Melatonin treatment induces chilling tolerance by regulating the contents of polyamine,γ-aminobutyric acid,and proline in cucumber fruit

Miilion P MADEBO,LUO Si-ming,WANG Li,ZHENG Yong-hua,JlN Peng

College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,P.R.China

Abstract The mechanism of melatonin (MT) induced chilling tolerance in harvested cucumber fruit was investigated at commercial maturity.In this study,cucumber fruits were treated with 100 µmol L-1 MT at 4°C and 90% relative humidity for 15 d of storage.In comparison with the control,cucumber treatment with MT resulted in reduced chilling injury (CI),decreased electrolyte leakage and enhanced firmness.The fruits treated with MT showed higher chlorophyll contents in storage conditions with suppressed chlorophyllase enzyme activity.MT treatment increased arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) enzyme activities.Moreover,enhanced expression of the Cucumis sativus ADC (CsADC)and C. sativus ODC (CsODC) genes resulted in the accumulation of polyamine contents.Similarly,proline levels exhibited higher levels among treated fruits.Meanwhile,the proline synthesizing enzymes △1-pyrroline-5-carboxylate syntheses (P5CS) and ornithine aminotransferase (OAT) were significantly increased,while a catabolic enzyme of proline dehydrogenase (PDH) activity was inhibited by treatment.In addition,MT induced expression of C. sativus OAT (CsOAT)and C. sativus P5CS (CsP5CS) genes.Cucumber fruits treated with MT also exhibited higher γ-aminobutyric acid (GABA)content by enhanced GABA transaminase (GABA-T) and glutamate decarboxylase (GAD) enzyme activities and a higher C. sativus GAD (CsGAD) gene expression.To sum up,the results show that MT treatment enhanced chilling tolerance,which was associated with the regulation of polyamines,as well as proline and γ-aminobutyric acid.

Keywords:cucumber,melatonin,polyamine,proline,GABA,chilling injury

1.lntroduction

Cucumber (CucumissativusL.),which belongs toCucurbitaceaefamily,is consumed as a fresh fruit and contains a high nutritional value.However,several types of fresh produce,including cucumber,are susceptible to various problems upon cold storage below 10°C.Cold storage is one of the technologies used to reduce respiration,avoid deterioration and enable shelf life extension (Hakimet al.1999).

Melatonin (N-acetyl-5-methoxytryptamine;MT),a natural hormone and regulator exclusive to animals,has been found to be distributed in different parts of plants,predominantly the leaves,roots,stems,and flowers (Arnao and Hernández-Ruiz 2015).The application of MT acts as an effective temperature stress regulator and a freeradical scavenger in plants,as well as showing darkness signaling,growth promotion regulating,and evident anti-oxidative responses (Zhanget al.2014).Besides,exogenous MT application improved ripening behavior of banana in relation with ethylene (Huet al.2017).In pear fruit,MT treatment reduced chilling injury (CI) by inhibiting the synthesis of ethylenevianitric oxide regulation (Liuet al.2019).Moreover,MT exposure reduced CI and enhanced antioxidant activity in tomato (Aghdamet al.2019),peach (Gaoet al.2016),and grape (Xuet al.2018) fruits.It also delayed senescence and minimized browning of the pericarp tissue of litchi fruit (Zhanget al.2018).Chilling tolerance was reported in peach fruit by MT pretreatmentviaan increase of hydrogen peroxide(H2O2),so it might act as a signaling molecule to induce protective mechanismsviaup-regulation of anti-oxidative gene expression (Caoet al.2018b).

Polyamines (PAs) are one of the essential stress regulating biomolecules.Moreover,PAs are nitrogenous compounds with low molecular weight,and naturally occur in plants systems as different compounds,namely putrescine (Put),spermidine (Spd),and spermine (Spm)(Jahanet al.2019).Exogenous polyamine (putrescine and spermidine) treatments in different apricot cultivars minimized CI by enhancing antioxidant enzyme activity during the storage period (Koushesh Sabaet al.2012).

Proline is a vital amino acid which plays roles as a cellular osmotic regulator,protein stabilizer,free radical scavenger,and lipid peroxidation inhibitor in plants.Moreover,it was found to accumulate quickly in response to different abiotic stresses (Verbruggen and Hermans 2008).In addition,the application of nitric oxide regulated proline metabolism,which was found to enhance the tolerance level to cold stress in banana fruit (Wanget al.2013).

γ-Aminobutyric acid (GABA) has shown a considerable role in regulating stress tolerance in many harvested fruit and vegetable products,which is tightly synchronized with polyamine metabolism (Shelpet al.1999).Previous studies have shown that GABA treatments alleviated CI in cucumber fruit by increased contents of proline and GABA,as well as elevated antioxidant activities(Malekzadehet al.2017).

