Journl of Integrtive Agriulture 016, 15(0): 60345-7 Aville online t www.sienediret.om SieneDiret RESEARCH ARTICLE Sliyli id llevites posthrvest hilling injury of sponge gourd (Luff Cylindri) HAN Cong 1,, 3*, ZUO Jin-hu 1*, WANG Qing 1, DONG Hi-zhou, GAO Li-pu 1 1 Ntionl Engineering Reserh Center for Vegetles, Beijing Ademy of Agriulture nd Forestry Sienes, Beijing 100097, P.R.Chin College of Food Siene nd Engineering, Shndong Agriulturl University, Ti n 71018, P.R.Chin 3 College of Food Siene nd Tehnology, Nnjing Agriulturl University, Nnjing 10095, P.R.Chin Astrt Effet of sliyli id (SA) on hilling injury (CI) of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C) ws evluted in this study. SA tretment t the onentrtion of 1.5 mmol L 1 signifintly redued posthrvest CI of sponge gourds. Besides, the pplition of SA ould effetively derese the eletrolyte lekge, redue the umultion of mlondildehyde (MDA) nd totl phenolis, enhne the tivities of ntioxidnt enzymes suh s superoxide dismutse (SOD), tlse (CAT) nd sorte peroxidse (APX), nd inhiit the tivities of phenyllnine mmoni-lyse (PAL) nd polyphenol oxidse (PPO). The enefiil effets of SA ould e ttriuted to preserved memrne integrity, inhiited memrne peroxidtion, enhned ntioxidnt system nd suppressed tivities of rowning relted enzymes. In sense, SA s posthrvest tool my e ommerilly used in lleviting CI of sponge gourd. Keywords: sponge gourd, sliyli id, hilling injury, qulity, ntioxidnt enzyme 1. Introdution Sponge gourds, the fruit of Luff ylindril, re widely known nd/or used for their fiers (Ooh nd Aluyor 009). The young immture fruits, hrvested efore internl fiers hve strted to hrden, re edile nd onsumed muh like summer sqush (Porterfield 1955; Zong et l. 1993). As Reeived 11 Jnury, 016 Aepted April, 016 HAN Cong, E-mil: hnong6559@16.om; ZUO Jin-hu, E-mil: zuojinhu@16.om; Correspondene GAO Li-pu, Tel: +86-10-51503051, E-mil: golipu@nerv.org * These uthors ontriuted eqully to this study. 016, CAAS. All rights reserved. Pulished y Elsevier Ltd. doi: 10.1016/S095-3119(16)61390-4 summer seson vegetle, sponge gourd is good soure of nutrients nd ontins multiple enefiil elements espeilly sponin nd muus (Porterfield 1955). However, the fruit is highly perishle nd its shelf-life is no more thn one week t mient temperture euse of wilting nd yellowing in pperne nd lignifition nd firosis in texture. Low temperture storge hs een demonstrted the min posthrvest strtegy pplied in extending posthrvest life of fruits nd vegetles, while the enefiil effets my e limited in sponge gourd euse of hilling injury (CI). The visile hilling-indued symptoms in sponge gourd inlude disolortion, wtery lk or rown spots on nd under the skin, nd serious lk-olored rot fter removl of fruit from low temperture storge (Zong et l. 1993). Dmge used y low temperture severely redued the qulity nd storge life of sponge gourd. Sliyli id (SA), nturl nd sfe phenoli ompound, hs een found to generte wide rnge of metol-
HAN Cong et l. Journl of Integrtive Agriulture 016, 15(0): 60345-7 3 i nd physiologil responses in plnts nd exhiited high potentil in ontrolling posthrvest losses of hortiulturl rops (Asghri nd Aghdm 010). In reent yers, the pplition of exogenous SA t non-toxi onentrtions inreses resistne to posthrvest CI in fruits, vegetles nd flowers suh s peh (Co et l. 010; Yng et l. 01), tomto (Zhng et l. 011; Aghdm et l. 01), plum (Luo et l. 011), pomegrnte (Syyri et l. 009), pinepple (Lu et l. 011), moo shoot (Luo et l. 01) nd nthurium (Promyou et l. 01). The mehnisms of SA tretment in lleviting CI were extensively, whih ould e ttriuted to enhning memrne integrity nd ntioxidnt system