higher activity of sucrose hydrolyzing enzyme exhibited higher chilling tolerance (Cao et al., 2013). Higher chilling tolerance accompanied with higher content glucose, fructose were determined in heat treated fruit compared with control fruit (Shao et al., 2013; Lara et al., 2009). Moreover, glucose and fructose are now considered as crucial compounds that have several beneficial effects in protecting plants against oxidative stresses (Couée et al., 2006). Oxidative stress is known to be one key factor of inducing cellular damage, particularly reducing membrane stability, and leading to chilling injury of plants (Mittler, 2002; Gill and Tuteja, 2010). In our work, OA treatment enhanced the content of glucose and fructose, decreased electrolyte leakage, content of H2O2 and MDA, which contributed to enhance the chilling tolerance of fruit. Our presented results showed that the enhancement of chilling tolerance in OA-treated apricot was accompanied with increase of glucose and fructose contents, and decrease of sucrose content. These may suggested that the effect of OA enhancing chilling
tolerance in apricot fruit may be attributed to the higher level of glucose and fructose. This result supports the view of that accumulation of reducing sugars should be beneficial for enhancing chilling tolerance of fruit. Sorbitol and sucrose are major products of photosynthetic and forms of translocated carbon in many species of the Rosaceae (Sun et al., 2011; Yamaki, 2010; Zhang et al., 2010; Cao et al., 2013). Among the system of sugar metabolism, SPS and SS synthesis are involved in the sucrose biosynthesis (Sun et al., 2011). SS cleavage catalyzes the process that cleaves sucrose to fructose and UDPglucose, whereas SS synthesis involved in the reversed process (Mao et al., 2006). SS cleavage and invertases (including AI and NI) catalyzes the breakdown of sucrose, AI and NI catalyze the hydrolysis of sucrose to fructose and glucose. Moreover, this process is irreversible (Borsani et al., 2009). We found that OA treatment enhanced activities of SPS, AI, NI and SS cleavage but suppressed the activity of SS-synthesis in the apricot, which could cause the lower content of sucrose in fruit treated with OA compared with control fruit. Sorbitol, as one kind of photosynthetic product, can be used as fuel and converted to fructose and glucose (Borsani et al., 2009). NAD+-dependent SDH catalyzes sorbitol to fructose, while NADP+dependent SDH and SOX catalyzes sorbitol to glucose (Loescher, 1987). In our study, the fruit treated with OA showed higher activity of NAD+-SDH and SOX accompanied with higher activity of AI, NI and SS cleavage. OA treated fruit showed lower content of sorbitol and no sorbitol can be detected on 21th day of storage. Taken all of these together, OA treatment resulted in the accumulation of fructose and glucose. To the best of our knowledge, there is no literature on the effect of OA treatment on the profiles and metabolism of sugar in fruit. Our results suggest that increase in levels of fructose and glucose could account for the OA-enhanced chilling tolerance in the apricot fruit. 5. Conclusion Our results indicate that exogenous oxalic acid (OA) can significantly reduce chilling injury of the apricot fruit. The major soluble sugars of the fruit were sorbitol, sucrose, fructose and glucose. Contents of glucose and fructose were enhanced, meanwhile, the levels of sucrose and sorbitol decreased by OA during the storage. OA treatment enhanced the enzymes activities related to increasing glucose and fructose, and suppressed the activity of SS-synthesis that could cause the lower content of sucrose. Our results suggest that increase in levels of fructose and glucose could account for the OA-enhanced chilling tolerance in the apricot fruit.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Changes in sugar metabolism caused by exogenous oxalic acid related to chilling tolerance of apricot fruit Zhen Wang, Jiankang Cao, Weibo Jiang* College of Food Science and Nutritional Engineering, China Agricultural University, PO Box 111, 17 Qinghuadonglu Road, Beijing 100083, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 August 2015 Received in revised form 23 November 2015 Accepted 26 November 2015 Available online xxx
Enhancement of chilling tolerance has been observed in fruits treated with oxalic acid (OA). To learn how OA may play a role in modulating chilling injury in apricot, the fruit were treated with OA (5 mM) and stored at 2 1 C for five weeks. OA treatment significantly inhibited chilling injury, electrolyte leakage and accumulation of hydrogen peroxide and malondialdehyde in apricot fruit. Sorbitol, sucrose, fructose and glucose were the major soluble sugars in the fruit. Contents of glucose and fructose were enhanced, meanwhile, the levels of sucrose and sorbitole were decreased by OA treatment during storage. OA treatment enhanced activities of the enzymes related to increasing glucose and fructose, and suppressed sucrose synthase synthesis activity which could explain the lower content of sucrose. The activities of sucrose synthase cleavage function, acid invertase, NAD+-sorbitol dehydrogenase and sorbitol oxidase were significantly enhanced by OA. Meanwhile, OA-treated apricot fruit showed lower sucrose synthase synthesis activity. The degradation of sorbitol in apricot fruit was accelerated by OA treatment. Our results indicate that higher chilling tolerance of OA treated fruit was associated with higher content of reducing sugars (glucose and fructose). ã 2015 Elsevier B.V. All rights reserved.