Prior studies have suggested insight into the association of polyamines (PAs),proline and GABA levels,and resistance to CI in harvested fruit and vegetable crops.Furthermore,exogenous application of MT in harvested products reduced CI by increasing PAs and proline,accompanied by enzymes and the expression of crucial genes involved in metabolism (Aghdamet al.2019).Meanwhile,other post-harvest treatments on the fresh products induced chilling toleranceviaenhanced polyamine,GABA and proline levels.To illustrate this effect,chilling tolerance was enhanced by MT application in peach and cucumber plants (Caoet al.2016;Zhaoet al.2017).In addition,the application of nitric oxide in banana (Wanget al.2013),GABA treatment in muskmelon (Huet al.2012) and heat treatment in pomegranate (Mirdehghanet al.2007) fruits induced cold storage toleranceviaenhanced PAs,GABA and proline contents.

Additionally,the energy maintenance in stress tolerance of the tissue system was regulated by chlorophyll pigments.Moreover,the chlorophylls responsible for the green pigmentation of cucumber fruit are similar to those present in the green tissues of other plants,and their qualitative composition does not change during the stages of ripening.However,the concentrations of chlorophylls and carotenoids in the fruits decrease in the ripening period (Mfnguez-Mosqueraet al.1994).

However,it is our understanding there are no reports on an MT effect in CI tolerance of cucumber,specifically focusing on the polyamine,proline,GABA,and chlorophyll contents along with the enzymes involved in their synthesis.This study was therefore conducted to investigate whether MT was associated with cold tolerance,as affected by polyamine,GABA,and proline contents in cucumbers stored in cold conditions.Thus,the findings present a new insight into MT induced polyamine accumulation,GABA accumulation,and proline and chlorophyll contents in cucumber,along with the enzymatic activities and expression of genes behind these phenomena.

2.Materials and methods

2.1.Plant materials and treatments

The cucumber fruit was harvested at commercial maturity from a commercial farm in Nanjing,Jiangsu,China.The fruits were transported to the laboratory followed by selection for uniform size,color and healthy fruits.The selected fruits were randomly grouped into two lots of 310 each,which consisted of three replicates.The first group was dipped in 100 µmol L-1MT.The concentration was set by a preliminary experiment (data not shown).The second group was dipped in distilled water as a control(CK).Both groups were soaked for 15 min and allowed to dry for 1 h at room temperature conditions.Then,fruits were then stored in the refrigerator at (4±1)°C and 90-95% relative humidity for 15 d,with a 3-d sampling interval.The fruit samples were collected from exocarp (peel tissue) and mesocarp (pulp tissue) separately.Then,the samples were frozen in liquid nitrogen and preserved separately at -20°C for chemical and enzymatic analyses,and at -80°C for molecular analyses.Frozen samples of exocarp were used for ion leakage,chlorophylla(Chla),chlorophyllb(Chlb) and chlorophyllase enzyme measurements,whereas mesocarp was utilized for all other measurements.

2.2.Measurements of Cl,electrolyte leakage,and firmness

The CI was evaluated by the appearance of dark watery patches and surface pitting on the skin of a fruit.CI was calculated by the numbers of fruits in scores given as 0 to 4 as follows:0,no signs of surface pitting or dark watery patches;1,＜25% of the surface of fruits affected;2,25-50% of the surface of fruits affected;3,51-75% of the surface of fruits affected;and 4,＞75% of the surface affected.Then,the following formula was used to calculate the result,as:CI index (0 to 4)=Σ(CI scale×Number of fruit at the CI level)/4×Total number of fruit in the treatment×100%.

Membrane permeability was expressed as electrolyte leakage,which was measured by a method described previously (Yanget al.2011) with a few modifications.The peel tissue was excised from two sides of each fruit.Ten discs of 10 mm diameter were excised from peel using a stainless-steel borer and incubated in deionized water.Conductivity of the incubated solutions (E1) was measured after 30 min using a Conductance Bridge(DDS-307,Shanghai Scientific Instruments,China).The samples were then boiled in their incubation solutions for 10 min.After cooling,the solution conductivity (E2) was measured.The electrolyte leakage rate was described as the percentage of the total conductivity,and calculated as:E1/E2×100.

For the firmness measurement,a TA-XT2i texture analyzer (Stable Micro System,Guildford,UK) was used.A small slice of fruit skin was removed,and the firmness was recorded from three different pieces of fruit at three different points per fruit.This was conducted by a penetration test with a 5-mm cylindrical probe in 10 mm depth and 1.0 mm s-1speed of probe.The firmness was defined as the maximum penetration force expressed in Newtons (N).

2.3.Measurements of chlorophyll content and chlorophyllase activity

Total chlorophyll content (total Chl) was measured by extraction of peel tissue using acetone following the method described previously (Caiet al.2019) with limited modification.The spectrophotometer absorbance levels were measured at 663 and 645 nm.The absorbance values were then calculated as:Chla=12.73×OD663-2.59×OD645;Chlb=22.87×OD645-4.67×OD663and Total Chl=(Chla+Chlb)×v/1 000m),where,vis the volume of extract andmis the weight of the sample.Chlaandbcontents were defined as milligrams per gram of fresh weight.

