Article pubs.acs.org/JAFC
Low-Temperature Conditioning Alleviates Chilling Injury in Loquat Fruit and Regulates Glycine Betaine Content and Energy Status Peng Jin, Yu Zhang, Timin Shan, Yuping Huang, Jia Xu, and Yonghua Zheng* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China ABSTRACT: The influence of low-temperature conditioning (LTC) treatment on chilling injury, glycine betaine content, and energy metabolism in loquat fruit at 1 °C storage was investigated. The results indicated that LTC treatment significantly reduced chilling injury index, ion leakage, and malondialdehyde content in loquat fruit. Betaine aldehyde hydrogenase (BADH) activity and endogenous glycine betaine (GB) content in loquats treated with LTC were significantly higher than those in control fruit. Moreover, LTC treatment induced activities of energy metabolism-associated enzymes, including H+-adenosine triphosphatase, Ca2+-adenosine triphosphatase, succinic dehydrogenase, and cytochrome c oxidase. LTC treatment triggered obviously higher levels of adenosine triphosphate (ATP) content and energy charge in loquat fruit. These results showed that LTC possibly alleviated chilling injury and enhanced chilling tolerance of loquat fruit by enhancing endogenous GB content and energy status. KEYWORDS: loquat fruit, chilling injury, low-temperature conditioning, glycine betaine, energy status
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energy metabolism and regulate ATP synthesis.16 Azevedo et al.17 reported that ATPase could be a marker in papaya fruit ripening and senescence. In our previous study, we found that chilling tolerance of peach fruit is correlated with SDH and CCO activities.14,16 The objective of this study was to evaluate the GB metabolism in chilling tolerance-induced loquat fruit by LTC. Moreover, the effects of LTC treatment on energy status and its metabolism were also investigated to elucidate the underlying mechanism by inducing chilling tolerance in loquat fruit.
INTRODUCTION Chilling injury (CI) is a physiological disorder that appears in loquat fruit during refrigeration, which limits its long-term storage and long-distance transportation. The major symptoms of CI in loquat fruit include internal browning, leathery, juiceless flesh, and adhesion of peel to the pulp.1 Lowtemperature conditioning (LTC) is a useful method of enhancing chilling tolerance in some fruit, such as avocado,2 grapefruit, 3 and peaches.4 LTC treatment can induce physiological or biochemical changes related to chilling stress response in loquat fruit.5 Glycine betaine (GB) is an important compatible substance that is accumulated in some plants under environmental stress such as water, salt, or chilling.6 GB plays an important role in maintaining the integrity of membranes and stabilizing enzyme structure, which is related to stress resistance mechanisms.7 GB in plant tissue is synthesized from choline by two steps of dehydrogenation reaction. Choline monooxygenase (CMO) and betaine aldehyde hydrogenase (BADH) are two key enzymes during GB synthesis. Choline is oxidized to betaine aldehyde by CMO first, then betaine aldehyde is oxidized to GB by BADH.6 It has been reported that stress tolerance could be enhanced in barley or sugar beet by inducing expression of BADH gene through genetic engineering.8 Rajashekar et al.9 reported that exogenous abscisic acid treatment increased accumulation of GB and induced cold tolerance in strawberry plant. Recently, we found that exogenous GB could alleviate CI and maintain high quality in cold-stored loquat fruit.10 Energy status is considered an important factor during ripening and senescence of postharvest fruit.11 Increasing evidence has confirmed that low CI and flesh browning were associated with high levels of adenosine triphosphate (ATP) content and energy charge in pear,12 lychee,13 and peach fruit.14 Zhou et al.15 also found that sufficient available energy status contributed to alleviating pitting in cold-stored blueberries. Adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH), and cytochromec c oxidase (CCO) are key enzymes in © XXXX American Chemical Society
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MATERIALS AND METHODS
