Effects of Exogenous Glycine Betaine on Oxidation Metabolism in Cucumbers during Low-temperature Storage

Li XU,Xiangning CHEN,Haiying ZHANG,Tao HAN,Fugui WANG

College of Food Science and Engineering,Beijing University of Agriculture,Beijing 102206,China

Responsible editor:Xiaohui FAN Responsible proofreader:Xiaoyan WU

G lycine betaine (GB)is a quaternary ammonium compound that has been confirmed as an important osmoprotectant[1].Under normal ambient conditions,GB content in plants is not high.However,under cold,drought,salt and other environmental stresses,plants can accumulate a large amount of GB of cells[2].As a non-toxic cytoplasmic osmotic a gent and protective agent,GB can improve stress resistance of plants[3].GB accumulation in cells can improve the osmotic adjustment ability of cells and stabilize the structure and function of high molecular weight proteins and biological membrane in cells.At present,a large number of studies have been conducted on the biosynthesis and osmotic adjustment ability of GB in plants.It has been demonstrated that GB accumulation can improve drought resistance,saline-alkali resis tance and cold resistance of crops[4-6].Broad bean seedlings treated with exogenous GB could maintain better water condition than untreated plants during water stress[7].Under low temperature stress,cucumber seedlings accumulate GB to adapt to low temperature and enhance the tolerance to low temperature;exogenous GB could reduce the inhibitory effect of cold stress on the activities of superoxide dismutase (SOD),catalase (CAT) and peroxidase (POD),membrane damage,intracellular material leakage and production of malondialdehyde(MDA),thereby improving the cold resistance of cucumber seedlings[8].

Most studies are concentrated on the effect of GB on plant seedlings under stresses,but little information is available on the effect of GB on postharvest cold-sensitive fruits.Zhang et al.[9]found that treating postharvest cucumbers with exogenous GB could reduce the chilling injury of fruit under low temperature conditions,increase proline content,enhance SOD activity in various tissues and improve cold resistance.Currently,the effect of exogenous GB on plant resistance has attracted much attention[10-11].Based on that,in this study,the effects of exogenous GB on oxidation metabolism-related physiological indicators in cucumber during lowtemperature storage were further investigated,aiming at providing a theoretical basis for improving cold resistance in cold-sensitive fruits and vegetables under low temperature storage conditions.

Materials and Methods

Materials and reagents

Cucumber(Cucumis sativus Linn.)cultivar Zhongnong No.8 was selected as the experimental material.Plump and straight cucumbers with 1cm stalks,uniform thickness and without mechanical damage were purchased from Beijiao Market in Changping District of Beijing Municipalitiy and sent immediately to the laboratory for processing.

Catalase Activity Assay Kit and Hydrogen Peroxide Assay Kit were produced by Nanjing Jiancheng Biological Engineering Institute.

Instruments

High-speed refrigerated centrifuge was purchased from Shanghai Saite Xiangyi Centrifuge Instrument Co.,Ltd.;UV-visible spectrophotometer was purchased from Beijing Purkinje General Instrument Co.,Ltd.

Material processing

On the basis of pre-experiment,the obtained cucumbers were soaked with 0,5,10 and 15 mmol/L GB solutions for 15 min,respectively,air-dried at room temperature,transferred to polyethylene film bags,sealed,and preserved at 4 ℃.Fifty cucumbers were processed in each treatment,with three replications.

Indicator determination

During storage,samples were collected randomly every other day to determine various physiological indicators.

Determination of GB content[12]

Sample preparation:with water extraction method,cucumbers were quick-frozen by liquid nitrogen and ground into powder; 40 g of cucumber powder was weighed,transferred to an erlenmeyer flask,added with 40 ml of water,and placed at room temperature overnight.On the next day,samples were filtrated,and pH was adjusted to 1.0; the filtrate was added with water to a final volume in a 100 ml flask as sample stock solution,and preserved in a refrigerator before use.

