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Effect of brassinolide on energy status and proline metabolism in postharvest bamboo shoot during chilling stress
Postharvest Biology and Technology 111 (2016) 240–246
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Effect of brassinolide on energy status and proline metabolism in postharvest bamboo shoot during chilling stress Zhanli Liua,b , Li Lia , Zisheng Luoa,* , Fangfang Zenga , Lei Jianga , Kaichen Tanga a b
Zhejiang University, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou 310058, PR China School of Agricultural and Food Engineering, Shandong University of Technology, Zibo, Shandong 255049, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 March 2015 Received in revised form 5 September 2015 Accepted 12 September 2015 Available online xxx
In this study, the effects of brassinolide treatment on chilling injury, energy status and proline metabolism in postharvest bamboo shoots (Phyllostachys praecox f. prevernalis) at 1 C were investigated. It was shown that chilling injury of bamboo shoots during 42-day storage was effectively reduced by brassinolide treatment at the concentration of 0.5 mM. In terms of energy status, brassinolide treatment significantly inhibited the increase of electrolyte leakage and the accumulation of malondialdehyde. Meanwhile, brassinolide treatment markedly retarded the decline of ATP content and maintained higher energy charge. Enzyme activities of energy metabolism including H+-ATPase, Ca2+-ATPase, succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO) were significantly enhanced by brassinolide treatment. Bamboo shoots treated by brassinolide treatment promoted D1-pyrroline-5-carboxylate synthetase (P5CS) and ornithined-aminotransferase (OAT) activity and inhibited proline dehydrogenase (PDH) activity, which elevated proline accumulation. These results suggest that the alleviation in chilling injury by brasinolide may be caused by enhanced enzyme activities related to energy and proline metabolism. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Bamboo shoot Brassinolide Chilling injury Energy status Proline metabolism
1. Introduction Bamboo shoots are immature culms emerging from nodes of the (pseudo-) rhizome of bamboo plants. Bamboo shoots only have a short shelf life limited to one day at ambient temperature (20– 25 C), while the shelf life could be extended to 28 days under low temperature and packaging (Kleinhenz et al., 2000). However, bamboo shoots are susceptible to chilling injury (CI), which would limit the advantage of maintaining quality during long period of storage at low temperature (Gu et al., 2002; Luo et al., 2008, 2012). Cell membrane integrity is the primary cell structure affected by CI. Energy supply in cellular is an important factor in controlling fruit ripening and senescence after harvest. It is suggested that the enhancement of membrane integrity is associated with maintenance of higher ATP content and energy charge (Chen and Yang, 2013; Jin et al., 2013). Proline is a compatible osmolyte and a reserve source of carbon, nitrogen and energy during recovery from stress. The accumulation of proline results in increase in cellular osmolarity, which provides turgor necessary for cell expansion. Therefore, energy status and proline metabolism are
considered as the important factors being involved in the acquisition of chilling tolerance. Brassinolide is essential for plant growth and development. Brassinosteroids are considered to be a class of plant polyhydroxysteroids. It is reported that brassinosteroids may exhibit a high potential in regulating a range of physiological processs (Fujioka and Yokota, 2003; Kim and Wang, 2010; Sasse, 2003). In recent years, it is reported in a few studies that brassinosteroids may play a role in enhancing chilling resistance of plant cells. Xia et al. (2009) found that brassinosteroid levels in cucumber (Cucumis sativus) were correlated with the tolerance to cold stress. Wang et al. (2012) also reported that brassinolide reduced chilling injury of green bell pepper. However, to our knowledge, little information is available regarding the effect of brassinolide on energy status and proline metabolism in bamboo shoot. Therefore, the aim of this study was to investigate the effect of brassinolide treatment on energy status content, enzymes activities of energy and proline metabolism in bamboo shoot stored at low temperature. 2. Materials and methods 2.1. Bamboo shoot, treatment, and storage
* Corresponding author. E-mail address: [email protected] (Z. Luo). http://dx.doi.org/10.1016/j.postharvbio.2015.09.016 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
Bamboo shoots (Phyllostachys praecox f. prevernalis) were harvested from a plantation in Yuhang, Zhejiang Province of
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China. The shoots were then packed in fiberboard cartons, and transferred to the laboratory in 3 h, with shoots of uniform size and free from blemishes were selected. The shoots were divided into six sets of 120. Three replicates were used for each of the following treatments: control (distilled water) and brassinolide at 0.5 mM for 10 min. This brassinolide concentration was chosen as being optimal from preliminary experiments using 0.1, 0.3, 0.5, 0.7, 0.9 mM. Samples were collected from 10 shoots from each treatment at 14 days intervals for enzyme analysis and measurement of electrolyte leakage, MDA, energy content, and proline metabolism. Bamboo shoots were cool air-dried for about 30 min and stored at 1 C and relative humidity at about 95% for 42 days. Another six sets of bamboo shoots were treated in distilled water (control) or brassinolide at 0.5 mM for 10 min. Bamboo shoots were cool air-dried for about 30 min and stored at 20 C and relative humidity at about 95% for 12 days. These extra experiments only for measure the change of ATP, ADP and AMP contents and energy charge during storage at 20 C every 4 days. 2.2. Chilling injury evaluation The chilling injury incidence was recorded on three independent replicates (with each replicate containing 30 individual shoots) every 14 days of storage at 1 C. Chilling injury symptoms of flesh browning and water-soaked on the flesh or leaf sheaths were visually obvious and determining the percentage of shoots affected by chilling. 2.3. Electrolyte leakage determination
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sucrose, 1 mM EDTA, 0.1% (w/v) bovine serum albumin, 0.1% (w/v) cysteine and 5 g/L polyvinyl pyrrolidone at 4 C. The homogenate was centrifuged at 5000 g for 10 min at 4 C. The supernatant was collected and centrifuged at 15,000 g for 10 min at 4 C for sedimentation of mitochondria. The mitochondria pellet was then resuspended in washing buffer (10 mM Tris–HCl buffer, containing 0.3 M mannitol, 0.25 M sucrose and 1 mM EDTA) and again centrifuged 15,000 g for 10 min at 4 C. The final sediment was dissolved with 5 mL washing buffer as crude mitochondria extract for the enzyme assays. H+-ATPase and Ca2+-ATPase activities were determined by the method of Jin et al. (2013). For H+-ATPase activity, 3 mL of total reaction mixture containing 0.5 mL of crude mitochondria extract, 0.03 M Tris–HCl buffer (pH 8.0), 0.05 M KCl, 0.05 M NaNO3, 3 mM Mg2SO4, 0.1 mM Na3VO4 and 0.1 mM (NH4)2MoO4. For Ca2+-ATPase activity, 3 mL of total reaction mixture containing 0.5 mL of crude mitochondria extract, 0.03 M Tris–HCl buffer (pH 8.0), 0.05 M KCl, 0.05 M NaNO3, 0.1 mM Na3VO4, 3 mM Ca(NO3)2 and 0.1 mM (NH4)2MoO4. The reaction was initiated by the addition of 0.1 mL of 0.03 M ATP-Tris (pH 8.0). After incubation at 37 C for 20 min, 0.03 M trichloroacetic acid was added into the mixture to terminate the reaction. One unit of H+-ATPase and Ca2+-ATPase activities were expressed as the release of 1 mmol of phosphorus per minute at 660 nm. 2.6. Measurement of succinate dehydrogenase activity Succinate dehydrogenase (SDH) activity was measured according to the method of Acevedo et al. (2013). The substrate solution contained 0.05 M potassium phosphate buffer (pH 7.8), 4 mM sodium azide, 1 mM phenazine methosulphate, 0.08 mM dichlorophenolindophenol and 0.1 M sodium succinate, incubating at 30 C for 10 min. The activity was determined by adding 0.5 mL of crude mitochondria extract. One unit of SDH activity was defined as an increase of 0.01 in absorbance at 600 nm per minute.
