Effect of Maturation Stage and Storage Temperature and Duration on β Cryptoxanthin Content in Satsuma Mandarin (Citrus unshiu Marc.

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1 This rtile is n Advne Online Pulition of the uthors orreted proof. Note tht minor hnges my e mde efore finl version pulition. The Hortiulture Journl Preview doi:.3/hortj.utd-7 JSHS The Jpnese Soiety for Hortiulturl Siene Effet of Mturtion Stge nd Storge Temperture nd Durtion on β Cryptoxnthin Content in Stsum Mndrin (Citrus unshiu Mr.) Fruit Hikru Mtsumoto *, Yoshihiko Adhi, Yoshinori Ikom 3 nd Msy Kto Institute of Fruit Tree nd Te Siene, NARO, Shizuok -9, Jpn Institute of Fruit Tree nd Te Siene, NARO, Irki 3-86, Jpn 3 Hedqurters, NARO, Irki 3-87, Jpn Fulty of Agriulture, Shizuok University, Shizuok -89, Jpn In stsum mndrin fruit (Citrus unshiu Mr.), β-ryptoxnthin is mjor rotenoid nd n importnt qulity omponent in the juie ss. The stge of mturity nd storge onditions of stsum mndrin fruit shipped to mrkets vries. However, the effets of mturtion stge, storge temperture nd durtion on hnges in β-ryptoxnthin ontent hve not een fully studied. In the present study, fruits were hrvested t different mturtion stges, nd hnges in β-ryptoxnthin ontent in the juie ss were investigted during storge t tempertures from to C. At C, in fruit hrvested while β-ryptoxnthin is still eing umulted on the tree, the ontent ontinued to inrese following dys of storge. However, in the fruit without β-ryptoxnthin umultion on the tree, the ontent did not inrese. At C, in the fruit umulting β-ryptoxnthin on the tree, the ontent ontinued to inrese fter dys storge, wheres in the fruit without β-ryptoxnthin umultion, the ontent did not inrese fter dys storge ut inresed fter 3 dys storge. At 8 C, the inrese in ontent ws lso oserved in the fruit when stored for 8 dys. In ontrst, t C, the ontent did not hnge notly t ny mturtion stge regrdless of the experimentl period. The hnges in the rotenoid ontent nd gene expression suggest tht rotenoid umultion during on-tree mturtion ontinues fter hrvest t C, ut not t C. The present results suggest tht β-ryptoxnthin ontent will not derese t rnge from to C irrespetive of the mturtion stge. Moreover, t 8 C nd ove, the β-ryptoxnthin ontent will grdully inrese, nd the rte will e more rpid in fruit hrvested when rotenoid umultion is in progress on the tree. Key Words: β-ryptoxnthin, rotenoid, mturtion stge, stsum mndrin, storge temperture. Introdution Before onsumption, posthrvest itrus fruit is exposed to vrious tempertures (low to high) in storge rooms, on store shelves, nd during trnsporttion (Grierson nd Miller, 6; Kder, ). Posthrvest temperture ffets itrus fruit qulity (e.g., olor, tste, nd flvor), s well s qulity omponents suh s rotenoids, sugrs, ids, nd voltiles (Alquézr et l., 8; Ldniy, 8; Oenlnd et l., ; Tietel et l., ). Crotenoids re importnt qulity omponents in it- Reeived; Septemer 9, 8. Aepted; Novemer, 8. First Pulished Online in J-STAGE on Jnury 9, 9. This work ws supported y grnt from the Reserh Projet on Development of Agriulturl Produts nd Foods with Helthpromoting Benefits (NARO), Jpn. * Corresponding uthor (E-mil: hikruoo@ffr.go.jp). rus fruit nd ontriute to the olor of the peel nd pulp (Gross, 987). In ddition, some rotenoids, suh s β- rotene nd β-ryptoxnthin, re preursors of vitmin A, whih serve s humn nutrients, nd redue the risk of hroni diseses (Olson, 989; Ymguhi, ). The posthrvest temperture effet on rotenoid umultion hs een widely studied in the peel of itrus fruit (Alquézr et l., 8; Brry nd Vn Wky, 6; Vn Wky et l., 9; Wheton nd Stewrt, 973). However, studies on rotenoids in the edile juie ss of the fruit hve een limited nd onduted for only few ultivrs nd storge onditions. Reent studies hve reported tht in the pulp of grpefruit nd ornges, the rotenoid ontent in fruit stored t C ws higher thn tht in fruit stored elow C (Crmon et l., ; Chudhry et l.,,, 7). Previously, we reported tht in Aoshim stsum mndrin fruit hrvested t the fully-olored stge, the rotenoid ontent in the juie ss did not hnge notly during 9 The Jpnese Soiety for Hortiulturl Siene (JSHS), All rights reserved.

