Co-ordination of early and late ripening events in apples is regulated through differential sensitivities to ethylene

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1 Journl of Experimentl Botny, Vol. 6, No. 9, pp , 29 doi:.93/jx/erp22 Advne Aess pulition 8 My, 29 This pper is ville online free of ll ess hrges (see for further detils) RESEARCH PAPER Co-ordintion of erly nd lte ripening events in pples is regulted through differentil sensitivities to ethylene Json W. Johnston, Kulrjthven Gunseeln, Pul Pidkl, Mindy Wng nd Roert J. Shffer* The New Zelnd Institute For Plnt & Food Reserh Limited, Privte Bg 9269, Auklnd, New Zelnd Reeived 8 Jnury 29; Revised 22 Mrh 29; Aepted 24 Mrh 29 Astrt In this study, it is shown tht nti-sense suppression of Mlus domesti -AMINO-CYCLOPROPANE-CARBOXYLASE OXIDASE (MdACO) resulted in fruit with n ethylene prodution suffiiently low to e le to ssess ripening in the sene of ethylene. Exposure of these fruit to different onentrtions of exogenous ethylene showed tht flesh softening, voltile iosynthesis, nd strh degrdtion, hd differing ethylene sensitivity nd dependeny. Erly ripening events suh s the onversion of strh to sugrs showed low dependeny for ethylene, ut high sensitivity to low onentrtions of ethylene (. ml l 2 ). By ontrst, lter ripening events suh s flesh softening nd ester voltile prodution showed high dependeny for ethylene ut were less sensitive to low onentrtions (needing. ml l 2 for response). A sustined exposure to ethylene ws required to mintin ripening, inditing tht the role of ethylene my go eyond tht of ripening initition. These results suggest oneptul model for the ontrol of individul ripening hrters in pple, sed on oth ethylene dependeny nd sensitivity. Key words: ACC oxidse (MdACO), pple, ethylene, fruit ripening, Mlus. Introdution The importne of ethylene for fruit ripening hs mde the iosyntheti pthwy for this hormone n ttrtive trget for studies iming to mnipulte nd understnd the ontrol of fruit ripening. Among the first trnsgeni fruit ever produed trgeted ethylene synthesis in tomto. These inluded the reduing levels of the enzymes ACC SYNTHASE (ACS) (Oeller et l., 99) nd ACC OXIDASE (ACO) (Piton et l., 993), nd over-expressing ACC DEAMINASE to remove ACC from the ripening fruit (Klee et l., 99).In other fruit speies, reserhers hve trgeted ACO in melon (Ayu et l., 996), nd ACO (Ross et l., 992) in the pple ultivrs, Greensleeves nd Royl Gl (Dndekr et l., 24; Shffer et l., 27). These studies demonstrted reduing ethylene in the limteri fruit redues fruit ripening, n effet reversed y dding exogenous ethylene. Mesuring the sensitivity of tissue to hormone gives insight into how the hormone ts. Due to the miguous nture of the word sensitivity, Firn (986) proposed five mesures of sensitivity. In this study, the term sensitivity is used for tegory III sensitivity, nmely the minimum onentrtion needed to hieve signifint response, dependeny is used for the tegory II sensitivity, nmely the rtio of the mximum response reltive to the response to in the sene of the hormone, nd responsivity is used to desrie tegory I sensitivity the mgnitude of mximum response whih n e evoked. The signifine of different sensitivities to the ripening hormone ethylene ws not ppreited in erly studies, s dependeny ws not onsidered longside sensitivity. ACS tomtoes, demonstrting less thn.5% of pek ethylene prodution, showed dely in mny ripening ttriutes, inluding hlorophyll loss nd reddening (Oeller et l., 99). It ws lso found tht levels of the ell wll gene POLYGALACTURONASE (PG) were of similr level to ontrol fruit suggesting tht this ws regulted independently to ethylene (Oeller et l., 99). This expression result ws lter found to e used y the sensitivity of PG to ethylene nd it hs now een estlished tht this PG is tully strongly regulted y ethylene in tomtoes, s suppression of ethylene prodution to.5% of the ontrols * To whom orrespondene should e ddressed. E-mil: rshffer@hortreserh.o.nz ª 29 The Author(s). This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Non-Commeril Liense ( whih permits unrestrited non-ommeril use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