tivity, inresing rginine pthwys nd het shok proteins (HSPs) gene expression, tivtion of C-repet inding ftor (CBF) pthwy nd ltertion in phenyllnine mmoni-lyse (PAL) nd polyphenol oxidse (PPO) enzymes tivities (Aghdm nd Bododk 013). To the est of our knowledge, the effet of SA tretment on physiologil nd iohemil hnges ssoited with CI nd posthrvest qulity of sponge gourd is not yet fully understood. The ojetives of this study were to exmine the effets of SA on ontrolling posthrvest CI of sponge gourd, nd to investigte the underlying mehnisms y SA in reduing dmge on ell memrnes integrity, inhiiting oxidtive stress nd induing ltertions in tissue rowning relted sustnes nd enzymes.. Mterils nd methods.1. Plnt mterils, tretments nd storge Sponge gourds Bngng gu (Luff ylindril (L.) Roem. Bng nggu) were hnd-hrvested t ommeril mturity from n orgni orhrd in Fngshn, Beijing, then wrpped with mesh g (void ruise) nd pked in seled fom oxes (hold moisture), nd trnsferred t mient temperture to the lortory within 3 h. Fruits were seleted for uniformity of size (length: out 5 m), olor, firmness, shpe nd free from lemishes, then rndomly divided into lots (150 fruits per lot). The first lot of fruits ws immersed in 1.5 mmol L 1 SA solution for 15 min, while distilled wter ws used s ontrol. The SA onentrtion ws hosen s eing optiml from preliminry experiments of CI evlution. Susequently, ll fruits were ir-dried for pproximtely 30 min nd stored t 9 C. Following 1, 3, 5, 7 or 9 dys of old storge, fruit were moved to ontrolled environment hmer nd mintined t 0 C for dys (shelf life). Fruit CI, eletrolyte lekge, mlondildehyde (MDA) nd H O ontent were ssessed fter dys of shelf-life. Smples of fruit flesh (pprox. 50 g) were frozen in liquid nitrogen nd stored t 80 C for mesurements of other prmeters... CI evlution Upon removl from old storge, the hilling symptoms of sponge gourd developed rpidly. It ppered s surfe rowning t the first severl dys of storge period, nd then serious lk-olored rot ws oserved on nd under the skin. The severity of the symptoms ws individully ssessed in eh fruit with the sore from 0 to 4 (no symptom), 1 (1 5% surfe rowning), (6 50% surfe rowning), 3 (lk-olored rot ppered on nd under the skin), 4 (serious lk-olored rot ppered on nd under the skin). CI index ws lulted y using the following formul: CI index= (The level of CI sored in sponge gourd) (Numer of fruit with the orresponding degree)/(totl numer of fruit in the smple).3. Eletrolyte lekge Eletrolyte lekge ws determined ording to Hung et l. (01) with some modifitions. Diss were removed with ork orer (10 mm in dimeter) from 10 fruits. Thirty diss were rinsed twie nd then inuted in 5 ml of 0.3 mol L 1 mnnitol in distilled wter t 5 C, nd shken for 30 min. Eletrolyte lekge ws determined with ondutivity meter (Model DDSJ-308A, Shnghi Sientifi Instruments, Chin). Another 30 diss ws oiled for 15 min in 5 ml of 0.3 mol L 1 mnnitol in distilled wter nd then ooled to 5 C to ssess totl eletrolytes. The reltive lekge ws expressed s perentge of the totl eletrolytes..4. Memrne oxidtion MDA onentrtion ws mesured ording to Luo et l. (011). Tissue smples (.0 g) were ground in liquid nitrogen nd extrted in 5 ml 10% (w/v) trihloroeti id (TCA). After entrifugtion t 10 000 g for 15 min, ml liquot of the superntnt ws mixed with ml 10% (w/v) TCA ontining 0.6% (w/v) TBA. The mixture ws heted to 100 C for 0 min, quikly ooled nd entrifuged t 10 000 g for 10 min. The superntnt ws olleted nd sornes t 53, 600 nd 450 nm were then mesured y Hithi U-1800 spetrophotometer (Hithi High-Tehnologies Corp., Tokyo, Jpn). The MDA onentrtion ws lulted ording to the formul: 6.45 (OD 53 OD 600 ) 0.56 OD 450. All mesurements were performed in triplite..5. Enzyme tivity Frozen tissue smples ( g) from 10 fruits were homogenized with 10 ml of ie-old extrtion uffers ontining 0. g of PVPP nd ground t 4 C. For superoxide dismutse