2 H. Mtsumoto, Y. Adhi, Y. Ikom nd M. Kto storge t or C for three weeks (Mtsumoto et l., 9). The stsum mndrin (C. unshiu. Mr.) hs mny ultivrs with different mturity sesons: very erly ripening (mture in lte Septemer to Otoer), erly ripening (Otoer to Novemer), mid-ripening (Novemer), nd lte ripening (lte Novemer to Deemer) (Hodgson, 967; Iwms, 976). The hrvest time of these ultivrs is generlly determined on the sis of peel olor nd mturity index (totl solule solid/idity), ut differs mong prodution res nd growers (Grierson, 6; Iwms, 976). For exmple, some very erly ripening ultivrs re hrvested t the stge of olor rek in severl prodution res, ut t the fully olored stge in others. In ddition, lte ripening ultivrs re normlly stored, ut storge tempertures nd durtions differ mong growers, shipping oopertives, nd so on. Thus, the mturtion stge nd storge onditions of stsum mndrin fruit shipped to mrkets vry. It is thought tht these ftors ffet hnges in the rotenoid ontent fter hrvest. However, in stsum mndrin fruit, the effets of mturtion stge nd storge temperture nd durtion on rotenoid ontent hve not yet een fully studied. In, the system of Foods with Funtion Clims ws estlished y the Consumer Affirs Ageny in Jpn. Stsum mndrin fruit ws registered s the first fresh produt with funtion lims sed on β- ryptoxnthin s funtionl sustne (Med- Ymmoto, 7), nd the funtion lims re leled on the pkging y severl griulturl oopertives. Thus, it is importnt to understnd ftors ffeting the β-ryptoxnthin ontent of the fruit shipped to mrket. The im of the present study ws to investigte the effets of posthrvest tempertures, storge durtion, nd mturtion stge on hnges in β-ryptoxnthin ontent in the juie ss of stsum mndrin fruit. The temperture effet inluded rnge from to C, ssuming storge t old or mient temperture nd room temperture (e.g., store shelf). Sine the stsum mndrin hs mny ultivrs, the experiments were onduted using severl ultivrs to exmine vritions in these effets. In experiment, the effet of temperture ( nd C) nd mturtion stge on rotenoid umultion nd gene expression relted to rotenoid iosynthesis during storge for dys ws exmined in Aoshim s n experimentl model. In experiment, vrietl differenes in rotenoid umultion t C storge for dys were exmined in stsum mndrin ultivrs. In experiment 3, the effet of n intermedite temperture rnging from to C, nd the omined effet of mturtion stge nd prolonged storge durtion were exmined in Nihinn No. s n experimentl model. In experiment, temperture effets during long-term storge were exmined in Aoshim lte ripening ultivrs. Mterils nd Methods Experiment : Effet of Temperture on rotenoid umultion in Aoshim stsum mndrin hrvested t different stges of mturtion To exmine the effet temperture on rotenoid ontent nd its iosynthesis t different mturtion stges, Aoshim stsum mndrin (C. unshiu Mr.) fruits were used s n experimentl model. The fruit ws hrvested t different mturtion stges, Ot. (8 dys fter flowering (DAF), drk green), Ot. (63 DAF, light green), Nov. (78 DAF, olor rek), Nov. 9 (93 DAF, ornge-yellow), De. ( DAF, fully olored), De. (8 DAF, overripe), nd Jn. 9 ( DAF, overripe) from tree t the Institute of Fruit Tree nd Te Siene, Citrus Reserh Division, Okitsu (Shizuok, Jpn). Fruit tht were uniform in size nd olor were seleted. The fruit were divided into 3 groups nd stored in the drk for dys t nd C under 8 9% reltive humidity with ontinuous ventiltion. After nd dys storge, three to six fruits per tretment were olleted, nd three smples per tretment were prepred y lending one or two fruit. The juie ss were seprted from the fruit, immeditely frozen in liquid nitrogen, nd stored t 8 C until use (Mtsumoto et l., 9). The hnges in rotenoid ontent nd gene expression relted to rotenoid iosynthesis in the juies ss of eh smple were nlyzed s desried elow. Experiment : Vrietl differenes in β-ryptoxnthin umultion fter storge t C The fruit of stsum mndrin ultivrs with different mturity sesons were hrvested t the ommeril hrvest dte. Hrvest dys were s follows: Ot. 9 for very erly ripening ultivrs ( Nihinn No., Iwski, Dowki, nd Ueno-wse ), Nov. 7 for erly ripening ultivrs ( Okitsu-wse nd Miygwwse ), Nov. 9 for mid ripening ultivrs ( Nnkn No. nd Seto ), nd Nov. 7 for lte ripening ultivrs ( Otsu No. nd Aoshim ). Fruit were lmost fully olored; however, the peel hd some green res. The fruit ws stored for dys t C under 8 9% reltive humidity. After nd dys storge, six fruits per tretment were olleted, nd three smples per tretment were prepred s desried ove. In ddition, t dys fter storge, the fruit stored on the tree were hrvested. The verge temperture in the field during on-tree storge for dys ws lulted sed on the AMeDAS (Automted Meteorologil Dt Aquisition System) equipment in the field t our institute. The verge tempertures were s follows: 8 C for very erly ripening, C for erly ripening, 3 C for mid ripening, nd C for lte ripening ultivrs, respetively. The hnges in rotenoid ontent in the juie ss of eh smple were nlyzed s desried elow.

