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Effects of hydrogen sulfide on yellowing and energy metabolism in broccoli
Postharvest Biology and Technology 129 (2017) 136–142
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Effects of hydrogen sulfide on yellowing and energy metabolism in broccoli a
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Dong Li , Li Li , Zhiwei Ge , Jarukitt Limwachiranon , Zhaojun Ban , Dongmei Yang , ⁎ Zisheng Luoa,
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a Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling Ministry of Agriculture, Zhejiang Key Laboratory for Agro-Food Processing, Yuhangtang Road 866, Hangzhou, 310058, People's Republic of China b Zhejiang University of Science and Technology, School of Biological and Chemical Engineering/School of Light Industry, Liuhe Road 318, Hangzhou, 310058, People's Republic of China c Hangzhou Wanxiang Polytechnic, Huawu Road 3, Hangzhou, 310023, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: Hydrogen sulfide Broccoli Yellowing Energy metabolism
The effects of H2S on yellowing and energy metabolism of broccoli florets treated with hydrogen sulfide (H2S) or DL-propargylglycine (PAG) were investigated after four days of storage at 20 °C. Our study showed that H2S treatment enhanced endogenous H2S content by 28.19% and 49.78% in comparison to the control and PAGtreated group respectively. This result might be related to the increase of L-cysteine desulfhydrase (LCD) and Dcysteine desulfhydrase (DCD) activities. Meanwhile, H2S treatment can maintain chlorophyll content at 0.329 g kg−1, whereas the control at 0.298 g kg−1 and PAG-treated group at 0.275 g kg−1. This led to an alleviation of the yellowing in broccoli florets. In addition, high endogenous H2S content also activated the key enzymes, involved in energy metabolism, including ATPases, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), glucokinase, fructokinase, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). As a result, significantly higher energy charge was observed in H2S-treated group (p < 0.05). These results suggest that H2S can effectively inhibit the yellowing and maintain high energy charge, therefore prolong the shelf life of postharvest broccoli.
1. Introduction Broccoli (Brassica oleracea var. Italica) is a high nutritive vegetable which contains significant content of vitamins, antioxidant substances, and anticarcinogenic compounds (Yuan et al., 2010). However, it turns to senescence quickly at ambient temperatures and thus reduces customer acceptance. Yellowing is a characteristic symptom of senescence in broccoli and occurs with chlorophyll breakdown (Fukasawa et al., 2010). Although several technologies such as 6-benzylaminopurine (Xu et al., 2012), sucrose treatment (Xu et al., 2016a) and 1-MCP (Xu et al., 2016b) have been reported to alleviate yellowing during storage, introduction and development of new methods to prolong the shelf life of broccoli are still in need. H2S is generally regarded as a toxic gas that smells like rotten eggs. However, accumulating evidences have indicated that only high sulfide concentrations cause toxic effects in cell, whereas low concentration of endogenous H2S plays a variety of physiological functions in both plants and animals (Doeller et al., 2001; Hancock and Whiteman, 2014) Consequently, H2S is qualified as the third gasotransmitter besides nitric oxide and carbon monoxide (Wang, 2003). Li et al. (2014)
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suggested that H2S could modulate antioxidant defense by maintaining high levels of metabolites and low accumulation of reactive oxygen species and malondialdehyde in broccoli, which delayed the postharvest senescence as a result. In addition, the effect of H2S on extending shelf life of grape (Ni et al., 2016), strawberry (Hu et al., 2012) and lotus root (Sun et al., 2015) has also been demonstrated. Recently energy supply, as a vital factor in controlling ripening and senescence in postharvest, has been wildly noticed. It was suggested that inadequate supplies of energy might be associated with membrane damage, which caused physiological disorders of postharvest horticultural crops and thus led to senescence and deterioration (Chen and Yang, 2013; Wang et al., 2013). Furthermore, glycolysis, the pentose phosphate pathway (PPP), the mitochondrial tricarboxylic acid (TCA) cycle, as well as electron transport chain are the central respiratory pathways, related to the supply of energy in plants (Vanlerberghe, 2013). Our previous study showed that H2S treatment enhanced the chilling tolerance of banana fruit by increasing the activities of enzymes, involved in energy metabolism in order to maintain energy charge during cold storage (Li et al., 2016a). However, to the best of our knowledge, H2S function in the yellowing and energy metabolism in
Corresponding author. E-mail address: [email protected] (Z. Luo).
http://dx.doi.org/10.1016/j.postharvbio.2017.03.017 Received 16 January 2017; Received in revised form 22 March 2017; Accepted 26 March 2017 0925-5214/ © 2017 Elsevier B.V. All rights reserved.
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2.5. L-Cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD) activities assay
postharvest broccoli is still unclear. The aim of the present study was to investigate the possible roles of H2S in yellowing of harvest broccoli as well as its regulation of energy status.
The LCD and DCD activities were measured using the method described by Riemenschneider et al. (2005) with some modifications. Two grams of frozen samples from each tray were ground in liquid nitrogen, followed by an addition of 8 mL of 20 mM Tris-HCl (pH 8.0). After centrifugation at 12,000 × g for 20 min, the soluble protein content of supernatant was collected and used for the following assay. For LCD activity measurement, 1 mL of supernatant was mixed with 1 mL of mixture solution containing 0.1 M Tris-HCl (pH 9.0), 2.5 mM dithiothreitol (DTT) and 0.8 mM L-cysteine and incubated at 37 °C for 15 min The reaction was terminated by adding 100 μL of 20 mM N,Ndimethyl-p-phenylenediamine dihydrochloride, dissolved in 7.2 M HCl and 100 μL of 30 mM FeCl3, dissolved in 1.2 M HCl. The absorbance of methylene blue at 667 nm was measured and Na2S solution was used to prepare a calibration curve. Similarly, the DCD activity was analyzed following the same methods with slight modifications: L-cysteine was replaced by D-cysteine in mixture solution, and the pH of the Tris-HCl was adjusted to 8.0.
2. Material and methods 2.1. Plant material and treatment Broccoli (B. oleracea L. var. Italica, cv. Lvyan) were harvested in Cixi District of Ningbo City and transported to the laboratory in Zhejiang University within two hours. After eliminating diseased and mechanical damaged ones, broccoli with uniform maturity and size were finally selected and randomly divided into three groups. In preliminary experiments, the concentration of H2S were 0.2, 0.4, 0.6, 0.8 and 1.0 mM in which 0.8 mM H2S was selected for this experiment according to the color evaluation. In this experiment, the first group was fumigated with 0.8 mM of H2S (NaHS as a donor) for 30 min at 20 °C, while the second group was sprayed with 0.5 mM of DLpropargylglycine (PAG) under the same condition. Distilled water was used as control (the third group). Every three broccoli florets were put on one plastic tray and wrapped with polyethylene films. All samples were stored in incubator (SANYO MIR-254, Panasonic Co., Ltd., Japan) at 20 °C with 85–90% relative humidity for 4 days in dark. Physiological parameters were measured every day. There were fifteen trays for each treatment, and each treatment conducted independently for three biological replications.