The disks were excised from the shoots with a 3-mm diameter cork borer. Twelve disks were then incubated in 30 mL of 0.3 M mannitol for 30 min in a 50 mL capped polypropylene centrifuge tube. The conductivity of the bathing solution was measured at 25 C using a DJS-1C conductivity meter (Shanghai Analytical Instrument Co., Shanghai, China). Total electrolyte conductivity was determined after freezing (24 h at 20 C), and heating the disks and bathing solutions in a boiling water bath for 30 min. The disks and bathing solution were then stored at 20 C for at least 24 h, boiled in water for 20 min, cooled to room temperature, and conductivity measurement was measured once again. The electrolyte leakage was expressed as percentage of the conductivity of total tissue electrolyte. Three independent replicates were conducted in each treatment.
Cytochrome C oxidase (CCO) activity was assayed by the method of Jin et al. (2013). The assay mixture contained 0.05 M phosphate buffer (pH 7.5), 0.3 mM reduced cytochrome C and 0.02 M dimethyl phenylenediamine. The reaction was initiated by adding 0.5 mL of crude mitochondria extract. One unit of CCO activity was defined as an increase of 0.01 in absorbance at 510 nm per minute.
2.4. Determination of malondialdehyde content
2.8. Measurement of ATP, ADP and AMP contents and energy charge
The content of malondialdehyde (MDA) was measured by the thiobarbituric acid (TBA) reaction method (Dhindsa et al., 1981) with some modifications. About 1.0 g samples were homogenized in 4.0 mL of 5% (w/v) trichloroacetic acid and centrifuged at 10,000 g for 15 min. The supernatant was mixed with 2.0 mL of 0.67% TBA, heated at 100 C for 20 min and then immediately cooled down on ice. After centrifugation at 5000 g for 10 min, absorbance of the supernatant was measured in spectrophotometer (Shimadzu UV-1750) at 450, 532 and 600 nm, respectively. Three independent replicates were conducted in each treatment.
ATP, ADP and AMP were extracted and assayed according to Zhou et al. (2014) with a minor modification. About 2 g of frozen shoots samples were ground in liquid nitrogen and extracted with 5 mL of 0.6 M perchloric acid. The homogenate was centrifuged at 15,000 g for 10 min at 4 C. A 3 mL aliquot of the supernatant was quickly neutralized to pH 6.5 with 1 M KOH, and then diluted to 5 mL and finally passed through a 0.45 mm filter. Measurements of ATP, ADP and AMP contents were conducted by a highperformance liquid chromatography (HPLC) system (Waters 2695 separation module, Waters Corp.) equipped with a reverse-phase Nova-Pak C18 column (4.6 mm 250 mm, Agilent Corp.) and a ultraviolet detector. Mobile phase A consisted of 0.03 M K2HPO4 and 0.02 M KH2PO4 dissolved in deionized water and adjusted to pH 7.0 with 0.1 M KOH. Mobile phase B was methyl alcohol. Elution was conducted by a linear gradient program as follows: 0 min, 100% A; 7 min, 80:20 (A:B); 9 min, 75:25 (A:B); and 10 min, 100% A. It takes a further 5 min for the program to return to the initial conditions and stabilise. The flow rate was 1.0 mL/min.
2.5. Extraction of mitochondria and measurement of mitochondrial ATPase Crude mitochondria were extracted from bamboo shoot by the method of Zhou et al. (2014) with a slight modification. Frozen bamboo shoots samples (10 g) were homogenized in 20 mL of 0.05 M Tris–HCl buffer (pH 7.8), containing 0.3 M mannitol, 0.25 M
2.7. Measurement of cytochrome C oxidase activity
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Sample aliquots (20 mL) were injected into the HPLC, and peaks were detected at 254 nm. ATP, ADP, and AMP contents were determined according to the external standard program and expressed on a fresh weight basis. Energy charge was calculated as: [ATP + 1/2 ADP]/[ATP + ADP + AMP]. 2.9. Proline content determination The proline content was assayed according to the method of Zhao et al. (2009) with modification. About 1 g of frozen bamboo shoots samples were homogenized in 5 mL of 3% (v/v) sulfosalicylic acid and centrifuged at 1,2000 g for 10 min. 2 mL of glacial acetic acid and 3 mL of ninhydrin reagent were mixed with supernatant (2 mL) and boiled for 10 min. Then 4 mL of toluene was added into the reaction mixture after the solution was cooled. The absorbance of the organic phase was recorded at 520 nm.. The results were expressed as mg proline per gram fresh material. 2.10. Measurement of OAT, P5CS and PDH activity Ornithine-d-aminotransferase (OAT) was assayed following the method of Zhang et al. (2012) with minor modification. The reaction system contained 35 mM L-ornithine, 0.05 mM phosphopyridoxal, 25 mM a-ketoglutarate and the enzyme extract. The mixture was incubated at 37 C for 20 min before adding 3 M HClO4. Then 2% ninhydrin was added into the mixture and boiled for 20 min. After cooling, the mixture was centrifuged at 15,000 g for 10 min. The precipitate dissolved in 2 mL toluene and quantified by measuring the absorbance at 510 nm. The activities of D1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (PDH) were assayed as described by Shang et al. (2011). About 1 g of frozen shoots samples were homogenized in 5 mL of 0.05 M Tris–HCl buffer (pH 7.4) containing 0.6 M KCl, 7 mM MgCl2, 1 mM DTT, 3 mM EDTA and 5% (w/v) insoluble polyvinylpyrrolidone. One unit of P5CS activity was defined as the change in absorbance per minute at 340 nm. One unit of PDH activity was defined as the amount of enzyme causing a change of 0.01 in absorbance per hour at 340 nm.