Fruit Material and Treatment. Fresh loquat fruits (Eriobotrya japonica L. cv. ‘Jiefangzhong’) were harvested at commercial maturity (firmness was about 2.0 N; total soluble solids content was about 9.5%) with light orange color from a commercial orchard in Fujian, China. The loquat fruits were selected for uniformity without any damage and then divided into two groups randomly (180 fruits per group). In the control group, loquat fruits were directly stored at 1 °C for 5 weeks after selection. In the LTC treatment group, loquat fruits were conditioned at 10 °C for 6 days and then stored at 1 °C for up to 5 weeks. The conditioning temperature was chosen according to our privious study (data not shown). All treatment and storage conditions were at 90−95% relative humidity. Ten fruits from each treatment were taken for CI index. Flesh samples of another 20 fruits from each treatment were collected with mixtion, frozen in liquid nitrogen, and stored at −80 °C until analysis of GB content, enzyme activities, and energy status. CI Index, Ion Leakage, and MDA Content Assay. CI index of loquats was visually evaluated using 10 fruits from each of three replicates by three evaluators according to the browning area of flesh.18 CI index was calculated using the formula CI index = Σ[(browning scale) × (number of fruits at the browning scale)]/(4 × total number of fruits in the treatment) × 100%. Received: February 1, 2015 Revised: March 26, 2015 Accepted: March 30, 2015
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DOI: 10.1021/acs.jafc.5b00605 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Ion leakage was measured with a conductivity meter (DDS-11A, Shanghai, China) according to our previous method.14 Ion leakage was expressed as relative conductivity. MDA content was measured according to the method of Hodges et al.,19 and the result was expressed as nanomoles per gram of fresh weight (FW). GB Content and BADH Activity Measurement. For GB content measure, 10 g of tissue sample was homogenized with 20 mL of methanol−chloroform extraction buffer (60% methanol, 25% chloroform) and then centrifuged at 10000g for 20 min at 4 °C. The aqueous phase was purified by an anionic resin (Dowex AG1 OH−, 200−400 mesh, Dow Chemical Co., Midland, MI, USA). The GB fraction was eluted with 6 mol L−1 NH4OH and then dried under nitrogen at 45 °C. The residue was dissolved with 2 mL of methanol. The GB content was assayed using high-performance liquid chromatography (Agilent 1100, Agilent Corp., Santa Clara, CA, USA) according to the method of Bessieres et al.20 GB content was expressed as micrograms per gram of FW. BADH activity was assayed according to the method of Arakawa et al.21 with some modification. Ten grams of tissue sample was homogenized with 20 mL of 0.2 mmol L−1 potassium phosphate buffer (pH 7.4) and centrifuged at 13000g for 20 min at 4 °C. The assay mixture consisted of 0.5 mL of crude enzyme extract, 2.9 mL of 0.2 mmol L−1 potassium phosphate buffer (pH 7.4), and 0.5 mL of 10 mmol L−1 betaine aldehydede. One unit of BADH activity was defined as an increase of 0.01 in absorbance per minute at 340 nm. Energy Metabolism Enzyme Assays. Crude mitochondria of loquat fruit samples were extracted for enzyme assay. H+-ATPase and Ca2+-ATPase activities were measured using our previous method.16 One unit of H+-ATPase and Ca2+-ATPase activities was defined as the release of 1 μmol of phosphorus per minute under the assay conditions. Succinic dehydrogenase (SDH) activity was measured according to the method of Ackrell et al.22 One unit of SDH activity was defined as an increase of 0.01 of absorbance at 600 nm per minute. CCO activity was measured according to our previous method.16 One unit of CCO activity was defined as an increase of 0.1 of absorbance at 510 nm per minute. ATP, ADP, and AMP Contents and Energy Charge (EC) Measurements. To analyze energy status, 2 g of loquat flesh tissue was ground with 5 mL of 0.6 mol L−1 perchloric acid. ATP, ADP, and AMP assays used high-performance liquid chromatography (Agilent 1100) according to the Jin et al.16 method. ATP, ADP, and AMP contents were expressed as micrograms per gram of FW. EC was calculated using the formula EC = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]. Protein Assay. Protein content of enzyme was measured using the Bradford23 method. Specific activity of enzymes was expressed as units per milligram of protein. Statistical Analysis. These experiments were carried out using a completely randomized design. Each treatment in the study was replicated three times. The experiment was carried out for twice and got the similar results. The data were from one experiment and expressed as the mean ± SE (standard error) of three replications. All statistical analyses were performed using SPSS software (version 14.0, SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance. Mean separations were compared by Duncan’s multiplerange tests. Differences at p < 0.05 were considered as significant.
