Changes in Metabolite Contents in Melon Seedling Leaves under Low Temperature Stress

Jingjing SHANG,Hongmei TIAN,Tongguang LIU*,Qi’an ZHANG,Ling FANG,Baohui DUAN,Chao WU,Jiashuang YAN

1.School of Horticulture,Anhui Agricultural University,Hefei 230036,China;2.Institute of Horticulture,Anhui Academy of Agricultural Sciences,Hefei 230031,China;3.Anqing Institute of Agricultural Sciences,Anqing 246003,China

Melon(Cucumis meloL.;Cucurbitaceae:Cucumis)is an annual trailing herb originating from tropical Africa.Melon is one of the world's top ten fruits.It is rich in nutrients and perfect in color,aroma and taste.Regular intake of melon is beneficial to human heart,spleen and intestinal activities,so it is loved by the people.

Melon lives in a warm climate and is hot tolerant.The optimal temperature for melon growth is 25-30℃,but it can grow in the temperature range of 14-45℃.When temperature is lower than 10℃,melon plants will be damaged by low temperature in varying extents.In recent years,with the rapid development of China’s horticulture,protected cultivation is becoming the main form of cultivation and production of melon.In winter and spring,melon production often encounters cold damage in China.Long-term low temperature is very negative for melon seedling growth,making cold damage problem increasingly prominent in the off-season production.At present,in the lower reaches of the Yangtze River,the nursing of melon seedlings has been in advanced,and the earliest has even been advanced to January,thus the transplanting of melon seedlings has been brought forward to February.During this period,the outside temperature is low,and facility cultivation will encounter long-term low temperature(＜15 ℃)if the weather is rainy continuously,seriously affecting the growth of young melon seedlings,thereby reducing the yield and quality of melon.The low temperature in winter and spring will severely affect the quality,yield and economic benefit of melon in facility cultivation. Therefore, researches on cold resistance of melon are of great significance for stable development of off-season and facility cultivation and improvement of economic efficiency of melon.

Material and Methods

Material

The selected melon cultivar was Zaotian No.1,which was provided by the Institute of Horticulture,Anhui Academy of Agricultural Sciences.

Methods

The full and size-uniform melon seeds were selected.They were soaked in water at 55℃for 15 min and then at 30℃for 8 h.And then,they were transferred into an incubator at 30℃.After the seeds germinated,they were sowed in plastic trays(5 cm×5 cm).The seedling management was the same as conventional management.

Atthe three-leafstage,the melon seedlings were transferred into a light incubator (SPX-250B-G,Shanghai Boxun Industry&Commerce Co.,Ltd.).The photoperiod was 12L∶12D,and the light intensity was 100 μmol/(m2·s).The treatments were arranged as low temperature treatment(day/night temperature of 5℃/5℃)and control(day/night temperature of 25℃/25℃).For the melon seedlings in the treatment groups,they were pre-treated at day/night temperature of 15℃/10℃for 2 d and then treated at day/night temperature of 5℃/5℃for 8 d.The size-uniform top second and third leaves of melon seedlings in the low temperature treatment group were sampled at 9:00-10:00 on day 2,4,6 and 8.There were three replications for each treatment.

The sampled melon leaves were weighed timely,packed and preserved at-80℃.After the sampling was completed,the determination of various indexes was carried out in a laboratory.

Determination of H2O2contentThe H2O2was extracted using acetone and determined using Ti(IV)-PAR colorimetry[1].A certain amount(1.5 g)of stored melon leaves was taken out.After cut into pieces,the melon leaves were mixed with 3 ml of pre-cooled acetone.The mixture was centrifuged at 10 000 r/min for 10 min at 4℃.A certain volume(1 ml)of supernatant was first mixed with a certain volume (3 ml)of extraction agent(CCl4∶CHCl3=3∶1,v∶v)and then mixed with a certain volume(5 ml)of distilled water.The new mixture was centrifuged at 4 000 r/min for 1 min.The obtained supernatant was used for determination of H2O2content.A certain volume(1 ml)of the obtained supernatant was mixed with appropriate amount of catalase(CAT)with final concentration of 3 U/ml and incubated at 30℃for 10 min.At the same time,a certain volume (1 ml)of the obtained supernatant was mixed with equivalent-volume of inactivated CAT solution.Both the two mixtures were mixed with certain volumes(1 ml for each)of phosphate buffer(0.2 mol/L,pH 7.8)and Ti(IV)-PAR chromogenic reagent(0.2 mol/L).The two new mixtures were bathed in water at 45℃for 20 min and stood at room temperature for 1 h.Their absorbances were determined at wavelength of 508 nm.Based on the absorbances,the H2O2content was determined.