Fig. 1. Effect of brassinolide treatment on chilling injury incidence (a), electrolyte leakage (b) and MDA (c) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
was 39.7% and 49.0% respectively at the end of storage. Lipid peroxidation can be indicated by measuring the content of MDA. As shown in Fig. 1c, the MDA content increased gradually, and the MDA content in brassinolide treated shoots was lower than that in the control. At the end of the storage period, the MDA content of shoots treated with brassinolide at 0.5 mM was 7.14 nmol g 1 FW, while the MDA content of the control shoots was 8.47 nmol g 1 FW.
2.11. Statistical design 3.2. ATP, ADP, AMP contents and energy charge Experiments were performed using a completely randomized design. All statistical analyses were performed with SPSS (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance (ANOVA). The overall least significant difference (LSD) at p = 0.05 was calculated and used to detect significant differences among treatments. 3. Results 3.1. Chilling injury incidence, electrolyte leakage and MDA content As shown in Fig. 1a, chilling injury incidence increased rapidly both in brassinolide treated and the control bamboo shoots stored at 1 C. Chilling injury symptoms in control shoot were observed only after 14 days of storage. Chilling injury incidence increased markedly after 14 days. However, chilling injury incidence for brassinolide treatment was significantly lower than that of control treatment from 14 to 42 days during storage (P < 0.05). On the 42nd day, chilling injury incidence in control was 37.9%, which was 3-fold higher than that in brassinolide treatment. The membrane permeability changes during storage were evaluated by determining the relative electrolyte leakage. The changes in electrolyte leakage of bamboo shoots are presented in Fig. 1b. The electrolyte leakage increased gradually with storage time. The electrolyte leakage of brassinolide and control treatment
As illustrated in Fig. 2a, the ATP content decreased gradually during the storage at 20 or 1 C. The ATP content in brassinolide treatment was significantly higher than that in the control from 14 to 42 days during the cold storage. The ATP content decreased more quickly during the storage at 20 C than that in 1 C. Brassinolide treatment retarded the decline of ATP content during the storage at 20 or 1 C. The changes of ADP content is shown in Fig. 2b. The ADP content decreased gradually during the storage at 20 or 1 C. Brassinolide treatment maintained higher ADP content in comparison with the control. The AMP levels increased gradually during the storage (Fig. 2c). The AMP cotnent in brassinolide treated shoots was significantly lower than that of the control during the storage at 20 or 1 C. Energy charge decreased gradually during the storage with the changes of ATP, ADP and AMP (Fig. 2d). Brassinolide treated shoots maintained higher energy charge than that of control during the storage at 20 or 1 C, but the changes of energy charge during the storage at 1 C were more extensively than that of storage at 20 C. 3.3. H+-ATPase and Ca2+-ATPase activities The effect of brassinolide on H+-ATPase activity of shoots stored at 1 C is shown in Fig. 3a. H+-ATPase activity decreased gradually in both treatments. However, brassinolide treatment significantly
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Fig. 2. Effect of brassinolide treatment on contents of ATP (a), ADP (b), AMP (c) and energy charge (d) of bamboo shoot during storage at 20 C or 1 C. Values are the means SD of triplicate assays.
(P < 0.05) inhibited the decrease of H+-ATPase activity. At the end of storage, with the control, H+-ATPase activity experienced a reduction of 60% over the initial value, while H+-ATPase activity in brassinolide treatment was observed to be significantly higher than that in the control.
Fig. 3. Effect of brassinolide treatment on activities of H+-ATPase (a) and Ca2 + -ATPase (b) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
As shown in Fig. 3b, Ca2+-ATPase activity increased slightly before 28 days and decreased afterwards. Ca2+-ATPase activity in the control shoots was 184.16 U g 1 FW, while the activity in brassinolide treated shoots was 195.65 U g 1 FW. Therefore, brassinolide treatment induced the increase of Ca2+-ATPase activity before 28 days and retarded the decline.