3 Hort. J. Preview 3 Experiment 3: Effet of intermedite temperture rnging from to C nd omined effet of mturtion stge nd prolonged storge durtion on β- ryptoxnthin umultion in Nihinn No. stsum mndrin To exmine effet of intermedite temperture rnging from to C nd the omined effet of mturtion stge nd prolonged storge durtion on β- ryptoxnthin umultion, Nihinn No. ws used s n experimentl model. The fruit ws hrvested in erly- nd lte-otoer. The fruit ws divided into four groups nd stored in the drk for 3 dys t,, nd C under 8 9% reltive humidity with ontinuous ventiltion. After, nd 3 dys storge, three to six fruit per tretment were olleted, nd three smples per tretment were prepred s desried ove. The hnges in rotenoid ontent in the juie ss of eh smple were nlyzed s desried elow. Experiment : Effet of temperture on β-ryptoxnthin umultion during long-term storge of Aoshim stsum mndrin fruit To exmine temperture-effet on β-ryptoxnthin umultion during long-term storge, the fruit of Aoshim, lte ripening ultivr, ws hrvested from tree in erly Deemer. The fruit ws divided into three groups nd stored without pre-storge onditioning in the drk for 9 dys t,, nd C under 8 9% reltive humidity with ontinuous ventiltion. After,, 3, 6, nd 9 dys storge, six fruits per tretment were olleted, nd three smples per tretment were prepred s desried ove. In nother yer, to exmine the temperture-effet during longterm storge nd the effet of the intermedite tempertures of to C, lrge-sle experiment ws onduted. The fruit of Aoshim ws hrvested from three different trees in erly Deemer. The fruit ws onditioned t mient temperture until the weight ws redued y % to prevent fruit dey during longterm storge. After pre-storge onditioning, the fruit ws divided into three groups nd stored in the drk for 8 dys t, 8, nd C under 8 9% reltive humidity with ontinuous ventiltion. After nd 8 dys storge, t lest three fruits per tree were olleted nd lended, nd three smples derived from three different trees were prepred s desried ove. A totl of nine fruits were used. The hnges in rotenoid ontent in the juie ss of eh smple were nlyzed s desried elow. Crotenoid nlysis In experiment., rotenoids, phytoene, ζ-rotene, β-rotene, β-ryptoxnthin, zexnthin, 9-isviolxnthin, nd ll-trns-violxnthin in the juie ss were extrted nd nlyzed ording to previously desried method (Mtsumoto et l., 9). The rotenoid ontent of eh smple ws nlyzed using HPLC (Agilent ; Agilent Tehnologies, Plo Alto, CA, USA) with mm.6 mm i.d., μm, YMC Crotenoid C3 olumn (YMC Europe GMBH, Dinslken, Germny) with ternry grdient elution of wter, methnol, nd MTBE pumped t flow rte of ml min nd photodiode rry detetion (Kto et l., ). In experiments, 3, nd, onventionl nlysis of β-ryptoxnthin ws performed ording to previously desried method. Briefly, β-ryptoxnthin ws extrted with ethnol nd nlyzed using HPLC with inry grdient elution (Kumgi et l., 6). The rotenoids were identified y ompring the retention times nd spetr with uthenti stndrds or literture dt (Kto et l., ). Crotenoid ontent ws determined y referene to the stndrd urve prepred for eh rotenoid. Totl RNA extrtion nd rel-time quntittive RT- PCR. Totl RNA ws extrted from the flvedo nd juie ss ording to previously reported method nd lened up with on-olumn DNse digestion (Ikom et l., 996). The retions of reverse trnsription (RT) were performed with purified RNA nd rndom hexmer using TqMn Reverse Trnsription Regents (Applied Biosystems, Foster City, CA) (Kto et l., 6). The gene expression nlyses for PSY (phytoene synthse), PDS (phytoene desturse), ZDS (ζ-rotene desturse), LCY (lyopene-β-ylse), HY (β-ring hydroxylse), ZEP (zexnthin epoxidse), nd NCED (nine-is-epoxyrotenoid dioxygense) were performed ording to previously reported method (Kto et l., 6). TqMn MGB proes nd sets of primers for CitPSY, CitPDS, CitZDS, CitLCY, CitHY, CitZEP, CitNCED3, nd CitNCED (previously desried s CitNCED), designed on stsum mndrin DNAs, hve een desried in previous studies (Ikom et l., 6). TqMn rel-time PCR ws rried out with TqMn Universl PCR Mster Mix (Applied Biosystems) ording to the mnufturer s instrutions. As n endogenous ontrol, the TqMn Riosoml RNA Control Regents VIC Proe (Applied Biosystems) ws used. The levels of gene expression were nlyzed with ABI PRISM 7 Sequene Detetion System Softwre (Applied Biosystems) nd normlized with the results of 8S riosoml RNA. Rel-time quntittive RT-PCR ws performed in three replites for eh smple. Sttistil dt nlysis The sttistil signifine of the results ws nlyzed using Tukey s HSD test t the % level using the pplition softwre JMP 8 (JMP relese 8.; SAS Institute In., Cry, NC, USA).