2.6. Assay of enzymes involved in energy metabolism The procedures of enzyme extract were conducted at 4 °C. For ATPase, cytochrome C oxidase (CCO) and succinate dehydrogenase (SDH), 20 g of frozen samples from each tray were ground in ice-bath with 30 mL of 50 mM Tris-HCl containing 0.25 M sucrose, 0.3 M mannitol, 1 M EDTA, 0.1% bovine serum albumin, 0.1% cysteine and 5 g L−1 polyvinyl pyrrolidone. After centrifugation at 4000 × g for 10 min, the supernatant was collected and centrifuged again at 12,000 × g for 10 min to precipitate the mitochondria. The sediment was washed twice with 5 mL of 10 mM Tris-HCl containing 1 mM EDTA, 0.3 M mannitol and 0.25 M sucrose, and centrifuged at 12,000 × g for 10 min Finally, the sediment was collected and dissolved with 4 mL of washing buffer. For glucokinase and fructokinase, 3.0 g of frozen samples from each tray were ground in ice-bath with 6 mL of 0.1 M Tris-HCl (pH 8.0) containing 10 mM KCl, 5 mM EDTA, 2 mM DTT, 1 mM MgCl2 and 10% glycerine, and centrifuged at 12,000 × g for 20 min The supernatant was collected. For glucose-6phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), 2.0 g of frozen samples from each tray were ground in ice-bath with 6 mL of 0.1 M Tris-HCl (pH 8.0) containing 2 mM MgCl2, 1 mM DTT, 1 mM EDTA and 1% PVP, and centrifuged at 12,000 × g for 20 min The supernatant was collected. H+-ATPase and Ca2+-ATPase activities were assayed according to the method of Jin et al. (2013). One unit of H+-ATPase or Ca2+-ATPase was defined as production of 1 μmol phosphorus/min. CCO activity was determined using the method of Jin et al. (2013). One unit of CCO was defined as an increase of 0.01 in absorption at 510 mm/min. SDH activity was assayed by the method of Acevedo et al. (2013). One unit of SDH was defined as an increase of 0.01 in absorption at 600 mm/min. Glucokinase and fructokinase activities were estimated according to the method of Schaffer and Petreikov (1997). One unit of glucokinase and fructokinase was defined as the amount of enzyme that causes a change of 0.01 in absorption at 340 nm/min when fructose and glucose were used as the substrate respectively. G6PDH and 6PGDH activities were carried out using the Sgherri et al. (2002) method. One unit of G6PDH and 6PGDH was calculated from the change of 0.01 in absorption at 340 mm/min when glucose-6phosphate and 6-phosphogluconate were used as the substrate respectively.
2.2. Color evaluation The value of L*, a* and b* was measured by a colorimeter (KONICA MINOLTA, CR-400, Japan). Three broccoli florets from each replicate were randomly selected and each broccoli floret was measured three times on different areas. The value of H* was calculated as H* = arctan (b*/a*). 2.3. Total chlorophyll content assay Total chlorophyll content was measured using the method described by Zhang et al. (2015) with some modifications. Five grams of frozen samples from each tray were ground in liquid nitrogen and extracted by adding 10 mL of 80% chilled acetone for 30 min in darkness. After centrifugation at 12,000 × g for 10 min, the supernatant was collected. This procedure was repeated until the residue turned white. Finally, the volume was made up to 25 mL using 80% acetone and the absorbance at 663 nm and 645 nm was measured by ultraviolet and visible spectrophotometer (UV-1750, Shimadzu Co., Ltd., Japan). The total chlorophyll content was presented as 20.29A663 + 8.05A645. 2.4. Endogenous H2S content assay The endogenous H2S was determined according to the method of Sekiya et al. (1982). Five grams of frozen samples from each tray were ground in liquid nitrogen, followed by an addition of 20 mL of 50 mM phosphate buffered saline (PBS, pH 6.8), containing 0.2 M ascorbic acid and 0.1 M EDTA. After centrifugation at 10,000 × g for 20 min, the supernatants were mixed in a test cube containing 0.1 M PBS (pH 7.4), 2 mM phosphopyridoxal and 10 mM L-cysteine. The released H2S was absorbed in a zinc acetate trap which located at the bottom of the test tube. After 30 min of reaction, 0.3 mL of 5 mM dimethyl-p-phenylenediamine in 3.5 mM H2SO4 was injected into the trap. Afterwards, 0.3 mL of 50 mM NH4Fe(SO4)2 in 0.1 M H2SO4 was injected to form methylene blue. The absorbance at 667 nm was measured after an incubation of 15 min at ambient temperature and Na2S solution was used to prepare a calibration curve. The endogenous H2S content was presented as μmol kg−1 fresh weight. 137
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2.7. ATP, ADP and AMP contents and energy charge assay The ATP, ADP and AMP contents were measured according to the method described by Liu et al. (2006). Two grams of frozen samples from each tray were ground in 5 mL of 0.6 M perchloric acid in an icebath. After centrifugation at 12,000 × g for 20 min, 3 mL of supernatant was collected and neutralized to pH 6.8 with 1 M KOH. Afterwards, the supernatant was allowed to stand for 30 min in an ice-bath before centrifuged at 10,000 × g for 10 min to remove the potassium perchlorate. Then, the supernatant was adjusted to 4 mL and filtered through a 0.45-μm filter for the following assays. The content of ATP, ADP and AMP was determined by a high-performance liquid chromatography (HPLC, LC-2010A, Shimadzu Corporation, Kyoto, Japan) equipped with an ultraviolet detector and a reverse-phase Luna 5 μm C 18 column (4.6 mm × 250 mm, Phenomenex, Torrance, CA). The detecting parameters were as follows: the mobile phase A was the phosphate buffer (pH 7.0), the mobile phase B was methyl alcohol, the injection volume was 20 μL and the flow rate of mobile phase was 1.0 mL min−1. The peaks at 254 nm were detected and the contents of ATP, ADP and AMP were analyzed using the external standard method. Energy charge was presented as [ATP + 1/2ADP]/[ATP + ADP + AMP]. 2.8. Statistical analysis Experiments were conducted using a completely randomized design. All statistical analyses were processed using SPSS (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA) was performed and the differences were considered significant at p < 0.05. 3. Result 3.1. Changes in color index and total chlorophyll content The value of L* increased and the value of H* decreased with storage time. H2S treatment postponed the change of color index while PAG treatment aggravated it. After four days of storage, the values of L* in H2S-treated group, PAG-treated group and control group were 49.63, 57.06 and 53.69 respectively, and the values of H* were 111.63, 98.26 and 104.69 respectively (Fig. 1A and B). Chlorophyll in florets degraded gradually over the storage period. However, the decrease was significantly delayed by H2S treatment (p < 0.05). The content of total chlorophyll in H2S-treated group was 10.40% higher than that in control on day 4. On the contrary, PAG treatment accelerated the degradation of chlorophyll and 7.72% lower of total chlorophyll was observed on day 4 compared with control (Fig. 1C). 3.2. Changes in endogenous H2S content, and the activities of LCD and DCD As shown in Fig. 2A, the endogenous H2S content decreased gradually during the storage while PAG, an inhibitor mainly responsible for H2S synthesis, accelerated the decrease. However, H2S treatment increased the endogenous H2S initially and then decreased after the second day. After four days of storage, the endogenous H2S in H2Streated broccoli florets was 10.14 μmol kg−1, significantly higher than that in control and PAG-treated samples (p < 0.05). Activities of LCD and DCD, the two key enzymes responsible for H2S synthesis, decreased during storage time in all groups. However, significantly higher activities were observed in H2S-treated group (p < 0.05). LCD and DCD activities in H2S-treated group were 36.09% and 19.19%, respectively which were higher than those in control at the end of the storage. Conversely, PAG treatment accelerated the decrease of LCD and DCD activities compared with control (Fig. 2B and C).