Figure 1. Effect of LTC treatment on CI index of loquat fruit during cold storage. Data are expressed as the mean of triplicate samples ± standard errors. Vertical bars represent the standard errors of the means. Significant differences are shown by different letters.
fruit increased with storage time, which was associated with CI development during cold storage. The increasing tendencies of ion leakage and MDA were significantly (p < 0.05) prevented by LTC (Figure 2). Effect of LTC Treatment on GB Content and BADH Activity in Loquat Fruit. The content of GB in control fruit decreased steadily during the whole storage time (Figure 3B). LTC treatment increased GB content after the first week in loquat fruit. GB contents were 30.1 and 27.5% higher in LTCtreated fruit than in control fruit in the third and fifth weeks, respectively. BADH activity increased until the third week and decreased slowly afterward in control fruits. LTC treatment significantly (p < 0.05) increased the activity of BADH during the whole storage time, which was 69.2% higher than in control loquat fruit at the end of storage. Effect of LTC Treatment on Energy Metabolism Enzyme Activities in Loquat Fruit. H+-ATPase and Ca2+ATPase activities decreased gradually during the whole storage time. LTC treatment significantly (p < 0.05) increased the activities of H+-ATPase and Ca2+-ATPase activities after the first week (Figure 4A,B). SDH activity increased until the second week and then decreased rapidly in control fruit. LTC treatment maintained higher activity of SDH, which was 32.7% higher than that in control loquat fruit at the end of the storage (Figure 4C). CCO activity declined slowly with storage time. LTC treatment could prevent the decrease of CCO activity in loquat fruit (Figure 4D). Effect of LTC Treatment on Energy Status in Loquat Fruit. ATP content declined gradually with storage time, whereas ADP content increased mildly until the first week and decreased rapidly after the second week (Figure 5). In general, LTC treatment maintained higher contents of ATP and ADP in comparison with the control fruit. AMP content increased gradually in control loquat during the whole storage time. The increasing tendency was significantly (p < 0.05) prevented by LTC treatment during the whole storage time except the fourth week. According to the difference in ATP and ADP contents, energy charge in loquat fruit decreased during storage. LTC treatment maintained the high level of energy charge in loquat fruit.
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RESULTS Effect of LTC Treatment on CI, Ion Leakage, and MDA Content in Loquat Fruit. CI usually develops in loquat fruit during cold storage for a long time. Flesh internal browning was the most obvious symptom of CI in loquat (shown in the Tabove of Contents graphic). The CI index increased gradually after the first week in cold-stored loquat fruit. LTC treatment significantly (p < 0.05) inhibited CI increase during storage. As shown in Figure 1, CI index values were 57.3 and 36.2% lower in LTC-treated loquat than in control fruit in the third and fifth weeks, respectively. Ion leakage and MDA content in loquat
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DISCUSSION LTC is a useful technique to maintain quality and reduce CI in postharvest industry of fruit and vegetables. Increasing evidence has shown that CI symptoms were alleviated in avocado, B
DOI: 10.1021/acs.jafc.5b00605 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Effect of LTC treatment on ion leakage (A) and MDA content (B) of loquat fruit during cold storage. Data are expressed as the mean of triplicate samples ± standard errors. Vertical bars represent the standard errors of the means. Significant differences are shown by different letters.
Figure 3. Effect of LTC treatment on BADH activity (A) and GB content (B) of loquat fruit during cold storage. Data are expressed as the mean of triplicate samples ± standard errors. Vertical bars represent the standard errors of the means. Significant differences are shown by different letters.
Figure 4. Effect of LTC treatment on activities of H+-ATPase (A), Ca2+-ATPase (B), SDH (C), and CCO (D) of loquat fruit during cold storage. Data are expressed as the mean of triplicate samples ± standard errors. Vertical bars represent the standard errors of the means. Significant differences are shown by different letters.
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DOI: 10.1021/acs.jafc.5b00605 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Effect of LTC treatment on contents of ATP (A), ADP (B), AMP (C), and energy charge (D) of loquat fruit during cold storage. Data are expressed as the mean of triplicate samples ± standard errors. Vertical bars represent the standard errors of the means. Significant differences are shown by different letters.