4 H. Mtsumoto, Y. Adhi, Y. Ikom nd M. Kto Results nd Disussion Experiment : The effet of temperture nd mturtion stge on rotenoid umultion in the juie ss of Aoshim stsum mndrin To exmine the effet of temperture nd mturtion stge on rotenoid umultion, fruit ws hrvested t 7 different stges of mturtion from Ot. to Jn. 9 nd stored t or C for dys. The rotenoid ontent ws nlyzed efore nd fter storge. Seven rotenoids, phytoene, ζ-rotene, β-rotene, β- ryptoxnthin, lutein, zexnthin, nd violxnthin, were quntified. The totl rotenoid ontent ws the sum of them. The violxnthin ontent ws the sum of 9-is-violxnthin nd ll-trns-violxnthin. During on-tree mturtion, the totl rotenoid ontent nd β-ryptoxnthin, mjor rotenoid in the juie ss (6 7% of the totl rotenoids), linerly inresed from Ot. to Nov. 9 or De., nd then remined t similr levels until Jn. 9 (Fig. ). At C, in the fruit hrvested efore Nov., the ontent of totl rotenoids nd β-ryptoxnthin signifintly inresed (.- to -fold) t dys fter hrvest, nd the inrese in ontent ws similr to tht in fruit stored on the tree for dys ( efore storge for dys lter, Fig. ). To e more speifi, the β-ryptoxnthin ontent of the fruit hrvested on Ot. nd stored until Nov. (.9 μg g ) t C ws similr to tht of the fruit hrvested on Nov. (. μg g ). In ontrst, in fruit hrvested fter Nov. 9, the inrese in the β-ryptoxnthin ontent fter hrvest t C ws smll nd sttistilly insignifint. At C, the inrese in β-ryptoxnthin ontent ws smll nd sttistilly insignifint ross ll mturtion stges (Fig. ). Likewise, β-rotene nd zexnthin signifintly inresed fter hrvest t C, nd the inrese ws greter in the erly thn the lter stge of mturtion (Fig. ). For exmple, in fruit hrvested efore Nov., β-rotene inresed.- to.9-fold fter hrvest t C, wheres fter Nov. 9, the ontent inresed only.- to.6-fold (Fig. ). At C, the hnge in ontent fter hrvest ws smll nd not sttistilly signifint t most mturtion stges. Similr hnges were oserved in the juie ss of the erly ripening ultivr Miygw-wse (dt not shown). These results showed tht in stsum mndrin fruit µg g Phytoene / / / /9 / / /9 ζ-crotene / / / /9 / / /9 β-crotene / / / /9 / / / β-cryptoxnthin / / / /9 / / /9 Zexnthin / / / /9 / / /9 Violxnthin / / / /9 / / / Lutein / / / /9 / / /9 Totl rotenoids / / / /9 / / /9 efore storge storge t for dys storge t for dys Hrvest dy Fig.. Chnges in rotenoid ontent efore nd fter storge t C nd C in the juie ss of Aoshim hrvested t different mturtion stges. Different letters indite signifint differenes mong smples hrvested on the sme dy t P <. y Tukey s HSD test. Content is expressed on fresh-weight sis, μg g. Dys fter flowering (DAF) were s follows: Ot. (8 DAF), Ot. (63 DAF), Nov. (78 DAF), Nov. 9 (93 DAF), De. ( DAF), De. (8 DAF), nd Jn. 9 ( DAF). The vlues re the men ± SE of three replites.