Keywords: Oxalic acid Sugar metabolism Glucose Fructose Chilling injury Apricot fruit
1. Introduction Apricots are popular around the world as a result of their high nutritional value and aroma (Solis-Solis et al., 2007). Various phenolic compounds including caffeic acid, ferulic acid, catechin, epicatechin, p-coumaric acid have been detected in apricot fruit (Sochor et al., 2010). Apricot fruit is usually stored at low temperatures to extend shelf life and maintain quality. During storage at low temperature, apricot fruit show some physiological disorders because apricot fruit is sensitive to low temperatures. Internal browning is one major symptom of chilling injury (CI) in apricot fruit (Saba et al., 2012). Recently, the metabolism of soluble sugars of fruit during the postharvest storage attracted the interest of numerous scientists (Borsani et al., 2009; Sun et al., 2011). The reason is that the profiles and concentrations of soluble sugars not only contribute to the organoleptic quality of fruit (Borsani et al., 2009), but also affect the chilling tolerance of fruit during cold storage (Agopian et al., 2011; Wang et al., 2013; Cao et al., 2013). In mandarin fruit, the
* Corresponding author. E-mail address: [email protected] (W. Jiang). http://dx.doi.org/10.1016/j.postharvbio.2015.11.015 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
level of sucrose is considered to enhance chilling tolerance (Holland et al., 2002). However, a higher level of fructose and glucose is believed to enhance the chilling tolerance of loquat fruit (Shao et al., 2013; Cao et al., 2013). Oxalic acid (OA), a natural organic acid, has been reported to play an important role in systemic resistance and response to environment (Zheng et al., 2012; Jin et al., 2014; Liang et al., 2009). Prestorage application with OA enhanced the antioxidant capacities of pomegranate (Sayyari et al., 2010) and inhibited the decay of mango (Zheng et al., 2012). Moreover, the application of OA enhanced the chilling tolerance of litchi, peach and plum fruit during the postharvest storage under low temperatures (Zheng and Tian, 2006; Jin et al., 2014; Wu et al., 2011). Some work has been reported on the effect of OA on the chilling tolerance of various fruits (Jin et al., 2014; Wu et al., 2011; Zheng and Tian., 2006). The effect of OA on the chilling tolerance of various fruits has been explained by various mechanisms (Jin et al., 2014; Wu et al., 2011; Zheng and Tian., 2006). To the best of our knowledge, there is no literature focus on the changes of soluble sugar content and metabolism of sugar in fruits caused by OA treatment. Then no work focuses on illuminating the relationship of changes of soluble sugar content caused by OA treatment and higher chilling tolerance of OA-treated fruit during
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the postharvest storage. The objective of this study was to determine changes in sugar content and metabolism caused by exogenous oxalic acid related to chilling tolerance of apricot fruit. 2. Materials and methods 2.1. Materials and treatments Apricot fruit (Prunus armeniaca L. cv. Diaogan) of commercial maturity were obtained from local market in Beijing, PR China. The fruit were packaged in fibreboard cartons, transferred to our laboratory (Beijing, PR China) within 5 h via truck. During the transportation, the fruit was kept at low temperature (4–8 C). Fruit sorted by hand (uniform size and color, without physical damage) were used in the experiment. Apricots were dipped in 5 mM OA solution for 10 min at room temperature. Control fruit were treated with distilled water. The fruit were then air-dried at room temperature for about 30 min. For each treatment, forty apricot fruit were placed into a plastic boxes (600 100 70 mm), and then stored at 2 1 C. The relative humidity (RH) was 85–90%. Each treatment contained 360 apricots. About 60 fruits were collected in each sampling time, and 18 of the collected fruits were used for the biochemical analysis, and 18 of the fruits used for measuring chilling injury (CI) and electrolyte leakage (EL). The measurement of CI was done in three replicates (per replicate consisted of six fruits). For the measurement of EL, flesh tissues from six fruits were used in one measurement. For the biochemical analysis, the flesh from sampling fruits were mixed, immediately frozen in liquid nitrogen and then stored at 80 C. The flesh from 6 sampling fruits were used as one repeat (3 repeats for each biochemical measurement). 