Fig. 1. Effect of H2S treatment on the value of L* (A) and H* (B), and chlorophyll content (C) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
3.3. Changes in enzymes involved in energy metabolism During the storage, the activities of H+-ATPase, Ca2+-ATPase, SDH, glucokinase and fructokinase decreased continuously while the activity of CCO increased (Fig. 3A–F). G6PDH activity increased initially and then decreased after the third day (Fig. 3G). Similar tendency was observed in 6PGDH and the maximum activity appeared at the end of 138
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lower than those in control (p < 0.05). However, there were no significant differences in H+-ATPase, Ca2+-ATPase, CCO, SDH, glucokinase and fructokinase activities between PAG-treated group and control on day 4. 3.4. Changes in ATP, ADP and AMP contents, and energy charge After harvest, contents of ATP and ADP level decreased gradually while AMP content increased in all groups (Fig. 4A–C). As a result, the energy charge in broccoli florets decreased during storage (Fig. 4D). H2S treatment maintained high levels of ATP and ADP, and inhibited the increase of AMP. The level of energy charge was 18.81% higher in H2S-treated broccoli florets compared with that in control (p < 0.05). PAG treatment, however, accelerated the degradation of ATP, ADP and the accumulation of AMP during initial storage. At the end of the storage, PAG-treated group showed significantly lower ATP content compared with control (p < 0.05), while there were no significant differences in ADP and AMP contents between two groups. The level of energy charge in PAG-treated group was 67.78, significantly lower than control (p < 0.05). 4. Discussion Recent studies have demonstrated that H2S was a signaling gaseous transmitter involved in many important biological roles such as chilling tolerance (Fu et al., 2013), heat tolerance (Li et al., 2013), antioxidant capacities (Hu et al., 2014), and thus related closely to the ripening and senescence in plants. In our present study, H2S treatment enhanced the endogenous H2S content in broccoli florets during storage (Fig. 2A). Meanwhile, the activities of LCD and DCD were also induced by H2S treatment (Fig. 2B and C). LCD and DCD are two key enzymes responsible for H2S synthesis. H2S treatment triggered endogenous H2S accumulation probably by increasing the activities of LCD and DCD (Fu et al., 2013). To further confirm the effect of endogenous H2S in broccoli florets, PAG-treated group was also observed. As H2S synthesis inhibitor, PAG inhibited the activities of LCD and DCD, which resulted in the decrease of endogenous H2S content (Fig. 2A–C). Similar results were reported by Hu et al. (2015) in water spinach leaves treated with exogenous H2S and PAG. Yellowing in broccoli is a characteristics symptom of senescence, which can be observed by an increase of L* and a decrease of H* during storage (Fig. 1A and B). However, H2S treatment postponed the change of color index. Previous study indicated that H2S fumigation could retard the decline of chlorophyll content in climacteric fruit, and prolong the postharvest life (Zhu et al., 2014). As illustrated in Fig. 1C, H2S treatment inhibited the degradation of total chlorophyll while PAG treatment accelerated it conversely. Hu et al. (2015) pointed out the activities of chlorophyll-degrading enzyme, chlorophyllase and Mg-dechelatase, might be impaired by H2S, thereby H2S fumigation alleviated the degradation of chloroplast after harvest. PAG treatment effectively inhibited the synthesis of endogenous H2S, leading to the enhancement of the chlorophyll-degrading enzymes activities, and finally accelerated chlorophyll degradation and yellowing as a result. Similarly, Xu et al. (2016a,b) showed that sucrose (12 g L−1) treatment inhibited an increase of L* value, retained a high hue angle and chlorophyll content in broccoli florets, resulted in extended shelf life. Energy metabolism plays vital roles in regulation of ripening and senescence in postharvest. It suggested that energy deficiency could induce lipid peroxidation and damage the integrity of membrane, which may account for the reduction of disease resistance and senescence (Yi et al., 2010; Shan et al., 2016). Pervious study indicated endogenous H2S remains a regulator of energy production in eukaryote cells under stress conditions, which enables the creature to deal with energy demand (Fu et al., 2012). In present study, energy charge in broccoli florets decreased gradually during storage along with the decrease of ATP, ADP and the increase of AMP. H2S with low
Fig. 2. Effect of H2S treatment on endogenous H2S content (A), L-cysteine desulfhydrase (LCD) activity (B) and D-cysteine desulfhydrase (DCD) activity (C) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
first day (Fig. 3H). H2S treatment enhanced all of the above enzymes significantly (p < 0.05). After four days of storage, the activities of H+-ATPase, Ca2+-ATPase, CCO, SDH, glucokinase, fructokinase, G6PDH and 6PGDH in H2S-treated group were 18.05%, 23.41%, 24.73%, 41.52%, 37.23%, 12.09%, 21.28% and 33.95% higher than those in control respectively. Conversely, PAG treatment inhibited these enzymes activities in the initial storage. At the end of storage, the activities of G6PDH and 6PGDH were 19.42% and 12.84%, respectively which were 139
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Fig. 3. Effect of H2S treatment on the activities of H+-ATPase (A), Ca2+-ATPase (B), cytochrome C oxidase (CCO) (C), succinate dehydrogenase (SDH) (D), glucokinase (E), fructokinase (F), glucose-6-phosphate dehydrogenase (G6PDH) (G) and 6-phosphogluconate dehydrogenase (6PGDH) (H) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
To further clarify the participation of H2S in the regulation of energy metabolism, eight enzymes involved in ATP synthesis and degradation were investigated. H+-ATPase, a proton pump energized by ATP hydrolysis, extrudes H+ and forms the membrane potential, which helps transport nutrients into cell (Palmgren, 2001). Ca2+ acts as a “second messenger” in plant signaling while high level of Ca2+ might cause structural damage and metabolism dysfunction. Ca2+-ATPase is responsible for active Ca2+ transport driven by hydrolysis of ATP, which maintains the low level of cytosolic Ca2+ (Muchhal et al., 1997).
