Diurnal Temperature Range Compression Hastens Berry Development and Modifies Flavonoid Partitioning in Grapes

Diurnl Temperture Rnge Compression Hstens Berry Development nd Modifies Flvonoid Prtitioning in Grpes Seth D. Cohen, 1 Julie M. Trr, 2 nd Jmes A. Kennedy 1,3 * Astrt: Tempertures during the dy nd night re known to influene grpe erry metolism nd resulting omposition. In this study, the flvonoid omposition of field-grown Vitis vinifer L. v. Merlot erries ws investigted s funtion of diurnl temperture rnge (DTR). The DTR ws ompressed y ooling erries during the dy nd heting them t night. Before verison, there were minor differenes in pronthoynidin (PA) omposition in the skins nd seeds due to temperture tretments, most notly derese in gllte-esterifition of seed flvn- 3-ols with ompressed DTR. Compressing the DTR signifintly hstened erry development nd the ineption of verison. Tretments imposed fter verison hd miniml impt on skin nd seed PAs; however, ompressed DTRs fvored the prtitioning of nthoynins nd flvonols towrd B-ring di-sustitution. Compressing the DTR of grpe erries hd onsistent effet on erry development nd prtitioning of flvonoid metolites while totl flvonoid ontent ws not signifintly ltered. Key words: Vitis vinifer, tnnin, nthoynin, flvonol, phenoli, limte The suessful ultivtion of winegrpes (Vitis vinifer L.) involves mthing ultivr to geogrphy nd limte. While ll elements of limte should e onsidered, temperturesed preditions re most ommon. This is illustrted in the degree dy lssifition system for grpe ultivtion in the United Sttes (Amerine nd Winkler 1944). The sis for this system is the generl temperture requirement for fruit to reh eptle mturity, defined primrily y sugr onentrtion. Differenes in temperture rnges n e ttriuted to vritions in ltitude, topogrphy, nd the proximity to lrge odies of wter, whih moderte nery lnd tempertures due to the high het pity of wter, s evident in nnul nd dily temperture flututions or the diurnl temperture rnge (DTR). The response of primry metolites to temperture hs een estlished. Generlly, higher tempertures during fruit ripening led to n inrese in the rtes of sugr umultion nd orgni id degrdtion while lower tempertures n led to redution in the rtes of sugr umultion nd 1 Deprtment of Food Siene nd Tehnology, Oregon Stte University, Corvllis, OR 97331; 2 United Sttes Deprtment of Agriulture Agriulturl Reserh Servie, Hortiulturl Crops Reserh Unit, 24106 N. Bunn Rd., Prosser, WA 99350; nd (urrently) 3 Deprtment of Vitiulture nd Enology, Cliforni Stte University, 2360 Est Brstow Ave., Fresno, CA 93740. *Corresponding uthor (emil: jkennedy@sufresno.edu; fx: 559 278-4795) Aknowledgments: This work ws funded y the Amerin Vineyrd Foundtion, the Vitiulture Consortium West, nd USDA-ARS CRIS no. 5358-21000-00D. Mention of trde nmes or ommeril produts in this rtile is solely for the purpose of providing speifi informtion nd does not imply reommendtion or endorsement y the USDA. The uthors thnk John Ferguson, USDA-ARS, for mintining the field equipment. Mnusript sumitted Fe 2011, revised My 2011, Sept 2011, epted Ot 2011 Copyright 2012 y the Amerin Soiety for Enology nd Vitiulture. All rights reserved. doi: 10.5344/jev.2011.11015 orgni id degrdtion (Kliewer 1964, 1965, Buttrose et l. 1971, Kliewer nd Torres 1972). While the onentrtion of sugrs nd ids n e djusted to reh desired sweetness, lohol potentil, nd idity during wine proessing, the onentrtions of seondry metolites re more diffiult to mnipulte. Pronthoynidins (PAs), or flvn-3-ol polymers, re responsile for wine stringeny; they umulte in grpe skins nd seeds erly in development efore lg period in erry growth, fter whih there is deline in the rte of umultion (Downey et l. 2003). The ineption of ripening (verison) is hrterized y rpid umultion of skin nthoynins in red grpes, the soure of olor in red wine (Pirie nd Mullins 1980). Flvonols, whih re thought to provide some stility to oth PAs nd nthoynins, umulte in the skin erly in development nd ontinue through erry ripening (Boulton 2001, Downey et l. 2003). While flvonol ontents inrese in response to UV