Fig. 4. Effect of brassinolide treatment on activities of CCO (a) and SDH (b) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
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3.4. CCO and SDH activities CCO activity in bamboo shoots stored at 1 C increased gradually (Fig. 4a). At the end of storage, CCO activity in brassinolide treated shoots was 33.09 U g 1 FW, which was higher than that in the control (29.93 U g 1 FW). In general, brassinolide treatment maintained higher CCO activity in comparison with the control from 14th to 42nd days during the storage. As shown in Fig. 4b, SDH activity decreased quickly before the 28th day and decreased slightly afterwards. At the end of storage, SDH activity in treated shoots was 72% higher than that in control shoots. SDH activity was significantly (P < 0.05) higher than that in control shoots. 3.5. Proline content and OAT, P5CS and PDH activity The change in proline content in brassinolide treated and control shoots is shown in Fig. 5a. The proline content increased gradually over time. The proline content in shoots treated with brassinolide showed significantly higher level during storage, relative to the control (P < 0.05). OAT activity increased to a peak on the 14th day and then decrease slightly for the remainder of the storage period in brassinolide and control treatment (Fig. 5b). However, OAT activity in brassinolide treatment was significantly higher than that in the control (P < 0.05). As shown in Fig. 5c, a continuous increase in P5CS activity was observed, both in control and brassinolide treated shoots. The application of brassinolide to shoots significantly increased the P5CS activity. PDH activity increased gradually during the first 28 days and decreased thereafter (Fig. 5d). In general, brassinolide treatment showed lower PDH activity in comparison with the control (P < 0.05). 4. Discussion Chilling injury (CI) is a major factor in reducing the quality and limiting the storage time of bamboo shoots. In this study, we found that brassinolide treatment significantly reduces CI of bamboo
shoots during the storage at 1 C. The results showed that chilling injury was inhibited by brassinolide, which are consistent with those in a previous study of green bell pepper (Wang et al., 2012). The mechanism by which the brassinolide induced chilling injury in bamboo shoots was investigated. It was reported that the cell membrane was the primary site for the development of chilling injury. Transition of cell membranes phase from a flexible liquidcrystalline to a solid-gel structure that occurs at chilling temperature will increase the risk of loss of controlled cell membrane semi-permeability (Lyons, 1973). Electrolyte leakage was used as the indicator of membrane damage. CI occurrence is often accompanied by oxidative damage, one of whose biological markers is MDA content, the final product of lipid peroxidation (Xu et al., 2009). Huang and Guo (2005) suggested that the increase in antioxidant enzymes activity would contribute to the adaptation of plants to cold stress and ameliorated oxidative damage such as lipid peroxidation (MDA as indicator) and electrolyte leakage. In our study, there was a continuous increase in electrolyte leakage and MDA in both treatments, but the application of brassinolide significantly delayed the increase of electrolyte leakage and MDA. Similar results were also found in green bell pepper (Wang et al., 2012). Previous reports have revealed that the development of chilling injury is partially attributed to limited availability of energy or low energy production, whereas maintaining of higher levels of ATP and energy charge alleviates chilling injury (Chen and Yang, 2013). It was suggested in previous works that energy depletion is associated with membrane damage (Su et al., 2005; Yi et al., 2008). In the present study, ATP content and energy charge in control and brassinolide treatment decreased gradually. However, brassinolide treatment maintained a higher level of ATP and energy charge. It has been reported that the improvement of chilling tolerance is related to enhancement in activities of energy metabolism enzymes. ATPase is a class of enzyme that catalyzes the decomposition of ATP into ADP and a free phosphate ion, which produce more energy (Heyes and Townsend, 1992). The plasma membrane H+-ATPase acts as a primary transporter by pumping protons out of the cell or into the lumen of the vacuole and thus creating pH and electrical potential differences across the plasma
Fig. 5. Effect of brassinolide treatment on proline content (a) and activities of OAT (b), P5CS (c) and PDH (d) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
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membrane (Azevedo et al., 2008). ATPase is linked with a specific role in protecting chilling-sensitive fruit from the injury effects of low temperature (Ghasernnezhad et al., 2008). Higher activities of H+-ATPase and Ca2+-ATPase were observed in brassinolide treated bamboo shoot (Fig. 3). The enhanced activities of ATPase in brassinolide treatment coincided with the higher ATP content, which is crucial to reduce CI in bamboo shoots under cold stress. Our studies are in accordance with those in peach fruit where the application of MeJA increased the chilling tolerance of peach fruit during cold storage, accompanied by enhanced activities of H+ATPase and Ca2+-ATPase (Jin et al., 2013). SDH catalyzes the oxidation of succinate to fumarate, which is associated with the generation of ATP in mitochondria. CCO is the last enzyme in the respiratory electron transport chain of mitochondria and plays a critical role in energy metabolism (Millar et al., 1995). Alterations in SDH and CCO activities may lead to disturbance of electron flow in the mitochondrial respiratory chain, thus resulting in chilling injury symptoms (Zhou et al., 2014). The activities of SDH and CCO in brassinolide treated bamboo shoot were significantly higher than that in control bamboo shoot. These results suggested that as important enzymes involved in energy metabolism, the enhanced activities of SDH and CCO may contribute to the alleviation in chilling injury by brassinolide treatment. Therefore, maintaining of energy metabolism enzymes activities of mitochondria to provide sufficient energy is an important factor in chilling stress. The role of proline in increasing cellular osmolarity, stabilising membrane and subcellular structures, and protecting cells against oxidative damage under stresses was suggested. (Kavi-Kishor et al., 2005; Posmyk and Janas, 2007; Kumar and Yadav, 2009). It was also reported that the stressed plants treated with proline showed considerable reduction in reactive oxygen species (Kaya et al., 2007; Posmyk and Janas, 2007). The proline accumulation is mainly attributed to its synthesis from glutamic acid and ornithine catalysed by P5CS and OAT respectively, and to its degradation catalysed by PDH (Verbruggen and Hermans, 2008). Our results indicated that brassinolide treatment significantly increased P5CS and OAT activity, while reduced the PDH activity, which in turn increased the content of proline. Li et al. (2014) reported that oxalic acid treatment significantly increased P5CS activity and reduced PDH activity with concomitant increase in proline levels, but did not activate OAT activity in mango fruit during cold storage. Thus, our study suggested that chilling-sensitive bamboo treated by brassinolide shoots exhibit increase in P5CS and OAT activity and inhibition in PDH activity, which elevated proline accumulation. The proline metabolism plays a key role in alleviating CI during long term cold storage and maintaining the quality of bamboo shoots. Therefore, brassinolide treatment maintained the quality of bamboo shoots and reduced the impact of chilling injury. Kaur et al. (2011a,b) demonstrated that exogenous proline was significantly effective in reducing the impact of chilling injury on reproductive growth in chickpea. Zhu et al. (2010) also found that brassinosteroids significantly delayed jujube fruit senescence and maintained the quality. The accumulation of proline and higher energy was two important factors for maintaining the membrane integrity. Proline may interact with ATP and energy metabolism enzymes to alleviate chilling injury of bamboo shoots. Our results showed that brassinolide treatment triggered a response that maintained the integrity of membranes, raised the energy supply and proline accumulation and inhibited lipid peroxidation. In conclusion, brassinolide treatment promoted postharvest bamboo shoots resistance to low temperature stress. Higher resistance of bamboo shoots to chilling stress was related to higher levels of ATP and energy charge, which was of benefit to membrane integrity. Furthermore, elevated proline accumulation was also an important factor in enhancing chilling resistance of bamboo shoots.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effect of brassinolide on energy status and proline metabolism in postharvest bamboo shoot during chilling stress Zhanli Liua,b , Li Lia , Zisheng Luoa,* , Fangfang Zenga , Lei Jianga , Kaichen Tanga a b