2 269 Johnston et l. ws still suffiient to indue % expression of this gene (Sitrit nd Bennett, 998). While the PG is oviously highly sensitive to low onentrtions of ethylene, the ACS mutnt tomtoes demonstrted slower loss of hlorophyll ( 2 d) thn the ontrols nd, finlly, they never turned right red, suggesting tht the olour response is less sensitive to ethylene. Work on the trnsgeni melon hs demonstrted tht mny ripening ttriutes pper to e independent of ethylene (Ayu et l., 996; Guis et l., 997; Peh et l., 28). In melon, it ws found tht the prodution of rom voltiles nd rind yellowing re onsidered to e ethylenedependent ripening events (Guis et l., 997), while strh rekdown, sugr umultion, loss of idity, nd flesh olourtion re onsidered ethylene-independent events. Fruit softening showed oth dependent nd independent effets with the knokout ACO fruit showing signifint softening leit t redued rte. This ethylene-independent softening hs een explined y the ft tht some ell wllmodifying enzymes re strongly regulted y ethylene while others ppered to hve n ethylene-independent expression (Nishiym et l., 27). Currently, there is little known out the ripening sensitivity of pples to different ethylene onentrtions. There is some evidene tht the ripening sensitivity to ethylene inreses during on-tree mturtion, ut these studies were limited to mesurements of endogenous ethylene prodution nd respirtory tivity (Hrkett et l., 97; Knee et l., 987). Ethylene onentrtions inside the ore vity of pples inrese from <. ll l to >5 ll l during ripening (Johnston et l., 2), ut it is not known if this entire rnge of onentrtions is physiologilly tive or if prodution is in exess of tht required during the lte stges of ripening. Apple ripening onsists of the onversion of strh to sugrs, redued idity, yellowing of the skin, redued flesh firmness, nd n inrese in flvour voltiles. Inhiitors of ethylene tion suh s - MCP hve een shown to redue softening nd voltile prodution, ut hve inonsistent or no effet on the onversion of strh to sugrs (Fn et l., 999). If similr proesses were ourring in pple s in melon, this vrition in effiy ould e explined y eh ripening omponent hving differing dependenies or sensitivities to ethylene. The ontrolled pplition of different onentrtions of ethylene to determine ripening thresholds is not possile using stndrd pple ultivrs, s most ultivrs produe too muh endogenous ethylene. This mkes it diffiult to determine if ripening responses re due to the dded ethylene, or to hnges in endogenous ethylene. The pplition of inhiitors of ethylene iosynthesis nd tion lso do not suffiiently suppress endogenous prodution in pple to llow suh studies. The present study irumvents this prolem y using reently developed Royl Gl ACO ntisense line tht only produes kground levels of ethylene (Shffer et l., 27). Using this system, the present study determines the sensitivity, responsivity, nd dependeny of erly nd lte ripening events in pple. New knowledge is presented on the ethylene ripening thresholds for individul ripening hrters of pples, nd new oneptul model is presented for the ethylene-medited oordintion of erly nd lte ripening events in pples. Mterils nd methods Apple growth onditions A single trnsgeni Mlus3domesti Royl Gl tree, ontining the ACC OXIDASE gene (Ross et l., 992) in n ntisense orienttion (ACOs), ws seleted s it produed only.3 ng l levels of ethylene in its fruit t full limteri (Shffer et l., 27). Seven sions were grfted onto M9 rootstoks nd grown next to three Royl Gl untrnsformed lines tht t s ontrol in greenhouse. At full loom, flowers were pollinted y hnd with M.3domesti Grnny Smith pollen. Three hundred nd forty three MdACOs pples nd 56 ontrol pples were hrvested t the sme time sed on the kground olour of the ontrol pples. The MdACOs pples were rndomly lloted into 4 groups of 4 pples, with 3 of these groups lloted to the ripening tretments desried in Tle, nd one group ssessed for