2.2. Determination of internal browning index (IBI), electrolyte leakage (EL), content of hydrogen peroxide (H2O2) and malondialdehyde (MDA) The symptom of chilling injury was expressed as internal browning index (IBI). IBI was determined according to previous method with slight modification (Saba et al., 2012). The severity of browning was visually assessed and classified as 5 grades: 0 = none; 1 = slightly (browning area < 5%); 2 = moderate (browning area 5–25%); 3 = moderate severe (browning area 25–50%); 4 = severe (browning area > 50%). The results were calculated as P follows: IBI = ( IBI level) (the number of fruits at this IBI level)/ (4 total number of fruits) 100%. A procedure previously reported was modified to determine the EL (Jin et al., 2014). For the determination of EL, ten flesh disks from fruits (the total weight of flesh was kept about 3 g) were collected with a 3-mm diameter cork borer, rinsed in 25 mL double distilled water for 30 min and was employed to measure the initial conductivity by one DJS-1C conductivity meter (Shanghai Analytical Instrument Co., Shanghai, China). Then the disks were placed at 20 C for 24 h, incubated in a boiling water for 30 min and then used to determine total electrolyte conductivity. The EL was expressed as relative conductivity: (the initial conductivity/the final conductivity) 100%. The content of MDA was determined according to method described by Jin et al. (2014) with some modification. Flesh tissue (2 g) was homogenized on ice with 3 mL trichloroacetic acid (100 g L 1) and then centrifuged at 12,000 g for 20 min at 4 C. A 0.5 mL supernatant was mixed with 2.5 mL thiobarbituric acid and then incubated in boiling water for 20 min. The reaction mixture was centrifuged at 12,000 g for 7 min at 4 C and absorbance of the supernatant was recorded at 450 nm, 532 nm, 600 nm respectively. MDA content in the supernatant was and
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calculated as follow: c (mM) = 6.45 (OD532–OD600)–0.56 OD450. The MDA content of flesh were expressed as nmol g 1 fresh weight. The content of H2O2 was assayed using the method previously described with slight modification (Patterson et al., 1984). The modification was that we used titanium tetrachloride-hydrochloric acid instead of titanium sulphate. Flesh tissue (2 g) was homogenized with 4 mL acetone (pre-chilled at 20 C) and then centrifuged at 12,000 g for 20 min at 4 C. A 1.0 mL supernatant with 0.1 mL titanium tetrachloride–hydrochloric acid and 0.2 mL ammonia (25%, v/v) was centrifuged at 12,000 g for 15 min at 4 C. The precipitation was collected and sufficiently suspended in 3 mL H2SO4 (10%, v/v), centrifuged at 12,000 g for 10 min at 4 C. Absorbance of the supernatant was recorded at 410 nm and the H2O2 content was expressed as nmol g 1 fresh weight. 2.3. Analysis of soluble sugar content Extraction and assays of soluble sugars were carried out with some modification according to a previous method (Shao et al., 2013). Five grams of frozen fresh from fruit samples were homogenized with 25 mL extraction solution on ice and kept still for 20 min. Special solution contained 3.2 g L 1 acetic acid and 2.4 g L 1 potassium ferrocyanide (dissolved with double distilled water). The mixture was centrifuged at 12,000 g for 20 min at 4 C. The supernatant was collected and diluted to 150 mL with double distilled water. Before the HPLC analysis, the resulting extract was filtered through a 0.22 mm membrane filter. A 10 mL sample was injected into an ultrafast liquid chromatography (UFLC). The UFLC contained HPLC (Agilent 1260, Agilent Corp, America., USA; column: Hedera, NH2; column temperature: 50 C) and an evaporative light-scattering detector (Agilent 1260, Agilent Corp, America). The mobile phase composition was acetonitrile and water (80:20). The total flow rate of mobile phase composition was 1 mL min 1. Individual sugars were identified by the retention of time and quantified based on standard curves of individual sugars. 