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Effect of 24-epibrassinolide on sugar metabolism and delaying postharvest senescence of kiwifruit during ambient storage
Scientia Horticulturae 253 (2019) 1–7
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Effect of 24-epibrassinolide on sugar metabolism and delaying postharvest senescence of kiwifruit during ambient storage
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Zemian Lu, Xiaolu Wang, Maomao Cao, Yaoyao Li, Jinlong Su, Hui Gao College of Food Science and Technology, Northwest University, Xi’an 710069, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: 24-Epibrassinolide Sugar metabolism Kiwifruit Postharvest senescence
Effect of 5 μM 24-epibrassinolide (EBR) on delaying the senescence of ‘Huayou’ kiwifruit and possible defense mechanisms were elucidated during ambient storage. Results revealed that EBR treatment retarded decrease in firmness as well as increase in weight loss and total soluble solid content in kiwifruit. EBR treatment prevented the increase of membrane permeability and suppressed the accumulation of malondialdehyde. EBR treatment postponed the degradation of starch to soluble sugars, resulting from the inactivation of amylase activity. EBR treatment also inhibited the activity of acid invertase, neutral invertase, sucrose phosphate synthase, sucrose synthase, hexokinase and fructokinase, and subsequently EBR-treated fruit exhibited the lower contents of sucrose, glucose and fructose. These results suggest that EBR treatment could be an innovative solution to delay the senescence of kiwifruit by regulating sugar metabolism.
1. Introduction Kiwifruit is a kind of fascinating fruit native to China, and has now become popular around the world due to its bright flesh color, enjoyable taste and abundant phytonutrients. The fruit is characterized as a typical climacteric fruit, which makes it susceptible to senescence and decay after harvest, leading to a shortened postharvest life (Crisosto, and Crisosto, 2001). Therefore, it is urgent to explore feasible solutions to retard senescence and extend shelf life of kiwifruit, and satisfactory results have been obtained by some chemical treatments, such as salicylic acid and its derivates (Zhang et al., 2004; Aghdam et al., 2010), 1methylcyclopropene (Koukounaras, and Sfakiotakis, 2007; Jhalegar et al., 2011; Park et al., 2015; Sharma et al., 2015; Lim et al., 2016), polyamines (Jhalegar et al., 2012), nitric oxide (Zhu et al., 2008; Zheng et al., 2017) and hydrogen-rich water (Hu et al., 2014). 2 -Epibrassinolide (EBR) is an important steroidal phytohormone with a favorable safety profile and strong biological activity that can elicit a broad range of cellular responses when applied exogenously, with success demonstrated by the fact that EBR allows the unique possibility of improving crop plants yields and overcoming environmental stresses (Ikekawa and Zhao, 1991; Malíková et al., 2008; Divi and Krishna, 2009). For this reason, recently, increasing attention has been centered on its application for postharvest horticultural products to enhance postharvest sensory and nutritional quality. It has been reported that EBR treatment is effective in increasing the
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biotic tolerance of Satsuma mandarin, and the stress-related metabolites, including ornithine, proline, γ-aminobutyric acid, d-xylose and dgalactose, were accumulated in EBR-treated fruit (Zhu et al., 2015). In table grape, Liu et al. (2016) gave similar results that EBR plays a protective role in attenuating the development of grey mould, which has partly been attributed to increased defense-related superoxide dismutase, peroxidase, catalase and phenylalanine ammonialyase activity. Besides, EBR has been shown to be conductive to inducing chilling tolerance in peach and eggplant fruits by influencing phenolic metabolism (Gao et al., 2015, 2016). Also, EBR treatment is suggested as a promising attempt by which cut surface browning of fresh-cut lotus root slices was suppressed, corresponding to an inhibited lipoxygenase activity, resulted in higher membrane integrity (Gao et al., 2017). It might be expected, based on the above, that EBR exposure will be a promising and efficient solution for postharvest kiwifruit against senescence. However, there is no existing information on the effect of EBR on kiwifruit. It has been proposed that sugar metabolism participates in the regulation of plant senescence (Rolland et al., 2002). To take leaf senescence for an example, glucose and fructose contents usually increase during senescence, with concomitant decrease in starch content, and senescence-like symptoms, such as leaf yellowing, can be induced by feeding glucose to leaf discs in light or can be inhibited by feeding melatonin to two‐year‐old plant of Malus hupehensis Rehd (Wingler et al., 1998; Wang et al., 2013). A similar rapid increase in glucose and fructose contents and decrease in starch content was recorded during
Corresponding author. E-mail address: [email protected] (H. Gao).
https://doi.org/10.1016/j.scienta.2019.04.028 Received 3 January 2019; Received in revised form 16 March 2019; Accepted 11 April 2019 0304-4238/ © 2019 Published by Elsevier B.V.
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as 6.45 × (OD 532 − OD 600) − 0.56 × OD450 and expressed on a fresh weight basis as μmol kg−1.
kiwifruit ripening and softening. Acetylsalicylic acid treatment slowed down the hydrolysis of starch and the accumulation of hexoses, and thus delayed senescence of kiwifruit (Zhang et al., 2004). Hu et al. (2019) got a similar result that 2% lacquer wax delays senescence in kiwifruit in relation to the regulation of soluble sugar. In such a case, the effect of applying EBR on the sugar metabolism of kiwifruit would be interesting to study. The aim of this study was to examine whether EBR treatment plays a role in senescence stress response in kiwifruit during ambient storage, and the emphasis was the regulatory activity of EBR on sugar metabolism. The results can contribute to providing a new solution for kiwifruit to maintain quality and extend shelf life and offering an evidence for the practical application of EBR.
2.4. Measurement of starch content and α-amylase activity Starch content was evaluated as previously described by Xu et al., 1998. Kiwifruit flesh tissue (1 g) was homogenized with 8 mL of 80% ethanol and filtered. This operation was repeated 3 times. After that, the residue was washed 3 times with ether solution, filtered and transferred to a beaker using a small amount of distilled water and boiled until the starch was gelatinized completely. The solution was set to 100 mL with distilled water and used for starch analysis. For starch content determination, starch solution of 2 mL was mixed with 0.2 mL of iodine solution and the total volume of the mixture was adjusted to 10 mL with distilled water. After placing the mixture for 10 min, the absorbance was read at 660 nm. Starch content was calculated using starch as a standard and expressed on a fresh weight basis as g kg−1. For determining the activity of α-amylase, kiwifruit flesh tissue (1 g) was homogenized with 10 mL of 100 mM sodium phosphate (pH 5.6) and centrifuged at 12, 000 × g for 20 min at 4 °C. α-Amylase activity was assayed by the 3,5-dinitrosalicylic acid (DNS) method at 540 nm using maltose as standard (Rick, and Stegbauer, 1974), and expressed on a fresh weight basis as U kg-1, where U is the amount of α-amylase that gives the formation of 1 mg maltose in 1 min.