mturity hrteristis t hrvest. The ontrol pples were rndomly lloted into four groups of 4 pples, with three of these groups used for ripening tretments (Tle ), nd one group ssessed for mturity t hrvest. Fruit ripening ssessments were performed 4 d fter hrvest for ll ripening tretments. Post-hrvest tretments Eh group of pples ws sujeted to different tretment (Tle ) eh in seprte single-lyered try. Ethylene tretments were onduted t 2 o C in 34 l ripening ins with ontinuous ir movement nd lime to sor CO 2. Nil ethylene tretments were lso rried out in seled ins with lime nd Purfilä (Multimix MM-, Cirul-Aire, Montrel, Cnd) to remove ny residul tmospheri ethylene. Ethylene ws injeted into the injetion ports nd ssessed for orret onentrtion h fter injetion. -Methylylopropne (-MCP) (SmrtFreshä, AgroFresh Ltd) tretments t onentrtion of 6 nl l were lso performed for 24 h in 34 l seled ins ontining lime. In ll tretments, the 34 l in ontined mximum of 4 pples ( omined mss of 2 g). Assessment of fruit mturity nd ripening For eh fruit, flesh firmness ws evluted using Texture Anlyser TAXT plus (Stle Mirosystems, United Kingdom) fitted with 7.9 mm Effegi penetrometer proe. The proe ws driven into the flesh t 4 mm s to depth of 9 mm, nd the mximum fore reorded s flesh firmness. Two redings were mde on opposite sides of eh fruit with the skin removed. Solule solids onentrtion ws used to estimte the sugr ontent, nd ws determined y pling n liquot of the juie relesed during firmness mesurement onto digitl refrtometer (Atgo, model PAL-, Jpn). Strh pttern index ws determined y

3 Tle. Summry of ripening tretments for MdACOs nd ontrol Royl Gl pples Ethylene sensitivity nd pple ripening 269 Ripening tretments inluded ethylene dosge, durtion of ethylene tretment, -methylylopropene (MCP) tretment, nd ripening temperture. Assessments for eh tretment were performed 4 d fter hrvest. Tretment no. Genotype Ethylene dosge (ll l ) Ethylene tretment durtion Ripening temperture ( C) 6 nl l MCP tretment Control 2 No 2 2 Yes 3 4 No 4 ACOs 2 No 5 4 No 6 2 Yes 7. 4 d 2 No 8. 4 d 2 No 9 4 d 2 No 4 d 2 No 4 d 2 No 2 4 d 2 No 3 3 min 2 Yes 4 4 h 2 Yes 5 d 2 Yes 6 7 d 2 Yes pling the ut surfe of n pple into iodine solution (2.5 g l iodine nd g l potssium iodide in distilled wter) for 6 s nd then rting the stining pttern ording to the ENZAFRUIT New Zelnd Interntionl sle from (ut surfe ompletely stined, high strh) to 6 (no surfe stining, low strh). Longitudinl qurter setions of fruit were held t 2 o C for the determintion of titrtle idity. Titrtions were performed ording to Hrker et l. (22). Bkground skin olour ws mesured using hrommeter (Minolt, model CR-3, Jpn), with two redings mde per fruit on lush-free res of skin. Internl ethylene onentrtion ws determined y extrting ml ore vity gs smple nd injeting it into gs hromtogrph (Hewlett Pkrd, 589 series II) equipped with n injetor t 6 o C, n tivted lumin F olumn (Allteh, glss.5 m36 mm32 mm, mesh 8/) set isothermlly t 3 o C, N 2 s the rrier gs (2 ml min ), flme ioniztion detetor set t 2 o C(H 2 t 2 ml min, ir t 2 ml min ), nd n integrtor (Hewlett Pkrd, model 3395) lirted with ertified gs stndrds. Voltiles were mesured s desried in Shffer et l. (27). Dt nlysis The signifine of tretment effets were determined using nlysis of vrine, nd men seprtion using proteted lest signifint differene (5%). Ethylene response dt were nlysed y non-liner regression nd logisti funtion: Ripening response ¼ þ A B ½ethyleneŠ D C þb where A is the lower symptote, B is the upper symptote, C is the [ethylene] for 5% response, nd D is the rte of hnge. Prmeter estimtes nd stndrd errors for C were interpreted s sensitivity, nd prmeter D s responsivity. All sttistil proedures were performed using Genstt, 9th edition. Ethylene dependeny ws lulted s: Dependeny ð%þ¼ ll l tretment ll l tretment ll l 3 tretment Results Mturity hrteristis t hrvest At hrvest, the ontrol fruit hd internl ethylene onentrtions rnging from.5 to 528 lll, while the MdACOs fruit hd levels rnging from undetetle to.8 ll l (Fig. A). Both the ontrol