5 Hort. J. Preview hrvested t the stge in whih β-ryptoxnthin ontinuously umultes on the tree, the ontent in the juie ss ontinued to inrese t C for dys fter hrvest nd tht the inresed ontent ws similr to tht in fruit left on the tree. In ontrst, in fruit hrvested t the stge in whih umultion of β-ryptoxnthin stgntes on the tree, the inrese in the ontent ws negligile t C for t lest dys (Fig. ). These results suggest tht the effets of temperture on the umultion of β-ryptoxnthin in the juie ss ws mturtion stge dependent nd tht the umultion on the tree would ontinue in posthrvest fruit t C, ut not t C, for t lest dys fter hrvest. Reent studies hve shown tht storge temperture ffet the rotenoid ontent in the pulp of severl itrus vrieties, nd the higher ontent during storge tended to e mintined t C ompred to elow C (Crmon et l., ; Chudhry et l.,,, 7; Mtsumoto et l., 9). In stsum mndrin fruit hrvested in erly Deemer, we previously reported tht the rotenoid ontent in the juie ss did not lerly inrese t C, ut the ontent ws higher t C thn t C for three weeks (Mtsumoto et l., 9). The present results re onsistent with these previous findings. To onfirm our hypothesis tht β-ryptoxnthin umultion on the tree ontinues fter hrvest t C, ut not t C, the gene expression of rotenoid metoli enzymes ws nlyzed in the fruit hrvested on Nov., De., nd De.. Gene expressions for CitPSY (phytoene synthse), CitPDS (phytoene desturse), CitZDS (ζ-rotene desturse), CitLCY (lyopene-βylse), CitHY (β-ring hydroxylse), CitZEP (zexnthin epoxidse), CitNCED3 (nine-is-epoxyrotenoid dioxygense), nd CitNCED exmined in the present study re onsidered to ontriute to rotenoid umultion during itrus fruit mturtion nd fter hrvest (Alquézr et l., 8; Ikom et l., 6; Kto et l.,, 6; Mtsumoto et l., 9). CitNCED isolted from the stsum mndrin ws previously desried s CitNCED (Ikom et l., 6). In the fruit hrvested on Nov., the gene expression of CitPSY, CitZDS, nd CitLCY, relted to β- ryptoxnthin iosynthesis, did not notly hnge fter storge t C (Fig. ). During on-tree mturtion from Nov. to De., these hnges were lso negligile (Blk rs in Fig. ). The gene expression of CitHY, CitZEP, nd CitNCED, relted to β- ryptoxnthin tolism, deresed oth fter storge t C of the fruit hrvested on Nov. nd during ontree mturtion from Nov. to De. (Fig. ). The ontent of β-ryptoxnthin inresed oth during storge t C nd on-tree mturtion (Fig. ). In ontrst, t C, gene expression relted to β-ryptoxnthin iosynthesis (CitPSY, CitPDS, CitZDS, nd CitLCY) deresed during the storge of fruit hrvested on Nov., ut did not lerly hnge during on-tree mturtion mrna reltive undne CitPSY / / / CitPDS / / / CitZDS / / / CitLCY / / / from Nov. to De. (Fig. ). The gene expression of CitHY did not hnge t C, ut deresed during ontree mturtion. The β-ryptoxnthin ontent did not hnge t C, ut inresed during on-tree mturtion (Fig. ). Thus, in the fruit hrvested on Nov. nd stored t C, hnges in the gene expression nd β- ryptoxnthin ontent fter hrvest were similr to those during on-tree mturtion from Nov. to De. ; however, they were different when stored t C. In the fruit hrvested on De., the β-ryptoxnthin ontent nd most of the gene expression did not hnge notly fter storge t C (Figs. nd ). These hnges were lso smll during on-tree mturtion from De. to De. (Fig. ). In ontrst, t C, gene expression relted to β-ryptoxnthin synthesis did not notly hnge fter hrvest; however, the gene expression of CitNCED3 nd CitNCED, relted to β CitHY / / / CitZEP / / / Hrvest dy / / / CitNCED3 CitNCED / / / Fig.. Chnges in the expression of rotenoid iosynthesisrelted genes efore (lk rs) nd fter storge t C (white rs) nd C (gry rs) for dys in the juie ss of Aoshim hrvested t different mturtion stges. An expression vlue of the smple immeditely fter hrvest on Nov. is ritrrily ssigned vlue of. The vlues re the men ± SE of three replites.