Determination of production rate of superoxide anion radical(O2.-) A certain amount(0.2 g)of melon leaves was placed in a pre-cooled mortar.A certain volume (1.6 ml)of pre-cooled phosphate buffer(50 mmol/L,pH 7.8)was added in the mortar.The grinding was carried out on ice.The mixture was transferred to a centrifuge tube and centrifuged at 12 000 r/min for 20 min at 4℃[2].A certain volume(0.5 ml)of supernatant was transferred into a new centrifuge tube.Subsequently,certain volumes of PBS buffer(0.5 ml;0.05 mol/L,pH 7.8)and hydroxylamine hydrochloride solution(1 ml;1 mmol/L)were added.After stood at 25℃for 1 h,the mixture was mixed with certain volumes of sulfanilic acid(1 ml;17 mmol/L)and α-naphthylamine (1 ml;17 mmol/L).After stood at 25℃for 20 min,the absorbance of the mixture was determined at wavelength of 530 nm.Distilled water was used for zero adjustment[3].

Determination of MDA contentA certain amount(0.5 g)of melon leaves was mixed with a certain volume(10 ml)of phosphate buffer(50 mmol/L,pH7.5)and then ground on ice.The DMA content was determined as described by Li and Mei[4].

Determination ofsoluble sugar contentA certain amount(2 g)of stored melon leaves was mixed with a certain volume(10 ml,added by several times)of ethanol and ground.The mixture was transferred to a centrifuge tube and bathed in water at 80℃for 1 h.Subsequently,the mixture was centrifuged at 4 000 r/min,and the supernatant was collected.The precipitate was suspended with 5 ml of 80% ethanol(added by two times)and bathed in water at 80℃for 10 min.The mixture was also centrifuged at 4 000r/min.Thetwosupernatantswere mixed and diluted to 25 ml.A certain volume (10 ml,added by several times)of the obtained solution was transferred to an evaporating dish and dried in an oven.The obtained residue was dissolved in 1 ml of ultrapure water,and the solution was passed through a 0.22 μm filter membrane.The contents of sucrose,glucose and fructose were determined with high performance liquid chromatography.

Determination of solubleprotein contentThe protein content was determined using Bradford assay[2].The standard curve was prepared using bovine serum albumin.A certain amount(0.2 g)of melon leaves was placed in a pre-cooled mortar.Subsequently,1.6 ml of pre-cooled phosphate buffer(50 mmol/L,pH 7.8)was added to the mortar.The melon leaves were ground on ice.The mixture was transferred to a centrifuge tube and centrifuged at 12 000 r/min for 20 min at 4 ℃.A certain amount(20 μl)of supernatant was mixed with 80 μl of phosphate buffer(50 mmol/L,pH 7.8)and 2.9 ml of Coomassie brilliant blue G-250 solution in succession.After 2-min reaction,the absorbance was determined at the wavelength of 595 nm.Referring to the prepared standard curve,the protein content was obtained.The mixture of 100 μl of phosphate bufferand 2.9 ml of Coomassie brilliant blue G-250 solution was used for zero adjustment.The soluble protein contentin melon leaves was calculated according to following formula:

In the formula (1),Crepresents the obtained protein content by referring to the standard curve(μg);VTrepresents the volume of extract after dilution(ml);VSrepresents the volume of extract used for determination (ml,20 μl);Wrepresents the fresh weight of sample(g).

Results and Analysis

Changes in H2O2content

Due to strong oxidation,H2O2is also one of the active oxygen molecules.Low temperature stress can cause oxidative damage to plants,leading to excessive production of H2O2.If excess H2O2accumulated in cells cannot be promptly removed,it will cause toxic effects,increasing the damage to cells.The results showed that with the proceeding of low temperature stress,the H2O2content in melon seedling leaves was first increased and then decreased(Fig.1).