fruit nd MdACOs fruit on exposed prts of the trees developed red lush t hrvest (Fig. 2A, B), lthough the intensity nd skin overge of reddening ws more pronouned on ontrol fruit. Both fruit types hd similr flesh firmness, ut the MdACOs fruit hd lower onentrtions of solule solids nd titrtle idity, less strh lerne, nd greener kground skin olour (Fig. B E). The strh pttern index for ontrol fruit rnged from to 6 nd to 3 in MdACOs fruit. MdACOs fruit hve improved wter retention nd redued inidene of ripening-relted rots Fourteen MdACOs-suppressed pples nd 4 Royl Gl untrnsformed fruit (ontrol fruit) were kept t 2 C for 3 weeks. These were weighed nd visully inspeted every 2 weeks for shrivel nd ripening-relted rots. After 6 8 weeks the ontrol fruit egn to show signs of shrivel muh erlier thn the MdACOs fruit tht egn to shrivel 2 26 weeks during this tretment (Fig. 2C, D). By 2 weeks five of the ontrol fruit hd ompletely rotted while there were no visul

4 2692 Johnston et l. Internl ethylene onentrtion (µl.l - )... 6 A C green Bkground olour (Hue o ) yellow 5 B D 4 Strh pttern index 4 2 Solule solids (%) E.45 F Firmness (N) Titrtle idity (%) RG ACOs RG ACOs Fig.. Untrnsformed Royl Gl (RG, wild type) nd MdACC oxidse (ACOs) fruit t hrvest. Conentrtion of internl ethylene in the ore vity (A), kground skin olour (B), strh pttern index (C), solule solids onentrtion (D), flesh firmness (E), nd titrtle idity (F). rots on ny of the MdACOs fruit for the whole of this period. In oth the MdACOs lines nd ontrol lines the wter loss from the fruit ws liner (Fig. 2E), with slower rte of wter loss from the MdACOs lines (verge.6 mg g FW d ) ompred to the ontrols (2.2 mg g FW d ). Comprison of ripening hrters etween MdACOs nd untrnsformed fruit Three groups of 4 ontrol fruit nd three groups of 4 MdACOs fruit were ssessed following 4-d period of ripening. The first group of ontrol nd MdACOs pples were stored with no tretment t 2 C. After this time the Royl Gl ontrol fruit hd higher internl ethylene onentrtion, hd softened y pproximtely 2 N, hd ner-omplete strh lerne, more yellow kground olour, nd deresed titrtle idity (Fig. 3). The MdACOs fruit hd no detetle inrese in internl ethylene onentrtion fter 4 d t 2 C, with ethylene levels rnging from undetetle to.94 ll l, nd only hnged slightly in skin kground olour nd firmness. Both the untrnsformed fruit nd the MdACOs fruit hd lrge degree of strh lerne, n inrese in solule

5 Ethylene sensitivity nd pple ripening 2693 Fig. 2. Apples with MdACC oxidse (MdACOs) knoked out nd untrnsformed Royl Gl. Royl Gl pples on tree t hrvest (A). MdACOs fruit t hrvest (B). MdACOs pple fter storge t room temperture for 6 months (C). Royl Gl pples fter storge t room temperture for 6 months (D). Weight loss from Royl Gl (filled oxes) nd MdACOs pples (open oxes) over 4 week period (E). solids onentrtion, nd loss of titrtle idity during the 4 d ripening period. To ssess whether there were ny ripening effets used y the very low ethylene onentrtions deteted in some of the MdACOs fruit, the seond group of MdACOs fruit were treted with the inhiitor of ethylene tion, -methylylopropene (-MCP) efore eing left to ripen for 4 d t 2 C. A group of ontrol fruit were lso treted. Tretment of the ontrol fruit with -MCP redued the internl ethylene onentrtion to etween.23 nd 4.2 ll l. There ws lso redued loss of firmness ompred with the untreted fruit ut -MCP hd little effet on the lerne of strh nd the ssoited inrese in solule solids onentrtion. MdACOs fruit treted with -MCP showed no signifint differenes from the untreted fruit suggesting tht the MdACOs lines hve omplete ethylene iosynthesis knokout with respet to ripening (Fig. 3). The third group of MdACOs nd ontrol pples were old-stored t 4 C for 4 d to ssess the effet of old storge on ripening hrters. MdACOs fruit showed no signifint differenes in ripening ompred with the fruit stored t 2 C for internl ethylene onentrtion, firmness, nd skin olour. Cold-stored MdACOs fruit hd slightly less strh lerne nd n inrese in solule solids onentrtion (Fig. 