6 6 H. Mtsumoto, Y. Adhi, Y. Ikom nd M. Kto ryptoxnthin tolism, mrkedly inresed (Fig. ). The inresed expression of CitNCED3 nd CitNCED fter storge t C ws not oserved during on-tree mturtion from De. to De.. Thus, the present results support our hypothesis tht rotenoid iosynthesis during on-tree mturtion ontinues fter hrvest t C, ut not t C, s the pttern of hnges in the gene expression nd β-ryptoxnthin ontent during ontree mturtion were similr to those t C fter hrvest, ut not t C. Previous studies lso reported tht in Nvelin ornges, hnges in the rotenoid ontent nd expression of rotenoid iosyntheti genes during storge t C were similr to those ourring nturlly during the mturtion of itrus fruit (Crmon et l., ; Rodrigo nd Zrís, 7). The results re onsistent with our hypothesis tht rotenoid iosynthesis ontinues fter hrvest t norml tempertures, ut not t low tempertures. Similr results were lso reported in posthrvest wtermelons; the results showed tht rotenoid iosynthesis in wtermelons ontinued long fter hrvest, nd rotenoid iosynthesis ws enhned during storge t C, ut inhiited t C (Perkins-Vezie nd Collins, 6). Experiment : Vrietl differenes in tempertureresponsive β-ryptoxnthin umultion t C To exmine whether the inrese in β-ryptoxnthin ontent t C is lso oserved in other ultivrs with different mturity sesons, the fruits of different stsum mndrin ultivrs were hrvested t their ommeril hrvest times nd stored t C for dys (Fig. 3). In ll the very erly ripening ultivrs ( Nihinn No., Iwski, Dowki, nd Uenowse ), the ontent signifintly inresed (.- to.- fold) t dys fter hrvest, nd the inrese in ontent ws similr to tht of fruit stored on the tree (Fig. 3). In ll the erly ripening ( Okitsu-wse nd Miygw-wse ) nd mid-ripening ultivrs ( Nnkn No. nd Seto ), hnges in the ontent fter hrvest or on the tree were smll or negligile (Fig. 3). In the lte-ripening ultivrs, the hnge ws negligile in Aoshim, ut in Otsu No., the ontent signifintly inresed (.-fold) fter hrvest. These results suggest tht the effets of β-ryptoxnthin umultion fter storing t C re different mong stsum mndrin ultivrs, proly euse the progress of β- ryptoxnthin umultion on the tree t hrvest time is different mong ultivrs. Experiment 3: Effet of intermedite temperture ( to C) nd omined effet of mturtion stge nd prolonged storge durtion on β-ryptoxnthin umultion in Nihinn No. stsum mndrin To exmine the effet of intermedite temperture etween nd C nd the omined effet of mturtion stge nd prolonged storge durtion on β- µg g efore storge storge t for dys storge on the tree for dys A A A3 A B B C C D D Stsum mndrin ultivrs with different hrvest sesons Fig. 3. Chnges in β-ryptoxnthin ontent efore nd fter storge t C for dys nd on-tree storge in the juie ss of stsum mndrin ultivrs. (A) Very erly ripening ultivrs hrvested on Ot. 9: A, Nihinn No. ; A, Iwski ; A3, Dowki ; A, Ueno-wse. (B) Erly ripening ultivrs hrvested on Nov. 7: B, Okitsu-wse ; B, Miygw-wse. (C) Mid-ripening ultivrs hrvested on Nov. 9: C, Nnkn No. ; C, Seto. (D) Lte-ripening ultivrs hrvested on Nov. 7: D, Aoshim ; D, Otsu No.. Content is expressed on fresh-weight sis, μg g. The vlues re the men ± SE of three replites. ryptoxnthin ontent, Nihinn No. were hrvested t two different mturtion stges, nd hnges in the β- ryptoxnthin ontent during storge were exmined t,,, nd C for 3 dys. Fruits were hrvested in erly Otoer, when the umultion of β- ryptoxnthin ws proeeding on the tree nd the peel hd some green res, s well s in lte Otoer when the umultion of β-ryptoxnthin hd lmost stgnted on the tree nd the peel ws fully olored. At C, hnges in the ontent fter hrvest were negligile for 3 dys irrespetive of hrvest dy (Fig. ). At C, in the erly Otoer hrvest, β-ryptoxnthin ontent inresed (.3-fold) t dys fter hrvest nd ws mintined until 3 dys fter hrvest (Fig. A). In ontrst, in the lte Otoer hrvest, t C the ontent did not hnge fter dys ut ws inresed (.3- fold) t 3 dys fter hrvest (Fig. B). Irrespetive of the hrvest dy, the ontent t 3 dys fter hrvest ws higher t,, nd C thn t C (Fig. ). These results showed tht, under posthrvest onditions rnging from to C, β-ryptoxnthin ontent does not derese for t lest 3 dys fter hrvest. Moreover, the present results suggest tht t C nd ove, the β-ryptoxnthin ontent inreses fter hrvest, nd more rpidly in the fruit hrvested when β- ryptoxnthin is umulting s opposed to when the umultion of β-ryptoxnthin stgntes on the tree. Previous studies hve shown tht in the pulp of ornges nd grpefruit, the β,β-xnthophyll ontent (e.g., β-rotene nd β-ryptoxnthin) grdully inresed during long-term storge t pproximtely C (Crmon et l., ; Chudhry et l.,, 7). In Nvelin ornges, during storge for 7 weeks, the rotenoid ontent remined lmost unhnged t C,