2. Material and methods 2.1. Plant material and treatments Kiwifruit (Actinidia chinesis cv. Huayou) was harvested at commercial maturity from an orchard in Xi'an City (Shaanxi Province, China) with uniform size, shape and color and free from mechanical damage. The selected kiwifruit were randomly divided into 27 groups of 15 fruit each. Twelve groups were immersed in an aqueous solution containing 5 μM EBR for 10 min at ambient temperature (20 °C) and another 12 groups treated with distilled water only, served as the controls and the remaining 3 groups were also sampled before treatment to determine the characteristics at harvest (0 day). The EBR concentration of 5 μM was selected based on an optimum in preliminary experiments using 0, 3, 5, 10 and 15 μM EBR (data not shown). All kiwifruit were air-dried after treatment, packed in low-density polyethylene bags and then placed at 20 °C for 20 d. Fifteen samples per replication were taken at intervals of 5, 10, 15 and 20 d and used for measurement of weight loss, flesh firmness, soluble solids content (SSC) and membrane permeability. At the same intervals, samples of flesh tissue were collected and then rapidly frozen at -80 °C for subsequent analysis. Each treatment was performed in triplicate.
2.5. Measurement of fructose, glucose and sucrose content Fructose, glucose and sucrose content were estimated using a modified method of Usenik et al. (2008). Kiwifruit flesh tissue (1 g) was homogenized with 5 mL of acetonitrile-water solution, ultrasound-extracted for 30 min and centrifugated at 5000 × g for 15 min. The supernatant was filtered through 0.45 μm Millipore filter 2 times and the filtrate was collected. A Shodex RI-201H liquid chromatography system (Hanbon Science & Technology Co., Ltd., Jiangsu, China) equipped with a carbohydrate column (4.6 × 250 mm, 4 μm, Cartridge) and a differential refractive detector was used for fructose, glucose and sucrose analysis. Acetonitrile-water solution (acetonitrile:water = 78:22, v/v) was employed as the mobile phase at a flow-rate of 1.0 mL min–1. The column temperature was kept at 35 °C and an aliquot of 10 μL of sample solution was injected into the liquid chromatography system. The contents of the three sugars were calculated by comparison of the peak areas obtained with those of sugar standards and expressed on a fresh weight basis as g kg−1.
2.2. Measurement of weight loss, firmness and SSC Weight loss was defined as the ratio of final sample weights to initial sample weights and expressed as %. Firmness was evaluated at two points of two different positions around the equator of 15 kiwifruit from each replicate (skin removed) using a fruit firmness tester (GY-3, Aidebao Instrument Co., Ltd, Leqing, China) equipped with an 8 mm diameter probe and expressed as N. SSC was estimated by measuring the refractive index with a WYT-4 hand-held refractometer (Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) calibrated with distilled water at 20 °C. SSC was expressed as %.
2.6. Measurement of enzymes activity related to sugar metabolism For determining the activity of acid invertase (AI), neutral invertase (NI), sucrose phosphate synthase (SPS), sucrose synthase synthesis activity (SS), hexokinase (HK) and fructokinase (FK), kiwifruit flesh tissue (1 g) were homogenized with 10 mL of 100 mM Tris-HCl buffer containing 1 mM ethylenediaminetetraacetic acid, 10 mM magnesium chloride, 10 mM ascorbic acid, 0.01% (v/v) TritonX-100 and 3% (w/v) polyvinyl pyrrolidone and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used as crude enzyme extract. Reaction mixtures for AI activity measurement contained 100 mM sodium acetate buffer (pH 5.0), 100 mM sucrose and supernatant. The mixture was incubated at 30 °C for 35 min and the reaction was terminated by keeping in boiling water for 10 min. AI activity was determined on the basis of the production of glucose at 540 nm, and expressed on a fresh weight basis as U kg−1, where U is the amount of AI that gives the formation of 1 mg glucose in 1 min. NI activity was similar to that of AI, except that the reaction was conducted at 50 mM HEPES-NaOH (pH 7.5). SPS activity was assayed according to Stitt et al. (1988). The reaction mixture contained 50 mM HEPES-NaOH buffer (pH 7.5), 15 mM MgCl2, 25 mM glucose-6-phosphate, 25 mM fructose-6-phosphate,
2.3. Measurement of electrolyte leakage and malondialdehyde content Electrolyte leakage was measured according to a method of Côté et al. (1993) with some modification. Kiwifruit flesh tissue (2 g) was cut into small pieces and then immersed and incubated in 30 mL deionized water; subsequently conductivity of the solution was recorded with a conductimeter (DDS-11 A, Youke Instrument Co., Ltd., Shanghai, China). The solution was then boiled at 100 °C for 10 min and quickly cooled thereafter and the total electrical conductivity was obtained. Electrolyte leakage was expressed as a percentage of total electrical conductivity. Malondialdehyde (MDA) content was evaluated following a modified method of Dhindsa et al. (1981). Kiwifruit flesh tissue (1 g) was homogenized with 6 mL of 10% trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The homogenate was then heated at 100 °C for 10 min. After cooling quickly by running water, the mixture was centrifuged at 5000 × g for 20 min. The absorbance of the supernatant was read at 450, 532 and 600 nm. MDA content was calculated 2
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25 mM uridine diphosphate glucose and 20 μl supernatant. The mixture was incubated at 30 °C for 35 min and the reaction was stopped by adding 0.1 mL 30% (w/v) KOH and placed in boiling water for 10 min. SPS activity was determined on the basis of the production of sucrose at 620 nm, and expressed on a fresh weight basis as U kg−1, where U is the amount of SPS that gives the formation of 1 mg sucrose in 1 min. SS activity was similar to that of SPS, except that 10 mM fructose was used instead of glucose-6-phosphate. Reaction mixtures for HK activity determination contained 50 mM Tris-HC1 buffer (pH 8.0), 4 mM MgCl2, 2.5 mM ATP, 0.33 mM NAD+, 1 U glucose-6-phosphate dehydrogenase, 1 mM glucose and supernatant. HK activity was estimated by the decrease in absorbance at 340 nm, and expressed on a fresh weight basis as U kg−1, where U = 0.01 ΔA340 nm per min. FK activity was similar to that of HK, except that the replacement of glucose with 1 mM fructose, and glucose-6-phosphate dehydrogenase with 1 U phosphoglucose isomerase.
2.7. Statistical analysis The experiments were performed in a completely randomized design. All experiments were conducted in triplicate and average values with standard errors are reported. Analysis of variance was carried out, and means were compared using Duncan’s multiple range tests at a significance level of 0.05 with SPSS 23.0.
3. Results 3.1. Effect of EBR treatment on weight loss, firmness and SSC Both control and EBR-treated fruit showed an increase in weight loss with storage duration, but EBR treatment delayed this process. During the whole storage, weight loss in EBR-treated fruit was 1.65%, showing about 1.3 times lower than that in control (Fig. 1A). Firmness in control fruit decreased slowly during the first 10 d of storage and declined rapidly thereafter, reaching a minimum value on day 15 (Fig. 1B). Fruit treated with EBR exhibited a similar trend, but the loss of firmness was inhibited. On day 15, firmness in EBR-treated fruit was about 95 N, and the rapid firmness loss was only just beginning. SSC in EBR-treated fruit was lower than that of control fruit during the most of storage time (Fig. 1C). About 30% decrease in SSC was recorded after 15 d of storage in comparison to control fruit.