zucchini squash, and sweet potatoes by LTC treatment.24 As to loquat fruit, Cai et al.5 reported that ‘Luoyangqing’ loquats conditioned at 5 °C for 6 days had better quality and lower CI symptoms than control fruits. Our previous study found that conditioning at 10 °C for 6 days could be the optimum condition for ‘Jiefangzhong’ loquat. This different temperature condition for pretreatment is probably due to the different loquat cultivar response to low-temperature stress. The beneficial effects of LTC have been attributed to enhancing antioxidant system, reducing electrolyte leakage, and inducing lipid membrane unsaturation. Moreover, LTC induced chilling tolerance by regulating the expression of transcripts related to secondary metabolism, hormone biosynthesis, and transcription factors.25 Our study provided new evidence that LTC alleviated CI in cold-stored loquat fruit associated with accumulation of GB and energy status. It has been indicated that GB accumulates in some plants responding to environmental stresses, including low temperature, water stress, or drought.6 Some plants accumulate small molecular substances with cryoprotectant activity against cold stress. Glycine betaine is one of several such osmotic adjustment substances that has a protective function to proteins and enzyme activities and even stabilizes membranes under environmental stresses.8 Previous evidence has suggested that there is a correlation between GB content and cold tolerance in higher plants. For example, endogenous GB was accumulated by cold acclimation treatment, which induced freezing tolerance in Arabidopsis.7 Similarly, tolerance to low temperatures was also improved in tomato plant GB treatment.26 In addition,
Rajashekar et al.9 found that exogenous abscisic acid treatment induced cold tolerance and improved freezing survival and regrowth in whole strawberry plants, which triggered endogenous GB accumulation. In our study, GB accumulation was associated with CI development in loquat fruit during cold storage. LTC treatment significantly reduced CI and a maintained higher level of GB content than those in control fruits. These results suggest that the accumulation of GB might contribute to the reduced CI in loquat fruits treated with LTC. BADH is the key enzyme that plays the important role during GB metabolism. It had been reported that heat treatment resulted in a high expression of BADH gene in transgenic tomato, which was correlated with up-regulated GB accumulation.27 Yang et al.28 found that GB accumulation as a result of the introduction of the BADH gene into tobacco enhances the tolerance against high-temperature stress. Chen et al.29 obtained similar results in maize, which indicated that GB accumulation caused by cold stress was associated with an increase in BADH activity. Our study showed that treatment with LTC maintained higher activity of BADH in loquat fruit. The decline of BADH activity in control fruit after the third week was possibly due to the senescence of loquat fruit, which may account for the low level of GB content. This suggested that the accumulation of GB in LTC-treated loquat fruit could be due to increased activity of BADH, the key enzyme involved in GB biosynthesis. It is worth mentioning that GB plays an important physiological role in stress tolerance involved in osmotic regulation, detoxication of reactive oxygen radicals, and protection of membrane integrity. These results indicated that D
DOI: 10.1021/acs.jafc.5b00605 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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postharvest chilling injury in loquat fruit. Postharvest Biol. Technol. 2006, 41, 252−259. (6) Ashraf, M.; Foolad, M. R. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 2007, 59, 206−216. (7) Xing, W.; Rajashekar, C. B. Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana. Environ. Exp. Bot. 2001, 46, 21−28. (8) Sakamoto, A.; Murata, N. The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant, Cell Environ. 2002, 25, 163−171. (9) Rajashekar, C. B.; Zhou, H.; Marcum, K. B.; Prakash, O. Glycine betaine accumulation and induction of cold tolerance in strawberry plants. Plant Sci. 1999, 148, 175−183. (10) Sun, Y. J.; Jin, P.; Shan, T. M.; Xu, J.; Zheng, Y. H. Effect of glycine betaine treatment on postharvest chilling injury and active oxygen metabolism in loquat fruits. Food Sci. 2014, 35, 210−215 (in Chinese with English abstract). (11) Jiang, Y. M.; Jiang, Y. L.; Qu, H. X.; Duan, X. W.; Luo, Y. B.; Jiang, W. B. Energy aspects in ripening and senescence of harvested horticultural crops. Stewart Postharvest Rev. 2007, 4, 1−5. (12) Saquet, A. A.; Streif, J.; Bangerth, F. Energy metabolism and membrane lipid alterations in relation to brown heart development in ’Conference’ pears during delayed controlled atmosphere storage. Postharvest Biol. Technol. 