Melatonin treatment alleviates chilling injury in mango fruit 'Keitt'by modulating proline metabolism under chilling stress

Mariama KEBBEH,DONG Jing-xian,HUAN Chen,SHEN Shu-ling,LIU Yan,ZHENG Xiao-lin

College of Food Science and Biotechnology,Zhejiang Gongshang University,Hangzhou 310018,P.R.China

Abstract Mangoes often suffer from low temperature-induced chilling injury (CI) during postharvest cold storage. Therefore,advanced techniques are crucial and in high demand to solve the chilling stress of mango fruit for a higher value. This study addresses chilling stress modulation by investigating the effects of melatonin treatment on CI,proline metabolism,and related gene expressions of ‘Keitt’ mango during cold storage after dipped in 0 (control),0.1 (MT1),and 0.2 mmol L-1 (MT2) melatonin solution for 30 min. The results revealed that melatonin treatment in MT1 significantly reduced CI development and increased proline content in mango fruit during cold storage compared to the control. These changes were along with increases in the activity of critical enzymes as well as the expression of encoding genes involved in proline biosynthesis,such as pyrroline-5-carboxylate synthetase (P5CS),pyrroline-5-carboxylate reductase (P5CR),ornithine D-aminotransferase (OAT),P5CS2,P5CR2,and OAT3. Additionally,proline dehydrogenase (PDH) activity and the expression of the PDH3 gene associated with proline dehydrogenation were lower in MT1-treated mangoes than the controlled group. Thus,melatonin treatment has regulated proline metabolism resulting in the accumulation of proline,subsequently contributing to enhancing the chilling tolerance of ‘Keitt’ mango fruit.

Keywords: chilling injury (CI),cold storage,mango fruit,melatonin,proline metabolism

1.Introduction

Mangoes (Mangiferaindica) are among the main fruit of tropical and subtropical origin and are very perishable during storage at ambient temperature. Cold storage is an effective method to prolong the storage life of fruit(Zhenget al.2007). However,mangoes are subjected to chilling injury (CI) under certain low temperatures. When the chilled fruit is transferred to ambient temperatures,CI results in greyish skin discoloration,browning,poor aroma and flavor,surface pitting,uneven ripening,wooliness,an increase in susceptibility to postharvest rot,and severe deterioration in a short period. These damages cause a reduction in fruit quality and commodity value (Nuneset al.2007).

Melatonin is a naturally occurring indoleamine that acts as an endogenous elicitor and signaling molecule for plants’ growth and development responses to biotic and abiotic stresses (Arnao and Hernández 2014,2020;Wanget al.2020). The effects of melatonin application on the postharvest preservation of fruits and vegetables have attracted significant attention,and some of its roles in preservation are well established. For example,melatonin application has proven to display exemplary performance against the chilling stress in mango fruit (Bhardwajet al.2021),peaches (Caoet al.2018),tomato (Aghdamet al.2019),litchi (Liuet al.2021),apricot (Medina-Santamarinaet al.2021),red bell papers (Konget al.2020),and pomegranate (Jannatizadeh 2019) by extending the shelflife,reducing deterioration,delaying the ripening and successfully reducing CI. However,the mechanism of the multifarious role of melatonin involved in CI alleviation in mangoes is not fully known.

Proline is one of the 20 protein-forming amino acids extensively distributed in plants in a free state,and it is biosynthesized from either glutamate or ornithine proline (Caoet al.2012). These two pathways in proline metabolism are biosynthesisviapyrroline-5-carboxylate synthetase (P5CS),which catalyzes glutamate and ornithine-delta-aminotransferase (OAT)catalyzing ornithine,whereas proline degradationviaproline dehydrogenase (PDH) catalysis (Szabados and Savouré 2010). In addition,proline is an osmoregulatory compound (Gomeset al.2010) and a proteinogenic amino acid with exceptional conformational rigidity. It can protect plants from abiotic stresses by stabilizing cellular homeostasis,elevating cell osmotic pressure,detoxifying reactive species,protecting and stabilizing membrane protein integrity,and triggering specific gene expression. This protection is essential for plant recovery from stresses (Liuet al.2020). Studies have suggested that proline accumulation enhances cold tolerance associated with CI attenuation in various fruits such as mango and litchi during postharvest (Liet al.2014;Liuet al.2020).