concentration acted as an electron donor and induced the synthesis of ATP (Goubern et al., 2007). Thus, exogenous H2S treatment maintained high ATP contents which help inhibited a decrease of energy charge. Simultaneously, PAG treatment further confirmed the speculation. PAG treatment inhibited the synthesis of endogenous H2S, reduced the production of ATP, and finally resulted in energy deficiency (Fig. 4A–D). Li et al. (2016b) reported controlled atmospheres (50% O2 + 50% CO2) delayed the senescence of broccoli and extended the storage period by inhibiting reduction of ATP and energy charge. 140
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Fig. 4. Effect of H2S treatment on the contents of ATP (A), ADP (B), AMP (C), and energy charge (D) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
In present study, high levels of H+-ATPase and Ca2+-ATPase activities were observed in broccoli florets treated with H2S while PAG aggravated the decrease of both ATPases during initial storage (Fig. 3A and B). The results suggested that H2S inhibited the decrease of ATPases by improving energy status, and thus maintained the integrity of membrane, and alleviated metabolism dysfunction as a result (Azevedo et al., 2008). These might at least partially protect plants against stress after harvest. Jin et al. (2014) reported enhanced activities of H+ATPase and Ca2+-ATPase retarded the energy charge decline and therefore increased chilling tolerance. Respiratory enzymes are essential for different respiration pathways, which can provide energy and support biological reaction. CCO, the terminal enzyme of the electron transport chain, acts as an electrondriven proton pump and plays important roles in energy production (Soto et al., 2012). SDH is a TCA circle as well as aerobic electron transport chain enzyme, which catalyzes the reduction of ubiquinone to the oxidation of succinate (Acevedo et al., 2013). Changes in SDH and CCO activities might disturb electron flow through electron transport chain and thus impact on energy metabolism (Zhou et al., 2014). Significantly higher activities of CCO and SDH were observed in H2Streated group, compared with control. Opposite effect was obtained in broccoli florets treated with PAG due to an inhibition of H2S production (Fig. 3C and D). Previous study indicated low-temperature condition treatment played positive roles in maintaining high levels of CCO and SDH in loquat fruit during cold storage (Jin et al., 2015). Glycolysis, a multi-enzyme pathway, responsible for the conversion of monosaccharides to pyruvic acid, is a ubiquitous feature of energy metabolism (Givan, 1999). Hexokinase as an essential enzyme catalyzes phosphorylation of hexose by ATP, which is the first step in utilization of hexose in all eukaryotic cells. Glucokinase and fructokinase exist as separate enzymes that act on glucose and fructose, respectively (Cárdenas et al., 1998). As depicted in Fig. 3E and F, the activities of glucokinase and fructokinase were significantly higher in H2S-treated broccoli florets than those in control. Simultaneously, PAG treatment accelerated the decrease of glucokinase and fructokinase activities and reached a low level rapidly by decreasing endogenous H2S content.
Wang et al. (2015) found the elevated activities of glucokinase and fructokinase might contribute to induce chilling tolerance of NO-treated banana fruit. PPP is another fundamental component of cellular metabolism that can reduce NADP+ to NADPH while converting glucose-6-phosphate to CO2 and pentose phosphate (Wamelink et al., 2008). G6PDH, the NADP+-dependent oxidoreductase, has been quoted as being rate limiting for the oxidative branch of the PPP, and 6PGDH is the second dehydrogenase of the hexose monophosphate shunt, which is vital for the flow through the PPP (Rosemeyer, 1987; Stincone et al., 2015). In our present study, high levels of G6PDH and 6PGDH activities in broccoli florets were induced by H2S treatment while PAG treatment showed the opposite effect (Fig. 3G and H). The activation of enzymes involved in the PPP might contribute to several important functions in cellular metabolism, including providing of precursors for nucleotide and amino acid biosynthesis, maintaining of carbon homoeostasis, providing of reducing molecules for anabolism, as well as defeating of oxidative stress, and thus postponing of senescence in postharvest (Stincone et al., 2015).
5. Conclusions In conclusion, our present study showed that H2S treatment induced the synthesis of endogenous H2S by activating LCD and DCD in broccoli florets during storage at 20 °C. High level of H2S content inhibited the degradation of chlorophyll, thus alleviated yellowing of broccoli florets as a result. Meanwhile, H2S treatment may also play important roles in maintaining high levels of ATP and energy charge, which was ascribed to the activation of enzymes involved in energy metabolic pathways including glycolysis, TCA circle, electron transport chain and PPP, which finally postponed senescence in postharvest. However, further investigation on how energy metabolism dysfunction directly related to the yellowing in broccoli florets is needed.
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Acknowledgements The research was financially supported by the National Natural Science Foundation of China (31371856, 31571895), the National Key Research and Development Program of China (2016YFD0401201) and Hangzhou Science and Technology Development Program (20160432B23). References Acevedo, R.M., Maiale, S.J., Pessino, S.C., Bottini, R., Ruiz, O.A., Sansberro, P.A., 2013. A succinate dehydrogenase flavoprotein subunit-like transcript is upregulated in Ilex paraguariensis leaves in response to water deficit and abscisic acid. Plant Physiol. Biochem. 65, 48–54. Azevedo, I.G., Oliveira, J.G., Da Silva, M.G., Pereira, T., Correa, S.R., Vargas, H., Facanha, A.R., 2008. P-type H+-ATPases activity, membrane integrity, and apoplastic pH during papaya fruit ripening. Postharvest Biol. Technol. 48, 242–247. Cárdenas, M.L., Cornish-Bowden, A., Ureta, T., 1998. Evolution and regulatory role of the hexokinase. Biochim. Biophys. Acta: Mol. Cell Res. 1401, 242–264. Chen, B., Yang, H., 2013. 6-Benzylaminopurine alleviates chilling injury of postharvest cucumber fruit through modulating antioxidant system and energy status. J. Sci. Food Agric. 93, 1915–1921. Doeller, J.E., Grieshaber, M.K., Kraus, D.W., 2001. Chemolithoheterotrophy in a metazoan tissue: thiosulfate production matches ATP demand in ciliated mussel gills. J. Exp. Biol. 204, 3755–3764. Fu, M., Zhang, W., Wu, L., Yang, G., Li, H., Wang, R., 2012. Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production. Proc. Natl. Acad. Sci. U.S.A. 109, 2943–2948. Fu, P., Wang, W., Hou, L., Liu, X., 2013. Hydrogen sulfide is involved in the chilling stress response in Vitis vinifera L. Acta Soc. Bot. Pol. 82, 295–302. Fukasawa, A., Suzuki, Y., Terai, H., Yamauchi, N., 2010. Effects of postharvest ethanol vapor treatment on activities and gene expression of chlorophyll catabolic enzymes in broccoli florets. Postharvest Biol. Technol. 55, 97–102. Givan, C.V., 1999. Evolving concepts in plant glycolysis: two centuries of progress. Biol. Rev. 74, 277–309. Goubern, M., Andriamihaja, M., Nubel, T., Blachier, F., Bouillaud, F., 2007. Sulfide, the first inorganic substrate for human cells. FASEB J. 21, 1699–1706. Hancock, J.T., Whiteman, M., 2014. Hydrogen sulfide and cell signaling: team player or referee? Plant Physiol. Biochem. 