Zhejiang University, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou 310058, PR China School of Agricultural and Food Engineering, Shandong University of Technology, Zibo, Shandong 255049, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 March 2015 Received in revised form 5 September 2015 Accepted 12 September 2015 Available online xxx
In this study, the effects of brassinolide treatment on chilling injury, energy status and proline metabolism in postharvest bamboo shoots (Phyllostachys praecox f. prevernalis) at 1 C were investigated. It was shown that chilling injury of bamboo shoots during 42-day storage was effectively reduced by brassinolide treatment at the concentration of 0.5 mM. In terms of energy status, brassinolide treatment significantly inhibited the increase of electrolyte leakage and the accumulation of malondialdehyde. Meanwhile, brassinolide treatment markedly retarded the decline of ATP content and maintained higher energy charge. Enzyme activities of energy metabolism including H+-ATPase, Ca2+-ATPase, succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO) were significantly enhanced by brassinolide treatment. Bamboo shoots treated by brassinolide treatment promoted D1-pyrroline-5-carboxylate synthetase (P5CS) and ornithined-aminotransferase (OAT) activity and inhibited proline dehydrogenase (PDH) activity, which elevated proline accumulation. These results suggest that the alleviation in chilling injury by brasinolide may be caused by enhanced enzyme activities related to energy and proline metabolism. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Bamboo shoot Brassinolide Chilling injury Energy status Proline metabolism
1. Introduction Bamboo shoots are immature culms emerging from nodes of the (pseudo-) rhizome of bamboo plants. Bamboo shoots only have a short shelf life limited to one day at ambient temperature (20– 25 C), while the shelf life could be extended to 28 days under low temperature and packaging (Kleinhenz et al., 2000). However, bamboo shoots are susceptible to chilling injury (CI), which would limit the advantage of maintaining quality during long period of storage at low temperature (Gu et al., 2002; Luo et al., 2008, 2012). Cell membrane integrity is the primary cell structure affected by CI. Energy supply in cellular is an important factor in controlling fruit ripening and senescence after harvest. It is suggested that the enhancement of membrane integrity is associated with maintenance of higher ATP content and energy charge (Chen and Yang, 2013; Jin et al., 2013). Proline is a compatible osmolyte and a reserve source of carbon, nitrogen and energy during recovery from stress. The accumulation of proline results in increase in cellular osmolarity, which provides turgor necessary for cell expansion. Therefore, energy status and proline metabolism are
considered as the important factors being involved in the acquisition of chilling tolerance. Brassinolide is essential for plant growth and development. Brassinosteroids are considered to be a class of plant polyhydroxysteroids. It is reported that brassinosteroids may exhibit a high potential in regulating a range of physiological processs (Fujioka and Yokota, 2003; Kim and Wang, 2010; Sasse, 2003). In recent years, it is reported in a few studies that brassinosteroids may play a role in enhancing chilling resistance of plant cells. Xia et al. (2009) found that brassinosteroid levels in cucumber (Cucumis sativus) were correlated with the tolerance to cold stress. Wang et al. (2012) also reported that brassinolide reduced chilling injury of green bell pepper. However, to our knowledge, little information is available regarding the effect of brassinolide on energy status and proline metabolism in bamboo shoot. Therefore, the aim of this study was to investigate the effect of brassinolide treatment on energy status content, enzymes activities of energy and proline metabolism in bamboo shoot stored at low temperature. 2. Materials and methods 2.1. Bamboo shoot, treatment, and storage
* Corresponding author. E-mail address: [email protected] (Z. Luo). http://dx.doi.org/10.1016/j.postharvbio.2015.09.016 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
Bamboo shoots (Phyllostachys praecox f. prevernalis) were harvested from a plantation in Yuhang, Zhejiang Province of
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China. The shoots were then packed in fiberboard cartons, and transferred to the laboratory in 3 h, with shoots of uniform size and free from blemishes were selected. The shoots were divided into six sets of 120. Three replicates were used for each of the following treatments: control (distilled water) and brassinolide at 0.5 mM for 10 min. This brassinolide concentration was chosen as being optimal from preliminary experiments using 0.1, 0.3, 0.5, 0.7, 0.9 mM. Samples were collected from 10 shoots from each treatment at 14 days intervals for enzyme analysis and measurement of electrolyte leakage, MDA, energy content, and proline metabolism. Bamboo shoots were cool air-dried for about 30 min and stored at 1 C and relative humidity at about 95% for 42 days. Another six sets of bamboo shoots were treated in distilled water (control) or brassinolide at 0.5 mM for 10 min. Bamboo shoots were cool air-dried for about 30 min and stored at 20 C and relative humidity at about 95% for 12 days. These extra experiments only for measure the change of ATP, ADP and AMP contents and energy charge during storage at 20 C every 4 days. 2.2. Chilling injury evaluation The chilling injury incidence was recorded on three independent replicates (with each replicate containing 30 individual shoots) every 14 days of storage at 1 C. Chilling injury symptoms of flesh browning and water-soaked on the flesh or leaf sheaths were visually obvious and determining the percentage of shoots affected by chilling. 2.3. Electrolyte leakage determination
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sucrose, 1 mM EDTA, 0.1% (w/v) bovine serum albumin, 0.1% (w/v) cysteine and 5 g/L polyvinyl pyrrolidone at 4 C. The homogenate was centrifuged at 5000 g for 10 min at 4 C. The supernatant was collected and centrifuged at 15,000 g for 10 min at 4 C for sedimentation of mitochondria. The mitochondria pellet was then resuspended in washing buffer (10 mM Tris–HCl buffer, containing 0.3 M mannitol, 0.25 M sucrose and 1 mM EDTA) and again centrifuged 15,000 g for 10 min at 4 C. The final sediment was dissolved with 5 mL washing buffer as crude mitochondria extract for the enzyme assays. H+-ATPase and Ca2+-ATPase activities were determined by the method of Jin et al. (2013). For H+-ATPase activity, 3 mL of total reaction mixture containing 0.5 mL of crude mitochondria extract, 0.03 M Tris–HCl buffer (pH 8.0), 0.05 M KCl, 0.05 M NaNO3, 3 mM Mg2SO4, 0.1 mM Na3VO4 and 0.1 mM (NH4)2MoO4. For Ca2+-ATPase activity, 3 mL of total reaction mixture containing 0.5 mL of crude mitochondria extract, 0.03 M Tris–HCl buffer (pH 8.0), 0.05 M KCl, 0.05 M NaNO3, 0.1 mM Na3VO4, 3 mM Ca(NO3)2 and 0.1 mM (NH4)2MoO4. The reaction was initiated by the addition of 0.1 mL of 0.03 M ATP-Tris (pH 8.0). After incubation at 37 C for 20 min, 0.03 M trichloroacetic acid was added into the mixture to terminate the reaction. One unit of H+-ATPase and Ca2+-ATPase activities were expressed as the release of 1 mmol of phosphorus per minute at 660 nm. 2.6. Measurement of succinate dehydrogenase activity Succinate dehydrogenase (SDH) activity was measured according to the method of Acevedo et al. (2013). The substrate solution contained 0.05 M potassium phosphate buffer (pH 7.8), 4 mM sodium azide, 1 mM phenazine methosulphate, 0.08 mM dichlorophenolindophenol and 0.1 M sodium succinate, incubating at 30 C for 10 min. The activity was determined by adding 0.5 mL of crude mitochondria extract. One unit of SDH activity was defined as an increase of 0.01 in absorbance at 600 nm per minute.