4 HAN Cong et l. Journl of Integrtive Agriulture 016, 15(0): 60345-7 (SOD), the extrtion uffer ws 100 mmol L 1 sodium phosphte (ph 7.8). For tlse (CAT) nd sorte peroxidse (APX), the extrtion uffer ws 100 mmol L 1 sodium phosphte (ph 7.0) while the uffer of APX ontining 0.1 mmol L 1 EDTA, 1 mmol L 1 sori id nd 1% polyvinyl-pyrrolidone. For lipoxygense (LOX), the extrtion uffer ws 100 mmol L 1 Tris-HCl (PH 8.0). For the ssy of polyphenol oxidse (PPO), the extrtion uffer ws 100 mmol L 1 sodium phosphte (ph 6.4), while the 50 mmol L 1 sodium orte uffer (ph 8.8, ontining 5 mol L 1 β-merptoethnol) ws used for PAL. The homogente ws entrifuged t 15 000 g for 30 min t 4 C nd the superntnt ws used for the enzyme ssy. Eh tretment ontined three replites nd the experiment ws repeted three times. Superoxide dismutse (SOD) tivity ws determined y the method of Jin et l. (009). One unit of SOD tivity ws defined s hnge of 1 per min in OD 560 nd results were expressed s U mg 1 FW. Ctlse (CAT) tivity ws nlyzed sed on Wng nd Tin (005) with ertin modifitions. The retion mixture onsisted of 1 ml sodium phosphte uffer (50 mmol L 1, ph 7.0), 1 ml H O (40 mmol L 1 ) nd 1 ml enzyme extrt. One unit of CAT tivity ws defined s hnge of 0.01 per min in OD 40 nd results were expressed s U g 1 FW. Asorte peroxidse (APX) tivity ws determined y the method of Nkno nd Asd (1989). One unit of APX ws defined hnge of 0.01 per min in OD 90, nd results were expressed s U g 1 FW. Polyphenol oxidse (PPO) tivity ws nlyzed ording to Liu et l. (007). One unit of PPO ws defined hnge of 0.001 per min in OD 460, nd results were expressed s U g 1 FW. Phenyllnine mmoni-lyse (PAL) tivity ws ssyed following the method of Meng et l. (008). One unit of PAL ws defined hnge of 0.01 per min in OD 90, nd results were expressed s U g 1 FW..6. Totl phenolis For nlysis of totl phenolis ontent, frozen tissue smples (4 g) from 6 fruits were extrted using mortr nd pestle with 35 ml of old 70% methnol, nd the smples were then entrifuged t 13 000 r min 1 nd 4 C for 15 min. A smple of the rude extrt (500 μl) ws dded to 1.5 ml of distilled wter nd nd 1 ml of Folin-Ciolteu regent; 1 ml of solution of 10% (w/v) N CO 3 ws dded. After inuting for h t 5 C, the sorne of the resulting stndrd (10, 0, 50, nd 100 mg L 1 ) ws mesured. The totl phenoli ontent ws expressed s milligrms of glli id per kilogrm of fresh weight (FW)..7. Sttistil nlysis All sttistil nlyses were performed with SPSS ver. 13.0 (SPSS In., Chigo, IL, USA). Dt t eh time point were nlyzed y one-wy ANOVA, nd men seprtions were performed y Dunn s new multiple rnge test. Differenes t P<0.05 were onsidered signifint. There were no signifint intertions etween tretments nd experiments, the vlues were reorded s mens with stndrd errors. 3. Results nd disussion As key signling moleule, SA hs een reported to ply n importnt role in triggering the plnt response to dverse environmentl onditions, suh s UV light, drought, slinity, hilling stress nd het shok (Ding et l. 001; Ding nd Wng 003; Asghri nd Aghdm 010). This is the first report on the effet of SA tretment on low temperture storge of sponge gourd. In the present study, the severity of hilling symptoms of sponge gourd ws evluted through the CI index prmeter. No visile CI symptoms were oserved in oth ontrol nd SA treted sponge gourds in the first 3 dys of storge (Fig. 1). However, sine the 5th dy, the