78, 37–42. Hu, L., Hu, S., Wu, J., Li, Y., Zheng, J., Wei, Z., Liu, J., Wang, H., Liu, Y., Zhang, H., 2012. Hydrogen sulfide prolongs postharvest shelf life of strawberry and plays an antioxidative role in fruits. J. Agric. Food Chem. 60, 8684–8693. Hu, K., Wang, Q., Hu, L., Gao, S., Wu, J., Li, Y., Zheng, J., Han, Y., Liu, Y., Zhang, H., 2014. Hydrogen sulfide prolongs postharvest storage of fresh-cut pears (Pyrus pyrifolia) by alleviation of oxidative damage and inhibition of fungal growth. PLoS One 9. Hu, H., Liu, D., Li, P., Shen, W., 2015. Hydrogen sulfide delays leaf yellowing of stored water spinach (Ipomoea aquatica) during dark-induced senescence by delaying chlorophyll breakdown, maintaining energy status and increasing antioxidative capacity. Postharvest Biol. Technol. 108, 8–20. Jin, P., Zhu, H., Wang, J., Chen, J., Wang, X., Zheng, Y., 2013. Effect of methyl jasmonate on energy metabolism in peach fruit during chilling stress. J. Sci. Food Agric. 93, 1827–1832. Jin, P., Zhu, H., Wang, L., Shan, T., Zheng, Y., 2014. Oxalic acid alleviates chilling injury in peach fruit by regulating energy metabolism and fatty acid contents. Food Chem. 161, 87–93. Jin, P., Zhang, Y., Shan, T., Huang, Y., Xu, J., Zheng, Y., 2015. Low-temperature conditioning alleviates chilling injury in loquat fruit and regulates glycine betaine content and energy status. J. Agric. Food Chem. 63, 3654–3659. Li, Z., Ding, X., Du, P., 2013. Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline. J. Plant Physiol. 170, 741–747. Li, S., Hu, K., Hu, L., Li, Y., Jiang, A., Xiao, F., Han, Y., Liu, Y., Zhang, H., 2014. Hydrogen sulfide alleviates postharvest senescence of broccoli by modulating antioxidant defense and senescence-related gene expression. J. Agric. Food Chem. 62, 1119–1129. Li, D., Limwachiranon, J., Li, L., Du, R., Luo, Z., 2016a. Involvement of energy metabolism to chilling tolerance induced by hydrogen sulfide in cold-stored banana fruit. Food Chem. 208, 272–278. Li, L., Lv, F., Guo, Y., Wang, Z., 2016b. Respiratory pathway metabolism and energy metabolism associated with senescence in postharvest Broccoli (Brassica oleracea L. var. italica) florets in response to O2/CO2 controlled atmospheres. Postharvest Biol. Technol. 111, 330–336.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effects of hydrogen sulfide on yellowing and energy metabolism in broccoli a
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Dong Li , Li Li , Zhiwei Ge , Jarukitt Limwachiranon , Zhaojun Ban , Dongmei Yang , ⁎ Zisheng Luoa,
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a Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling Ministry of Agriculture, Zhejiang Key Laboratory for Agro-Food Processing, Yuhangtang Road 866, Hangzhou, 310058, People's Republic of China b Zhejiang University of Science and Technology, School of Biological and Chemical Engineering/School of Light Industry, Liuhe Road 318, Hangzhou, 310058, People's Republic of China c Hangzhou Wanxiang Polytechnic, Huawu Road 3, Hangzhou, 310023, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: Hydrogen sulfide Broccoli Yellowing Energy metabolism
The effects of H2S on yellowing and energy metabolism of broccoli florets treated with hydrogen sulfide (H2S) or DL-propargylglycine (PAG) were investigated after four days of storage at 20 °C. Our study showed that H2S treatment enhanced endogenous H2S content by 28.19% and 49.78% in comparison to the control and PAGtreated group respectively. This result might be related to the increase of L-cysteine desulfhydrase (LCD) and Dcysteine desulfhydrase (DCD) activities. Meanwhile, H2S treatment can maintain chlorophyll content at 0.329 g kg−1, whereas the control at 0.298 g kg−1 and PAG-treated group at 0.275 g kg−1. This led to an alleviation of the yellowing in broccoli florets. In addition, high endogenous H2S content also activated the key enzymes, involved in energy metabolism, including ATPases, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), glucokinase, fructokinase, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). As a result, significantly higher energy charge was observed in H2S-treated group (p < 0.05). These results suggest that H2S can effectively inhibit the yellowing and maintain high energy charge, therefore prolong the shelf life of postharvest broccoli.
1. Introduction Broccoli (Brassica oleracea var. Italica) is a high nutritive vegetable which contains significant content of vitamins, antioxidant substances, and anticarcinogenic compounds (Yuan et al., 2010). However, it turns to senescence quickly at ambient temperatures and thus reduces customer acceptance. Yellowing is a characteristic symptom of senescence in broccoli and occurs with chlorophyll breakdown (Fukasawa et al., 2010). Although several technologies such as 6-benzylaminopurine (Xu et al., 2012), sucrose treatment (Xu et al., 2016a) and 1-MCP (Xu et al., 2016b) have been reported to alleviate yellowing during storage, introduction and development of new methods to prolong the shelf life of broccoli are still in need. H2S is generally regarded as a toxic gas that smells like rotten eggs. However, accumulating evidences have indicated that only high sulfide concentrations cause toxic effects in cell, whereas low concentration of endogenous H2S plays a variety of physiological functions in both plants and animals (Doeller et al., 2001; Hancock and Whiteman, 2014) Consequently, H2S is qualified as the third gasotransmitter besides nitric oxide and carbon monoxide (Wang, 2003). Li et al. (2014)
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suggested that H2S could modulate antioxidant defense by maintaining high levels of metabolites and low accumulation of reactive oxygen species and malondialdehyde in broccoli, which delayed the postharvest senescence as a result. In addition, the effect of H2S on extending shelf life of grape (Ni et al., 2016), strawberry (Hu et al., 2012) and lotus root (Sun et al., 2015) has also been demonstrated. Recently energy supply, as a vital factor in controlling ripening and senescence in postharvest, has been wildly noticed. It was suggested that inadequate supplies of energy might be associated with membrane damage, which caused physiological disorders of postharvest horticultural crops and thus led to senescence and deterioration (Chen and Yang, 2013; Wang et al., 2013). Furthermore, glycolysis, the pentose phosphate pathway (PPP), the mitochondrial tricarboxylic acid (TCA) cycle, as well as electron transport chain are the central respiratory pathways, related to the supply of energy in plants (Vanlerberghe, 2013). Our previous study showed that H2S treatment enhanced the chilling tolerance of banana fruit by increasing the activities of enzymes, involved in energy metabolism in order to maintain energy charge during cold storage (Li et al., 2016a). However, to the best of our knowledge, H2S function in the yellowing and energy metabolism in
Corresponding author. E-mail address: [email protected] (Z. Luo).
http://dx.doi.org/10.1016/j.postharvbio.2017.03.017 Received 16 January 2017; Received in revised form 22 March 2017; Accepted 26 March 2017 0925-5214/ © 2017 Elsevier B.V. All rights reserved.