2.4. Extraction and assays of activity of sucrose phosphate synthase (SPS), sucrose synthase synthesis activity (SS-synthesis), sucrose synthase cleavage activity (SS-cleavage), acid invertase (AI) and neutral invertase (NI) For the assay of relative activity of SPS, SS-synthesis, SScleavage, AI and NI, the extraction process was modified from a previous method (Shao et al., 2013). Flesh tissues (3 g) were homogenized on ice with 0.5 g crosslinking polyvingypyrrolidone (PVPP) and 6 mL buffer containing 50 mM HEPES–NaOH (pH 8.5), 10 mM MgCl2, 2.5 mM DTT, 0.1% TritonX-100 (v:v) and 10 mM vitamin C, 10 mM b-mercaptoethanol, 20% (v:v) glycerol, 10 mg mL 1 leupeptin, 10 mg mL 1 chymostatin. The homogenate was centrifuged at 12,000 g for 30 min at 4 C. A cold pre-equilibrated Sephadex G-25 column was used to desalt the supernatant. These crude extracts were used for the measurement of activity of enzymes. For the assay of activity of SPS, the reaction system consisted of HEPES–NaOH buffer (50 mM, pH 7.5; containing 15 mM MgCl2), 25 mM fructose 6-phosphate, 25 mM glucose 6-phosphate, 25 mM UDP-glucose and crude enzyme extract. The reaction system was incubated for 30 min at 37 C. At last, 0.1 mL 30% (w:v) KOH was added and then the mixture was incubated in boiling water for 5 min to terminate the reaction. The content of sucrose produced by this reaction was determined using the anthrone assay (van Handel, 1963). SS-synthesis was assayed as SPS but with 60 mM fructose instead of fructose-6-phosphate, and in the absence of glucose-6phosphate. The content of sucrose was determined using the
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anthrone assay (van Handel, 1963). The activities of SPS and SSsynthesis were expressed as katal kg 1 protein. For the assay of activity of SS-cleavage, the reaction system consisted of acetic acid-sodium acetate buffer (100 mM, pH 5.5; containing 5 mM NaF), 100 mM sucrose and 20 mM UDP and enzyme extract. The reaction system was incubated for 30 min at 25 C and then terminated by transferring to a water bath (80 C) for 10 min. The content of glucose produced by this reaction was determined using the DNS method (Miller, 1959). Activity of AI was analyzed by a previously described method (Cao et al., 2013). The reaction mixture contained 100 mM sucrose, 100 mM acetic acid-sodium acetate (pH 5.0) and enzyme solution. The reaction mixture was incubated at 37 C for 1 h, and then terminated by incubating in boiling water for 5 min. 1 mL 3, 5dinitrosalicylic acid (DNS) was added to the mixture to determine the content of glucose. The amount of reducing sugar produced was calculated based on a standard glucose curve. The determent of activity of NI was similar to AI. The only difference was that the pH of 100 mM potassium phosphate buffer was 7.5. The activities of SS-cleavage, AI and NI were expressed as katal kg 1 protein. The quantity of protein was determined using a previous method (Bradford, 1976). 