ontrol smples developed hilling symptoms severely, nd the index ws 3.4 fter 9 dys t 9 C plus dys t 0 C. SA tretment signifintly inhiited the inrese of CI index, nd the lrgest redution ws otined when the onentrtion rehed 1.5 mmol L 1. On ount of this, we determined the effetive dose of SA ws 1.5 mmol L 1. It is well estlished tht eletrolyte lekge is mesurement of loss of semipermeility of ell memrnes, nd hs een widely used s n inditor of CI (MCollum nd MDon- CI index 4 3 1 0.5 mmol L 1 SA 1.0 mmol L 1 SA.0 mmol L 1 SA 0 Dys of storge (d) Fig. 1 Effet of sliyli id (SA) on hilling injury (CI) index of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). The sme s in Fig..
HAN Cong et l. Journl of Integrtive Agriulture 016, 15(0): 60345-7 5 ld 1991). As shown in Fig., the inrese of eletrolyte lekge in SA-treted fruits ws oviously lower ompred with ontrol ones, whih suggested tht SA n derese old dmge to ell memrnes nd therefore preserve ell memrne integrity of sponge gourd. Thus, our results demonstrted tht SA s posthrvest tool n e used to llevite CI of sponge gourd. One of the min use of CI is the overprodution of retive oxygen speies (ROS, suh s O -., H O nd OH), if not effetively nd rpidly removed, the redundnt ROS ould use lipid peroxidtion (Chen nd Li 001; Liu et l. 010). MDA, produed from the peroxidtion of memrne lipids, is often used s n inditor of oxidtive stress nd dmge (Ohkw et l. 1979). As shown in Fig. 3, the inrese of Eletril ondutivity (%) 80 70 60 50 40 30 0 10 0 Dys of storge (d) Fig. Effet of SA on eletril ondutivity of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). MDA ontent ws losely ssoited with the development of CI, nd signifint differene (P<0.05) ws oserved sine the 5th dy. In order to mintin the onentrtion of ROS t reltively low levels, plnts hve evolved mehnisms to svenge these toxi nd retive speies y ntioxidnt ompounds nd y enzymti ntioxidnt systems, suh s SOD, CAT nd APX (Wise 1995). SOD, the mjor O -. svenging enzyme, tlyzes the disproportiontion of O -. rdils into H O nd O, while, oth CAT nd APX tlyze the degrdtion of H O to H O nd O. In onsistent with the previous findings in peh (Wng et l. 006; Yng et l. 01) nd uumer (Co et l. 009) fruit, SA tretment mintined higher tivities of SOD, CAT nd APX in sponge gourd ompred with ontrol smples (Figs. 4, 5 nd 6). This oservtion indited tht the enefiil effet of SA in lleviting CI of sponge gourd is involved in elevting ntioxidnt enzymes tivities. Interestingly, unlike with the deresing trend of CAT nd APX, the tivity of SOD hd slight inrese t the 5th dy in ontrol ones nd 7th dy in SA treted smples, respetively. Suh result my due to the defene mehnism of plnt whih indued the tivity of SOD, nd therey detoxified superoxide ion to H O. With the extension of low temperture storge, fresh rowning of sponge gourd ws ourred under the skin. PAL nd PPO re two importnt enzymes relted to the rowning proess of fruits nd vegetles. PAL prtiiptes in the iosynthesis of phenoli ompounds (Rösler et l. 1997), while PPO oxidizes these phenols to quinines (Mrtinez nd Whitker 1995) nd therey uses tissue rowning. In the present study, the ontent of phenoli ompounds nd tivities of PAL nd PPO in oth ontrol nd SA treted smples inresed throughout the whole storge period. MDA (nm g 1 FW).0 1.8 1.6 1.4 1. 1.0 0.8 0.6 0.4 Dys of storge (d) SOD tivity (U mg 1 FW) 1.8 1.6 1.4 1. 1.0 0.8 Dys of storge (d) Fig. 3 Effet of SA on mlondildehyde (MDA) ontent of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). The sme s elow. Fig. 4 Effet of SA on superoxide dismutse (SOD) tivity of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C).