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2.5. L-Cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD) activities assay
postharvest broccoli is still unclear. The aim of the present study was to investigate the possible roles of H2S in yellowing of harvest broccoli as well as its regulation of energy status.
The LCD and DCD activities were measured using the method described by Riemenschneider et al. (2005) with some modifications. Two grams of frozen samples from each tray were ground in liquid nitrogen, followed by an addition of 8 mL of 20 mM Tris-HCl (pH 8.0). After centrifugation at 12,000 × g for 20 min, the soluble protein content of supernatant was collected and used for the following assay. For LCD activity measurement, 1 mL of supernatant was mixed with 1 mL of mixture solution containing 0.1 M Tris-HCl (pH 9.0), 2.5 mM dithiothreitol (DTT) and 0.8 mM L-cysteine and incubated at 37 °C for 15 min The reaction was terminated by adding 100 μL of 20 mM N,Ndimethyl-p-phenylenediamine dihydrochloride, dissolved in 7.2 M HCl and 100 μL of 30 mM FeCl3, dissolved in 1.2 M HCl. The absorbance of methylene blue at 667 nm was measured and Na2S solution was used to prepare a calibration curve. Similarly, the DCD activity was analyzed following the same methods with slight modifications: L-cysteine was replaced by D-cysteine in mixture solution, and the pH of the Tris-HCl was adjusted to 8.0.
2. Material and methods 2.1. Plant material and treatment Broccoli (B. oleracea L. var. Italica, cv. Lvyan) were harvested in Cixi District of Ningbo City and transported to the laboratory in Zhejiang University within two hours. After eliminating diseased and mechanical damaged ones, broccoli with uniform maturity and size were finally selected and randomly divided into three groups. In preliminary experiments, the concentration of H2S were 0.2, 0.4, 0.6, 0.8 and 1.0 mM in which 0.8 mM H2S was selected for this experiment according to the color evaluation. In this experiment, the first group was fumigated with 0.8 mM of H2S (NaHS as a donor) for 30 min at 20 °C, while the second group was sprayed with 0.5 mM of DLpropargylglycine (PAG) under the same condition. Distilled water was used as control (the third group). Every three broccoli florets were put on one plastic tray and wrapped with polyethylene films. All samples were stored in incubator (SANYO MIR-254, Panasonic Co., Ltd., Japan) at 20 °C with 85–90% relative humidity for 4 days in dark. Physiological parameters were measured every day. There were fifteen trays for each treatment, and each treatment conducted independently for three biological replications.
2.6. Assay of enzymes involved in energy metabolism The procedures of enzyme extract were conducted at 4 °C. For ATPase, cytochrome C oxidase (CCO) and succinate dehydrogenase (SDH), 20 g of frozen samples from each tray were ground in ice-bath with 30 mL of 50 mM Tris-HCl containing 0.25 M sucrose, 0.3 M mannitol, 1 M EDTA, 0.1% bovine serum albumin, 0.1% cysteine and 5 g L−1 polyvinyl pyrrolidone. After centrifugation at 4000 × g for 10 min, the supernatant was collected and centrifuged again at 12,000 × g for 10 min to precipitate the mitochondria. The sediment was washed twice with 5 mL of 10 mM Tris-HCl containing 1 mM EDTA, 0.3 M mannitol and 0.25 M sucrose, and centrifuged at 12,000 × g for 10 min Finally, the sediment was collected and dissolved with 4 mL of washing buffer. For glucokinase and fructokinase, 3.0 g of frozen samples from each tray were ground in ice-bath with 6 mL of 0.1 M Tris-HCl (pH 8.0) containing 10 mM KCl, 5 mM EDTA, 2 mM DTT, 1 mM MgCl2 and 10% glycerine, and centrifuged at 12,000 × g for 20 min The supernatant was collected. For glucose-6phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), 2.0 g of frozen samples from each tray were ground in ice-bath with 6 mL of 0.1 M Tris-HCl (pH 8.0) containing 2 mM MgCl2, 1 mM DTT, 1 mM EDTA and 1% PVP, and centrifuged at 12,000 × g for 20 min The supernatant was collected. H+-ATPase and Ca2+-ATPase activities were assayed according to the method of Jin et al. (2013). One unit of H+-ATPase or Ca2+-ATPase was defined as production of 1 μmol phosphorus/min. CCO activity was determined using the method of Jin et al. (2013). One unit of CCO was defined as an increase of 0.01 in absorption at 510 mm/min. SDH activity was assayed by the method of Acevedo et al. (2013). One unit of SDH was defined as an increase of 0.01 in absorption at 600 mm/min. Glucokinase and fructokinase activities were estimated according to the method of Schaffer and Petreikov (1997). One unit of glucokinase and fructokinase was defined as the amount of enzyme that causes a change of 0.01 in absorption at 340 nm/min when fructose and glucose were used as the substrate respectively. G6PDH and 6PGDH activities were carried out using the Sgherri et al. (2002) method. One unit of G6PDH and 6PGDH was calculated from the change of 0.01 in absorption at 340 mm/min when glucose-6phosphate and 6-phosphogluconate were used as the substrate respectively.