2.5. Extraction and assays of activity NAD+-dependent sorbitol dehydrogenase (NAD+-SDH) and sorbitol oxidase (SOX) For the assay of relative activity of NAD+-SDH and SOX, the extraction process was modified from a previous method (Sun et al., 2011). For NAD+-SDH, flesh tissue (2 g) was homogenized on ice with 0.5 g PVPP. And then the mixture was extracted with 6 mL buffer containing 0.1 M Tris–HCl (pH 9.0), 20 mM b-mercaptoethanol, 8% (v/v) glycerol, 0.1% (v/v) Tween 20. For SOX, flesh tissue (2 g) was homogenized on ice with 0.5 g PVPP and 6 mL of 0.1 M HEPES–NaOH buffer (pH 7.5). The buffer contained 10 mM dithiothreitol (DTT), 3 mM acetate, 8% (v/v) glycerol, 0.1% (v/v) Tween 20. The homogenate was centrifuged at 12,000 g for 30 min at 4 C. The supernatant was desalted with Sephadex G-25 column. Activity of NAD+-SDH was analyzed by a previously described method (Cao et al., 2013). The reaction mixture contained 0.1 M Tris–HCl (pH 9.0), 1 mM NAD and crude enzyme extract. Before the start of reaction, the mixture was incubated at 25 C for 5 min. Then the mixture was added 300 mM sorbitol to start the reaction, and the change of absorbance was monitored at 340 nm during 5 min. The relative activity of NAD+-SDH was expressed as katal kg 1 protein. Activity of SOX was analyzed by a previously described method (Sun et al., 2011). The reaction mixture contained 100 mM acetic acid-sodium acetate (pH 4.5), crude enzyme extract and 300 mM sorbitol. The reaction mixture was incubated at 30 C for 30 min and then transferred to boiling water for 10 min to terminate the action. The content of glucose produced by this reaction was determined using the DNS method (Miller, 1959). The relative activity of SOX was expressed as katal kg 1 protein. The quantity of protein was determined using a previous method (Bradford, 1976). 2.6. Statistical analysis For each treatment, the determination was done in triplicate. Analyses of data were performed by one-way analysis of variance (ANONA) from SPSS 12.0 window. Significant differences were calculated by least significant difference (LSD) test and defined as P < 0.05.
3. Results 3.1. Effect of OA on internal browning index (IBI), electrolyte leakage (EL), content of hydrogen dioxide (H2O2) and malondialdehyde (MDA) For control fruit, IBI (the symptom of chilling injury in apricot fruit) was detected at 14th day of storage (Fig. 1A). OA treatment delayed the time that chilling occurred and induced the increase of chilling tolerance of apricot fruit (P < 0.05). EL (Fig. 1B) of apricot fruit (including control and OA treated fruit) showed a continuous increase during the storage and OA treated fruit showed lower EL than that of control fruit (P < 0.05). The content of H2O2 in all fruit gradually increased to the end of storage (Fig. 1C). Fruit treated with OA showed significantly lower content of H2O2 than control fruit (P < 0.05). The content of MDA in fruit showed an intense increase during the storage which was similar to the pattern of H2O2 in fruit (Fig. 1D). The accumulation of MDA in fruit was significantly inhibited by OA treatment (P < 0.05). 3.2. Effect of OA on content of soluble sugar Sucrose content in control fruit slightly increased during the early 21 d of storage and then decreased (Fig. 2A). During the early 21 d of storage, OA treatment suppressed the increase of sucrose content in fruit and then enhanced the decrease of sucrose content at the following days. As a consequence, the content of sucrose in fruit treated with OA was significantly lower than that in control fruit (P < 0.05). Content of sorbitol in control fruit continually decreased during the storage and OA treatment enhanced the degradation of sorbitol (Fig. 2B). OA treated fruit showed significantly lower content of sorbitol than control fruit (P < 0.05). No sorbitol was detected in fruit treated with OA at 21th day and the following day of storage. Levels of glucose in control fruit decreased during the early 14 days of storage and then increased (Fig. 2C). However, the levels of glucose in fruit treated with OA decreased during the early 7 d of storage and then increased. OA treated fruit showed significantly higher content of glucose than control fruit (P < 0.05). Content of fructose in control fruit slightly decreased during the early 7 d of storage, whereas the content of OA treated fruit continually increased during the storage (Fig. 2D). OA treated fruit showed profoundly higher content of fructose than control fruit (P < 0.05). 