Chlorophyllase activity was measured according to Yanget al.(2004) with limited modification.A 3-g frozen peel sample was homogenized in 5 mL acetone extraction buffer containing potassium phosphate buffer(5 mmol L-1,pH 7.0),50 mmol L-1KCl and 0.24% Triton X-100 for 60 min at 30°C.The mixture was centrifuged at 12 000×g at 4°C for 15 min.Then,a mixture of 0.3 mL of supernatant,0.2 mL of substrate (1 µmol mL-1Chlaor Chlb) and 2 mL of reaction buffer containing 100 mmol L-1sodium phosphate (pH 7.0) and 0.24% triton X-100 was incubated for 30 min at 30°C.Then,the reaction was stopped with 0.5 mL of 10 mmol L-1KOH.After the reaction,1 mL of the mixture was further mixed with 5 mL of hexane/acetone (3:2,v/v) solvent to eliminate the interference of chlorophyll.The chlorophyllase activity was measured by spectrophotometric absorbance at 667 nm.One unit of chlorophyllase was defined as the amount of enzyme required to catalyze the production of 1 µmol L-1Chlaor Chlbper minute.

2.4.Determination of polyamine content,and arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities

The polyamine content was determined following a modified method (Wanget al.2017).The frozen sample(4.0 g) was ground followed by a 1-h incubation with 1 mL of 5% perchloric acid (PCA) at room temperature,then centrifuged for 20 min at 12 000×g at 4°C.Then,2 mL of supernatant was incubated with 10 µL of benzoyl chloride and 1 mL of NaOH (2 mol L-1) for 25 min.Next,2 mL NaCl and 2 mL ether was added,and the mixture was centrifuged at 5 000×g at 4°C for 5 min.A 1-mL ether fraction phase was collected for hot air drying.After drying,the residue was dissolved in 1-mL methanol.The target compounds were detected by Kromasil high performance liquid chromatography (HPLC) with a photo-diode detector equipped with a C18 column.For separation,65% methanol was used at 0.8 mL min-1flow rate at 30°C,and an injection volume of 20 µL,Polyamines were detected by 254 nm absorbance and quantified by external standards.The results were expressed as nanomoles per gram of fresh weight.

The enzymatic activities of ADC and ODC were measured by a described method (Wanget al.2017)with a few changes.The frozen samples (3.0 g) were homogenized with 5 mL of sodium phosphate 100 mmol L-1,pH 8.0 (comprising 0.1 mmol L-1phenylmethylsulfonyl fluoride (PMSF),1 mmol L-1pyridoxal phosphate buffer (PLP),5 mmol L-1dithiothreitol (DTT),5 mmol L-1ethylenediaminetetraacetic acid (EDTA),25 mmol L-1ascorbic acid and 1% polyvinyl pyrrolidone(PVPP).Then,0.2 mL of L-arginine (25 mmol L-1) and L-ornithine (25 mmol L-1) were added for ADC and ODC determination,respectively.The absorbance at 254 nm was measured.One unit of ADC or ODC activity was defined as the amount of enzyme that produced 1 µmol Put.The results were expressed as nanomoles per gram of fresh weight.

2.5.Determination of GABA content,and γ-aminobutyric acid transaminase (GABA-T) and glutamate decarboxylase (GAD) activities

GABA content was assayed according to a previous method (Deewatthanawonget al.2010a) with a few modifications.The samples of 3.0 g were ground in liquid nitrogen.The ground samples were mixed with 1 mL of methanol for 10 min for extraction.Then,1 mL of 70 mmol L-1lanthanum oxide was added to the extracts and centrifuged at 1 000×g for 10 min at 4°C.A supernatant of 0.5 mL was collected and mixed with 0.16 mL of potassium phosphate buffer (0.1 mol L-1) at pH 8.6 (containing 1 mmol L-1α-ketoglutaric acid,0.1 U GABase,and 0.6 mmol L-1NADP+).Then,1 mmol L-1α-ketoglutaric acid was added,followed by 10 min incubation at 25°C.The absorbance at 340 nm was taken and quantified in the reduction of NADP+.The result was expressed in micrograms per gram of fresh weight.

GABA-T and GAD activities were assayed according to a previous method (Deewatthanawonget al.2010b)after a few changes.Samples (3.0 g) were homogenized with 5 mL of Tris-HCL buffer (100 mmol L-1,pH 7.0)containing 10% glycerin,1 mmol L-1phenylmethylsulfonyl fluoride (PMSF),1 mmol L-1dithiothreitol (DTT),0.5 mmol L-1pyridoxal phosphate (PLP),and 5 mmol L-1ethylenediaminetetraacetic acid (EDTA),and then centrifuged at 12 000×g for 25 min at 4°C.For GAD measurement,the reaction mixture of potassium phosphate buffer (0.1 mol L-1,pH 5.8) comprising pyridoxal phosphate (PLP) 400 µmol L-1,glutamate 3 mmol L-1and 0.1 mL of supernatant was mixed,then incubated at 30°C for 1 h.The reaction was stopped by adding hydrochloric acid (0.5 mol L-1).One unit of GAD activity was defined as the amount of enzyme catalyzing the formation of one microgram of GABA per hour.For the GABA-T activity assay,0.3 mL supernatant and 1 mL of reaction mixture Tris-HCL (50 mmol L-1,pH 8.2),10% (v/v) glycerol,1.5 mmol L-1dithiothreitol (DTT),0.75 mmol L-1ethylenediaminetetraacetic acid (EDTA),0.1 mmol L-1pyridoxal phosphate (PLP),1.6 mmol L-1GABA and 4 mmol L-1pyruvate were mixed,then incubated at 30°C for 1 h.The reaction was stopped by adding 0.1 mL of sulfosalicylic acid (10 mmol L-1).Then,0.1 mL of terminated sample and 1 mL of the reaction mixture of sodium carbonate buffer (50 mmol L-1,pH 10)containing 1.5 mmol L-1NAD+and 0.02 U of L-alanine dehydrogenase was incubated at 25°C for 15 min.The spectrophotometric absorbance at 340 nm was taken.The enzyme activity unit was expressed as the production of one micromole of alanine per hour.