3.2. Effect of EBR treatment on electrolyte leakage and MDA content Electrolyte leakage in both control and EBR-treated fruit presented an almost linear increase during storage, but EBR treatment prevented this increase. At the end of storage, electrolyte leakage in EBR-treated fruit was 60.51%, being about 1.2 times lower than that in control (Fig. 2A). And, similarly, MDA content in EBR-treated fruit was lower during the whole storage and this inhibition averaged about 27% at the end of storage (Fig. 2B). Fig. 1. Effect of EBR treatment on weight loss (A), firmness (B) and SSC (C) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
3.3. Effect of EBR treatment on starch content and α-amylase activity Starch content in control fruit decreased rapidly and reached a minimum value on day 15 (Fig. 3A). EBR-treated fruit exhibited a similar change pattern, but the decrease of starch was delayed by EBR treatment. On day 15, starch content in EBR-treated fruit was more than 51 times higher than that of control fruit. Differing from starch content, α-amylase activity in control fruit increased during storage. EBR treatment inhibited α-amylase activity. At the end of storage, α-amylase activity in control and EBR-treated fruit were 940 U kg−1 and 210 U kg−1, respectively (Fig. 3B).
3.4. Effect of EBR treatment on glucose, fructose and sucrose content Glucose and fructose contents increased continuously regardless of treatment during storage (Fig. 4A and B). EBR treatment delayed the increase of these two sugars and resulted in an average decrease in both glucose and fructose contents of about 36% on day 15. For sucrose
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Fig. 2. Effect of EBR treatment on electrolyte leakage (A) and MDA content (B) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
concern in kiwifruit storage. The current results showed that EBR treatment at a concentration of 5 μM prevented the decrease of firmness, as well as the increase of weight loss and SSC in ‘Huayou’ kiwifruit (Fig. 1). This indicated that EBR might be an effective solution for kiwifruit to delay senescence during ambient storage and thus, can be implemented for prolonging shelf life. Similar effects of EBR on postharvest quality have also been reported on eggplant, Satsuma mandarin, peach, table grape and lotus root (Gao et al., 2015; Zhu et al., 2015; Gao et al., 2016; Liu et al., 2016; Gao et al., 2017). Membrane damage or deterioration is a characteristic physiological feature of plant senescence, and the process is known to be accompanied by greatly increased electrolyte leakage (Thompson et al., 1998). This leakage change has been correlated with the peroxidation of membrane lipid thereby with the accumulation of MDA, a secondary end-product of oxidative damage of membrane lipid (Blokhina et al., 2003). In postharvest kiwifruit, increase of electrolyte leakage and MDA content contribute to the risk of accelerating senescence has been reported. And that some postharvest treatments as mentioned in introduction have been used to defense senescence induced membrane damage or deterioration (Zhang et al., 2004; Zhu et al., 2008; Hu et al., 2014). We herein the application of EBR reduced electrolyte leakage in kiwifruit, and a simultaneous decrease in MDA content was also observed during storage (Fig. 2). This indicated that EBR treatment could provide kiwifruit a preventive effect against membrane lipid peroxidation, which in turn would, help maintaining membrane integrity and delaying senescence, as it has been proposed by Liu et al. (2016) for the regulation of this action in table grape by EBR treatment during ambient storage. Kiwifruit is a typical fruit that accumulates carbohydrate in the form of starch. During senescence, starch is hydrolyzed and converted into soluble sugars, resulting in sweeter flavor and softer texture of kiwifruit (MacRae et al., 1992). It is true, in the current study, trajectory analysis showed that starch content had a high negative correlation with SSC (r = -0.9985), but a high positive correlation with firmness (r = 0.9525)
content, there was a rapid increase occurred in control fruit in the middle stage of storage, but then it fell sharply until the end of storage (Fig. 4C). EBR treatment not only inhibited the increase of sucrose but also delayed the time to the peak of sucrose content. 3.5. Effect of EBR treatment on the activity of enzymes involved in sugar metabolism AI activity in control fruit increased during the first 5 d of storage and then remained stable and finally increased with an accelerated speed until the end of storage (Fig. 5A). EBR treatment reduced AI activity, showing about 34% decrease at the end of storage compared with control fruit. For NI activity, there was a rapid increase occurred in control fruit during the first 15 d of storage, but then dropped until the end of storage (Fig. 5B). EBR treatment led to a decrease in NI activity and the activity of NI in EBR-treated fruit was always lower during the whole storage. SS activity in control fruit increased and peaked on day 5 (Fig. 5C), and was higher than that in EBR-treated fruit during the first 10 d of storage. On day 5, SS activity in control was 1.5 times higher than that in EBR-treated fruit. EBR treatment reduced SPS activity as well during most of storage (Fig. 5D). HK activity in control fruit increased during the first 15 d of storage and then decreased (Fig. 5E). EBR-treated fruit showed a lower HK activity during the most of storage time and HK activity in EBR-treated fruit was about 43% lower than that in control on day 15. FK activity increased from day 0 to day 15 and then declined in control fruit but increased continuously in EBR-treated fruit (Fig. 5F). EBR treatment suppressed FK activity during the most of storage time. On day 15, FK activity in EBR-treated fruit was 10.64 × 103 U kg−1, showing about 1.32 times lower than that in control. 4. Discussion Weight loss, firmness and SSC are primary parameters of greatest
Fig. 3. Effect of EBR treatment on starch content (A) and α-amylase activity (B) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest. 4
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therefore be reflections of senescence in kiwifruit. In the current study, faster starch conversion and higher α-amylase activity were observed in control kiwifruit (Fig. 3). However, increases in α-amylase activity were inhibited by EBR treatment during the most of storage time, coinciding with the higher starch content and the lower SSC (Figs. 1C and 3), which indicated that EBR-induced inhibition of starch degradation might be essential for kiwifruit to withstand senescence. Consistent with our findings, a previous report has proposed that Adα-amylase1 showed the potential to regulate starch degradation; moreover, postharvest treatments-induced senescence delay in kiwifruit have been ascribed partially to their ability to inhibit α-amylase activity during ambient storage (Hu et al., 2016, 2019). Glucose, fructose and sucrose are major soluble sugars form for kiwifruit (MacRae et al., 1992). We came to the same conclusion in ‘Huayou’ kiwifruit. Moreover, we found that the contents of glucose and fructose increased in control kiwifruit during storage, while sucrose changed drastically, rising and falling rapidly within 5–20 days. Not quite in line with our findings, Mack et al. (2017) reported that sucrose remained constant in ‘Hayward’ kiwifruit during the most of storage time and increased slightly