2.2. Color evaluation The value of L*, a* and b* was measured by a colorimeter (KONICA MINOLTA, CR-400, Japan). Three broccoli florets from each replicate were randomly selected and each broccoli floret was measured three times on different areas. The value of H* was calculated as H* = arctan (b*/a*). 2.3. Total chlorophyll content assay Total chlorophyll content was measured using the method described by Zhang et al. (2015) with some modifications. Five grams of frozen samples from each tray were ground in liquid nitrogen and extracted by adding 10 mL of 80% chilled acetone for 30 min in darkness. After centrifugation at 12,000 × g for 10 min, the supernatant was collected. This procedure was repeated until the residue turned white. Finally, the volume was made up to 25 mL using 80% acetone and the absorbance at 663 nm and 645 nm was measured by ultraviolet and visible spectrophotometer (UV-1750, Shimadzu Co., Ltd., Japan). The total chlorophyll content was presented as 20.29A663 + 8.05A645. 2.4. Endogenous H2S content assay The endogenous H2S was determined according to the method of Sekiya et al. (1982). Five grams of frozen samples from each tray were ground in liquid nitrogen, followed by an addition of 20 mL of 50 mM phosphate buffered saline (PBS, pH 6.8), containing 0.2 M ascorbic acid and 0.1 M EDTA. After centrifugation at 10,000 × g for 20 min, the supernatants were mixed in a test cube containing 0.1 M PBS (pH 7.4), 2 mM phosphopyridoxal and 10 mM L-cysteine. The released H2S was absorbed in a zinc acetate trap which located at the bottom of the test tube. After 30 min of reaction, 0.3 mL of 5 mM dimethyl-p-phenylenediamine in 3.5 mM H2SO4 was injected into the trap. Afterwards, 0.3 mL of 50 mM NH4Fe(SO4)2 in 0.1 M H2SO4 was injected to form methylene blue. The absorbance at 667 nm was measured after an incubation of 15 min at ambient temperature and Na2S solution was used to prepare a calibration curve. The endogenous H2S content was presented as μmol kg−1 fresh weight. 137
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2.7. ATP, ADP and AMP contents and energy charge assay The ATP, ADP and AMP contents were measured according to the method described by Liu et al. (2006). Two grams of frozen samples from each tray were ground in 5 mL of 0.6 M perchloric acid in an icebath. After centrifugation at 12,000 × g for 20 min, 3 mL of supernatant was collected and neutralized to pH 6.8 with 1 M KOH. Afterwards, the supernatant was allowed to stand for 30 min in an ice-bath before centrifuged at 10,000 × g for 10 min to remove the potassium perchlorate. Then, the supernatant was adjusted to 4 mL and filtered through a 0.45-μm filter for the following assays. The content of ATP, ADP and AMP was determined by a high-performance liquid chromatography (HPLC, LC-2010A, Shimadzu Corporation, Kyoto, Japan) equipped with an ultraviolet detector and a reverse-phase Luna 5 μm C 18 column (4.6 mm × 250 mm, Phenomenex, Torrance, CA). The detecting parameters were as follows: the mobile phase A was the phosphate buffer (pH 7.0), the mobile phase B was methyl alcohol, the injection volume was 20 μL and the flow rate of mobile phase was 1.0 mL min−1. The peaks at 254 nm were detected and the contents of ATP, ADP and AMP were analyzed using the external standard method. Energy charge was presented as [ATP + 1/2ADP]/[ATP + ADP + AMP]. 2.8. Statistical analysis Experiments were conducted using a completely randomized design. All statistical analyses were processed using SPSS (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA) was performed and the differences were considered significant at p < 0.05. 3. Result 3.1. Changes in color index and total chlorophyll content The value of L* increased and the value of H* decreased with storage time. H2S treatment postponed the change of color index while PAG treatment aggravated it. After four days of storage, the values of L* in H2S-treated group, PAG-treated group and control group were 49.63, 57.06 and 53.69 respectively, and the values of H* were 111.63, 98.26 and 104.69 respectively (Fig. 1A and B). Chlorophyll in florets degraded gradually over the storage period. However, the decrease was significantly delayed by H2S treatment (p < 0.05). The content of total chlorophyll in H2S-treated group was 10.40% higher than that in control on day 4. On the contrary, PAG treatment accelerated the degradation of chlorophyll and 7.72% lower of total chlorophyll was observed on day 4 compared with control (Fig. 1C). 3.2. Changes in endogenous H2S content, and the activities of LCD and DCD As shown in Fig. 2A, the endogenous H2S content decreased gradually during the storage while PAG, an inhibitor mainly responsible for H2S synthesis, accelerated the decrease. However, H2S treatment increased the endogenous H2S initially and then decreased after the second day. After four days of storage, the endogenous H2S in H2Streated broccoli florets was 10.14 μmol kg−1, significantly higher than that in control and PAG-treated samples (p < 0.05). Activities of LCD and DCD, the two key enzymes responsible for H2S synthesis, decreased during storage time in all groups. However, significantly higher activities were observed in H2S-treated group (p < 0.05). LCD and DCD activities in H2S-treated group were 36.09% and 19.19%, respectively which were higher than those in control at the end of the storage. Conversely, PAG treatment accelerated the decrease of LCD and DCD activities compared with control (Fig. 2B and C).
Fig. 1. Effect of H2S treatment on the value of L* (A) and H* (B), and chlorophyll content (C) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
3.3. Changes in enzymes involved in energy metabolism During the storage, the activities of H+-ATPase, Ca2+-ATPase, SDH, glucokinase and fructokinase decreased continuously while the activity of CCO increased (Fig. 3A–F). G6PDH activity increased initially and then decreased after the third day (Fig. 3G). Similar tendency was observed in 6PGDH and the maximum activity appeared at the end of 138
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lower than those in control (p < 0.05). However, there were no significant differences in H+-ATPase, Ca2+-ATPase, CCO, SDH, glucokinase and fructokinase activities between PAG-treated group and control on day 4. 3.4. Changes in ATP, ADP and AMP contents, and energy charge After harvest, contents of ATP and ADP level decreased gradually while AMP content increased in all groups (Fig. 4A–C). As a result, the energy charge in broccoli florets decreased during storage (Fig. 4D). H2S treatment maintained high levels of ATP and ADP, and inhibited the increase of AMP. The level of energy charge was 18.81% higher in H2S-treated broccoli florets compared with that in control (p < 0.05). PAG treatment, however, accelerated the degradation of ATP, ADP and the accumulation of AMP during initial storage. At the end of the storage, PAG-treated group showed significantly lower ATP content compared with control (p < 0.05), while there were no significant differences in ADP and AMP contents between two groups. The level of energy charge in PAG-treated group was 67.78, significantly lower than control (p < 0.05). 4. Discussion Recent studies have demonstrated that H2S was a signaling gaseous transmitter involved in many important biological roles such as chilling tolerance (Fu et al., 2013), heat tolerance (Li et al., 2013), antioxidant capacities (Hu et al., 2014), and thus related closely to the ripening and senescence in plants. In our present study, H2S treatment enhanced the endogenous H2S content in broccoli florets during storage (Fig. 2A). Meanwhile, the activities of LCD and DCD were also induced by H2S treatment (Fig. 2B and C). LCD and DCD are two key enzymes responsible for H2S synthesis. H2S treatment triggered endogenous H2S accumulation probably by increasing the activities of LCD and DCD (Fu et al., 2013). To further confirm the effect of endogenous H2S in broccoli florets, PAG-treated group was also observed. As H2S synthesis inhibitor, PAG inhibited the activities of LCD and DCD, which resulted in the decrease of endogenous H2S content (Fig. 2A–C). Similar results were reported by Hu et al. (2015) in water spinach leaves treated with exogenous H2S and PAG. Yellowing in broccoli is a characteristics symptom of senescence, which can be observed by an increase of L* and a decrease of H* during storage (Fig. 1A and B). However, H2S treatment postponed the change of color index. Previous study indicated that H2S fumigation could retard the