The techniques used to study an organism’s transcriptome are the sum of its RNA transcripts that enable the study of how gene expression changes in different organisms to be instrumental in understanding fruits and vegetables (Baiet al.2023). Previous gene expression analysis of several mango cultivars has provided insights into the fundamental molecular biology of this plant in the postharvest of mangoes (Khanet al.2019). The mango transcriptome of ‘Langra’,‘Zill’,‘Shelly’,‘Kent’,‘Dashehari’,and ‘Keitt’ (Azimet al.2014;Dautt-Castroet al.2015;Tafolla-Arellanoet al.2017) has been reported from China,Israel,Mexico,India,and the USA. However,there is no existing systematic study on the expression of genes for proline metabolism in mango,and the primary obstacle to further progress in mango genetic research is the limited availability of genomic data (Tafolla-Arellanoet al.2017).

Therefore,this study investigated the effect of melatonin on CI regarding proline metabolism and the expression of its genes in ‘Keitt’ mango fruit.The objective was to clarify further the mechanism of melatonin alleviating CI in mangoes during cold storage.

2.Materials and methods

2.1.Materials and treatments

Mature green mangoes ‘Keitt’ were harvested at maturity in a commercial orchard in Panzhihua,Sichuan Province,China. About 300 uniform-sized fruit were selected for uniformity without any bruises,cuts,disease,decay,impurities,and other imperfection of physical damage,lack of disease,or infection. They were randomly divided into three groups CK (control),melatonin treatment 1(MT1),and 2 (MT2). The CK fruit was dipped in distilled water containing 0.05% Tween 80 for 30 min,MT1 and MT2 were dipped in 0.1 and 0.2 mmol L-1melatonin solution containing 0.05% Tween 80,respectively,for 30 min,and then all the fruit were air-dried at room temperature (Donget al.2021).

Each nine fruit per group in a replicate were placed inside a clean,soft layered plastic basket and wrapped with a 0.05-mm polyethylene bag without a seal. They were stored in low-temperature humidity chambers(Sanyo,MIR 554) at (5±0.5)°C with 85-95% relative humidity (RH) for 21 d and then transferred to room temperature (25°C) for 4 d. Samples of the fruit’s peels were collected in 7-and 2-d intervals during storage at low and room temperatures,respectively.All the samples were diced,chopped,and preserved by liquid nitrogen and stored at -80°C for further analysis.

2.2.Determination of CI index

The CI incidence was recorded on three independent replicates every 7 d at cold storage and every 2 d at room temperature storage. CI symptoms such as flesh browning,peel pitting,and water-soaked on the fruit surface were visually observed to determine the percentage of fruit affected by chilling (Liet al.2014). The scale was visually evaluated as follows: 0,0%;1,＜10%;2,10-25%;3,25-50%;4,＞75% of fruit surface area.

The CI index was calculated by the equation: CI index(%)=∑(Scale×The number of fruit at that scale)/(4×Total number of fruit)×100 (Bhardwajet al.2021).

2.3.Determination of firmness,total soluble solids(TSS),titratable acidity (TA),and respiration rate

Fruit firmness was determined using a handheld sclerometer (STEPS 41050,Germany) equipped with an 8-mm diameter probe. Two measurements were carried out at two equidistant points on the equatorial axis of each peeled mango fruit. The firmness was expressed as Newton (N).

TSS and TA content from the juice of each fruit were measured using a Pocket Brix-Acidity Meter (PAL-BX/ACID 8,Atago,Japan),and the result was expressed as a percentage of Brix and citric acid,respectively (dos Santos Netoet al.2017).

The respiration rate of the fruit was expressed as CO2mg kg-1fresh weight (FW) h-1according to the method of Huanet al.(2021) using a respiration meter (Dansensor?CheckMate,Denmark). Five fresh fruit were enclosed in a 4.7-L glass jar at (25±1)°C for 15 min and 1 h,respectively. Three biological replicates were used.