The disks were excised from the shoots with a 3-mm diameter cork borer. Twelve disks were then incubated in 30 mL of 0.3 M mannitol for 30 min in a 50 mL capped polypropylene centrifuge tube. The conductivity of the bathing solution was measured at 25 C using a DJS-1C conductivity meter (Shanghai Analytical Instrument Co., Shanghai, China). Total electrolyte conductivity was determined after freezing (24 h at 20 C), and heating the disks and bathing solutions in a boiling water bath for 30 min. The disks and bathing solution were then stored at 20 C for at least 24 h, boiled in water for 20 min, cooled to room temperature, and conductivity measurement was measured once again. The electrolyte leakage was expressed as percentage of the conductivity of total tissue electrolyte. Three independent replicates were conducted in each treatment.
Cytochrome C oxidase (CCO) activity was assayed by the method of Jin et al. (2013). The assay mixture contained 0.05 M phosphate buffer (pH 7.5), 0.3 mM reduced cytochrome C and 0.02 M dimethyl phenylenediamine. The reaction was initiated by adding 0.5 mL of crude mitochondria extract. One unit of CCO activity was defined as an increase of 0.01 in absorbance at 510 nm per minute.
2.4. Determination of malondialdehyde content
2.8. Measurement of ATP, ADP and AMP contents and energy charge
The content of malondialdehyde (MDA) was measured by the thiobarbituric acid (TBA) reaction method (Dhindsa et al., 1981) with some modifications. About 1.0 g samples were homogenized in 4.0 mL of 5% (w/v) trichloroacetic acid and centrifuged at 10,000 g for 15 min. The supernatant was mixed with 2.0 mL of 0.67% TBA, heated at 100 C for 20 min and then immediately cooled down on ice. After centrifugation at 5000 g for 10 min, absorbance of the supernatant was measured in spectrophotometer (Shimadzu UV-1750) at 450, 532 and 600 nm, respectively. Three independent replicates were conducted in each treatment.
ATP, ADP and AMP were extracted and assayed according to Zhou et al. (2014) with a minor modification. About 2 g of frozen shoots samples were ground in liquid nitrogen and extracted with 5 mL of 0.6 M perchloric acid. The homogenate was centrifuged at 15,000 g for 10 min at 4 C. A 3 mL aliquot of the supernatant was quickly neutralized to pH 6.5 with 1 M KOH, and then diluted to 5 mL and finally passed through a 0.45 mm filter. Measurements of ATP, ADP and AMP contents were conducted by a highperformance liquid chromatography (HPLC) system (Waters 2695 separation module, Waters Corp.) equipped with a reverse-phase Nova-Pak C18 column (4.6 mm 250 mm, Agilent Corp.) and a ultraviolet detector. Mobile phase A consisted of 0.03 M K2HPO4 and 0.02 M KH2PO4 dissolved in deionized water and adjusted to pH 7.0 with 0.1 M KOH. Mobile phase B was methyl alcohol. Elution was conducted by a linear gradient program as follows: 0 min, 100% A; 7 min, 80:20 (A:B); 9 min, 75:25 (A:B); and 10 min, 100% A. It takes a further 5 min for the program to return to the initial conditions and stabilise. The flow rate was 1.0 mL/min.
2.5. Extraction of mitochondria and measurement of mitochondrial ATPase Crude mitochondria were extracted from bamboo shoot by the method of Zhou et al. (2014) with a slight modification. Frozen bamboo shoots samples (10 g) were homogenized in 20 mL of 0.05 M Tris–HCl buffer (pH 7.8), containing 0.3 M mannitol, 0.25 M
2.7. Measurement of cytochrome C oxidase activity
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Sample aliquots (20 mL) were injected into the HPLC, and peaks were detected at 254 nm. ATP, ADP, and AMP contents were determined according to the external standard program and expressed on a fresh weight basis. Energy charge was calculated as: [ATP + 1/2 ADP]/[ATP + ADP + AMP]. 2.9. Proline content determination The proline content was assayed according to the method of Zhao et al. (2009) with modification. About 1 g of frozen bamboo shoots samples were homogenized in 5 mL of 3% (v/v) sulfosalicylic acid and centrifuged at 1,2000 g for 10 min. 2 mL of glacial acetic acid and 3 mL of ninhydrin reagent were mixed with supernatant (2 mL) and boiled for 10 min. Then 4 mL of toluene was added into the reaction mixture after the solution was cooled. The absorbance of the organic phase was recorded at 520 nm.. The results were expressed as mg proline per gram fresh material. 2.10. Measurement of OAT, P5CS and PDH activity Ornithine-d-aminotransferase (OAT) was assayed following the method of Zhang et al. (2012) with minor modification. The reaction system contained 35 mM L-ornithine, 0.05 mM phosphopyridoxal, 25 mM a-ketoglutarate and the enzyme extract. The mixture was incubated at 37 C for 20 min before adding 3 M HClO4. Then 2% ninhydrin was added into the mixture and boiled for 20 min. After cooling, the mixture was centrifuged at 15,000 g for 10 min. The precipitate dissolved in 2 mL toluene and quantified by measuring the absorbance at 510 nm. The activities of D1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (PDH) were assayed as described by Shang et al. (2011). About 1 g of frozen shoots samples were homogenized in 5 mL of 0.05 M Tris–HCl buffer (pH 7.4) containing 0.6 M KCl, 7 mM MgCl2, 1 mM DTT, 3 mM EDTA and 5% (w/v) insoluble polyvinylpyrrolidone. One unit of P5CS activity was defined as the change in absorbance per minute at 340 nm. One unit of PDH activity was defined as the amount of enzyme causing a change of 0.01 in absorbance per hour at 340 nm.