2003, 30, 123−132. (13) Song, L. L.; Jiang, Y. M.; Gao, H. Y.; Li, C. T.; Liu, H.; You, Y. L. Effects of adenosine triphosphate on browning and quality of harvested litchi fruit. Am. J. Food Technol. 2006, 1, 173−178. (14) Jin, P.; Zhu, H.; Wang, L.; Shan, T. M.; Zheng, Y. H. Oxalic acid alleviates chilling injury in peach fruit by regulating energy metabolism and fatty acid contents. Food Chem. 2014, 161, 87−93. (15) Zhou, Q.; Zhang, C. L.; Cheng, S. C.; Wei, B. D.; Liu, X. Y.; Ji, S. J. Changes in energy metabolism accompanying pitting in blueberries stored at low temperature. Food Chem. 2014, 164, 493− 501. (16) Jin, P.; Zhu, H.; Wang, J.; Chen, J. J.; Wang, X. L.; Zheng, Y. H. Effect of methyl jasmonate on energy metabolism in peach fruit during chilling stress. J. Sci. Food Agric. 2013, 93, 1827−1832. (17) Azevedo, I. G.; Oliveira, J. G.; da Silva, M. G.; Pereira, T.; Correa, S. F.; Vargas, H. P-type H+-ATPases activity, membrane integrity, and apoplastic pH during papaya fruit ripening. Postharvest Biol. Technol. 2008, 48, 242−247. (18) Cao, S.; Zheng, Y.; Wang, K.; Jin, P.; Rui, H. Methyl jasmonate reduces chilling injury and enhances antioxidant enzyme activity in postharvest loquat fruit. Food Chem. 2009, 115, 1458−1463. (19) Hodges, D. M.; Delong, J. M.; Forney, C.; Prange, R. K. Improving the thiobarbituric acid reactive-substances assay for estimating lipid peroxidation in plant tissue containing anthocyanin and other interfering compounds. Planta 1999, 207, 604−611. (20) Bessieres, M. A.; Gibon, Y.; Lefeuvre, J. C.; Larher, F. A singlestep purification for glycinebetaine determination in plant extracts by isocratic HPLC. J. Agric. Food Chem. 1999, 47, 3718−3722. (21) Arakawa, K.; Katkyama, M.; Takabe, T. Levels of betain and betaine aldehyde dehydrogenase activity in green leaves and etiolated leaves and roots of barley. Plant Cell Physiol. 1990, 31, 797. (22) Ackrell, B. A.; Keamery, E. B.; Singer, T. P. Mammalian succinate dehydrogenase. Methods Enzymol. 1978, 53, 466−483. (23) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 1976, 72, 248−254. (24) Jin, P.; Cao, S. F.; Zheng, Y. H. Managing chilling injury in fruits. Acta Hortic. 2013, 1012, 1087−1095. (25) Maul, P.; McCollum, G. T.; Popp, Mick; Guy, C. L.; Porat, R. Transcriptome profiling of grapefruit flavedo following exposure to low temperature and conditioning treatments uncovers principal molecular components involved in chilling tolerance and susceptibility. Plant, Cell Environ. 2008, 31, 752−768.
LTC induced cold tolerance of loquat due in part to regulating endogenous GB metabolism. Energy supply in cells plays an important role in controlling fruit ripening, senescence, and physiological disorder after harvest.11 Many studies have suggested that postharvest CI symptoms of various kinds of fruits, such as pears,12 litchis,13 longans,30 and peaches,14 mainly resulted from membrane damage related to the energy deficit under cold stress. In the present study, LTC treatment effectively maintained high levels of ATP and energy charge and reduced CI in cold-stored loquat fruit. Moreover, activities of energy metabolism enzymes were significantly higher in loquat fruits treated with LTC than in control fruits. This suggests that these enzymes played positive roles in the process of energy production. Similar results were also found in peach fruit in our previous study.31 ATP and energy might play important roles in cell membrane protection and fatty acid biosynthesis. Under stress conditions, stressresistant cultivars could maintain higher energy levels, resulting in a decrease in membrane leakage. Thus, our results indicated that LTC could enhance chilling tolerance in cold-stored loquat fruit by means of enhancing energy accumulation, preventing ion leakage increase, and protecting membrane function. In conclusion, the results of this work suggest that treatment with LTC decreased CI in loquat fruit contributed to enhancing endogenous GB content and energy status. The GB accumulation plays an important role in enhancing cold tolerance in loquat fruit by LTC treatment, which was attributed to increasing BADH activity. Furthermore, the reduction of CI by LTC treatment may be due to induced enzyme activity related to energy metabolism. However, further studies are needed to evaluate other related enzyme activities and gene expression involved in metabolism of GB or energy.
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AUTHOR INFORMATION
Corresponding Author
*(Y.Z.) Phone: +86-25-84399080. Fax: +86-25-84395618. Email:
[email protected]. Funding
This study was supported by the National Natural Science Foundation of China (No. 31000824 and 31371862), the Jiangsu Provincial Scientific and Technical Supporting Program (SBE201330109), and the Qinglan Project of Jiangsu Province. Notes
The authors declare no competing financial interest.
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REFERENCES
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