when the fruit was overripe. Based on these results, it can be inferred that the three soluble sugars participate in triggering and regulating the senescence of kiwifruit. Such a subjective inference was demonstrated by the relationship between membrane permeability, a predictor of plant senescence, and the three soluble sugars in control kiwifruit. Trajectory analysis for control kiwifruit showed high positive correlations of 0.9303 and 0.9264, respectively, between electrolyte leakage and glucose and fructose during the whole storage, while a high positive correlation of 0.9197 between electrolyte leakage and sucrose during the first 20 d of storage. Since sugar signaling, especially those of glucose and sucrose are related to the control of oxidative stress, it is considered that glucose and sucrose display a synergistic action in senescence delay, and the former one might be more dominant than that of the latter in senescent kiwifruit. Conflicting results have been given; for example, Jia et al. (2013) demonstrated that exogenous sucrose accelerated strawberry fruit ripening, whereas glucose showed a much smaller effect. The difference respond to glucose and sucrose between the current study and Jia et al. (2013) is probably due to different species, maturity stages, sugar-modulated gene expression and/or signaling transduction pathway. EBR-treated kiwifruit exhibited lower contents of glucose, fructose and sucrose during the most of storage time, which might be partial of the mechanism whereby EBR retards senescence (Fig. 4). A possible explanation is that soluble sugars can be involved in, or associated with, reactive oxygen species-generating metabolic pathways. In yeast and in mammalian cell, it has been confirmed that glucose autooxidation increased the generation of reactive oxygen species. Bolouri-Moghaddam et al. (2010) recently summarized a hypothetical sugar-antioxidant network operating in plant cells, with a central role appointed to glucose. For postharvest fruit, sucrose degradation and synthesis are the dominant parts of sugar metabolism; in this process, AI, NI, and SS are responsible for the cleavage of sucrose, while SPS catalyzes the synthesis of sucrose (Sturm, 1999; Miron, and Schaffer, 1991). MacRae et al. (1992) has proposed that there is a futile cycle of sucrose synthesis and degradation in senescing kiwifruit. Briefly, through the action of SPS, glucose produced by starch degradation is used to synthesize sucrose, that is then transported into the vacuole, where it is cleaved by AI to glucose and fructose. We herein the application of EBR inhibited the activity of SPS (Fig. 5D); furthermore, a positive correlation existed between SPS activity and sucrose content in EBR-treated kiwifruit (r = 0.9920), all of which explained the suppressed synthesis of sucrose by EBR treatment during storage. Also, EBR treatment was observed to be able to restrain AI, NI and SS activity (Fig. 5A, B and C), together a negative correlation of 0.8555 between SS activity and sucrose content, which were beneficial to less sucrose cleavage. There is good reason, then, to believe that EBR treatment weakens the futile cycle of sucrose
Fig. 4. Effect of EBR treatment on the content of glucose (A), fructose (B) and sucrose (C) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
in control fruit during storage. α-Amylase is a key enzyme for starch conversion, and the reaction it catalyzes is the hydrolysis of the internal α-1, 4-glucosidic linkage in the starch to produce soluble sugars; in kiwifruit, α-amylase activity has been studied in relation to starch degradation (Wegrzyn, and MacRae, 1995; Zhang et al., 2004). A rapid starch degradation or a higher α-amylase activity or both might 5
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Fig. 5. Effect of EBR treatment on the activity of AI (A), NI (B), SS (C), SPS (D), HK (E) and FK (F) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
degradation. In conclusion, EBR at a concentration of 5 μM delayed the senescence of kiwifruit during ambient storage. EBR-mediated senescence may be derived from its capacity to maintain membrane lipid integrity, inhibit starch conversion as well as weaken the futile cycle of sucrose synthesis and degradation. Therefore, EBR treatment is a promising solution for kiwifruit to control postharvest senescence.
synthesis and degradation, and that SPS and SS tend to prevail over those of AI and NI in the regulation of sucrose metabolism, indicative that EBR treatment makes a significant contribution to senescence delay in kiwifruit through regulating SPS and SS activity. Similarly, Zhang et al. (2004) suggested that SPS may play a key role in sugar metabolism of kiwifruit, and it could be activated by hexoses and feedbackinhibited by sucrose; therefore, the inhibitory effect of acetylsalicylic acid treatment on the rapid increases in glucose, fructose and sucrose contents were related to inactivation of SPS and AI activity. It has been reported that the futile cycle of sucrose synthesis and degradation amplified the activation of SPS promoting the conversion of starch to soluble sugars (MacRae et al., 1992). This is likely to be the case in the current study since, as discussed above, both starch degradation and SPS activity were inhibited by EBR treatment. HK and FK are two enzymes responsible for glucose and fructose cleavage. In the current study, HK and FK activity were suppressed in EBR-treated kiwifruit (Fig. 5E and F), which theoretically should be more likely to cause accumulation of glucose and fructose. What is true, however, is that the contents of glucose and fructose in EBR-treated kiwifruit were lower than those in control during the whole storage; this might be because EBR treatment simultaneously weakens the conversion of starch and the futile cycle of sucrose synthesis and
Declarations of interest None. Acknowledgement This work was supported by the Project of Scientific and Technological of Shaanxi Province [grant numbers 2018NY-116]. References Aghdam, M.S., Motallebiazar, A., Mostofi, Y., Moghaddam, J.F., Ghasemnezhad, M., Erkan, M., Aksoy, U., 2010. Effects of MeSA vapor treatment on the postharvest quality of’ Hayward’ kiwifruit. Acta Hortic. 877 (877), 743–748. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage and
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oxygen deprivation stress: a review. Ann. Bot. 91 (2), 179–194. Bolouri-Moghaddam, M.R., Le Roy, K., Xiang, L., Rolland, F., Van den Ende, W., 2010. Sugar signalling and antioxidant network connections in plant cells. FEBS J. 277 (9), 2022–2037. Côté, F., Thompson, J.E., Willemot, C., 1993. Limitation to the use of electrolyte leakage for the measurement of chilling injury in tomato fruit. Postharvest Biol. Technol. 3 (2), 103–110. Crisosto, C.H., Crisosto, G.M., 2001. Understanding consumer acceptance of early harvested ‘Hayward’ kiwifruit. Postharvest Biol. Technol. 22 (3), 205–213. Dhindsa, R.S., Plumb, Dhindsa P., Thorpe, T.A., 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32 (126), 93–101. Divi, U., Krishna, P., 2009. Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance. N. Biotechnol. 26 (3), 131–136. Gao, H., Kang, L.N., Liu, Q., Cheng, N., Wang, B.N., Cao, W., 2015. Effect of 24-epibrassinolide treatment on the metabolism of eggplant fruits in relation to development of pulp browning under chilling stress. J. Food Sci. Technol. 