2.4.Measurement of proline content

Proline content was determined as described by Liet al.(2014) with slight modification. Proline was extracted from 1.0 g frozen peel that was grounded in liquid nitrogen to a fine powder. The proline content was extracted with 10 mL of 3% (w/v) sulfosalicylic acid at 100°C for 10 min with shaking;after cooling for 10 min,it was centrifuged at 12 000×g for 10 min,and the supernatant was collected as the proline extract. Pipette 2.0 mL of the extract into a test tube with a stopper,add 2.0 mL of glacial acetic acid,3.0 mL of acid ninhydrin reagent,and incubate in a water bath at 90°C for 30 min. Add 4.0 mL toluene after rapid cooling in an ice bath for 10 min and vortexed for 15 s. Allow tubes to stand for at least 20 min in the dark at room temperature to allow the separation of the toluene and aqueous phase. The toluene phase was collected into test tubes after the solution was stratified.The upper proline-toluene fraction was gently pipetted into the cuvette and performed colorimetric determination at a wavelength of 520 nm. The proline content was expressed as mg kg-1FW.

2.5.Measurement of OAT,P5CS,PDH and P5CR activity involved in proline metabolism

The activities of P5CS,OAT,and PDH were determined as described by Shanget al.(2011). P5CS and PDH were extracted from a 1.2-g frozen peel with 3.6 mL of 50 mmol L-1Tris-HCl buffer (pH 7.4) containing 7 mmol L-1MgCl2,600 mmol L-1KCl,3 mmol L-1EDTA,1 mmol L-1dithiothreitol (DTT),and 5% (w/v) insoluble polyvinylpyrrolidone. After centrifuging at 15 000×g for 20 min,the supernatants were the crude enzyme solutions of P5CS and PDH.

For the P5CS activity determination,the enzyme extract was mixed with 100 mmol L-1Tris-HCl (pH 7.2),75 mmol L-1sodium glutamate,25 mmol L-1MgCl2,and 5 mmol L-1ATP.

The reaction was initiated by the addition of 0.4 mmol L-1NADPH. For PDH activity determination,the reaction mixture was 150 mmol L-1Na2CO3-HCl buffer (pH 10.3)containing 2.67 mmol L-1proline and 10 mmol L-1NAD+.The P5CS and PDH activity was expressed as U kg-1FW,where one U of enzyme activity was defined as a change of 0.01 in absorbance at 340 nm min-1.

OAT was extracted from 1.0 g frozen peel with 3.0 mL of 100 mmol L-1potassium phosphate buffer (pH 7.9)containing 1 mmol L-1EDTA,15% (v/v) glycerol,and 10 mmol L-1β-mercaptoethanol. After centrifuging at 15 000×g for 20 min,the supernatant was the crude OAT enzyme.

The reaction mixture was 200 mmol L-1Tris-HCl buffer (pH 7.8) containing 12.5 mmol L-1α-ketoglutarate,46.8 mmol L-1ornithine,and 0.125 mmol L-1NADH. The OAT activity was expressed as U kg-1FW,where 1 U of OAT activity was defined as the amount of enzyme causing a decrease of 0.01 in absorbance at 340 nm min-1(Liuet al.2016).

P5CR activity was measured from the mango peel using the Pyrroline-5-carboxylate reductase (P5CR) Test Kit (Nanjing Jiancheng,Jiangsu,China) according to the manufacturer’s protocol. An enzyme activity unit was defined as 1 nmol NADH produced per gram of tissue per minute. The P5CR activity was expressed as nmol g-1FW min-1.

2.6.Expressions of the genes encoding the key enzymes involved in proline metabolism

The total RNA from the mango samples was isolated using the MiniBEST Plant RNA Extraction Kit (TaKaRa,Japan) according to the manufacturer’s protocol. The quality of the RNA sample was assessed by agarose gel electrophoresis after high-quality RNA samples were converted into first-strand cDNA (Thermo Scientific,USA).

Primers for the selected genes were designed using NCBI and mangobase database (https://mangobase.org/)designing tool (Appendix A).