3). Cold storge of the ontrol fruit suppressed internl ethylene onentrtions to ll l, nd redued softening, skin yellowing, nd strh lerne ompred with fruit stored t 2 C. Ethylene sensitivity for individul ripening hrteristis To estlish the level of ethylene needed to indue ripening response, MdACOs lines were exposed ontinuously to rnge of ethylene onentrtions from. to ll l for 4 d t 2 C. Ethylene response urves were then produed for the six min ripening trits nd for the six dominnt voltiles (Fig. 4). While there ws no ler response to ethylene for titrtle idity nd solule solids onentrtion, distint response urves were estlished for voltile prodution, firmness, skin kground olour, nd strh lerne. The generl pttern for these urves ws sigmoidl, with little or no hnge in ripening t low onentrtions, followed y rpid inrese in response from. to ll l, nd little or no hnge therefter t whih point the proess ws sturted. However, there were

6 2694 Johnston et l. Internl ethylene onentrtion (µl.l - ).. Firmness (N) 'Royl Gl' 'ACOs' 6 6,d d Strh pttern index 4 2 Solule solids (%) 4 2 Titrtle idity (%) Bkground olour (hue ) At hrvest 4 d t 2 oc -MCP + 4 d t 2 oc 4 d t 4 oc At hrvest 4 d t 2 oc -MCP + 4 d t 2 oc 4 d t 4 oc Fig. 3. Untrnsformed Royl Gl (wild type, unfilled rs) nd MdACOs fruit (filled rs) t hrvest nd fter ripening following three different storge tretments. Anlysis of vrine P vlues for ll ripening hrters were <.. Tretments lelled with the sme letter re similr ording to 5% lest signifint differene. sutle differenes in the responses of individul ripening trits, where the mjority of the response ourred etween. nd ll l for softening, kground olour nd totl voltiles, while in ontrst lower rnge of. to ll l ws required for strh lerne. Results from the. nd. ll l ethylene onentrtions were indistinguishle from the no-ethylene tretment ontrols for voltiles, firmness, nd kground olour suggesting tht these onentrtions re elow the response threshold for these proesses. A smll inrese in strh degrdtion ws oserved for the. ll l ethylene-treted fruit, suggesting lower response threshold for this trit. The differentil sensitivity of the different ripening trits ws further quntified using non-liner regression to estimte the ethylene onentrtion required to indue hlf-mximum response. This pproh showed tht the hlf-mximl response for strh lerne ourred t low ethylene

7 Ethylene sensitivity nd pple ripening 2695 Firmness (N) A Strh pttern index B Skin hue C 2.5 D. E 2 F Ethyl ette (ng.g - FW) Solule solids G Propyl ette (ng.g - FW) Titrtle idity (%) H Totl voltile prodution (ng.g - FW) methylutyl ette (ng.g - FW) I Butyl ette (ng.g - FW) J... -Frnesene (ng.g - FW) K... Ethylene dosge (µl.l - ) -Hexnol (ng.g - FW) 3 2 L... Fig. 4. Response urves of different ripening hrters for MdACOs fruit treted with different onentrtions of ethylene. Flesh firmness (A) strh pttern index (B), skin kground olour (C), solule solids onentrtion (D), titrtle idity (E), totl voltiles (F), nd individul voltiles (G L). Eh point depits the men of 4 fruit. onentrtions, with skin yellowing, softening, nd voltile prodution requiring progressively higher ethylene onentrtions to indue similr 5% response (Fig. 5A). The rte of hnge prmeter estimted from this nlysis ws similr for ll ripening hrters (Fig. 5B), suggesting the responsivity to ethylene ws similr despite differenes in sensitivity. The ethylene response urves lso differed etween voltiles. The prodution of -frnesene, utyl ette, nd 2-methylutyl ette ourred one the ethylene onentrtion exeeded. ll l, while the prodution of ethyl ette nd propyl ette only ourred one the ethylene onentrtion exeeded higher onentrtion of ll l (Fig. 4). The esters tended to hve n rupt inrese in prodution in response to inresed ethylene dosge, wheres voltiles suh s -frnesene nd hexnol hd more grdul inrese nd seemed to respond to wider rnge of ethylene onentrtions. Alph-frnesene nd - hexnol lso showed higher sturtion threshold onentrtion of > ll l, wheres most other voltiles were sturted t onentrtions > ll l.