2.6.Determination of proline content,and ornithine aminotransferase (OAT),Δ1-pyrroline-5-carboxylate syntheses (P5CS) and proline dehydrogenase(PDH) activities

Proline was assayed by a modified method (Zhaoet al.2009;Wanget al.2017).The samples (2.0 g) were homogenized with 5 mL of sulphosalicylic acid (3%,v/v).The mixture was kept in boiling water for 10 min,then centrifuged at 12 000×g at 4°C for 10 min.The supernatant of 2 mL was collected and mixed with 2 mL glacial acetic acid and 3 mL ninhydrin.The mixture was boiled for 30 min,then 4 mL of methylbenzene was added to the mixture for extraction followed by 30 s of shaking.Finally,absorbance at 520 nm was measured.The content unit was expressed as micrograms per gram of fresh weight.

The enzyme activities of OAT,P5CS and PDH were assayed as described by Caoet al.(2012) with modifications.OAT was assayed by homogenizing 2.0 g of sample in 5 mL of 50 mmol L-1potassium phosphate buffer,pH 8.0 (containing 1 mmol L-1dithiothreitol(DTT),1% w/v polyvinyl pyrrolidone (PVPP),1 mmol L-1α-ketoglutarate and 0.05 mmol L-1pyridoxal phosphate).The mixture was centrifuged at 12 000×g at 4°C for 10 min,and a supernatant of 1 mL was mixed with potassium phosphate buffer (50 mmol L-1,pH 8.0,containing 35 mmol L-1ornithine,0.05 mmol L-1pyridoxine and 5 mmol L-1α-ketoglutarate).Then,the mixture was incubated for 20 min at 30°C.The reaction was ended by adding 0.4 mL of HCLO4(3 mmol L-1).Then,0.2 mL of 2%ninhydrin was added,followed by boiling for 25 min,and the mixture was then cooled and centrifuged at 12 000×g at 4°C for 10 min.The sediment was dissolved in 2 mL of anhydrous alcohol,and the absorbance increase at 510 nm was measured.For the P5CS and PDH assays,3.0 g of sample was homogenized with 10 mL of Tris-HCL buffer (50 mmol L-1,pH 7.5) containing 7 mmol L-1MgCl2,0.6 mmol L-1KCl,3 mmol L-1ethylenediaminetetraacetic acid (EDTA),1 mmol L-1dithiothreitol (DTT),and 5% v/v polyvinyl pyrrolidone (PVPP).The mixture was then centrifuged at 12 000×g at 4°C for 15 min.For P5CS,0.3 mL of enzyme extract was mixed with 3 mL of Tris-HCL buffer (100 mmol L-1,pH 7.3,containing 25 mmol L-1MgCl2,75 mmol L-1sodium glutamate,5 mmol L-1ATP).The reaction was initiated by 0.2 mL of NADPH (0.4 mmol L-1),and the absorbance at 340 nm was measured.The enzyme activity was defined as the amount of 1 µmol NADPH formation per second.Regarding PDH activity,0.2 mL of enzyme extract was mixed with 1.6 mL sodium carbonate buffer (pH 10,0.15 mol L-1) and 0.1 mol L-1L-proline for the reaction.The PDH activity reaction was initiated with 0.2 mL of 10 mmol L-1NAD+,and the absorbance at 340 nm was measured.The activity was defined as a 0.001 decrease per minute in the assay condition

2.7.Total RNA extraction and quantitative real-time PCR analysis

Total RNA extraction was conducted as described(Jahanet al.2019) with modifications.Tissue samples were used for extraction following the manufacturer’s instructions for the RNA simple Total RNA Kit (TaKaRa,Tokyo,Japan).Then,cDNA was constructed (TaKaRa,Tokyo,Japan).The gene-specific primer sequences are listed in Table 1.Real-time PCR used a QuantStudio™(Applied Biosystems,Foster City,CA,USA) with Hieff qPCR SYBR Green Master Mix (Yeasen Biotech Co.,Ltd.,Shanghai,China).The reaction comprised 10 µL Hieff qPCR SYBR Master Mix (No Rox/Low Rox/High Rox) (1×),1 µL template cDNA (10×),0.4 µL each specific primer(10 µmol L-1),and 8.2 µL sterilized ddH2O.The cycling was conditioned as 95°C for 5 min,followed by 40 cycles of denaturation at 95°C for 10 s and annealing at 60°C for 30 s,and a final extension at 95°C for 15 s.Relative expression was quantified relative to Ef-1a using the 2-ΔΔCtformula for the comparison.