Quantitative reverse transcription-PCR (qRT-PCR)analysis was performed on various samples in a 20-μL reaction volume. The analysis was carried out using SYBR Green I dye (TaKaRa,Dalian,China) on an ABI 7500 Real-Time PCR Machine (Applied Biosystems,Foster,the USA).MiActin1was used as an endogenous reference gene for data normalization (Luoet al.2013).Data normalization was done using Ctvalues of the genes of experimental samples (varieties) with their respective reference gene (ΔCt) and comparison of expressed genes. Each was calculated as ΔΔCt,and the overall gene expression levels in fold change were calculated using the formula. Fold change=2-ΔΔCt(Bajpaiet al.2018).

2.7.Statistical analysis

All data were repeated by mean±standard error data system. The SPSS 23.0 Software was used for statistical analysis and the origin 8.5 Software was used for mapping. The differenceP＜0.05 is considered a significant difference.

3.Results

3.1.Effect of melatonin treatment on Cl

The CI symptoms on the fruit surface were observed until 14 d in cold storage,and then the symptoms gradually developed as the storage progressed. However,MT treatment significantly inhibited CI development in the mango fruit as MT1 and MT2 displayed lower CI index from 14 to 21+2 d and from 14 to 21+4 d compared to CK. MT1 predominantly remained lowest in the CI index for the whole storage time (Fig.1).

Fig.1 Effects of melatonin (MT) treatments on chilling injury (CI) (A and B) of mango during storage. 21+2 or 21+4,stored for 2 or 4 d at 25°C after storage at 5°C for 21 d. Vertical bars represent the standard errors of means (n=3). Statistical significance for the control was determined by LSD: P≤0.05.

3.2.Effect of melatonin treatment on firmness,TSS,TA,and respiration rate

Firmness in all the fruit was maintained steadily with a high value until d 21+2 and then drastically dropped at d 21+4. However,compared to CK,the firmness in the MT1-treated fruit was significantly higher during cold storage,whereas in MT2-treated fruit,it was significantly lower after it was transferred to room temperature(Fig.2-A).

The TSS in all the fruit was steadily maintained during the cold storage,then gradually increased during storage at room temperature. Although TSS in MT2 treated fruit was significantly higher than that in CK and MT1 at 7 and 21 d when the fruit was transferred to room temperature,TSS in MT1-treated fruit was significantly lower than that in CK,but no significant difference was presented between CK and MT2 (Fig.2-B).

Similarly,changes in TA for all the fruit were not apparent during storage,but TA in MT1-treated fruit was significantly higher than that in CK from 14 to 21+2 d,while in MT2,it was significantly lower at 7 and 21 d compared to CK (Fig.2-C).

Changes in the respiration rate of the fruit are shown in Fig.2-D,where the rate in MT1-and MT2-treated fruit were significantly lower than in CK during cold storage,but there was no significant difference among CK,MT1,and MT2 when the fruit was moved to room temperature.

Fig.2 Effects of melatonin (MT) treatments on firmness (A),total soluble solids (TSS) (B),titratable acidity (TA) (C) and respiration rate (D) of mango during storage. 21+2 or 21+4,stored for 2 or 4 d at 25°C after storage at 5°C for 21 d. Vertical bars represent the standard errors of means (n=3). Statistical significance for the control was determined by LSD: P≤0.05.

3.3.Effect of melatonin treatment on proline content

The proline content in the fruit increased to a peak at 14 d and then dropped at 21 d during cold storage,but it increased again to a peak at 21+2 d and decreased slightly at 21+4 d when the fruit was transferred to room temperature. Notably,the higher proline level in the treated fruit was observed before the CI was excessive. MT1-treated fruit amassed a higher level of proline accumulation than CK,thus showing the strongest induced by MT1 treatment. MT2-treated fruit showed higher proline levels at 7,14,and 21 d than CK and then dropped lower than CK on 21+2 and 21+4 d(Fig.3).

Fig.3 Effects of melatonin (MT) treatments on proline content in mango during storage. 21+2 or 21+4,stored for 2 or 4 d at 25°C after storage at 5°C for 21 d. Vertical bars represent the standard errors of means (n=3). Statistical significance for the control was determined by LSD: P≤0.05.