52 (6), 3394–3401. Gao, H., Zhang, Z.K., Lv, X.G., Cheng, N., Peng, B.Z., Cao, W., 2016. Effect of 24-epibrassinolide on chilling injury of peach fruit in relation to phenolic and proline metabolisms. Postharvest Biol. Technol. 111, 390–397. Gao, H., Chai, H.K., Cheng, N., Cao, W., 2017. Effects of 24-epibrassinolide on enzymatic browning and antioxidant activity of fresh-cut lotus root slices. Food Chem. 217, 45–51. Hu, H.L., Li, P.X., Wang, Y.N., Gu, R.X., 2014. Hydrogen-rich water delays postharvest ripening and senescence of kiwifruit. Food Chem. 156 (11), 100–109. Hu, X., Kuang, S., Zhang, A.D., Zhang, W.S., Chen, M.J., Yin, X.R., Chen, K.S., 2016. Characterization of starch degradation related genes in postharvest kiwifruit. Int. J. Mol. Sci. 17 (12), 2112. Hu, H.L., Zhou, H.S., Li, P.X., 2019. Lacquer wax coating improves the sensory and quality attributes of kiwifruit during ambient storage. Sci. Hortic. 244, 31–41. Ikekawa, N., Zhao, Y.J., 1991. Application of 24-epibrassinolide in agriculture. ACS Symp. Ser. 474 (24), 280–291. Jhalegar, M.J., Sharma, R.R., Pal, R.K., Arora, A., Dahuja, A., 2011. Analysis of physiological and biochemical changes in kiwifruit (Actinidia deliciosa cv. Allison) after the postharvest treatment with 1-methylcyclopropene. J. Plant Biochem. Biotechnol. 20 (2), 205–210. Jhalegar, M.J., Sharma, R.R., Pal, R.K., Rana, V., 2012. Effect of postharvest treatments with polyamines on physiological and biochemical attributes of kiwifruit (Actinidia deliciosa) cv. Allison. Fruits 67 (01), 13–22. Jia, H.F., Wang, Y.H., Sun, M.Z., Li, B.B., Han, Y., Zhao, Y.X., Li, X.L., Ding, N., Li, C., Ji, W.L., Jia, W.S., 2013. Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening. New Phytol. 198 (2), 453–465. Koukounaras, A., Sfakiotakis, E., 2007. Effect of 1-MCP prestorage treatment on ethylene and CO2 production and quality of ‘Hayward’ kiwifruit during shelf-life after short, medium and long term cold storage. Postharvest Biol. Technol. 46 (2), 174–180. Lim, S., Han, S.H., Kim, J., Lee, H.J., Lee, J.G., Lee, E.J., 2016. Inhibition of hardy kiwifruit (Actinidia aruguta) ripening by 1-methylcyclopropene during cold storage and anticancer properties of the fruit extract. Food Chem. 190, 150–157. Liu, Q., Xi, Z.M., Gao, J.M., Meng, Y., Lin, S., Zhang, Z.W., 2016. Effects of exogenous 24‐epibrassinolide to control grey mould and maintain postharvest quality of table grapes. Int. J. Food Sci. Technol. 51 (5), 1236–1243. Mack, C., Wefers, D., Schuster, P., Weinert, C.H., Egert, B., Bliedung, S., Trierweiler, B.,
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Effect of 24-epibrassinolide on sugar metabolism and delaying postharvest senescence of kiwifruit during ambient storage
T
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Zemian Lu, Xiaolu Wang, Maomao Cao, Yaoyao Li, Jinlong Su, Hui Gao College of Food Science and Technology, Northwest University, Xi’an 710069, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: 24-Epibrassinolide Sugar metabolism Kiwifruit Postharvest senescence
Effect of 5 μM 24-epibrassinolide (EBR) on delaying the senescence of ‘Huayou’ kiwifruit and possible defense mechanisms were elucidated during ambient storage. Results revealed that EBR treatment retarded decrease in firmness as well as increase in weight loss and total soluble solid content in kiwifruit. EBR treatment prevented the increase of membrane permeability and suppressed the accumulation of malondialdehyde. EBR treatment postponed the degradation of starch to soluble sugars, resulting from the inactivation of amylase activity. EBR treatment also inhibited the activity of acid invertase, neutral invertase, sucrose phosphate synthase, sucrose synthase, hexokinase and fructokinase, and subsequently EBR-treated fruit exhibited the lower contents of sucrose, glucose and fructose. These results suggest that EBR treatment could be an innovative solution to delay the senescence of kiwifruit by regulating sugar metabolism.
1. Introduction Kiwifruit is a kind of fascinating fruit native to China, and has now become popular around the world due to its bright flesh color, enjoyable taste and abundant phytonutrients. The fruit is characterized as a typical climacteric fruit, which makes it susceptible to senescence and decay after harvest, leading to a shortened postharvest life (Crisosto, and Crisosto, 2001). Therefore, it is urgent to explore feasible solutions to retard senescence and extend shelf life of kiwifruit, and satisfactory results have been obtained by some chemical treatments, such as salicylic acid and its derivates (Zhang et al., 2004; Aghdam et al., 2010), 1methylcyclopropene (Koukounaras, and Sfakiotakis, 2007; Jhalegar et al., 2011; Park et al., 2015; Sharma et al., 2015; Lim et al., 2016), polyamines (Jhalegar et al., 2012), nitric oxide (Zhu et al., 2008; Zheng et al., 2017) and hydrogen-rich water (Hu et al., 2014). 2 -Epibrassinolide (EBR) is an important steroidal phytohormone with a favorable safety profile and strong biological activity that can elicit a broad range of cellular responses when applied exogenously, with success demonstrated by the fact that EBR allows the unique possibility of improving crop plants yields and overcoming environmental stresses (Ikekawa and Zhao, 1991; Malíková et al., 2008; Divi and Krishna, 2009). For this reason, recently, increasing attention has been centered on its application for postharvest horticultural products to enhance postharvest sensory and nutritional quality. It has been reported that EBR treatment is effective in increasing the
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biotic tolerance of Satsuma mandarin, and the stress-related metabolites, including ornithine, proline, γ-aminobutyric acid, d-xylose and dgalactose, were accumulated in EBR-treated fruit (Zhu et al., 2015). In table grape, Liu et al. (2016) gave similar results that EBR plays a protective role in attenuating the development of grey mould, which has partly been attributed to increased defense-related superoxide dismutase, peroxidase, catalase and phenylalanine ammonialyase activity. Besides, EBR has been shown to be conductive to inducing chilling tolerance in peach and eggplant fruits by influencing phenolic metabolism (Gao et al., 2015, 2016). Also, EBR treatment is suggested as a promising attempt by which cut surface browning of fresh-cut lotus root slices was suppressed, corresponding to an inhibited lipoxygenase activity, resulted in higher membrane integrity (Gao et al., 2017). It might be expected, based on the above, that EBR exposure will be a promising and efficient solution for postharvest kiwifruit against senescence. However, there is no existing information on the effect of EBR on kiwifruit. It has been proposed that sugar metabolism participates in the regulation of plant senescence (Rolland et al., 2002). To take leaf senescence for an example, glucose and fructose contents usually increase during senescence, with concomitant decrease in starch content, and senescence-like symptoms, such as leaf yellowing, can be induced by feeding glucose to leaf discs in light or can be inhibited by feeding melatonin to two‐year‐old plant of Malus hupehensis Rehd (Wingler et al., 1998; Wang et al., 2013). A similar rapid increase in glucose and fructose contents and decrease in starch content was recorded during
Corresponding author. E-mail address: [email protected] (H. Gao).
https://doi.org/10.1016/j.scienta.2019.04.028 Received 3 January 2019; Received in revised form 16 March 2019; Accepted 11 April 2019 0304-4238/ © 2019 Published by Elsevier B.V.