8 2696 Johnston et l. A B C Tot. voltile prod. Softening Skin yellowing Strh degrdtion Tot. voltile prod. Softening Skin yellowing Strh degrdtion Tot. voltile prod. Softening Skin yellowing Inresed sol. solids Strh degrdtion Loss of idity Ethylene onentrtion (µl.l - ) Responsivity (ethylene onentrtion - ) Ethylene dependeny (%) Fig. 5. Sensitivity (A), responsivity (B), nd dependeny (C) of the MdACOs pples to exogenous ethylene. Sensitivity nd responsivity ws determined s the ethylene onentrtion required for 5% response nd the rte of hnge, respetively, using nonliner regression nd logisti funtion. Ethylene dependeny (%) ws lulted s the differene etween the ll l nd ll l ethylene tretments divided y the ll l ethylene tretment, nd multiplying y to give perentge: %¼ethylene independent, %¼ethylene dependent. Ethylene dependeny for individul ripening hrteristis To estlish the degree of ethylene dependene for eh ripening trit, MdACOs fruit treted with ll l ethylene, nd untreted fruit stored t 2 C, were ompred with the hrvest smples. Tking the vlue for eh trit exposed to ll l s % the perentge ripening for eh trit in the sene of ethylene (ethyleneindependent trits) ould e determined (Fig. 5C). Two groups of ripening trits emerged, those tht were strongly ethylene-dependent (inresed totl voltiles, loss of firmness nd skin yellowing), nd those tht were only wekly ethylene-dependent (strh lerne, loss of titrtle idity, nd inresed solule solids onentrtion). A ontinuous exposure to ethylene is required for ripening To ssess if ethylene ts s trigger for ripening or whether ontinuous exposure to ethylene is required, MdACOs fruit were exposed to ll l ethylene for different periods of time followed y tretment with 6 nl l -MCP to stop further ethylene tion. Fruit were exposed to ethylene for 3 min, 4 h, d, nd 7 d, nd the sme ripening hrters s ssessed ove were mesured fter 4 d t 2 o C. The results from these fruit were lso ompred with those from fruit tht hd een treted with ll l for 4 d in the previous experiment. For the ripening trits showing moderte-to-strong dependeny for ethylene (kground skin olour, firmness, nd voltiles), the responses of fruit reeiving short period of ethylene (3 min, 4 h, nd d) exposure followed y tretment of -MCP were indistinguishle from the responses of no-ethylene tretment MdACOs fruit. However, for strh lerne ( ripening proesses with wek dependene for ethylene), one dy ws suffiient to signifintly elerte strh loss. Fruit treted with ethylene for 7 d hd n intermedite level of ripening hrters ompred with fruit reeiving the 4 d tretment (Fig. 6), suggesting ontinued exposure of ethylene is needed for ripening to our. Disussion Ripening in pples is omplex progression of developmentl steps. These steps re not ompletely ontrolled y ethylene, s there is lwys degree of ethylene-independent progression. This suggests model of ethylene tion where ethylene is ting s modultor of ripening rther thn ripening trigger. While the morphologil hnges tht our in pples during ripening re well doumented, it is shown tht these trits seem to follow n overlpping sequene of events, strting with the onversion of strh to sugrs nd loss of idity, yellowing of the skin, softening of the flesh, nd finlly n inrese in rom voltiles. As this sequene progresses, the dependeny for ethylene eomes stronger nd the sensitivity to ethylene dereses (Fig. 7). For most pple ultivrs, rther thn hving onstnt level, there is n inrese in ethylene during the ripening proess (Johnston et l., 2). This inrese in ethylene onentrtion my llow the different ripening hrters to e o-ordinted so tht strh onversion lwys ours efore exessive fruit softening or the prodution of ttrtnt voltiles. The low ethylene dependeny for strh lerne suggests tht this proess is minly driven y other developmentl ftors, ut the high sensitivity of this proess to low ethylene onentrtions my provide kup stimulus should the non-ethylene developmentl ues fil to develop. When the MdACOs pples re left for onsiderle period of time, the gretest hnge tht ours is weight loss, leit t lower rte thn the ontrol pples. This