Construction of standard curve:0,1,2,3,4 and 5 ml of GB standard solution(1 mg/ml)were transferred to 15 ml centrifuge tubes,added with 5,4,3,2,1 and 0 ml of distilled water,respectively,placed in an ice bath for 15 min,added with 5 ml of 15 g/L Reinecke salt solution (freshly prepared),shaken,preserved at 0-4 ℃for at least 3 h,slightly shaken to suspend the crystals,and centrifuged at 8 000 r/min for 10 min;the supernatant was removed,and the precipitate was added with 5 ml of diethyl ether,shaken fully to wash the crystals,centrifuged at 8 000 r/min for 10 min,and air-dried; the crystals were added with 5 ml of acetone,shaken evenly to fully dissolve GB-Reinecke salt crystals,and centrifuged at 8 000 r/min for 10 min.The absorbance was measured at 525 nm to draw the standard curve.Subsequently,5 ml of prepared sample solution was collected,and cooled at room temperature for 15 min; pH was adjusted to 1.0 with concentrated hydrochloric acid; the following steps were consistent with the construction of standard curve,with three replications.The measured value was substituted into the standard curve to calculate GB content in samples.

Determination of lipoxygenase(LOX) activity[13]Accurately 2.75 ml of 0.1 mol/L acetic acid-sodium acetate buffer (pH 5.5) was collected,added with 50 μl of 0.1 mol/L sodium linoleate solution,incubated at 30 ℃for 10 min,added with 200 μl of crude enzyme solution,and mixed evenly.Distilled water was used as a reference for zero setting.At 15 s,the absorbance of the reaction system at 234 nm was recorded as the initial value;subsequently,the absorbance was recorded every 30 s for six times,with three replications.LOX activity was calculated according to the following formula:

Where,ΔOD234indicates the change in OD234within the reaction time; Vtindicates the total volume of extracted enzyme solution/ml; Windicates the fresh weight of plant samples; Vsindicates the volume of applied enzyme solution/ml; t indicates the reaction time.

Determination of peroxidase (POD)activityPOD activity was determined with guaiacol method[14]:37.5 ml of 0.2 mol/L phosphate buffer was added with 8.93 ml of 1.5% guaiacol,heated and stirred on a magnetic stirrer until guaiacol was dissolved,cooled to room temperature,added with 3.57 ml of 0.5% H2O2,mixed evenly,and preserved in a refrigerator.In determination process,2.1 ml of phosphate buffer was added with 0.5 ml of guaiacol and 0.2 ml of H2O2as the reaction mixture.Phosphate buffer(pH 6.0)was added into a cuvette as a reference for zero setting; 0.2 ml of supernatant of enzyme solution was added with 2.8 ml of reaction mixture in another cuvette.With accurate timing,OD470was measured every minute for five times using an ultraviolet spectrophotometer.POD activity was represented by the change in OD470of per gram of fruits and vegetables per minute,ΔOD470/(min·mg),with three replications.POD activity was calculated according to the following formula:

Where,ΔOD470indicates the change in OD470within the reaction time; Vtindicates the total volume of extracted enzyme solution/ml; Windicates the fresh weight of plant samples; Vsindicates the volume of applied enzyme solution/ml; t indicates the reaction time.

Determination of catalase (CAT)activityCAT activity was determined in accordance with the instructions of kits produced by the first branch of Nanjing Jiancheng Biological Engineering Institute.The kit comprises reagent 1,reagent 2,reagent 3 and reagent 4.According to the number of samples,each concentration was tested with three measuring tubes and one control tube.Firstly,0.05 ml of tissue homogenates were added into each measuring tube; 1.0 ml of reagent 1 and 0.1 ml of reagent 2 were added into the control tube and each measuring tube,mixed evenly,incubated at 37 ℃for 1 min,and added with 1.0 ml of reagent 3 and 0.1 ml of reagent 4; subsequently,0.05 ml of tissue homogenates were added into the control tube,and mixed evenly.The absorbance of each tube was measured with an optical path of 0.5 cm at 405 nm with three replications.CAT activity was calculated according to the following formula:

CAT activity in tissue homogenates=[(OD of control tube-OD of measuring tube) ×271]/(60×Sampling quantity×Homogenate protein content)

Where,271 is the inverse of the slope.