exposure, temperture does not pper to hve n influene (Spyd et l. 2002). Previous reserh hs demonstrted effets of oth high nd low tempertures on nthoynin umultion. Inhiition of nthoynin development ws reported t higher tempertures (e.g., 35 C), while t moderte dy tempertures (e.g., 25 C) nthoynin umultion ws inversely relted to DTR (Kliewer nd Torres 1972, Kliewer 1977). A hstening of erry olortion is ssoited with inresed preverison night temperture, while similr tempertures during ripening n redue nthoynin umultion (Koyshi et l. 1967). Susequent work hs onfirmed redued nthoynin umultion t higher dy (>30 C) nd night (30 C vs. 15 C) temperture nd temperture-relted prtitioning (Kliewer 1977, Mori et l. 2005, Ymne et l. 2006, Trr et l. 2008). Het-medited redution of nthoynin umultion ws relted to redutions in gene trnsripts nd enzyme tivities s well s degrdtion of existing nthoynins (Mori et l. 2005, 2007, Ymne et l. 2006). 112

Diurnl Temperture Rnge nd Berry Development 113 Most reserh regrding the umultion of PAs hs foused on the impt of solr rdition. For exmple, exposure to solr rdition, nd in some instnes UV speifilly, resulted in inresed PA umultion in severl ultivrs of V. vinifer (Downey et l. 2004, Cortell nd Kennedy 2006, Koym nd Goto-Ymmoto 2008). Little work hs een done to deouple the effets of light exposure nd temperture. It is doumented tht the surfe temperture of exposed erries my exeed ir temperture y s muh s 14 C, ringing into question the effets ttriuted to light exposure versus temperture lone (Smrt nd Sinlir 1976, Trr et l. 2000). The ojetive of this reserh ws to determine the effets of ompressing the DTR on flvonoid umultion in V. vinifer v. Merlot erries grown under field onditions. The first yer of experimentl dt (2006) ws pulished previously, speifilly to desrie n nlytil method for profiling PA metolism nd umultion in grpe erries (Cohen et l. 2008). Relevnt dt from 2006 re inluded here to provide referene nd sttistil nlysis for three yers of replited field experiment foused on the DTR. Dt presented here demonstrte the effets of DTR ompression on PAs from oth seeds nd skins nd on nthoynins nd flvonols from skins. Experiments were onduted in mture vineyrd from fruit set to verison nd gin from verison to fruit mturity to oinide with the tive periods of umultion of the different lsses of metolites ross three onseutive experimentl sesons. Berry tempertures were mnipulted suh tht the nturl diurnl pttern of temperture in the field ws onserved. Mterils nd Methods Field proedure. The three-yer study spnned 2006 to 2008 t the Irrigted Agriulture Reserh nd Extension Center ner Prosser, WA (46.30 N; 119.75 W). Own-rooted Merlot vines (plnted in 1999) were in rows oriented northsouth, with 2.3 m etween rows nd 1.8 m etween vines within row. Vines were trined to ilterl ordon t 1.2 m oveground nd winter-pruned nnully to 2-ud spurs. Vines were mnged ording to stndrd ommeril prtie for the distrit. Experimentl lusters were oriented on the est spet of the vine nd were exposed uniformly to irrdine (R s ) y tuking shoots nd leves ehind th wire t 1.5 m oveground. Tretments were pplied to individul lusters, nd eh luster ws treted s replite (n = 4). Three temperture-ontrol tretments were pplied: (1) no temperture ontrol (mient); (2) onvetive ontrol (low); nd (3) ompressed DTR (dmp). Tretments were pplied sed on stges of erry development using the modified E L system (Coome 1995): (1) etween stges 27 28 (2 to 4 mm erry dim) nd stges 34 35 (verison; onset of ripening), termed, nd (2) etween stges 34 35 nd ommeril mturity, termed I. Commeril mturity ws defined s erry solule solids 22 Brix in rndom smple of 100 erries olleted from untreted lusters on the experimentl vines. Fruit tempertures were ontrolled in oth Periods I nd II in 2006 nd 2007, while oservtions in 2008 were limited to. There ws no modifition of fruit mirolimte for mient lusters, exept to mintin uniform exposure to R s. Dmp ws omplished y fored onvetion without the use of enlosures (Trr et l. 2000) to minimize hnges in rdition nd humidity round the fruit. Chilled ir ws delivered during the