6 HAN Cong et l. Journl of Integrtive Agriulture 016, 15(0): 60345-7 3 700 CAT tivity (U g 1 FW) 8 4 0 16 1 8 4 0 Dys of storge (d) Totl phenolis ontent (mg kg 1 FW) 600 500 400 300 00 Dys of storge (d) Fig. 5 Effet of SA on tlse (CAT) tivity of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). Fig. 7 Effet of SA on totl phenolis ontent of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). APX tivity (U g 1 FW min 1 ) 800 750 700 650 600 550 500 450 PAL tivity (U g 1 FW ) 3.5 3.0.5.0 1.5 1.0 0.5 400 Dys of storge (d) 0.0 Dys of storge (d) Fig. 6 Effet of SA on sorte peroxidse (APX) tivity of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). Fig. 8 Effet of SA on phenyllnine mmoni-lyse (PAL) tivity of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). However, the pplition of SA signifintly restrined this inresing trend nd rought lower vlues of these three prmeters in SA treted sponge gourds (Figs. 7, 8 nd 9). This oservtion ws in gree with the previous reserhes of plum (Luo et l. 011), pinepple (Lu et l. 011), pomegrnte (Syyri et l. 009) nd moo shoot (Luo et l. 01), tht the suppression of tissue rowning y SA ws usully ssoited with diminished totl phenolis ontent nd inhiited PAL nd PPO tivities. 4. Conlusion Low-temperture indued CI in sponge gourd. This is the first study reporting on the use of SA tretment in lleviting posthrvest CI of sponge gourd. The dte otined in the present study showed tht the pplition of SA llevited CI symptoms, redued eletrolyte lekge, inhiited the inrese of MDA nd totl phenolis ontents, enhned the ntioxidnt enzymes tivities of SOD, CAT nd APX, nd suppressed PAL nd PPO tivities. The enefiil effets of SA my e ttriuted to mintining memrne integrity, inhiiting memrne peroxidtion, enhning ntioxidnt system nd suppressing rowning relted enzymes. These results suggested tht SA s posthrvest tool ould effetively llevite CI nd enhne hilling tolerne of sponge gourd. However, further studies re
HAN Cong et l. Journl of Integrtive Agriulture 016, 15(0): 60345-7 7 PPO tivity (U g 1 FW ) 800 700 600 500 400 300 00 needed to explore the ommeril use of SA in lleviting CI of sponge gourd. Aknowledgements This work ws supported y the Ministry of Agriulture of Chin (CARS-5), the Speil Fund for Agro-Sientifi Reserh in the Puli Interest, Chin (0103095), the Ntionl Nturl Siene Foundtion of Chin (31401536), the Nturl Siene Foundtion of Beijing (614400), the Young Investigtor Fund of Beijing Ademy of Agriulturl nd Forestry Sienes of Chin (01404) Referenes Aghdm M S, Asghri M, Mordeygi H, Mohmmdkhni N, Mohyeji M, Rezpour-Frd J. 01. Effet of posthrvest sliyli id tretment on reduing hilling injury in tomto fruit. Romnin Biotehnologil Letters, 17, 7466 7473. Aghdm M S, Bododk S. 013. Physiologil nd iohemil mehnisms regulting hilling tolerne in fruits nd vegetles under posthrvest sliyltes nd jsmontes tretments. Sienti Hortiulture, 156, 73 85. Asghri M, Aghdm M S. 010. Impt of sliyli id on posthrvest physiology of hortiulturl rops. Trends in Food Siene & Tehnology, 1, 50 509. Co S, Hu Z, Wng H. 009. Effet