Changes in production rate of superoxide anion radical(O2.-)

O2.-harms plants mainly through starting membrane lipid peroxidation or de-esterification[5],thereby destroying membrane structure.At the first period of low temperature stress,the production rate of O2.-was increased.However,the production rate of O2.-in the treatment group was lower than that in the control group.With the proceeding of low temperature stress,the production rate of O2.-reached the peak on day 4,and then started to decline.In the treatment group,the production rate of O2.-on day 8 was lower than that on day 2 by 12.24% (Fig.2).

Changes in MDA content

MDA is the end product of membrane lipid peroxidation in plants,and is also one of the major indices reflecting damage to membrane system[6].As shown in Fig.3,the DMA contents in melon seedling leaves in both treatment and control groups were all increased with the proceeding of low temperature stress,but the MDA content in the treatment group was higher than that in the control group.On day 2,the MDA content in the treatment group was 36.87% higher than that in the control group,and on day 8,the MDA content in the treatment group was 77.23% higher than that in the control group.

Changes in soluble sugar content

As shown in Table 1,the soluble sugarcontentin melon seedling leaves in the treatment group was higher than that in the control group.At the same time,among sucrose,glucose and fructose,the sucrose content was highest.With the proceeding of low temperature stress,the soluble sugar content was decreased first and then increased.

Changes in soluble protein content

As shown in Fig.4,the soluble protein content in the treatment group was higher than that in the control group,and it was increased with the proceeding of low temperature stress.On day 2 and 8,the soluble protein content in the treatment group was higher than that in the control group by 1.5 times and 1.9 times,respectively.It indicated that the soluble protein content was increased faster under low temperature stress than under normal conditions.

Conclusions and Discussion

Correlations between H2O2content and O2.-production rate and cold resistance of young melon seedlings

Table 1 Changes in soluble sugar content in melon seedling leaves under low temperature stress

SOD (superoxide dismutase),POD(peroxidase)and CAT(catalase)are active oxygen scavengers in plants.SOD is an important protective enzyme in enzymatic defense system of plants,and it activity is closely related to cold resistance of plants.SOD can defend against or reduce damage of active oxygen free radicals and peroxides to membrane system,protecting the normalmetabolism.Some studies have shown that POD,as a protective enzyme in plants under low temperature stress,can rapidly remove or reduce damage to plants,thereby improving cold resistance of plants.In the protective enzyme system of plant,POD is mainly to catalyze hydrogen peroxide,avoiding the damage of harmful substances to plants under low temperature stress.CAT is mainly present in peroxide molecules,and its function is to regulate H2O2content in plants under low temperature stress.As an important scavenger for H2O2,CAT plays an important role in maintaining homeostasis ofH2O2.SOD,POD and CAT coordinate with each other,and remove excess free radicals,maintaining free radicals in plants at a normal level,thereby improving cold resistance of plants[7].In this study,the content of H2O2and production rate of O2.-,as well as activities of SOD,POD and CAT,in melon seedling leaves were determined.The results showed that with the proceeding of low temperature stress,the content of H2O2and production rate of O2.-were all increased first and then decreased,and the activities of SOD,POD and CAT were also increased first and then decreased.The increased activities of SOD,POD and CAT might be caused by increased O2.-production rate and H2O2content in melon seedling leaves under low temperature stress.Therefore,it is speculated that increased O2.-production rate and H2O2content can increase activities of SOD,POD and CAT in melon seedling leaves.It indicates that increased H2O2content at the early period of low temperature stress can improve cold resistance of melon seedlings.The decreased O2.-production rate and H2O2content 4 d after the treatment might be caused by destroyed cell structure and slowed respiratory rate in melon seedling leaves under low temperature stress.