Table 1 Primers used for gene expression analysis

2.8.Statistical analysis

This experiment was conducted in a completely randomized design (CRD) with three biological replications.One-way analysis of variance (ANOVA)in SAS version 9.2 (SAS Institute,Inc,NC,USA) used for analysis.Mean separations were compared using Fisher’s least significant differences (LSD) tests at a 5%level of significance.

3.Results

3.1.Cl,firmness and electrolyte leakage

The CI level in cucumber fruit was reduced by MT treatment,compared with control fruits,in cold storage(Fig.1-A).Furthermore,the CI level was highly increased after 9 d of storage in control conditions.Hence,MT pretreatment in cold storage resulted in a 40% lower CI level at 15 d storage than that of the control fruits.

MT treated cucumber fruit showed significantly(P＜0.05) higher firmness throughout the storage durations(Fig.1-B).Moreover,the firmness showed a slight decrease in the first 9 d,and then a quick decline during the remaining cold storage.The firmness on the initialday was about 14.3 N,as compared with 12 and 10.4 N in treated and control fruits,respectively,at 15 d after storage.

A quick increase in ion leakage was recorded after 3 d of cold storage in both treated and control fruits (Fig.1-C).However,MT treated fruits exhibited a lower electrolyte leakage rate compared with control fruits.

Fig.1 Effects of melatonin (MT) treatment on chilling injury (CI) index (A),firmness (B) and electrolyte leakage (C) of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

3.2.Chl a and b contents,total Chl content and chlorophyllase activity

The Chlacontent exhibited a decreasing tendency in both treated and control fruits (Fig.2-A).The treatment of MT showed a significantly (P＜0.05) higher level of Chlacontent than that of control fruits.In addition,treated fruit showed higher Chlbcontent than the control.However,there was no significant (P＞0.05) difference in the Chlbcontent at the early stage of storage (Fig.2-B).Hence,the cumulative total Chl content in treated fruit showed higher level in cold storage as compared to control fruits(Fig.2-C).

Fig.2 Effects of melatonin (MT) treatment on Chl a content (A),Chl b content (B),total Chl content (C) and chlorophyllase activity (D)of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

Chlorophyllase activity was significantly (P＜0.05)suppressed during the entire storage period (Fig.2-D).The effects of MT on chlorophyllase enzyme activity demonstrated decreasing trends in both treated and control fruit,while the treated fruits showed lower activity than the control.The MT treatment resulted in lower chlorophyllase activity.

3.3.γ-Aminobutyric acid (GABA) content,GAD and GABA-T activities,and C.sativus GAD (CsGAD)expression

TheGABA content substantially increased across all storage periods of the fruits (Fig.3-A).The difference was statistically significant (P＜0.05) in treated and control fruits.However,the MT treated fruits showed a higher content than the control throughout the cold storage period.

GAD and GABA-T activities are shown in Fig.3-B and C during the storage duration.These two activities showed different patterns of changes.GAD activity exhibited in decreasing pattern,whereas GABA-T activity showed a gradual increase with the advancement of the storage period.However,there were clear differences in the levels of changes in the control and MT treated fruits.Treated fruits trended higher in GAD and GABA-T enzymatic activities than their respective control fruits after days of storage.As indicated in Fig.3-D,in conjunction with boosted GABA content and synthesizing enzymes,CsGADgene expression was up-regulated with a sharp increase up to 12 d of storage compared to the control.

Fig.3 Effects of melatonin (MT) treatment on γ-aminobutyric acid (GABA) content (A),GABA transaminase (GABA-T) activity (B),glutamate decarboxylase (GAD) activity (C) and relative expression of the Cucumis sativus GAD (CsGAD) gene (D) of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

3.4.Polyamine (PA) contents,ADC and ODC activities and C.sativus ADC (CsADC) and C.sativus ODC(CsODC) expression

In this study,levels of PAs,namely putrescine (Put),sermidine (Spd) and spermine (Spm),were measured in the low-temperature storage conditions.As shown inFig.4-A,B and C,Put content rose quickly under chilling conditions.After 9 d of storage,the Put level was elevated considerably higher in the MT treated fruits than in control fruits.The trend in Spd content was characterized by a pattern relatively similar to Put.Under chilling,MT treatment increased the Spd level in cold storage.Moreover,the Spd content showed a slow increase up to 9 d of storage,and thereafter declined in the later stage of cold storage.Concerning the Spm content,MT treatment stimulated the Spm level relative to the control.The Spm content increased slightly up to 9 d,and it then decreased by 15 d of cold storage.In general,the polyamine contents were considerably induced by MT treatment in chilling storage conditions.