Fig. 1. Effect of brassinolide treatment on chilling injury incidence (a), electrolyte leakage (b) and MDA (c) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
was 39.7% and 49.0% respectively at the end of storage. Lipid peroxidation can be indicated by measuring the content of MDA. As shown in Fig. 1c, the MDA content increased gradually, and the MDA content in brassinolide treated shoots was lower than that in the control. At the end of the storage period, the MDA content of shoots treated with brassinolide at 0.5 mM was 7.14 nmol g 1 FW, while the MDA content of the control shoots was 8.47 nmol g 1 FW.
2.11. Statistical design 3.2. ATP, ADP, AMP contents and energy charge Experiments were performed using a completely randomized design. All statistical analyses were performed with SPSS (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance (ANOVA). The overall least significant difference (LSD) at p = 0.05 was calculated and used to detect significant differences among treatments. 3. Results 3.1. Chilling injury incidence, electrolyte leakage and MDA content As shown in Fig. 1a, chilling injury incidence increased rapidly both in brassinolide treated and the control bamboo shoots stored at 1 C. Chilling injury symptoms in control shoot were observed only after 14 days of storage. Chilling injury incidence increased markedly after 14 days. However, chilling injury incidence for brassinolide treatment was significantly lower than that of control treatment from 14 to 42 days during storage (P < 0.05). On the 42nd day, chilling injury incidence in control was 37.9%, which was 3-fold higher than that in brassinolide treatment. The membrane permeability changes during storage were evaluated by determining the relative electrolyte leakage. The changes in electrolyte leakage of bamboo shoots are presented in Fig. 1b. The electrolyte leakage increased gradually with storage time. The electrolyte leakage of brassinolide and control treatment
As illustrated in Fig. 2a, the ATP content decreased gradually during the storage at 20 or 1 C. The ATP content in brassinolide treatment was significantly higher than that in the control from 14 to 42 days during the cold storage. The ATP content decreased more quickly during the storage at 20 C than that in 1 C. Brassinolide treatment retarded the decline of ATP content during the storage at 20 or 1 C. The changes of ADP content is shown in Fig. 2b. The ADP content decreased gradually during the storage at 20 or 1 C. Brassinolide treatment maintained higher ADP content in comparison with the control. The AMP levels increased gradually during the storage (Fig. 2c). The AMP cotnent in brassinolide treated shoots was significantly lower than that of the control during the storage at 20 or 1 C. Energy charge decreased gradually during the storage with the changes of ATP, ADP and AMP (Fig. 2d). Brassinolide treated shoots maintained higher energy charge than that of control during the storage at 20 or 1 C, but the changes of energy charge during the storage at 1 C were more extensively than that of storage at 20 C. 3.3. H+-ATPase and Ca2+-ATPase activities The effect of brassinolide on H+-ATPase activity of shoots stored at 1 C is shown in Fig. 3a. H+-ATPase activity decreased gradually in both treatments. However, brassinolide treatment significantly
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Fig. 2. Effect of brassinolide treatment on contents of ATP (a), ADP (b), AMP (c) and energy charge (d) of bamboo shoot during storage at 20 C or 1 C. Values are the means SD of triplicate assays.
(P < 0.05) inhibited the decrease of H+-ATPase activity. At the end of storage, with the control, H+-ATPase activity experienced a reduction of 60% over the initial value, while H+-ATPase activity in brassinolide treatment was observed to be significantly higher than that in the control.
Fig. 3. Effect of brassinolide treatment on activities of H+-ATPase (a) and Ca2 + -ATPase (b) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
As shown in Fig. 3b, Ca2+-ATPase activity increased slightly before 28 days and decreased afterwards. Ca2+-ATPase activity in the control shoots was 184.16 U g 1 FW, while the activity in brassinolide treated shoots was 195.65 U g 1 FW. Therefore, brassinolide treatment induced the increase of Ca2+-ATPase activity before 28 days and retarded the decline.