9 Ethylene sensitivity nd pple ripening P <. 6. P <. Firmness (N) 4 Strh pttern index ,, Skin Hue P <. Titrtle idity nd solule solids (%) Solule solids, P = n.s. Titrtle idity, P = n.s. 96 +MCP 3 min 4 h dy 7 dys 4 dys. +MCP Durtion of ethylene tretment 3 min 4 h dy 7 dys 4 dys Fig. 6. Fruit ripening in MdACOs fruit treted with ll l ethylene for different periods of time followed y tretment with -MCP. Fruit ws mesured fter 4 d. Anlysis of vrine P vlues re displyed, tretments lelled with the sme letter re similr ording to 5% lest signifint differene. suggests tht only ertin progression of ripening n e hieved in the sene of ethylene. The ethylene dependeny for strh lerne ppers less, s it would e likely tht there would e omplete lering of strh if the 2-week ripening period were extended. Beuse of pple numer onstrints, limittion to this study ws tht ripening ws mesured t single time point in pple fruit development. It hs een well doumented tht, s pples mture, they eome more sensitive to ethylene (Hrkett et l., 97). This mens it is possile tht the ethylene response urves my shift to the left or the right for fruit hrvested t different mturities. It is lso likely tht these response urves my shift for different ultivrs nd for fruit exposed to different onditions during growth; ftors whih wrrnt further investigtion. Nevertheless, despite these potentil shifts in the response urve, it is likely tht the reltive sensitivities nd dependenies of the different ripening hrters for ethylene would remin in the sme order, s most pple ultivrs tend to follow the sme sequene of ripening events (e.g. strh lerne is lwys n erly ripening event). The present reserh lso demonstrtes tht ethylene ws most physiologilly tive for ripening etween. nd ll l, whih is similr to those onentrtions mesured in most pple ultivrs during the hrvest period. These onentrtions lso fit with storge reommendtions for ethylene onentrtions to e less thn. ll l to redue ripening (Stow et l., 2). While reserh hs een initited to unrvel the ethylene dependeny of ripening pthwys (Peh et l., 28), there is lk of reserh on the moleulr sis for ethylene

10 2698 Johnston et l. et l., 99) tht ripening ould e stopped if ethylene fruit were exposed to only short period of ethylene. As ripening ours through enzymi tion, it is surprising tht residul ripening does not our even fter short period of ethylene exposure. This suggests tht enzyme turnover is high during ripening or tht there re dditionl lyers of regultion for enzyme tion to progress in the sene of ethylene. Fig. 7. Coneptul model for ontrol of individul ripening hrters in pples. Ethylene sensitivity lulted from the onentrtion of ethylene required for 5% ripening response. Ethylene dependeny mesured y the mount of ripening mesured in the sene of ethylene. sensitivity. The differing ethylene sensitivities of individul ripening hrters rises questions s to whether this is medited through different signlling systems for eh ripening pthwy, or through differenes in the numers of ethylene-inding domins for key genes ssoited with eh ripening proess. There is onsiderle vrition in the ripening proess in fleshy fruits, ut when the individul trits re exmined seprtely, there re remrkle onsistenies. The strh to sugr onversion is ommon to mny fruit. In some ultivrs of pple it hs een reported s ethylene independent, while in others it is more ethylene dependent (Thmmwong nd Arkw, 27). In melons, strh hs lso een suggested to e independent to ethylene (Ayu et l., 996), it would e interesting if this strh lerne ould e elerted in this fruit with exogenous ethylene. The dependeny on ethylene for hnges in skin olourtion hs een reorded in melon (Ayu et l., 996) nd tomto (Oeller et l., 99). The ethylene dependeny of fruit softening nd voltile prodution hs een extensively studied, with responses to ethylene y mny ndidte ell wll genes in pples responding to ethylene (Atkinson et l., 998; Wks et l., 23; Goulo et l., 28) nd in other fruit, (Brummell, 26), nd y rom iosynthesis genes (Defilippi et l., 25; Shffer et l., 27). Interestingly, work with melon hs shown tht mny of the ell-wll relted genes re regulted independently of ethylene, in ddition to those tht re regulted y ethylene (Nishiym et l., 27). While muh progress hs een mde there is still muh to lern out the moleulr ontrol of these genes. Finlly, it ppers from this nd previous work tht the ethylene signl is onstntly ontrolling the ripening proess. As well s this study, it ws shown in tomto (Oeller Conlusion This study hs hrterized for the first time the ethylene response urves for individul ripening hrters in pple. It hs reveled tht ethylene-medited ripening is ontrolled t two levels, firstly, through differing dependenies for ethylene, nd, seondly, through differing sensitivities to ethylene. This dul ontrol mehnism mens tht the sequene of