Fig.4 Effects of melatonin (MT) treatment on putrescine content (A),spermidine content (B),and spermine content (C) of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

The polyamine synthesizing ADC and ODC activities are shown in Fig.5-A and C for both control and treated fruits.ADC activity slowly decreased during the storage periods.ODC activity was also reduced quickly after 6 d of storage.For both ADC and ODC activities,the levels in MT treated fruits remained higher than in control fruits.MT pretreatment amplified ADC and ODC activities in response to chilling.As shown in Fig.5-B and D,the relative expression ofCsADCandCsODCgenes showed increased transcripts in MT treated fruit during storage,and both genes followed a decreasing trend in both treated and control fruits after 12 d of storage.

Fig.5 Effects of melatonin (MT) treatment on arginine decarboxylase (ADC) activity (A),relative expression of Cucumis sativus ADC (CsADC) gene (B),ornithine decarboxylase (ODC) activity (C) and relative expression of C.sativus ODC (CsODC) gene (D)of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

3.5.Proline content,P5CS,PDH and OAT activities and C.sativus P5CS (CsP5CS),C.sativus PDH(CsPDH) and C.sativus OAT (CsOAT) expression

The proline contents in both treated and control fruit showed increasing trends with the progression of the cold storage duration (Fig.6-A).The MT treatment of fruits significantly (P＜0.05) promoted the proline content relative to the control fruits.The P5CS exhibited an elevated trend during the entire storage period (Fig.6-B and C).P5CS activity achieved a rapid increase during cold storage in treated fruits.Besides,the part of proline synthesizingCsP5CSgene expression was significantly enhanced.Regarding OAT activity,both control and treated fruits showed gradual increases up to 9 d of storage and consequently exhibited rapid declines during the remaining storage duration.Treated fruits were induced with a higher OAT activity than the control fruits (Fig.7-A).TheCsOATgene expression was increased in treated fruits compared to the control(Fig.7-B).In contrast,PDH activity showed a different pattern of change than OAT and P5CS (Fig.7-C).In the control fruit,PDH activity was higher than in MT treated fruits.Whereas,in accordance with the proline catabolic enzyme,theCsPDHgene showed lower expression in treated fruit (Fig.7-D).

Fig.6 Effects of melatonin (MT) treatment on proline content (A),Δ1-pyrroline-5-carboxylate syntheses (P5CS) activity (B),and relative expression of the Cucumis sativus P5CS (CsP5CS) gene (C) of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

Fig.7 Effects of melatonin (MT) treatment on ornithine aminotransferase (OAT) activity (A),relative expression of the Cucumis sativus OAT (CsOAT) gene (B),proline dehydrogenase (PDH) activity (C) and relative expression of the C.sativus PDH (CsPDH)gene (D),of cucumber fruit during storage at 4°C for 15 d and subsequently transferred to 20°C for 3 d.Vertical bars represent the standard errors of the means of triplicate assays (n=3).Values with different letters for a given day were significantly different at P＜0.05.

4.Discussion

MT is a pleiotropic signaling molecule with diverse functions in regulating various aspects of stress tolerance in the plant system (Guptaet al.2013).Subsequent to its discovery in plants,research interest regarding MT has been augmented,due to its expanded biological role as a principal plant regulator and its defensive roles in unpredictable environmental conditions like cold stress(Arnao and Hernández-Ruiz 2015;Gaoet al.2016).

Cold storage is a widely used mechanism for prolonging the shelf life of fresh produce.Hence,CI may develop if the cucumber is stored at a temperature below 13°C,characterized by surface pitting,followed by the high tendency of decay.In this study,MT treatment significantly mitigated the CI level in cucumber stored at(4±1)°C for 15 d of cold storage.Similar to our findings,CI was alleviated by MT treatment (100 mmol L-1) in tomato (Aghdamet al.2019) and peach (Gaoet al.2016).

Among the quality characteristics,firmness is a crucial textural quality,and can be affected by an increased polygalacturonase pectin-esterase enzyme activity which results in the depolymerization of pectin substances(Maftoonazad and Ramaswamy 2005).Similarly,the exposure of peach fruit to MT induced chilling tolerance by depolymerization of cell wall polysaccharides (Caoet al.2018a).Therefore,the effect of the pretreatment of MT was to moderate the activities of the enzymes and delay the softening of cucumber.In our work,firmness was considerably induced by MT treatment relative to control fruits.In addition,in peach and strawberry,MT maintained higher firmness (Gaoet al.2016;Liuet al.2018) by MT exposure during the storage conditions.Furthermore,chitosan treatment improved the firmness of cucumber fruit in post-harvest storage conditions(Mohammadiet al.2016).

The ion leakage rate is essential for basic membrane integrity under chilling conditions,which affects membrane fluidity and enzymatic activities (Heidarvand and Amiri 2010).In our study,ion leakage continued to increase during the progress of storage.However,MT treatment minimized the ion leakage rate.Similarly,the rapid increase of electrolyte leakage was suppressed by MT treatment in fruit in comparison with the control (Zhaoet al.2017).Hence,it is quite reasonable to suggest that MT treatment protects the membrane and maintains integrity in the cellular units,thus resulting in the reduction of chilling damage in cold storage conditions.