decline of chlorophyll content in climacteric fruit, and prolong the postharvest life (Zhu et al., 2014). As illustrated in Fig. 1C, H2S treatment inhibited the degradation of total chlorophyll while PAG treatment accelerated it conversely. Hu et al. (2015) pointed out the activities of chlorophyll-degrading enzyme, chlorophyllase and Mg-dechelatase, might be impaired by H2S, thereby H2S fumigation alleviated the degradation of chloroplast after harvest. PAG treatment effectively inhibited the synthesis of endogenous H2S, leading to the enhancement of the chlorophyll-degrading enzymes activities, and finally accelerated chlorophyll degradation and yellowing as a result. Similarly, Xu et al. (2016a,b) showed that sucrose (12 g L−1) treatment inhibited an increase of L* value, retained a high hue angle and chlorophyll content in broccoli florets, resulted in extended shelf life. Energy metabolism plays vital roles in regulation of ripening and senescence in postharvest. It suggested that energy deficiency could induce lipid peroxidation and damage the integrity of membrane, which may account for the reduction of disease resistance and senescence (Yi et al., 2010; Shan et al., 2016). Pervious study indicated endogenous H2S remains a regulator of energy production in eukaryote cells under stress conditions, which enables the creature to deal with energy demand (Fu et al., 2012). In present study, energy charge in broccoli florets decreased gradually during storage along with the decrease of ATP, ADP and the increase of AMP. H2S with low
Fig. 2. Effect of H2S treatment on endogenous H2S content (A), L-cysteine desulfhydrase (LCD) activity (B) and D-cysteine desulfhydrase (DCD) activity (C) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
first day (Fig. 3H). H2S treatment enhanced all of the above enzymes significantly (p < 0.05). After four days of storage, the activities of H+-ATPase, Ca2+-ATPase, CCO, SDH, glucokinase, fructokinase, G6PDH and 6PGDH in H2S-treated group were 18.05%, 23.41%, 24.73%, 41.52%, 37.23%, 12.09%, 21.28% and 33.95% higher than those in control respectively. Conversely, PAG treatment inhibited these enzymes activities in the initial storage. At the end of storage, the activities of G6PDH and 6PGDH were 19.42% and 12.84%, respectively which were 139
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Fig. 3. Effect of H2S treatment on the activities of H+-ATPase (A), Ca2+-ATPase (B), cytochrome C oxidase (CCO) (C), succinate dehydrogenase (SDH) (D), glucokinase (E), fructokinase (F), glucose-6-phosphate dehydrogenase (G6PDH) (G) and 6-phosphogluconate dehydrogenase (6PGDH) (H) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
To further clarify the participation of H2S in the regulation of energy metabolism, eight enzymes involved in ATP synthesis and degradation were investigated. H+-ATPase, a proton pump energized by ATP hydrolysis, extrudes H+ and forms the membrane potential, which helps transport nutrients into cell (Palmgren, 2001). Ca2+ acts as a “second messenger” in plant signaling while high level of Ca2+ might cause structural damage and metabolism dysfunction. Ca2+-ATPase is responsible for active Ca2+ transport driven by hydrolysis of ATP, which maintains the low level of cytosolic Ca2+ (Muchhal et al., 1997).
concentration acted as an electron donor and induced the synthesis of ATP (Goubern et al., 2007). Thus, exogenous H2S treatment maintained high ATP contents which help inhibited a decrease of energy charge. Simultaneously, PAG treatment further confirmed the speculation. PAG treatment inhibited the synthesis of endogenous H2S, reduced the production of ATP, and finally resulted in energy deficiency (Fig. 4A–D). Li et al. (2016b) reported controlled atmospheres (50% O2 + 50% CO2) delayed the senescence of broccoli and extended the storage period by inhibiting reduction of ATP and energy charge. 140
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Fig. 4. Effect of H2S treatment on the contents of ATP (A), ADP (B), AMP (C), and energy charge (D) in broccoli florets during storage at 20 °C for 4 days. Values are presented as means ± SD (n = 3).
In present study, high levels of H+-ATPase and Ca2+-ATPase activities were observed in broccoli florets treated with H2S while PAG aggravated the decrease of both ATPases during initial storage (Fig. 3A and B). The results suggested that H2S inhibited the decrease of ATPases by improving energy status, and thus maintained the integrity of membrane, and alleviated metabolism dysfunction as a result (Azevedo et al., 2008). These might at least partially protect plants against stress after harvest. Jin et al. (2014) reported enhanced activities of H+ATPase and Ca2+-ATPase retarded the energy charge decline and therefore increased chilling tolerance. Respiratory enzymes are essential for different respiration pathways, which can provide energy and support biological reaction. CCO, the terminal enzyme of the electron transport chain, acts as an electrondriven proton pump and plays important roles in energy production (Soto et al., 2012). SDH is a TCA circle as well as aerobic electron transport chain enzyme, which catalyzes the reduction of ubiquinone to the oxidation of succinate (Acevedo et al., 2013). Changes in SDH and CCO activities might disturb electron flow through electron transport chain and thus impact on energy metabolism (Zhou et al., 2014). Significantly higher activities of CCO and SDH were observed in H2Streated group, compared with control. Opposite effect was obtained in broccoli florets treated with PAG due to an inhibition of H2S production (Fig. 3C and D). Previous study indicated low-temperature condition treatment played positive roles in maintaining high levels of CCO and SDH in loquat fruit during cold storage (Jin et al., 2015). Glycolysis, a multi-enzyme pathway, responsible for the conversion of monosaccharides to pyruvic acid, is a ubiquitous feature of energy metabolism (Givan, 1999). Hexokinase as an essential enzyme catalyzes phosphorylation of hexose by ATP, which is the first step in utilization of hexose in all eukaryotic cells. Glucokinase and fructokinase exist as separate enzymes that act on glucose and fructose, respectively (Cárdenas et al., 1998). As depicted in Fig. 3E and F, the activities of glucokinase and fructokinase were significantly higher in H2S-treated broccoli florets than those in control. Simultaneously, PAG treatment accelerated the decrease of glucokinase and fructokinase activities and reached a low level rapidly by decreasing endogenous H2S content.
Wang et al. (2015) found the elevated activities of glucokinase and fructokinase might contribute to induce chilling tolerance of NO-treated banana fruit. PPP is another fundamental component of cellular metabolism that can reduce NADP+ to NADPH while converting glucose-6-phosphate to CO2 and pentose phosphate (Wamelink et al., 2008). G6PDH, the NADP+-dependent oxidoreductase, has been quoted as being rate limiting for the oxidative branch of the PPP, and 6PGDH is the second dehydrogenase of the hexose monophosphate shunt, which is vital for the flow through the PPP (Rosemeyer, 1987; Stincone et al., 2015). In our present study, high levels of G6PDH and 6PGDH activities in broccoli florets were induced by H2S treatment while PAG treatment showed the opposite effect (Fig. 3G and H). The activation of enzymes involved in the PPP might contribute to several important functions in cellular metabolism, including providing of precursors for nucleotide and amino acid biosynthesis, maintaining of carbon homoeostasis, providing of reducing molecules for anabolism, as well as defeating of oxidative stress, and thus postponing of senescence in postharvest (Stincone et al., 2015).
5. Conclusions In conclusion, our present study showed that H2S treatment induced the synthesis of endogenous H2S by activating LCD and DCD in broccoli florets during storage at 20 °C. High level of H2S content inhibited the degradation of chlorophyll, thus alleviated yellowing of broccoli florets as a result. Meanwhile, H2S treatment may also play important roles in maintaining high levels of ATP and energy charge, which was ascribed to the activation of enzymes involved in energy metabolic pathways including glycolysis, TCA circle, electron transport chain and PPP, which finally postponed senescence in postharvest. However, further investigation on how energy metabolism dysfunction directly related to the yellowing in broccoli florets is needed.
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