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as 6.45 × (OD 532 − OD 600) − 0.56 × OD450 and expressed on a fresh weight basis as μmol kg−1.
kiwifruit ripening and softening. Acetylsalicylic acid treatment slowed down the hydrolysis of starch and the accumulation of hexoses, and thus delayed senescence of kiwifruit (Zhang et al., 2004). Hu et al. (2019) got a similar result that 2% lacquer wax delays senescence in kiwifruit in relation to the regulation of soluble sugar. In such a case, the effect of applying EBR on the sugar metabolism of kiwifruit would be interesting to study. The aim of this study was to examine whether EBR treatment plays a role in senescence stress response in kiwifruit during ambient storage, and the emphasis was the regulatory activity of EBR on sugar metabolism. The results can contribute to providing a new solution for kiwifruit to maintain quality and extend shelf life and offering an evidence for the practical application of EBR.
2.4. Measurement of starch content and α-amylase activity Starch content was evaluated as previously described by Xu et al., 1998. Kiwifruit flesh tissue (1 g) was homogenized with 8 mL of 80% ethanol and filtered. This operation was repeated 3 times. After that, the residue was washed 3 times with ether solution, filtered and transferred to a beaker using a small amount of distilled water and boiled until the starch was gelatinized completely. The solution was set to 100 mL with distilled water and used for starch analysis. For starch content determination, starch solution of 2 mL was mixed with 0.2 mL of iodine solution and the total volume of the mixture was adjusted to 10 mL with distilled water. After placing the mixture for 10 min, the absorbance was read at 660 nm. Starch content was calculated using starch as a standard and expressed on a fresh weight basis as g kg−1. For determining the activity of α-amylase, kiwifruit flesh tissue (1 g) was homogenized with 10 mL of 100 mM sodium phosphate (pH 5.6) and centrifuged at 12, 000 × g for 20 min at 4 °C. α-Amylase activity was assayed by the 3,5-dinitrosalicylic acid (DNS) method at 540 nm using maltose as standard (Rick, and Stegbauer, 1974), and expressed on a fresh weight basis as U kg-1, where U is the amount of α-amylase that gives the formation of 1 mg maltose in 1 min.
2. Material and methods 2.1. Plant material and treatments Kiwifruit (Actinidia chinesis cv. Huayou) was harvested at commercial maturity from an orchard in Xi'an City (Shaanxi Province, China) with uniform size, shape and color and free from mechanical damage. The selected kiwifruit were randomly divided into 27 groups of 15 fruit each. Twelve groups were immersed in an aqueous solution containing 5 μM EBR for 10 min at ambient temperature (20 °C) and another 12 groups treated with distilled water only, served as the controls and the remaining 3 groups were also sampled before treatment to determine the characteristics at harvest (0 day). The EBR concentration of 5 μM was selected based on an optimum in preliminary experiments using 0, 3, 5, 10 and 15 μM EBR (data not shown). All kiwifruit were air-dried after treatment, packed in low-density polyethylene bags and then placed at 20 °C for 20 d. Fifteen samples per replication were taken at intervals of 5, 10, 15 and 20 d and used for measurement of weight loss, flesh firmness, soluble solids content (SSC) and membrane permeability. At the same intervals, samples of flesh tissue were collected and then rapidly frozen at -80 °C for subsequent analysis. Each treatment was performed in triplicate.
2.5. Measurement of fructose, glucose and sucrose content Fructose, glucose and sucrose content were estimated using a modified method of Usenik et al. (2008). Kiwifruit flesh tissue (1 g) was homogenized with 5 mL of acetonitrile-water solution, ultrasound-extracted for 30 min and centrifugated at 5000 × g for 15 min. The supernatant was filtered through 0.45 μm Millipore filter 2 times and the filtrate was collected. A Shodex RI-201H liquid chromatography system (Hanbon Science & Technology Co., Ltd., Jiangsu, China) equipped with a carbohydrate column (4.6 × 250 mm, 4 μm, Cartridge) and a differential refractive detector was used for fructose, glucose and sucrose analysis. Acetonitrile-water solution (acetonitrile:water = 78:22, v/v) was employed as the mobile phase at a flow-rate of 1.0 mL min–1. The column temperature was kept at 35 °C and an aliquot of 10 μL of sample solution was injected into the liquid chromatography system. The contents of the three sugars were calculated by comparison of the peak areas obtained with those of sugar standards and expressed on a fresh weight basis as g kg−1.
2.2. Measurement of weight loss, firmness and SSC Weight loss was defined as the ratio of final sample weights to initial sample weights and expressed as %. Firmness was evaluated at two points of two different positions around the equator of 15 kiwifruit from each replicate (skin removed) using a fruit firmness tester (GY-3, Aidebao Instrument Co., Ltd, Leqing, China) equipped with an 8 mm diameter probe and expressed as N. SSC was estimated by measuring the refractive index with a WYT-4 hand-held refractometer (Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) calibrated with distilled water at 20 °C. SSC was expressed as %.
2.6. Measurement of enzymes activity related to sugar metabolism For determining the activity of acid invertase (AI), neutral invertase (NI), sucrose phosphate synthase (SPS), sucrose synthase synthesis activity (SS), hexokinase (HK) and fructokinase (FK), kiwifruit flesh tissue (1 g) were homogenized with 10 mL of 100 mM Tris-HCl buffer containing 1 mM ethylenediaminetetraacetic acid, 10 mM magnesium chloride, 10 mM ascorbic acid, 0.01% (v/v) TritonX-100 and 3% (w/v) polyvinyl pyrrolidone and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used as crude enzyme extract. Reaction mixtures for AI activity measurement contained 100 mM sodium acetate buffer (pH 5.0), 100 mM sucrose and supernatant. The mixture was incubated at 30 °C for 35 min and the reaction was terminated by keeping in boiling water for 10 min. AI activity was determined on the basis of the production of glucose at 540 nm, and expressed on a fresh weight basis as U kg−1, where U is the amount of AI that gives the formation of 1 mg glucose in 1 min. NI activity was similar to that of AI, except that the reaction was conducted at 50 mM HEPES-NaOH (pH 7.5). SPS activity was assayed according to Stitt et al. (1988). The reaction mixture contained 50 mM HEPES-NaOH buffer (pH 7.5), 15 mM MgCl2, 25 mM glucose-6-phosphate, 25 mM fructose-6-phosphate,
2.3. Measurement of electrolyte leakage and malondialdehyde content Electrolyte leakage was measured according to a method of Côté et al. (1993) with some modification. Kiwifruit flesh tissue (2 g) was cut into small pieces and then immersed and incubated in 30 mL deionized water; subsequently conductivity of the solution was recorded with a conductimeter (DDS-11 A, Youke Instrument Co., Ltd., Shanghai, China). The solution was then boiled at 100 °C for 10 min and quickly cooled thereafter and the total electrical conductivity was obtained. Electrolyte leakage was expressed as a percentage of total electrical conductivity. Malondialdehyde (MDA) content was evaluated following a modified method of Dhindsa et al. (1981). Kiwifruit flesh tissue (1 g) was homogenized with 6 mL of 10% trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The homogenate was then heated at 100 °C for 10 min. After cooling quickly by running water, the mixture was centrifuged at 5000 × g for 20 min. The absorbance of the supernatant was read at 450, 532 and 600 nm. MDA content was calculated 2
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25 mM uridine diphosphate glucose and 20 μl supernatant. The mixture was incubated at 30 °C for 35 min and the reaction was stopped by adding 0.1 mL 30% (w/v) KOH and placed in boiling water for 10 min. SPS activity was determined on the basis of the production of sucrose at 620 nm, and expressed on a fresh weight basis as U kg−1, where U is the amount of SPS that gives the formation of 1 mg sucrose in 1 min. SS activity was similar to that of SPS, except that 10 mM fructose was used instead of glucose-6-phosphate. Reaction mixtures for HK activity determination contained 50 mM Tris-HC1 buffer (pH 8.0), 4 mM MgCl2, 2.5 mM ATP, 0.33 mM NAD+, 1 U glucose-6-phosphate dehydrogenase, 1 mM glucose and supernatant. HK activity was estimated by the decrease in absorbance at 340 nm, and expressed on a fresh weight basis as U kg−1, where U = 0.01 ΔA340 nm per min. FK activity was similar to that of HK, except that the replacement of glucose with 1 mM fructose, and glucose-6-phosphate dehydrogenase with 1 U phosphoglucose isomerase.