3.4.Effect of melatonin treatment on the activity of its metabolic enzymes and the expression of its genes

The P5CS and P5CR activity of the fruit exhibited similar increasing tendencies. MT treatments promoted the enhancement of P5CS and P5CR activity while showing the highest enhancement in MT1 treatment against CK(Fig.4-A and B). Additionally,MT treatments improved the activity of OAT,showing a steadily increasing tendency across the storage period compared to CK,where MT1 recorded the uppermost increasing tendency (Fig.4-C).The PDH activity recorded an increasing and decreasing tendency,rising to a peak at 21 d and declining gradually during storage. However,MT1 recorded the lowest decreasing trend than CK and MT2. Thus,melatonin treatment suppressed the rises and enhanced the reductions in the activity of PDH in MT1-treated fruit compared to CK (Fig.4-D).

Fig.4 Effects of melatonin (MT) treatments on synthetase activities of pyrroline-5-carboxylate synthetase (P5CS) (A),pyrroline-5-carboxylate reductase (P5CR) (B),ornithine-delta-aminotransferase (OAT) (C) and proline dehydrogenase (PDH) (D). 21+2 or 21+4,stored for 2 or 4 d at 25°C after storage at 5°C for 21 d. Vertical bars represent the standard errors of means (n=3).Statistical significance for the control was determined by LSD: P≤0.05.

Similar to P5CS,P5CR,and OAT,the gene expressions ofP5CS2,P5CR2,andOAT3were increased during storage and upregulated by MT treatments.P5CR2andOAT3exhibited their ultimate peak at 21+4 d in the storage period. In all the samples,the expression of MT1 was the highest inP5CS2,P5CR2,andOAT3genes compared to CK (Fig.5-A-C). Equally,thePDH3expression tends to decrease in MT1 and MT2 during cold and room temperature storage,revealing it was suppressed by the MT treatments. Thus,thePDH3expression in MT treatments was significantly lower than in CK from 14 to 21+4 d compared to CK (Fig.5-D).

Fig.5 Effects of melatonin (MT) treatments on the relative expressions of P5CS2 (A),P5CR2 (B),OAT3 (C) and PHD3 (D) of mango during storage. 21+2 or 21+4,stored for 2 or 4 d at 25°C after storage at 5°C for 21 d. Vertical bars represent the standard errors of means (n=3). Statistical significance for the control was determined by LSD: P≤0.05.

4.Discussion

Cold storage is a generalized technology used to avoid quick deterioration,preserve quality and slow down many of the processes responsible for the deterioration and loss of quality in fruit. However,the fruit is susceptible to CI that disturbs cellular homeostasis,causing physiological disorders,such as a considerable rise in respiration rate,influencing firmness,TSS,and TA content,and irreversibly reducing the external and internal quality (Gaoet al.2018). Liuet al.(2020) have considered the increase in respiration rate as one of characteristics of CI in litchi fruit during cold storage. In this work,the melatonin treatment,particularly with 0.10 mmol L-1,significantly reduced the respiration rate in the mango fruit during cold storage and also decreased CI when the visible symptoms of CI appeared after 14 d in cold storage(Figs.1 and 2-D),which indicated that the change in respiratory activity might be a sign of chilling damage prior to the appearance of invisible symptoms under chilling stress,and the melatonin treatment alleviated CI that would be of benefit to the maintenance of fruit quality during cold storage.

CI has been the primary constraint to the quality upkeep of various fruit during low-temperature storage by declining its postharvest value,and pre-storage application of some chemicals/elicitors can improve the chilling tolerance of fruit during cold storage (Zhanget al.2021). Our previous work has documented that the application of oxalic acid apparently improves chilling tolerance in mango fruit ‘Zill’ which is associated with proline accumulation and maintenance of high energy status during storage at low temperatures (Liet al.2014).Recently,Bhardwajet al.(2021) have reported that CI alleviation in mango fruit by 0.1 mmol L-1melatonin treatment was cultivar-dependent,as among four cultivars including ‘Langra’,‘Dashehari’,‘Chaunsa’,and ‘Gulab Jamun’,the fruit ‘Langra’ responded best to 0.1 melatonin treatment by decreasing their CI along with proline accumulation during cold storage,while the fruit ‘Gulab Jamun’ did not experience any significant effect. In this work,the 0.1 mmol L-1MT treatment showed promising results in alleviating CI along with proline accumulation in mango fruit ‘Keitt’ compared to the control and 0.2 mmol L-1melatonin treatment. Thus,it was suggested that melatonin treatment also exerted a concentrationdependent inhibition on CI for mango fruit,and the proline accumulation was one of the important factors that contributed to chilling tolerance in the mango cultivars with melatonin treatment.