Fig. 4. Effect of brassinolide treatment on activities of CCO (a) and SDH (b) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
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3.4. CCO and SDH activities CCO activity in bamboo shoots stored at 1 C increased gradually (Fig. 4a). At the end of storage, CCO activity in brassinolide treated shoots was 33.09 U g 1 FW, which was higher than that in the control (29.93 U g 1 FW). In general, brassinolide treatment maintained higher CCO activity in comparison with the control from 14th to 42nd days during the storage. As shown in Fig. 4b, SDH activity decreased quickly before the 28th day and decreased slightly afterwards. At the end of storage, SDH activity in treated shoots was 72% higher than that in control shoots. SDH activity was significantly (P < 0.05) higher than that in control shoots. 3.5. Proline content and OAT, P5CS and PDH activity The change in proline content in brassinolide treated and control shoots is shown in Fig. 5a. The proline content increased gradually over time. The proline content in shoots treated with brassinolide showed significantly higher level during storage, relative to the control (P < 0.05). OAT activity increased to a peak on the 14th day and then decrease slightly for the remainder of the storage period in brassinolide and control treatment (Fig. 5b). However, OAT activity in brassinolide treatment was significantly higher than that in the control (P < 0.05). As shown in Fig. 5c, a continuous increase in P5CS activity was observed, both in control and brassinolide treated shoots. The application of brassinolide to shoots significantly increased the P5CS activity. PDH activity increased gradually during the first 28 days and decreased thereafter (Fig. 5d). In general, brassinolide treatment showed lower PDH activity in comparison with the control (P < 0.05). 4. Discussion Chilling injury (CI) is a major factor in reducing the quality and limiting the storage time of bamboo shoots. In this study, we found that brassinolide treatment significantly reduces CI of bamboo
shoots during the storage at 1 C. The results showed that chilling injury was inhibited by brassinolide, which are consistent with those in a previous study of green bell pepper (Wang et al., 2012). The mechanism by which the brassinolide induced chilling injury in bamboo shoots was investigated. It was reported that the cell membrane was the primary site for the development of chilling injury. Transition of cell membranes phase from a flexible liquidcrystalline to a solid-gel structure that occurs at chilling temperature will increase the risk of loss of controlled cell membrane semi-permeability (Lyons, 1973). Electrolyte leakage was used as the indicator of membrane damage. CI occurrence is often accompanied by oxidative damage, one of whose biological markers is MDA content, the final product of lipid peroxidation (Xu et al., 2009). Huang and Guo (2005) suggested that the increase in antioxidant enzymes activity would contribute to the adaptation of plants to cold stress and ameliorated oxidative damage such as lipid peroxidation (MDA as indicator) and electrolyte leakage. In our study, there was a continuous increase in electrolyte leakage and MDA in both treatments, but the application of brassinolide significantly delayed the increase of electrolyte leakage and MDA. Similar results were also found in green bell pepper (Wang et al., 2012). Previous reports have revealed that the development of chilling injury is partially attributed to limited availability of energy or low energy production, whereas maintaining of higher levels of ATP and energy charge alleviates chilling injury (Chen and Yang, 2013). It was suggested in previous works that energy depletion is associated with membrane damage (Su et al., 2005; Yi et al., 2008). In the present study, ATP content and energy charge in control and brassinolide treatment decreased gradually. However, brassinolide treatment maintained a higher level of ATP and energy charge. It has been reported that the improvement of chilling tolerance is related to enhancement in activities of energy metabolism enzymes. ATPase is a class of enzyme that catalyzes the decomposition of ATP into ADP and a free phosphate ion, which produce more energy (Heyes and Townsend, 1992). The plasma membrane H+-ATPase acts as a primary transporter by pumping protons out of the cell or into the lumen of the vacuole and thus creating pH and electrical potential differences across the plasma
Fig. 5. Effect of brassinolide treatment on proline content (a) and activities of OAT (b), P5CS (c) and PDH (d) of bamboo shoot during storage at 1 C. Values are the means SD of triplicate assays.
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membrane (Azevedo et al., 2008). ATPase is linked with a specific role in protecting chilling-sensitive fruit from the injury effects of low temperature (Ghasernnezhad et al., 2008). Higher activities of H+-ATPase and Ca2+-ATPase were observed in brassinolide treated bamboo shoot (Fig. 3). The enhanced activities of ATPase in brassinolide treatment coincided with the higher ATP content, which is crucial to reduce CI in bamboo shoots under cold stress. Our studies are in accordance with those in peach fruit where the application of MeJA increased the chilling tolerance of peach fruit during cold storage, accompanied by enhanced activities of H+ATPase and Ca2+-ATPase (Jin et al., 2013). SDH catalyzes the oxidation of succinate to fumarate, which is associated with the generation of ATP in mitochondria. CCO is the last enzyme in the respiratory electron transport chain of mitochondria and plays a critical role in energy metabolism (Millar et al., 1995). Alterations in SDH and CCO activities may lead to disturbance of electron flow in the mitochondrial respiratory chain, thus resulting in chilling injury symptoms (Zhou et al., 2014). The activities of SDH and CCO in brassinolide treated bamboo shoot were significantly higher than that in control bamboo shoot. These results suggested that as important enzymes involved in energy metabolism, the enhanced activities of SDH and CCO may contribute to the alleviation in chilling injury by brassinolide treatment. Therefore, maintaining of energy metabolism enzymes activities of mitochondria to provide sufficient energy is an important factor in chilling stress. The role of proline in increasing cellular osmolarity, stabilising membrane and subcellular structures, and protecting cells against oxidative damage under stresses was suggested. (Kavi-Kishor et al., 2005; Posmyk and Janas, 2007; Kumar and Yadav, 2009). It was also reported that the stressed plants treated with proline showed considerable reduction in reactive oxygen species (Kaya et al., 2007; Posmyk and Janas, 2007). The proline accumulation is mainly attributed to its synthesis from glutamic acid and ornithine catalysed by P5CS and OAT respectively, and to its degradation catalysed by PDH (Verbruggen and Hermans, 2008). Our results indicated that brassinolide treatment significantly increased P5CS and OAT activity, while reduced the PDH activity, which in turn increased the content of proline. Li et al. (2014) reported that oxalic acid treatment significantly increased P5CS activity and reduced PDH activity with concomitant increase in proline levels, but did not activate OAT activity in mango fruit during cold storage. Thus, our study suggested that chilling-sensitive bamboo treated by brassinolide shoots exhibit increase in P5CS and OAT activity and inhibition in PDH activity, which elevated proline accumulation. The proline metabolism plays a key role in alleviating CI during long term cold storage and maintaining the quality of bamboo shoots. Therefore, brassinolide treatment maintained the quality of bamboo shoots and reduced the impact of chilling injury. Kaur et al. (2011a,b) demonstrated that exogenous proline was significantly effective in reducing the impact of chilling injury on reproductive growth in chickpea. Zhu et al. (2010) also found that brassinosteroids significantly delayed jujube fruit senescence and maintained the quality. The accumulation of proline and higher energy was two important factors for maintaining the membrane integrity. Proline may interact with ATP and energy metabolism enzymes to alleviate chilling injury of bamboo shoots. Our results showed that brassinolide treatment triggered a response that maintained the integrity of membranes, raised the energy supply and proline accumulation and inhibited lipid peroxidation. In conclusion, brassinolide treatment promoted postharvest bamboo shoots resistance to low temperature stress. Higher resistance of bamboo shoots to chilling stress was related to higher levels of ATP and energy charge, which was of benefit to membrane integrity. Furthermore, elevated proline accumulation was also an important factor in enhancing chilling resistance of bamboo shoots.
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