Post-harvest senescence of fruits and vegetables is enhanced by the degradation of an intrinsic membrane protein related to chlorophyll content.The chlorophyllase enzyme is a determinant for the biosynthesis of chlorophyll(Mfnguez-Mosqueraet al.1994).It has been reported that exogenous application of MT delayed senescence of Chinese flowering cabbage,along with reduced expression of chlorophyll catabolic genes (Tanet al.2019).As mechanisms were determined,MT-mediated senescence involves inhibition of chlorophyll degradationviareduced activity of the chlorophyllase enzyme.The chloroplast development and photosynthetic efficiency are critically determined by chlorophyll content (Shuet al.2016).Different abiotic stresses accelerate chlorophyll degradation and negatively affect the consumption of stored energy in crop products (Yuanet al.2012).However,exogenous MT treatment significantly increased the chlorophyll contents.Our finding was complimentary with those of previous authors (Anwaret al.2018),who reported 2,4-epibrassinolide treatment was characterized by increased chlorophyll contents,thus improving seedling growth in cucumber.Chlorophyllase activities were reduced in this study,and since it is a vital enzyme that determines the content of chlorophyll,it functions in degrading pigments in biological systems.Showing a positive correlation with the chlorophyll contents,treated fruits showed lower chlorophyllase activity,which indicates a slow degradation potential of the chlorophyll content in MT treated fruits.The increase of chlorophyll content might be attributed to the stimulation of biosynthetic enzyme activities in stress tolerance by activating chlorophyll biosynthesis-related gene expression levels in stress conditions (Wuet al.2014).

Polyamines are aliphatic amines that maintain enzymatic activities,nucleic acid and protein structures,membrane integrity by ROS scavenging and phospholipid binding capacity (Aghdamet al.2019).Furthermore,polyamines are known to provide various functions in the physiological process and biotic/abiotic stress regulation(Guptaet al.2013).With polycationic characteristics at physiological pH,polyamines bind to negatively charged groups in cellular compartments.Consequently,this enables cellular homeostasis under stress conditions.The interactions of proteins,phospholipids,and nucleic acids serve the biochemical protection functions in harvested products (Groppa and Benavides 2008).The fruit CI tolerance induced by the accumulation of polyamines has been demonstrated in banana (Wanget al.2016),pomegranate fruit (Mirdehghanet al.2007),and tomato (Zhanget al.2011).Polyamine contents were accumulated in banana stored in cold conditions by elevated ADC and ODC activities (Wanget al.2016).Arginine and ornithine amino acids initiate the biosynthesis of polyamines.Furthermore,ornithine is converted to putrescine by ODC and arginine is transformed to Spm/Spd by ADC decarboxylation (Alcázaret al.2010).Polyamine treatments significantly reduced CI in the cucumber by increasing the content of synthesized Put,Spd,and Spm (Zhanget al.2009).Similarly,MT treatment improved the cold stress defense mechanisms in peach by enhancing polyamine (Put,Spd,and Spm)contents (Caoet al.2016).An increased transcript level ofLeARG1andLeARG2encoding arginase genes,along with arginine decarboxylase (LeADC),ornithine decarboxylase (LeODC) and ornithine aminotransferase(LeOAT) genes,enhanced chilling tolerance in cherry tomato (Zhanget al.2011).Also,it was found that transcripts of thePpADCandPpODCgenes were upregulated in peach treated with MT in chilling storage (Caoet al.2016).Our result is consistent with the findings mentioned above in the up-regulation ofCsADCandCsODCgene expression which coincided with increased levels of ADC and ODC enzyme activities.Besides,there was a report on the induction of an essential biosynthetic gene expression which modulated polyamine content to withstand stress in cold storage (Guptaet al.2013).TheZat12gene was involved in the accumulation of Putviaup-regulation ofADC1andADC2expression in cold stress by MT application.Moreover,theCsZat12gene showed stimulated antioxidant characteristics in cucumber plants (Zhaoet al.2017).Furthermore,polyamine metabolism is regulated by three synthesizing enzymes of ADC,ODC,and SAMDC and collectively regulated for mitigating cold stress in cucumber plants(Zhaoet al.2017).In addition,it is involved in the accumulation of polyamines by increased ADC and ODC enzymatic activities and transcript abundances (Caoet al.2016;Zhaoet al.2017).In short,MT treatment increased polyamines by enhancing enzymatic activities and the encoding gene ofCsADCandCsODCexpression levels and thus,this could be considered as one of the possible mechanisms of MT regulation of the chilling stress.