2.7. Statistical analysis The experiments were performed in a completely randomized design. All experiments were conducted in triplicate and average values with standard errors are reported. Analysis of variance was carried out, and means were compared using Duncan’s multiple range tests at a significance level of 0.05 with SPSS 23.0.
3. Results 3.1. Effect of EBR treatment on weight loss, firmness and SSC Both control and EBR-treated fruit showed an increase in weight loss with storage duration, but EBR treatment delayed this process. During the whole storage, weight loss in EBR-treated fruit was 1.65%, showing about 1.3 times lower than that in control (Fig. 1A). Firmness in control fruit decreased slowly during the first 10 d of storage and declined rapidly thereafter, reaching a minimum value on day 15 (Fig. 1B). Fruit treated with EBR exhibited a similar trend, but the loss of firmness was inhibited. On day 15, firmness in EBR-treated fruit was about 95 N, and the rapid firmness loss was only just beginning. SSC in EBR-treated fruit was lower than that of control fruit during the most of storage time (Fig. 1C). About 30% decrease in SSC was recorded after 15 d of storage in comparison to control fruit.
3.2. Effect of EBR treatment on electrolyte leakage and MDA content Electrolyte leakage in both control and EBR-treated fruit presented an almost linear increase during storage, but EBR treatment prevented this increase. At the end of storage, electrolyte leakage in EBR-treated fruit was 60.51%, being about 1.2 times lower than that in control (Fig. 2A). And, similarly, MDA content in EBR-treated fruit was lower during the whole storage and this inhibition averaged about 27% at the end of storage (Fig. 2B). Fig. 1. Effect of EBR treatment on weight loss (A), firmness (B) and SSC (C) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
3.3. Effect of EBR treatment on starch content and α-amylase activity Starch content in control fruit decreased rapidly and reached a minimum value on day 15 (Fig. 3A). EBR-treated fruit exhibited a similar change pattern, but the decrease of starch was delayed by EBR treatment. On day 15, starch content in EBR-treated fruit was more than 51 times higher than that of control fruit. Differing from starch content, α-amylase activity in control fruit increased during storage. EBR treatment inhibited α-amylase activity. At the end of storage, α-amylase activity in control and EBR-treated fruit were 940 U kg−1 and 210 U kg−1, respectively (Fig. 3B).
3.4. Effect of EBR treatment on glucose, fructose and sucrose content Glucose and fructose contents increased continuously regardless of treatment during storage (Fig. 4A and B). EBR treatment delayed the increase of these two sugars and resulted in an average decrease in both glucose and fructose contents of about 36% on day 15. For sucrose
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Fig. 2. Effect of EBR treatment on electrolyte leakage (A) and MDA content (B) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
concern in kiwifruit storage. The current results showed that EBR treatment at a concentration of 5 μM prevented the decrease of firmness, as well as the increase of weight loss and SSC in ‘Huayou’ kiwifruit (Fig. 1). This indicated that EBR might be an effective solution for kiwifruit to delay senescence during ambient storage and thus, can be implemented for prolonging shelf life. Similar effects of EBR on postharvest quality have also been reported on eggplant, Satsuma mandarin, peach, table grape and lotus root (Gao et al., 2015; Zhu et al., 2015; Gao et al., 2016; Liu et al., 2016; Gao et al., 2017). Membrane damage or deterioration is a characteristic physiological feature of plant senescence, and the process is known to be accompanied by greatly increased electrolyte leakage (Thompson et al., 1998). This leakage change has been correlated with the peroxidation of membrane lipid thereby with the accumulation of MDA, a secondary end-product of oxidative damage of membrane lipid (Blokhina et al., 2003). In postharvest kiwifruit, increase of electrolyte leakage and MDA content contribute to the risk of accelerating senescence has been reported. And that some postharvest treatments as mentioned in introduction have been used to defense senescence induced membrane damage or deterioration (Zhang et al., 2004; Zhu et al., 2008; Hu et al., 2014). We herein the application of EBR reduced electrolyte leakage in kiwifruit, and a simultaneous decrease in MDA content was also observed during storage (Fig. 2). This indicated that EBR treatment could provide kiwifruit a preventive effect against membrane lipid peroxidation, which in turn would, help maintaining membrane integrity and delaying senescence, as it has been proposed by Liu et al. (2016) for the regulation of this action in table grape by EBR treatment during ambient storage. Kiwifruit is a typical fruit that accumulates carbohydrate in the form of starch. During senescence, starch is hydrolyzed and converted into soluble sugars, resulting in sweeter flavor and softer texture of kiwifruit (MacRae et al., 1992). It is true, in the current study, trajectory analysis showed that starch content had a high negative correlation with SSC (r = -0.9985), but a high positive correlation with firmness (r = 0.9525)
content, there was a rapid increase occurred in control fruit in the middle stage of storage, but then it fell sharply until the end of storage (Fig. 4C). EBR treatment not only inhibited the increase of sucrose but also delayed the time to the peak of sucrose content. 3.5. Effect of EBR treatment on the activity of enzymes involved in sugar metabolism AI activity in control fruit increased during the first 5 d of storage and then remained stable and finally increased with an accelerated speed until the end of storage (Fig. 5A). EBR treatment reduced AI activity, showing about 34% decrease at the end of storage compared with control fruit. For NI activity, there was a rapid increase occurred in control fruit during the first 15 d of storage, but then dropped until the end of storage (Fig. 5B). EBR treatment led to a decrease in NI activity and the activity of NI in EBR-treated fruit was always lower during the whole storage. SS activity in control fruit increased and peaked on day 5 (Fig. 5C), and was higher than that in EBR-treated fruit during the first 10 d of storage. On day 5, SS activity in control was 1.5 times higher than that in EBR-treated fruit. EBR treatment reduced SPS activity as well during most of storage (Fig. 5D). HK activity in control fruit increased during the first 15 d of storage and then decreased (Fig. 5E). EBR-treated fruit showed a lower HK activity during the most of storage time and HK activity in EBR-treated fruit was about 43% lower than that in control on day 15. FK activity increased from day 0 to day 15 and then declined in control fruit but increased continuously in EBR-treated fruit (Fig. 5F). EBR treatment suppressed FK activity during the most of storage time. On day 15, FK activity in EBR-treated fruit was 10.64 × 103 U kg−1, showing about 1.32 times lower than that in control. 4. Discussion Weight loss, firmness and SSC are primary parameters of greatest
Fig. 3. Effect of EBR treatment on starch content (A) and α-amylase activity (B) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest. 4