3.3. Effect of OA on sucrose synthase synthesis activity (SS-synthesis), sucrose phosphate synthase activity (SPS), sucrose synthase cleavage activity (SS-cleavage), acid invertase activity (AI) and neutral invertase activity (NI) As shown in Fig. 3, activity of sugar metabolism enzymes in apricot fruit could be altered by the application of OA treatment during the postharvest storage. Activity of SS-synthesis in control fruit was stimulated to increase at early storage and reached the peak after 21 d of storage. Activity of SS-synthesis in OA treated fruit was significantly (P < 0.05) inhibited and kept in a lower status than that in the control fruit (Fig. 3A). Activity of SPS in control fruit showed increase during the early 14 d of storage and afterwards decreased continually (Fig. 3B). Our work indicated that activity of SPS in fruit was significantly (P < 0.05) enhanced by OA treatment (P < 0.05). As to SS-cleavage, the maximum value of activity in control fruit was observed at 28th day of storage (Fig. 3C). For fruit treated with OA, higher activity of SS-cleavage was observed during the storage than that in control fruit. As to control fruit, the activity of NI continually increased (Fig. 3D). In our work, the activity of NI was intensified by OA treatment (P < 0.05). The increase of activity of AI in control fruit
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Fig. 1. Effects of OA on internal browning index (A), electrolyte leakage (B), and contents of H2O2 (C), MDA (D) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
Fig. 2. Effects of OA on contents of sucrose (A), sorbitol (B), glucose (C), fructose (D) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
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Fig. 3. Effects of OA on activity of SS-synthesis (A), SPS (B), SS-cleavage (C), NI (D) and AI (E) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
did not stop until 28th day of storage (Fig. 3E). OA treatment profoundly enhanced the activity of AI (P < 0.05). 3.4. Effect of OA on the activity of sorbitol oxidase (SOX) and NAD+sorbitol dehydrogenase (NAD+-SDH) For control fruit, activity of SOX increased slightly and reached the peak after 14 d of storage (Fig. 4A). As to OA treated fruit, activity of SOX was significantly enhanced and kept in a higher status than that in the control fruit throughout the storage (P < 0.05). Activity of NAD+-SDH increased during the early 14 d of storage and then decreased (Fig. 4B). Activity of NAD+-SDH was significantly enhanced by OA treatment (P < 0.05). 4. Discussion Our present study showed that treatment with OA could reduce chilling injury of the apricot fruit during storage at 2 C for 5 weeks. Similar effects of OA on chilling tolerance have also been observed
in peach, litchi and plum fruits (Jin et al., 2014; Zheng and Tian et al., 2006; Wu et al., 2011). Damage of membrane system is a primary event of chilling injury in plant, which will lead to the loss of cellular compartmentalization and increase of electrolyte leakage and lipid peroxidation (Wongsheree et al., 2009; Cao et al., 2011). Hence, membrane permeability is usually used to assess of plant cell membrane stability. It is suggested that maintaining membrane stability can enhance chilling tolerance of fruit stored a chilling temperature (Zhang and Tian, 2009). Our study on the apricot fruit showed that the OA-treatment could prevent increase of electrolyte leakage from the flesh cells, as well as suppressing accumulations of MDA and H2O2. H2O2 can lead to peroxidation and breakdown of unsaturated fatty acids in membrane lipids because of its relatively long half-life, which will do harm to the membrane system (Quan et al., 2008). MDA, one product of lipid peroxidation, reflects the peroxidation of lipids and affects membrane structure, disturbing normal physiological metabolism (Zhang and Tian, 2009).
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Fig. 4. Effects of OA on activity of SOX (A) and SDH (B) in apricot fruit during storage at 2 C for 35 days. The values are expressed as means standard error (n = 3).