of sliyli id on the tivities of nti-oxidnt enzymes nd phenyllnine mmoni-lyse in uumer fruit in reltion to hilling injury. Journl of Hortiulturl Siene & Biotehnology, 84, 15 130. Co S, Hu Z, Zheng Y, Lu B. 010. Synergisti effet of het tretment nd sliyli id on lleviting internl rowning in old-stored peh fruit. Posthrvest Biology Dys of storge (d) Fig. 9 Effet of SA on polyphenol oxidse (PPO) tivity of sponge gourd during storge (9 dys, 9 C) plus shelf life ( dys, 0 C). nd Tehnology, 58, 93 97. Chen W, Li P. 001. Chilling-indued C + overlod enhnes prodution of tive oxygen speies in mize (Ze mys L.) ultured ells: The effet of sisi id tretment. Plnt Cell nd Environment, 4, 791 800. Ding C K, Wng C Y. 003. The dul effets of methyl sliylte on ripening nd expression of ethylene iosyntheti genes in tomto fruit. Plnt Siene, 164, 589 596. Ding C K, Wng C Y, Gross K C, Smith D L. 001. Redution of hilling injury nd trnsript umultion of het shok protein genes in tomtoes y methyl jsmonte nd methyl sliylte. Plnt Siene, 161, 1153 1159. Hung S, Li T, Jing G, Xie W, Chng S, Jing Y, Dun X. 01. 1-Methylylopropene redues hilling injury of hrvested okr (Hiisus esulentus L.) pods. Sienti Hortiulture, 141, 4 46. Jin P, Zheng Y, Tng S, Rui H, Wng C. 009. A omintion of hot ir nd methyl jsmonte vpor tretment llevites hilling injury of peh fruit. Posthrvest Biology nd Tehnology, 5, 4 9. Liu J, Tin S, Meng X, Xu Y. 007. Effets of hitosn on ontrol of posthrvest diseses nd physiologil responses of tomto fruit. Posthrvest Biology nd Tehnology, 44, 300 306. Liu Y, Jing H, Zho Z, An L. 010. Nitri oxide synthse like tivity-dependent nitri oxide prodution protets ginst hilling-indued oxidtive dmge in Chorispor ungen suspension ultured ells. Plnt Physiology nd Biohemistry, 48, 936 944. Luo Z, Chen C, Xie J. 011. Effet of sliyli id tretment on lleviting posthrvest hilling injury of Qingni plum fruit. Posthrvest Biology nd Tehnology, 6, 115 10. Luo Z, Wu X, Xie Y, Chen C. 01. Allevition of hilling injury nd rowning of posthrvest moo shoot y sliyli id tretment. Food Chemistry, 131, 456 461. Lu X, Sun D, Li Y, Shi W, Sun G. 011. Pre- nd post-hrvest sliyli id tretments llevite internl rowning nd mintin qulity of winter pinepple fruit. Sienti Hortiulture, 130, 97 101. Mrtinez M V, Whitker J R. 1995. The iohemistry nd ontrol of enzymti rowning. Trends in Food Siene & Tehnology, 6, 195 00. MCollum T G, MDonld R E. 1991. Eletrolyte lekge, respirtion, nd ethylene prodution s indies of hilling injury in grpefruit. HortSiene, 6, 1191 119. Meng X, Li B, Liu J, Tin S. 008. Physiologil responses nd qulity ttriutes of tle grpe fruit to hitosn prehrvest spry nd posthrvest oting during storge. Food Chemistry, 106, 501 508. Nkno Y, Asd K. 1989. Hydrogen peroxide is svenged y srote speifi peroxidse in spinh hloroplsts. Plnt nd Cell Physiology,, 867 880. Ooh I O, Aluyor E O. 009. Luff ylindri - An emerging sh rop. Afrin Journl of Agriulturl Reserh, 4, 684 688. Ohkw H, Ohishi N, Ygi K. 1979. Assy for lipid peroxides
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