Determination of hydrogen peroxide(H2O2)contentH2O2content was determined in accordance with the instructions of kits produced by the first branch of Nanjing Jiancheng Biological Engineering Institute.The kit comprises reagent 1,reagent 2 and 5% standard application solution.According to the number of samples,appropriate number of measuring tubes,one control tube and one standard tube were set.Firstly,each tube was added with 1 ml of reagent 1; control tube,standard tube and measuring tube were added with 0.1 ml of distilled water,0.1 ml of 0.5% standard application solution,and 0.1 ml of samples,respectively;the mixtures were mixed evenly,incubated in a water bath at 37 ℃for 1 min,added with 1 ml of reagent 2,and mixed evenly.The absorbance of each tube was measured at 405 nm with three replications.H2O2content was calculated according to the following formula:

H2O2contentin samples(mmol/L)=[(OD of measuring tube-OD of control tube)/(OD of standard tube -OD of control tube)]×Standard concentration(163 mmol/L)×Sample dilution

Determination of malondialdehyde(MDA) contentMDA content was determined in accordance with previous literature[15]:5 g of cucumbers were weighed,added with 2.5 ml of 5% TCAsolution and a small amount of quartz sand,ground into homogenates,added with 2.5 ml of TCA,further ground,and centrifuged at 8 000 r/min for 10 min; the supernatant was collected as the sample extract; 2 ml of supernatant was collected(2 ml of distilled water was used in control group),added with 2 ml of 0.6%TBAsolution,and mixed evenly.The tube was placed in a boiling water bath for 10 min (timing since the formation of small bubbles in the tube),cooled to room temperature,and centrifuged at 8 000 r/min for 15 min; the supernatant was collected to measure the volume.The absorbance was measured at 532 nm and 600 nm using 0.6% TBAsolution as a blank control with three replications.MDA content was calculated according to the following formula:

Where,V/v indicates the total volume of extract/total volume of measuring solution; R indicates the total volume of reaction solution; Windicates the fresh weight of plant samples/g; 0.155 is the molar extinction coefficient of MDA.

Data analysis

Experimental data were analyzed using SPSS statistical software.Significant difference analysis was performed,and mean±SE was marked.

Results and Analysis

Effect of exogenous GB on GB content in cucumbers during low-temperature storage

During low-temperature storage,GB content in cucumbers showed a stable rising trend (Fig.1).GB content in cucumbers treated with different concentrations of GB exhibited a similar variation trend to control.GB content in cucumbers rised with the increase of GB concentration.To be specific,GB content in cucumbers treated with 5 mmol/L GB was the closest to control; GB content in cucumbers treated with 10 mmol/L and 15 mmol/L was significantly higher than that in control(P＜0.05),indicating that exogenous GB was conducive to the accumulation of GB in cucumber tissues.

Effect of exogenous GB on lipoxygenase(LOX)activity in cucumbers during low-temperature storage

In the early period of low-temperature storage(first 4 d),LOX activity in cucumbers showed a slow upward trend(Fig.2);subsequently,LOX activity in cucumbers increased remarkably and reached the highest on the 8thd that was approximately doubled compared with the initial level; on the 12thd,LOX activity in cucumbers declined slightly.LOX activity in cucumbers treated with different concentrations of GB exhibited a similar variation trend to control.However,LOX activity in cucumbers treated with GB was lower than that in control.To be specific,LOX activity in cucumbers treated with GB on the 12thd was close to that in control on the 8thd.In addition,LOX activity in cucumbers exhibited no significant differences among different GB concentrations,indicating that GB treatment could reduce LOX activity in cucumbers under chilling stress.

Effect of exogenous GB on peroxidase (POD) activity in cucumbers during low-temperature storage

During low-temperature storage,POD activity in cucumbers showed a slow downward trend (Fig.3),which declined rapidly since the 10thd.POD activity in cucumbers treated with different concentrations of GB exhibited a similar variation trend to control.Moreover,POD activity in cucumbers treated with GB was higher compared with control since the 6thd.Especially,POD activity in cucumbers treated with 10 mmol/L GB (P ＜0.05) was 15%-25% higher than that in control,which indicated that appropriate GB treatment could effectively improve POD activity in cucumbers under low temperature stress.

Effect of exogenous GB on catalase(CAT) activity in cucumbers during low-temperature storage

During low-temperature storage,CAT activity in cucumbers showed a stable upward trend(Fig.4).CAT activity in cucumbers treated with different concentrations of GB exhibited a similar variation trend to control and was significantly higher compared with control (P＜0.05).To be specific,CAT activity in cucumbers treated with 10 mmol/L GB was significantly higher than that in cucumbers treated with 15 mmol/L GB (P＜0.05); CAT activity in cucumbers treated with 5 mmol/L GB was significantly higher than that in cucumbers treated with 10 mmol/L and 15 mmol/L GB within the first four days(P ＜0.05); however,since the 8thd,CAT activity in cucumbers treated with 5 mmol/L GB was significantly lower than that in cucumbers treated with 10 mmol/L GB but higher than that in cucumbers treated with 15 mmol/L GB(P＜0.05).In short,GB treatment could effectively improve CAT activity in cucumbers under low temperature stress.