Effect of low temperature stress on MDA content in melon seedling leaves and correlation between MDA content and cold resistance of melon seedlings

MDA is a product of membrane lipid peroxidation,and its accumulation can further damage membranes and cells.MDA is generally used as an index reflecting degree of membrane lipid peroxidation and strength of cold resistance of plants[8].The results of this study showed that with the proceeding of low temperature stress,MDA content was increased significantly, which might be due to strengthened membrane lipid peroxidation caused by overproduction of reactive oxygen species.It suggested that low temperature had destroyed cell structure of melon seedling leaves.However,the increment of MDA content in melon seedling leaves in the treatment group was smaller than that in the control group,indicating reduced sensitivity of melon seedlings to low temperature.Thus the cold resistance of melon seedlings was improved.

Correlation between soluble sugar content and cold resistance of melon seedlings

Soluble sugars are also a class of protective substance inside cells.They can reduce the freezing point to improve protection capacity of protoplasm.Soluble sugars can protect colloid protein against denaturation and coagulation caused by low temperature[9].This study found that the soluble sugar content in the treatment group was higher than that in the control group,and it was reduced first and then increased.Low temperature stress affected the growth of young melon seedlings,and the equilibrium between synthesis and transfer of sugars in melon seedling leaves was broken;when the sugar content was reduced to a certain level,plant adjusted its metabolism to rapidly increase sugar content and to adapt itself to stress conditions.Studies have shown that low temperature can increase starch hydrolase activity and accelerate starch hydrolysis,increasing soluble sugar content and cytolymph concentration,thereby improving cold resistance of plants[10].Jianet al.[11]considered that at the early period of low temperature stress,plants defended themselves against cold damage possibly through increasing soluble sugar content in cells.Therefore,appropriate low temperature stress can be conducted to increase soluble sugar content and improve osmotic adjustment ability,thereby improving cold resistance of melon seedlings.In addition,Xuet al.[12]found that with the decreased temperature,the soluble sugar content inCucurbita pepowas still higher than those in general varieties.Melon andCucurbita pepoall belong to the Cucurbitaceae,so their physiological and biochemical characteristics are similar.Therefore,in the earlyseason and facility cultivation of melon,selection of cultivar is also very important for cold resistance of melon seedlings.

Correlation between soluble protein content and cold resistance of melon seedlings

Studies have shown that low temperature stress leads to changes in proteins in plant cells,represented by changes in soluble proteins and enzymes and generation of cold resistant proteins[13-19].However,soluble proteins have strong hydrophilcity,and they can significantly enhance cell moisture holding capacity;increased soluble proteins can shackle more moisture,reducing the damage of low temperature to protoplasm,as well as death of protoplasm[20],thereby improving cold resistance of plants.In this study,the soluble protein content in melon seedling leaves was increased under low temperature stress,indicating synthesis of new proteins induced by low temperature stress.This might be the performance of body’s adaptation to low temperature,and synthesis of new proteins contributed to cold resistance of melon seedlings.

In summary,increased H2O2content at the early period of low temperature stress can stimulate increased activity of protection enzyme,reducing the damage of low temperature stress to melon.Therefore,spaying H2O2solution at an appropriate concentration can improve cold resistance of melon.Low temperature stress leads to increased MDA content,destroying cell membrane structure.However,with the proceeding of low temperature stress,the increase of MDA content is slowed, indicating melon leaves’adaptation to the low-temperature environment.Soluble sugars are a class of protective substances in cells under cold damage and freezing injury stresses,and their content is positively related to cold resistance of most plants.Increased soluble protein content can enhance osmotic adjustment ability of cells and maintain metabolic balance.The responses of plants to low temperature stress are represented by synthesis of stress-tolerant proteins,resulting in increased soluble protein content in plants.In this study,with the proceeding of low temperature stress,the soluble sugar content and soluble protein contentin melon seedling leaves were increased,indicating initiative synthesis and accumulation of soluble sugars and proteins to adapt melon seedlings to low temperature environment.This study only studied a few main metabolites in melon seedling leaves.The adaptation mechanism of melon seedlings to low temperature environment is extremely complex,and it requires comprehensive researches on a number of physiological and biochemical processes,and the interactions among above physiologicaland biochemical processes,as well as further mechanisms,still need further studies.Based on the obtained results above,it can be concluded that appropriate low temperature stress can enhance the cold resistance of melon.

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