individul ripening events is tightly oordinted so tht erly ripening events re less dependent on ethylene ut re lso highly sensitive to ethylene should the ethylene-independent ftors fil to develop. This model wrrnts further investigtion in reltion to ppliility to other fruits, prtiulrly in reltion to the ripening physiology of limteri nd non-limteri fruit. Aknowledgements The uthors would like to knowledge Nihl De Silv nd Mrk Wohlers for sttistil dvie, Andrew Alln nd In Ferguson for ritilly reding the mnusript, nd the New Zelnd Foundtion for Reserh, Siene nd Tehnology (C6X75; Pipfruit juiy future) for funding this reserh. Referenes Atkinson RG, Bolitho KM, Wright MA, Iturriggoiti-Bueno T, Reid SJ, Ross GS Apple ACC-oxidse nd polyglturonse: ripening-speifi gene expression nd promoter nlysis in trnsgeni tomto. Plnt Moleulr Biology 38, Ayu R, Guis M, BenAmor M, Gillot L, Roustn JP, Lthé A, Bouzyen M, Peh JC Expression of ACC oxidse ntisense gene inhiits ripening of ntloupe melon fruits. Nture Biotehnology 4, Brummell DA. 26. Cell wll disssemly in ripening fruit. Funtionl Plnt Biology 33, 3 9. Dndekr AM, Teo G, Defilippi BG, Urtsu SL, Pssey AJ, Kder AA, Stow JR, Colgn RJ, Jmes DJ. 24. Effet of downregultion of ethylene iosynthesis on fruit flvor omplex in pple fruit. Trnsgeni Reserh 3, Defilippi BG, Dndekr AM, Kder AA. 25. Reltionship of ethylene iosynthesis to voltile prodution, relted enzymes, nd preursor vilility in pple peel nd flesh tissues. Journl of Agriulturl nd Food Chemistry 53,

11 Ethylene sensitivity nd pple ripening 2699 Fn XT, Blnkenship SM, Mttheis JP Methylylopropene inhiits pple ripening. Journl of the Amerin Soiety for Hortiulturl Siene 24, Firn RD Growth sustne sensitivity: the need for lerer ides, preise terms nd purposeful experiments. Physiologi Plntrum 67, Goulo LF, Cosgrove DJ, Oliveir CM. 28. Cloning, hrteristion nd expression nlyses of DNA lones enoding ell wllmodifying enzymes isolted from ripe pples. Posthrvest Biology nd Tehnology 48, Guis M, Botondi R, BenAmor M, Ayu R, Bouzyen M, Peh JC, Lthé A Ripening-ssoited iohemil trits of Cntloupe Chrentis melons expressing n ntisense ACC oxidse trnsgene. Journl of the Amerin Soiety for Hortiulturl Siene 22, Hrker FR, Mrsh KB, Young H, Murry SH, Gunson FA, Wlker SB. 22. Sensory interprettion of instrumentl mesurements. 2. Sweet nd id tste of pple fruit. Posthrvest Biology nd Tehnology 24, Hrkett PJ, Hulme AC, Rhodes MJC, Wooltorton LSC. 97. The threshold vlue for physiologil tion of ethylene on pple fruits. Journl of Food Tehnology 6, Johnston JW, Hewett EW, Hertog MLATM, Hrker FR. 2. Temperture indues differentil softening responses in pple ultivrs. Posthrvest Biology nd Tehnology 23, Klee HJ, Hyford MB, Kretzmer KA, Brry GF, Kishore GM. 99. Control of ethylene synthesis y expression of teril enzyme in trnsgeni tomto plnts. The Plnt Cell 3, Knee M, Htfield GS, Brmlge WJ Response of developing pple fruits to ethylene tretment. Journl of Experimentl Botny 38, Nishiym K, Guis M, Rose JKC, et l. 27. Ethylene regultion of fruit softening nd ell wll disssemly in Chrentis melon. Journl of Experimentl Botny 58, Oeller PW, Wong LM, Tylor LP, Pike DA, Theologis A. 99. Reversile inhiition of tomto fruit senesene y ntisense RNA. Siene 254, Peh JC, Bouzyen M, Lthé A. 28. Climteri fruit ripening: ethylene-dependent nd independent regultion of ripening pthwys in melon fruit. Plnt Siene 75, 4 2. Piton S, Brton SL, Bouzyen M, Hmilton AJ, Grierson D Altered fruit ripening nd lef senesene in tomtoes expressing n ntisense ethylene-forming enzyme trnsgene. The Plnt Journl 3, Ross GS, Knighton ML, Lyyee M An ethylene-relted DNA from ripening pples. Plnt Moleulr Biology 9, Shffer RJ, Friel EN, Souleyre EJF, et l. 27. A genomis pproh revels tht rom prodution in pple is ontrolled y ethylene predominntly t the finl step in eh iosyntheti pthwy. Plnt Physiology 44, Sitrit Y, Bennett AB Regultion of tomto fruit polyglturonse mrna umultion y ethylene: re-exmintion. Plnt Physiology 6, Stow J, Dover CJ, Genge PM. 2. Control of ethylene iosynthesis nd softening in Cox s Ornge Pippin pples during low-ethylene, lowoxygen storge. Posthrvest Biology nd Tehnology 8, Thmmwong M, Arkw O. 27. Strh degrdtion of dethed pple fruit in reltion to ripening nd ethylene. Journl of the Jpnese Soiety for Hortiulturl Siene 76, Wks Y, Htsuym Y, Tkhshi A, Sto T, Niizeki M, Hrd T. 23. Divergent expression of six expnsin genes during pple fruit ontogeny. Europen Journl of Hortiulturl Siene 68,