7 Hort. J. Preview 7 ut inresed t C (Crmon et l., ). In Str Ruy grpefruit, β-rotene nd lyopene levels in the juie of fruit stored t C were higher thn tht in fruit stored t C fter 6 weeks of storge, ut no signifint differenes were oserved fter four nd eight weeks of storge (Chudhry et l., ). These results re onsistent with our present result tht t pproximtely C, prolonged storge inresed rotenoid ontent, nd the ontent t C nd ove ws higher thn tht t C. Experiment : Effet of temperture on β-ryptoxnthin umultion during long-term storge of Aoshim stsum mndrin fruit Aoshim, lte ripening ultivr, re usully stored t low or mient temperture for one to three months. Thus, the effets of temperture on β-ryptoxnthin umultion during storge were exmined t,, nd C for 9 dys. The fruit ws hrvested in the norml hrvest seson of erly Deemer, during whih rotenoid umultion on the tree stgntes, nd stored without pre-storge onditioning. At C, hnges in the β-ryptoxnthin ontent were negligile for 9 dys fter hrvest (Fig. ). At C, hnges in ontent were negligile t dys fter hrvest, ut the ontent inresed.3-fold t 3 dys fter hrvest nd ws mintined until 9 dys fter hrvest (Fig. ). At 3 nd 9 dys fter hrvest, the ontent in the fruit stored t C ws higher thn tht t C. Similr hnges were oserved t C, ut mny fruits hd deyed y 6 dys fter hrvest (Fig. ). To redue dey during storge, pre-storge onditioning nd low-temperture storge elow C is desirle (Hsegw nd I, 983, 98). In ddition, reent studies reported tht storge t 8 C ws desirle to mintin the flvor qulity of some mndrin ultivrs (Oenlnd et l., ; Tietel et l., ). Thus, to exmine the effet of tempertures etween nd C on β-ryptoxnthin umultion, Aoshim ws hr- vested in erly Deemer nd pre-storge onditioning ws onduted efore the fruit ws stored t, 8, nd C for 8 dys. After 8 dys storge, hnges in the β-ryptoxnthin ontent were negligile t C, ut the ontent inresed.3-fold t 8 nd C (Fig. 6). These results showed tht in Aoshim stsum mndrin fruit stored t onditions rnging from to C, the β-ryptoxnthin ontent does not derese for t lest 9 dys of storge. Moreover, the present results suggest tht t 8 C nd ove, the β-ryptoxnthin ontent would inrese fter hrvest even if the fruit ws hrvested t the stge in whih β-ryptoxnthin umultion stgntes on the tree. Another reent study on Aoshim stsum mndrin fruit lso reported tht the β-ryptoxnthin ontent in the pulp ws mintined during storge t 8 C for months (Hmski nd Ymg, 7). In onlusion, the results of the present study hve shown tht, in the juie ss of stsum mndrin fruit, the β-ryptoxnthin ontent does not derese under posthrvest onditions etween nd C, irrespetive of the mturtion stge. The present results suggest tht µg g - 3 d d 3d 6d 9d Dys fter hrvest Fig.. Chnges in β-ryptoxnthin ontent efore nd fter storge t,, nd C in the juie ss of Aoshim hrvested in erly Deemer. Different letters indite signifint differenes mong the storge tempertures t P <. y Turkey s HSD test. Content is expressed on fresh-weight sis, μg g. The vlues re the men ± SE of three replites. µg g (A) d d 3d Dys fter hrvest 6 8 (B) d d 3d Dys fter hrvest Fig.. Chnges in β-ryptoxnthin ontent efore nd fter storge t,,, nd C in the juie ss of Nihinn No. hrvested in erly Otoer (A) nd lte Otoer (B). Different letters indite signifint differenes mong the storge tempertures t P <. y Tukey s HSD test. Content is expressed on fresh-weight sis, μg g. The vlues re the men ± SE of three replites. µg g - 3 d 8 Fig. 6. Chnges in β-ryptoxnthin ontent efore nd fter storge t, 8, nd C for 8 dys in the juie ss of Aoshim hrvested in erly Deemer. Different letters indite signifint differenes mong the smples efore nd fter storge t different tempertures t P <. y Turkey s HSD test. Content is expressed on fresh-weight sis, μg g. The vlues re the men ± SE of three replites.