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therefore be reflections of senescence in kiwifruit. In the current study, faster starch conversion and higher α-amylase activity were observed in control kiwifruit (Fig. 3). However, increases in α-amylase activity were inhibited by EBR treatment during the most of storage time, coinciding with the higher starch content and the lower SSC (Figs. 1C and 3), which indicated that EBR-induced inhibition of starch degradation might be essential for kiwifruit to withstand senescence. Consistent with our findings, a previous report has proposed that Adα-amylase1 showed the potential to regulate starch degradation; moreover, postharvest treatments-induced senescence delay in kiwifruit have been ascribed partially to their ability to inhibit α-amylase activity during ambient storage (Hu et al., 2016, 2019). Glucose, fructose and sucrose are major soluble sugars form for kiwifruit (MacRae et al., 1992). We came to the same conclusion in ‘Huayou’ kiwifruit. Moreover, we found that the contents of glucose and fructose increased in control kiwifruit during storage, while sucrose changed drastically, rising and falling rapidly within 5–20 days. Not quite in line with our findings, Mack et al. (2017) reported that sucrose remained constant in ‘Hayward’ kiwifruit during the most of storage time and increased slightly when the fruit was overripe. Based on these results, it can be inferred that the three soluble sugars participate in triggering and regulating the senescence of kiwifruit. Such a subjective inference was demonstrated by the relationship between membrane permeability, a predictor of plant senescence, and the three soluble sugars in control kiwifruit. Trajectory analysis for control kiwifruit showed high positive correlations of 0.9303 and 0.9264, respectively, between electrolyte leakage and glucose and fructose during the whole storage, while a high positive correlation of 0.9197 between electrolyte leakage and sucrose during the first 20 d of storage. Since sugar signaling, especially those of glucose and sucrose are related to the control of oxidative stress, it is considered that glucose and sucrose display a synergistic action in senescence delay, and the former one might be more dominant than that of the latter in senescent kiwifruit. Conflicting results have been given; for example, Jia et al. (2013) demonstrated that exogenous sucrose accelerated strawberry fruit ripening, whereas glucose showed a much smaller effect. The difference respond to glucose and sucrose between the current study and Jia et al. (2013) is probably due to different species, maturity stages, sugar-modulated gene expression and/or signaling transduction pathway. EBR-treated kiwifruit exhibited lower contents of glucose, fructose and sucrose during the most of storage time, which might be partial of the mechanism whereby EBR retards senescence (Fig. 4). A possible explanation is that soluble sugars can be involved in, or associated with, reactive oxygen species-generating metabolic pathways. In yeast and in mammalian cell, it has been confirmed that glucose autooxidation increased the generation of reactive oxygen species. Bolouri-Moghaddam et al. (2010) recently summarized a hypothetical sugar-antioxidant network operating in plant cells, with a central role appointed to glucose. For postharvest fruit, sucrose degradation and synthesis are the dominant parts of sugar metabolism; in this process, AI, NI, and SS are responsible for the cleavage of sucrose, while SPS catalyzes the synthesis of sucrose (Sturm, 1999; Miron, and Schaffer, 1991). MacRae et al. (1992) has proposed that there is a futile cycle of sucrose synthesis and degradation in senescing kiwifruit. Briefly, through the action of SPS, glucose produced by starch degradation is used to synthesize sucrose, that is then transported into the vacuole, where it is cleaved by AI to glucose and fructose. We herein the application of EBR inhibited the activity of SPS (Fig. 5D); furthermore, a positive correlation existed between SPS activity and sucrose content in EBR-treated kiwifruit (r = 0.9920), all of which explained the suppressed synthesis of sucrose by EBR treatment during storage. Also, EBR treatment was observed to be able to restrain AI, NI and SS activity (Fig. 5A, B and C), together a negative correlation of 0.8555 between SS activity and sucrose content, which were beneficial to less sucrose cleavage. There is good reason, then, to believe that EBR treatment weakens the futile cycle of sucrose
Fig. 4. Effect of EBR treatment on the content of glucose (A), fructose (B) and sucrose (C) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
in control fruit during storage. α-Amylase is a key enzyme for starch conversion, and the reaction it catalyzes is the hydrolysis of the internal α-1, 4-glucosidic linkage in the starch to produce soluble sugars; in kiwifruit, α-amylase activity has been studied in relation to starch degradation (Wegrzyn, and MacRae, 1995; Zhang et al., 2004). A rapid starch degradation or a higher α-amylase activity or both might 5
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Fig. 5. Effect of EBR treatment on the activity of AI (A), NI (B), SS (C), SPS (D), HK (E) and FK (F) in kiwifruit. Vertical bars represent the standard errors of the means of triplicate assays. The symbol (*) shows significantly different at P < 0.05 between control and treatment at the same day after harvest.
degradation. In conclusion, EBR at a concentration of 5 μM delayed the senescence of kiwifruit during ambient storage. EBR-mediated senescence may be derived from its capacity to maintain membrane lipid integrity, inhibit starch conversion as well as weaken the futile cycle of sucrose synthesis and degradation. Therefore, EBR treatment is a promising solution for kiwifruit to control postharvest senescence.
synthesis and degradation, and that SPS and SS tend to prevail over those of AI and NI in the regulation of sucrose metabolism, indicative that EBR treatment makes a significant contribution to senescence delay in kiwifruit through regulating SPS and SS activity. Similarly, Zhang et al. (2004) suggested that SPS may play a key role in sugar metabolism of kiwifruit, and it could be activated by hexoses and feedbackinhibited by sucrose; therefore, the inhibitory effect of acetylsalicylic acid treatment on the rapid increases in glucose, fructose and sucrose contents were related to inactivation of SPS and AI activity. It has been reported that the futile cycle of sucrose synthesis and degradation amplified the activation of SPS promoting the conversion of starch to soluble sugars (MacRae et al., 1992). This is likely to be the case in the current study since, as discussed above, both starch degradation and SPS activity were inhibited by EBR treatment. HK and FK are two enzymes responsible for glucose and fructose cleavage. In the current study, HK and FK activity were suppressed in EBR-treated kiwifruit (Fig. 5E and F), which theoretically should be more likely to cause accumulation of glucose and fructose. What is true, however, is that the contents of glucose and fructose in EBR-treated kiwifruit were lower than those in control during the whole storage; this might be because EBR treatment simultaneously weakens the conversion of starch and the futile cycle of sucrose synthesis and
Declarations of interest None. Acknowledgement This work was supported by the Project of Scientific and Technological of Shaanxi Province [grant numbers 2018NY-116]. References Aghdam, M.S., Motallebiazar, A., Mostofi, Y., Moghaddam, J.F., Ghasemnezhad, M., Erkan, M., Aksoy, U., 2010. Effects of MeSA vapor treatment on the postharvest quality of’ Hayward’ kiwifruit. Acta Hortic. 877 (877), 743–748. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage and
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