GABA is a non-protein amino acid,which is a four carbon compound found in most living organisms (Shelpet al.1999).GABA biosynthesis is stimulated with a coordinated manner of enzymesviathe GABA shunt pathway (Deewatthanawonget al.2010b;Hyunet al.2013).The metabolism of GABA,regulatedviathe GABA shunt derived from glutamateviaCa2+,comprises three necessary enzymes,namely GAD,GABA-T activity,and SSADH (Deewatthanawonget al.2010a,b).The functional roles of GABA have been reported as signaling,energy balance,responses to stress,and nitrogen metabolism (Faitet al.2008).GABA plays a significant role in CI in most vegetables and fruits in post-harvest conditions (Palmaet al.2014).It was indicated that GABA is likely to accumulate in the response defense system to biotic and abiotic stresses in plants (Shelpet al.1999).The increased GABA shunt and GABA-T activity,provide carbon and nitrogen of NADH as a reduction molecule and of ATP as the energy currency,but also serves as a H2O2scavenger (Aghdamet al.2016).The GABA biosynthesis operates in the cytosolviadirect conversion of glutamate to GABA by the enzymatic activity of GAD(Faitet al.2008).Other reports of MeJA treatments in loquat (Caoet al.2012) and glycine betaine treatment in peach (Shanet al.2016) enhanced GABA content,and consequently reduced CI.Our study was consistent with the findings of Caoet al.(2016),who reported that MT treatment significantly up-regulatedPpGADexpression,and thus,increased GABA accumulation in peach.The finding in this experiment indicated a high accumulation of GABA during the progress of storage.Also,an increased GABA content in cold storage conditions has been reported by Caoet al.(2016) and Wanget al.(2016).A few researchers reported that GAD activity played a leading role in promoting GABA accumulation,which further contributes to the tolerance of chilling conditions in fruits (Shanget al.2011;Caoet al.2012).However,other research demonstrated it was mainly GABA-T,rather than the GAD enzyme,which led to GABA accumulation(Liet al.2013).Correspondingly,the level of GABA accumulation was enhanced compared with high GABA T activity and GAD activity (Deewatthanawonget al.2010a;Caoet al.2016;Wanget al.2016).Based on our result,we believe MT treatment could facilitate the GABA shunt and tolerance of cucumber fruit to the cold stress mediated by enhanced expression of theCsGADgene,GAD activity and GABA-T activity,resulting in GABA accumulation,which maintained the mechanisms of the fruit to resist chilling stress under prolonged cold storage

Proline is an amino acid useful for withstanding different stresses in plants.In higher plants,glutamateviaP5CS and ornithineviaOAT were found to be synthesizing mechanisms of proline (Ruizet al.2002;Verbruggen and Hermans 2008).Proline,used as an osmoprotectant,exhibited a control function for ROS scavenging enzyme stability.Besides,proline exhibited a direct ROS scavenging activity,which is essential in inducing CI tolerance by maintaining membrane integrity(Aghdam and Bodbodak 2013).In higher plants,P5CS,OAT and PDH are vital enzymes involved in proline metabolism.P5CS and OAT regulate the biosynthesis of proline,whereas PDH plays a proline degradation role.The previous studies on Loquat (Caoet al.2012)and Peach (Shanget al.2011) confirmed that proline metabolism contributed to the reduction of chilling stress in storage conditions.Similarly with our findings,higher P5CS and OAT with lower PDH activities were exhibited with proline accumulation in NO treated banana (Wanget al.2013).The synthesis of proline is believed to be from glutamate or ornithine by catalyst enzymes of P5CS and OAT,respectively.The ornithine (OAT) contributes to the synthesis of proline under various stresses in fruits by coordinated regulation of P5CS,OAT,and PDH.Upregulated expression and promotion ofPpP5CSandPpOATactivities,and inhibited expression ofPpPDHpromoted proline accumulation.The present experiment showed higher relative expressions ofCsP5CSandCsOATgenes and with a lower expression level of theCsPDHgene.In peach fruit stored in cold conditions,MT application induced proline accumulation (Caoet al.2016).Similar to our findings,enzymatic activities of P5CS and OAT were highly regulated with low PDH activities,further accounting for proline accumulation in banana (Wanget al.2013) and loquat (Caoet al.2012).Our experiment showed that MT induced a significant amount of tolerance to cold stress in cucumber through the coordination of P5CS,OAT,and PDH,with an accumulation of proline.Thus,inducing resistance to injury in cold storage cucumber.From the above results,the increased rate of proline synthesis and reduced degradation rate correlated for enhanced proline content in MT treated fruit,which might play an essential role in the MT regulated chilling tolerance in cucumber fruit during storage.

5.Conclusion

This study revealed the decisive role of MT treatment in reducing CI.The injury tolerance was enhanced by the mechanism of higher polyamine accumulation,triggered by increased synthesizing enzyme activities of ADC and ODC.Also,endogenous GABA levels were induced by high GAD and GABA-T activity.Proline content was triggered by P5CS and OAT activity,which was concurrent with lower PDH activity,resulting in a high proline level.In addition,CsADC,CsODC,CsGAD,CsP5CS,andCsOATexpression levels were induced by MT treatment.The chlorophyll content was conserved in treated fruits by lower chlorophyllase enzyme activity,resulting in lower chlorophyll degradation.In summary,100 µmol L-1MT was involved in tolerance to the development of CI in cucumber fruit,possibly associated with the increase in the metabolism of polyamines,GABA,and proline.

Acknowledgements

This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions and Program for Student Innovation through Research and Training (SRT),China (1918C12).

Declaration of competing interest

The authors declare that they have no conflict of interest.