Additionally,proline plays important roles in increasing cellular osmolarity,stabilizing membrane and subcellular structures,and protecting cells against oxidative damage under stresses (Kishoret al.2005;Szabados and Savouré 2010). Research has shown that the proline accumulationviaregulation of its metabolism triggered by different elicitors is involved in improving chilling tolerance for various fruit during cold storage (Liet al.2014;Liuet al.2020). For example,the application of γ-aminobutyric acid improves the chilling tolerance in banana fruit associated with proline accumulation from higher P5CS activity accompanied by lower PDH activity and then gives rise to the protection of safe membrane integrity and CI alleviation (Wanget al.2014). Equally,conferring chilling tolerance in peach fruit by exogenous melatonin application was ascribed to higherP5CSandOATexpression accompanied by lowerPDHexpression (Gaoet al.2016). Moreover,the proline accumulation arising from upregulating gene expressions ofOATandP5CSand loweringPDHexpression in tomato fruit contributes to the improvement of chilling tolerance in response to exogenous melatonin application at 100 μmol L-1(Aghdamet al.2019).Also,it has been indicated that melatonin treatment enhances the resistance of litchi fruit to chilling stress by inducing the synthesis of proline (Liuet al.2020). Our results also showed that the 0.1 mmol L-1melatonin treatment enhanced the activities and expressions ofP5CS,P5CR,andOAT(Figs.4-A-C and 5-A-C) and declined the activity and expression ofPDH(Figs.4-D and 5-D) in mango fruit during cold storage and subsequent transfer to normal temperature. Using a heat map comparison among the control,0.1 and 0.2 mmol L-1melatonin treatment,we further clarified that the proline accumulation triggered by melatonin treatment in ‘Keitt’ mango fruit was collectively attributed to the regulation of its synthesis and degradation during postharvest,and the 0.1 mmol L-1melatonin treatment displayed the most outstanding results (Fig.6). Thus,these facts,together with the evidence in our work,suggested that the melatonin treatment triggered proline accumulationviaregulation of its metabolism and contributed to improving chilling tolerance,thereby reducing CI and maintaining the quality of the mango fruit in cold storage. Also,the proline metabolism might be categorized as one of the most important reactions involved in inducing chilling tolerance against CI in various fruit during postharvest.

Fig.6 Heatmap of the contents of the major pathway identified in proline metabolism in ‘Keitt’ mango during storage. A-D,P5CS(A),P5CR (B),OAT (C) and PDH (D) between melatonin (MT) and CK. The blue,yellow and red denoted the low,middle,and high contents,respectively. 21+2 or 21+4,stored for 2 or 4 d at 25°C after storage at 5°C for 21 d. Asterisks (＊) indicated that mean values were significantly different between MT treatment and CK (P≤0.05).

5.Conclusion

The results obtained in this study showed evidence for the potential role of melatonin treatment in alleviating the CI of mango fruit ‘Keitt’ during cold storage. The melatonin treatments with 0.1 and 0.2 mmol L-1,particularly with 0.1 mmol L-1increased P5CS,P5CR,and OAT activities,upregulated their coding gene expressions in relation to proline biosynthesis,decreased PDH activity and downregulated the gene expression associated with proline degradation. The outcome of melatonin treatment might be due to its capability to enhance chilling tolerance in mango fruit,which was attributed to accumulating prolineviaregulation of proline metabolism. Additionally,it was suggested that 0.1 mmol L-1melatonin of treatment was the recommended concentration for alleviating CI in mango fruit during cold storage.

Acknowledgements

This research project was financially supported by the National Natural Science Foundation of China (32072280).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendix associated with this paper is available on http://www.ChinaAgriSci.com/V2/En/appendix.htm