8 8 H. Mtsumoto, Y. Adhi, Y. Ikom nd M. Kto the β-ryptoxnthin ontent grdully inreses fter hrvest t 8 C nd ove, nd tht the inrese rte is mturtion stge-dependent. The rte of inrese in the β-ryptoxnthin ontent will e more rpid in fruit hrvested when rotenoid umultion is in progress on the tree. Literture Cited Alquézr, B., M. J. Rodrigo nd L. Zrís. 8. Crotenoid iosynthesis nd their regultion in itrus fruit. Tree nd Forestry Siene nd Biotehnology : 3 3. Brry, G. H. nd A. A. Vn Wky. 6. Low-temperture old shok my indue rind olour development of Nules Clementine mndrin (Citrus retiult Blno) fruit. Posthrvest Biol. Teh. : Crmon, L., L. Zrís nd M. J. Rodrigo.. Stimultion of olortion nd rotenoid iosynthesis during posthrvest storge of Nvelin ornge fruit t C. Posthrvest Biol. Tehnol. 7: 8 7. Chudhry, P. R., G. K. Jyprksh, R. Port nd B. S. Ptil.. Degreening nd posthrvest storge influenes Str Ruy grpefruit (Citrus Prdisi Mf.) iotive ompounds. Food Chem. 3: Chudhry, P. R., G. K. Jyprksh, R. Port nd B. S. Ptil.. Low temperture onditioning redues hilling injury while mintining qulity nd ertin iotive ompounds of Str Ruy grpefruit. Food Chem. 3: 3 9. Chudhry, P. R., X. Yu, G. K. Jyprksh nd B. S. Ptil. 7. Influene of storge temperture nd low-temperture onditioning on the levels of helth-promoting ompounds in Rio Red grpefruit. Food Si. Nutr. : 3. Grierson, W. 6. Mturity nd grde stndrds. p In: W. F. Wrdowski, W. M. Miller, D. J. Hll nd W. Grierson (eds.). Fresh Citrus Fruit, nd edition, Florid Siene Soure: Longot Key, FL. Grierson, E. nd W. M. Miller. 6. Storge of itrus fruits. p In: W. F. Wrdowski, W. M. Miller, D. J. Hll nd W. Grierson (eds.). Fresh Citrus Fruit, nd edition, Florid Siene Soure: Longot Key, FL. Gross, J Pigments in Fruits. Ademi Press: London, UK. Hsegw, Y. nd Y. I The effets of storge temperture on the qulity of itrus fruit. I. Color hnge of the itrus peel during storge. Bull. Fruit Tree Res. Stn. B : 9 8 (In Jpnese with English strt). Hsegw, Y. nd Y. I. 98. The effets of storge temperture on the qulity of itrus fruit. II. The effets of the high temperture of pretretment. Bull. Fruit Tree Res. Stn. B : 3 6 (In Jpnese with English strt). Hmski, S. nd I. Ymg. 7. Effet of tehnique for improving qulity nd posthrvest tretment on β- ryptoxnthin ontent in Stsum mndrin fruit. Hort. Res. (Jpn) 6 (Suppl. ): 336 (In Jpnese). Hodgson, R. W Hortiulturl vrieties of itrus.: The mndrins: Stsum mndrin. p.. In: W. Reuther, H. J. Weer nd L. D. Bthelor (eds.). The Citrus industry. Vol.. University of Cliforni Agriulturl Siene, Oklnd, CA. Ikom, Y., H. Mtsumoto nd M. Kto. 6. Diversity in the rotenoid profiles nd the expression of genes relted to rotenoid umultion mong itrus genotypes. Breed. Si. : Ikom, Y., M. Yno, K. Ogw, T. Yoshiok, Z. C. Xu, S. Hisd, M. Omur nd T. Moriguhi Isoltion nd evlution of RNA from polyshride-rih tissues in fruit for qulity y DNA lirry onstrution nd RT-PCR. J. Jpn. So. Hort. Si. 6: Iwms, M Knkitsu no Hynsyu (In Jpnese). 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Development of funtionl griulturl produts nd use of new helth lim system in Jpn. Trends in Food Si. Tehnol. 69: Mtsumoto, H., Y. Ikom, M. Kto, N. Nkjim nd Y. Hsegw. 9. Effet of posthrvest temperture nd ethylene on rotenoid umultion in the flvedo nd juie ss of Stsum mndrin (Citrus unshiu Mr.) fruit. J. Agri. Food Chem. 7: Oenlnd, D., S. Collin, B. Mkey, J. Sievert nd M. L. Arpi.. Storge temperture nd time influenes sensory qulity of mndrins y ltering solule solids, idity nd rom voltile omposition. Posthrvest Biol. Tehnol. 9: Olson, J. A Provitmin-A funtion of rotenoids: The onversion of β-rotene into vitmin-a. J. Nutr. 9: 8. Perkins-Vezie, P. nd J. K. Collins. 6. Crotenoid hnges of intt wtermelons fter storge. J. Agri. Food Chem. : Rodrigo, M. J. nd L. Zrís. 7. Effet of posthrvest ethylene tretment on rotenoid umultion nd the expression of rotenoid iosyntheti flvedo of ornge (Citrus sinensis L. Osek) fruit. Posthrvest Biol. Tehnol. 3:. Tietel, Z., E. Lewinsohn, E. Fllik nd R. Port.. 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