Effect of exogenous GB on hydrogen peroxide (H2O2) content in cucumbers during low-temperature storage

During low-temperature storage,H2O2content in cucumbers showed an overall trend of gradual accumulation(Fig.5).H2O2content in cucumbers treated with different concentrations of GB exhibited a similar variation trend to control and was significantly lower compared with control since the 4thd(P ＜0.05).H2O2content in cucumbers treated with 10 mmol/L GB was the lowest since the 8th d,indicating that appropriate GB treatment could effectively reduce H2O2accumulation in cucumbers under low temperature stress.

Effect of exogenous GB on malondialdehyde (MDA) content in cucumbers during low-temperature storage

During low-temperature storage,MDA content in cucumbers showed an overall slight upward trend (Fig.6).MDA content in cucumbers treated with different concentrations of GB exhibited a similar variation trend to control and was significantly lower compared with control (P ＜0.05).To be specific,MDA content in cucumbers treated with 10 mmol/L reached the lowest,indicating that appropriate GB treatment could effectively reduce MDA accumulation in cucumbers under low temperature stress.

Discussions

Under low temperature stress,cell membrane transforms from normal liquid crystal structure into solid gel structure,thereby solidifying lipid materials,improving membrane permeability,damaging cell molecular function and leading to the imbalance of ions inside and outside the cell membrane,resulting in ion leakage and causing abnormal metabolic changes[16].

Lipoxygenase (LOX) is a key enzyme in fatty acid oxidation pathway,which catalyzes free unsaturated fatty acids to produce lipid peroxylradicals,thereby resulting in damages to the phospholipid bilayer of cell membrane,improving membrane permeability,causing metabolic disorders and aging[17-18].In this study,results show that GB treatment can reduce LOX activity in cucumbers during lowtemperature storage,which suggests that GB can decline the production of free radicals and peroxides by reducing LOX activity.

Peroxidase (POD)is a protective enzyme in plants that can scavenge free radicals generated constantly in vivo and maintain metabolic balance,which is extremely sensitive to various adverse conditions.POD activity can to some extent reflect the level of cold resistance in plants.In plants subjected to cold damage,POD can coordinate with superoxide dismutase(SOD)and catalase (CAT) to eliminate free radicals produced by cells,thereby improving stress resistance in plants and protecting the integrity of membrane structure[19].In this study,results show that GB treatment can significantly improve POD activity in cucumbers during low-temperature storage.Previous studies[9]have shown that GB can improve SOD activity to strengthen the scavenging effect on superoxide anion radicals.Zhang et al.[20]analyzed saline-alkali resistance in wheat and gained consistent results.

H2O2is not a free radical species but one of the main active oxygen species causing oxidative stress due to the strong oxidation property.H2O2can be catalyzed and decomposed by CAT to reduce membrane lipid peroxidation.Low temperature stress decreases CAT activity in plants,thus resulting in large-scale accumulation of various reactive oxygen species and causing membrane lipid peroxidation.In this study,results show that GB treatment can effectively maintain CAT activity and reduce H2O2accumulation.To be specific,cucumbers treated with 10 mmol/L GB exhibited the highest CAT activity and the lowest H2O2content.

Malondialdehyde (MDA) is the final product of membrane lipid peroxidation,and its content can to some extent reflect the level of stress resistance in plants[21].In this study,results show that GB treatment can effectively inhibit the production and accumulation of MDA in cucumbers during lowtemperature storage and enhance the stability of cell membrane.Chen et al.[22]reported that exogenous GB could effectively decline MDA content in maize seedlings under low temperature stress.Zhang et al.[20]investigated the effects of betaine on activities of membrane protective enzymes in wheat seedlings and found a similar mechanism.Zhang et al.[9]analyzed the effects of exogenous GB on chilling resistance in cucumber fruits and found that GB treatment could effectively stabilize cell membrane permeability,which may be related with the conclusion in this study that GB treatment improves GB content in cucumbers during low-temperature storage.

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