Our result showed that sorbitol, sucrose, fructose and glucose were the major soluble sugars in apricot fruit of cultivar ‘Diaogan’. A previous work has reported that sucrose, fructose and glucose are the major sugars in apricot fruit of cultivar ‘Shannong Kaixin’ and other nine cultivars of apricot, but no ‘Diaogan’ (Chen et al., 2006). The total sugar content in fruit of cultivar ‘Diaogan’ used in our experiment was more than 160 g kg 1, while the highest content of total sugar was less than 90 g kg 1 in fruit the cultivars in the previous study (Chen et al., 2006). Several the other studies on species of Rosaceae, such as peach, pear, apple and loquat, have shown that sucrose, fructose, sorbitol, glucose are major soluble sugars of these fruits (Sun et al., 2011; Yamaki, 2010; Zhang et al., 2010; Cao et al., 2013). An earlier study reported that grape fruit with a higher content of reducing sugars (including glucose and fructose) showed a higher chilling tolerance (Purvis and Grierson., 1982). Nowadays, it is widely believed that soluble sugars could be associated with chilling tolerance of plants at low temperature. There is a controversy on whether sucrose or reducing sugar (glucose and fructose) are responsible for the beneficial effect of sugar on the chilling tolerance of plant (Holland et al., 2002; Shao et al., 2013). It has been suggested that heat treatment prevented the decline of sucrose content and enhanced the chilling tolerance of mandarin fruit (Holland et al., 2002). However, our work indicated that the correlation between sucrose content and relative electrolyte leakage was positive and not significant (r = 0.27713, P > 0.05). Moreover, lower sucrose content accompanied with higher relative electrolyte leakage and chilling injury has been reported in other work (Shao et al., 2013; Lara et al., 2009). A study on loquat fruit storage at 1 C for 20 days showed that the cultivar having higher sucrose and glucose contents and higher activity of sucrose hydrolyzing enzyme exhibited higher chilling tolerance (Cao et al., 2013). Higher chilling tolerance accompanied with higher content glucose, fructose were determined in heat treated fruit compared with control fruit (Shao et al., 2013; Lara et al., 2009). Moreover, glucose and fructose are now considered as crucial compounds that have several beneficial effects in protecting plants against oxidative stresses (Couée et al., 2006). Oxidative stress is known to be one key factor of inducing cellular damage, particularly reducing membrane stability, and leading to chilling injury of plants (Mittler, 2002; Gill and Tuteja, 2010). In our work, OA treatment enhanced the content of glucose and fructose, decreased electrolyte leakage, content of H2O2 and MDA, which contributed to enhance the chilling tolerance of fruit. Our presented results showed that the enhancement of chilling tolerance in OA-treated apricot was accompanied with increase of glucose and fructose contents, and decrease of sucrose content. These may suggested that the effect of OA enhancing chilling
tolerance in apricot fruit may be attributed to the higher level of glucose and fructose. This result supports the view of that accumulation of reducing sugars should be beneficial for enhancing chilling tolerance of fruit. Sorbitol and sucrose are major products of photosynthetic and forms of translocated carbon in many species of the Rosaceae (Sun et al., 2011; Yamaki, 2010; Zhang et al., 2010; Cao et al., 2013). Among the system of sugar metabolism, SPS and SS synthesis are involved in the sucrose biosynthesis (Sun et al., 2011). SS cleavage catalyzes the process that cleaves sucrose to fructose and UDPglucose, whereas SS synthesis involved in the reversed process (Mao et al., 2006). SS cleavage and invertases (including AI and NI) catalyzes the breakdown of sucrose, AI and NI catalyze the hydrolysis of sucrose to fructose and glucose. Moreover, this process is irreversible (Borsani et al., 2009). We found that OA treatment enhanced activities of SPS, AI, NI and SS cleavage but suppressed the activity of SS-synthesis in the apricot, which could cause the lower content of sucrose in fruit treated with OA compared with control fruit. Sorbitol, as one kind of photosynthetic product, can be used as fuel and converted to fructose and glucose (Borsani et al., 2009). NAD+-dependent SDH catalyzes sorbitol to fructose, while NADP+dependent SDH and SOX catalyzes sorbitol to glucose (Loescher, 1987). In our study, the fruit treated with OA showed higher activity of NAD+-SDH and SOX accompanied with higher activity of AI, NI and SS cleavage. OA treated fruit showed lower content of sorbitol and no sorbitol can be detected on 21th day of storage. Taken all of these together, OA treatment resulted in the accumulation of fructose and glucose. To the best of our knowledge, there is no literature on the effect of OA treatment on the profiles and metabolism of sugar in fruit. Our results suggest that increase in levels of fructose and glucose could account for the OA-enhanced chilling tolerance in the apricot fruit. 5. Conclusion Our results indicate that exogenous oxalic acid (OA) can significantly reduce chilling injury of the apricot fruit. The major soluble sugars of the fruit were sorbitol, sucrose, fructose and glucose. Contents of glucose and fructose were enhanced, meanwhile, the levels of sucrose and sorbitol decreased by OA during the storage. OA treatment enhanced the enzymes activities related to increasing glucose and fructose, and suppressed the activity of SS-synthesis that could cause the lower content of sucrose. Our results suggest that increase in levels of fructose and glucose could account for the OA-enhanced chilling tolerance in the apricot fruit.
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