dy nd heted ir during the night. The verge temperture of untreted lusters (n = 4) ws used s referene from whih ir ws delivered to dmp lusters s needed, t 5-se intervls, to mintin n offset from mient lusters of -8 C during the dy nd +8 C t night. No hilled ir ws delivered if erry temperture (T ) ws 10 C, temperture found to inhiit photosynthesis in grpevines (Hendrikson et l. 2004). To ount for the effets of het trnsfer y fored onvetion, mient ir ws delivered to low lusters t the sme rte tht heted or ooled ir ws delivered to dmp lusters. During, T ws estimted y fine-wire thermoouples (0.13 mm dim; Type T [opper-onstntn]) eh enpsulted in 4 to 6 mm dim ed of silione. Four juntions were wired in prllel nd were positioned etween erries long the length of the rhis. During I, re thermoouples (2-mm long juntions; 0.13 mm dim) were inserted diretly under the erry skin nd the insertion point ws seled with wter-sed glue. Air temperture t ordon height (T ; 1.2 m oveground) ws mesured with shielded, spirted fine-wire thermoouple. Glol irrdine ws mesured y pyrnometer (model 8-48; Eppley Lortories, Newport, RI), nd irrdine in the fruiting zone ws mesured y 1-m long tue solrimeters (n = 3; model TSL; Delt-T Devies, Cmridge, UK) instlled prllel to the ordon t 1.2 m oveground. Before deployment, tue solrimeters were oriented north-south nd their output ws normlized to tht of the Eppley pyrnometer. Sensor signls were snned every 5 se nd verges reorded every 12 min y dt quisition system (AM-25T nd CR-10X, Cmpell Sientifi, Logn, UT). Clusters for experiments were hrvested when the first treted luster rehed verison, defined y visully deteted hnge in erry skin olor. I lusters were hrvested when smple of untreted erries rehed ommeril mturity. Hrvested erries were trnsported on ie nd kept under refrigertion efore eing exised from the rhis (pediels ut t flre nd reeptle left tthed), ounted, weighed, frozen in liquid nitrogen, nd stored t -80 C until nlyses. Chemil nlyses. Prior to mnul dissetion nd seprtion of skin from seed, erries were thwed nd men erry volume (50 or 100 erries) ws estimted y H 2 O displement. The numer of olored erries ws determined y visul ssessment for reporting perent erry olortion t verison. To estimte totl solule solids onentrtion, 15 to 20 frozen erries were rushed nd inuted t 75 C for 1 hr then entrifuged (3500 g) for 10 min, efore determintion with portle digitl refrtometer (WM-7; Atgo, Tokyo, Jpn). Skin extrts were prepred y merting lyophilized, whole

114 Cohen et l. erry skins in n etone:wter solution (2:1) t 20 C for 24 hr under N 2 gs. Seed extrts were prepred similrly exept tht in 2008, 1 g of dried seed ws extrted using 10 ml solvent. Following removl of etone, extrts were rought to volume in ultrpure wter nd stored t -30 C until nlysis. Flvonols, nthoynins, nd pronthoynidins (PAs) were nlyzed y high-performne liquid hromtogrphy (HPLC) (model 1100; Hewlett-Pkrd, Plo Alto, CA). The instrument ws equipped with diode rry detetor (DAD) nd n externl olumn oven when required (model CH-430; Eppendorf, Westury, NY). All dt were nlyzed with Chemsttion softwre (ver. A.08.03; Agilent, Snt Clr, CA). Anlysis of monomeri phenolis ws performed following pulished method (Lmuel-Rventos nd Wterhouse 1994). In 2006, queous extrts were filtered using syringe filter (Arodis PTFE [13 mm, 0.45 µm]; Pll Corportion, Est Hills, NY). In 2007 nd 2008, smples were entrifuged t 16,000 g for 15 min to remove solids. Queretin (Sigm Aldrih, St. Louis, MO) nd mlvidin-3-o-gluoside (Extrsynthèse, Geny, Frne) were used s quntittive stndrds for flvonols nd nthoynins, respetively. Compositionl nlysis of PAs ws rried out following id-tlyzed levge in the presene of phlorogluinol (phlorogluinolysis). Aliquots of queous extrts were lyophilized, dissolved in MeOH, nd then reted with phlorogluinol regent (1:1) ording to previous method (Kennedy nd Jones 2001). Following retion, smples were omined with queous sodium ette nd then nlyzed immeditely y HPLC (Kennedy nd Tylor 2003). Individul PA suunits were quntified s desried (Kennedy nd Jones 2001) using (+)-tehin quntittive stndrd (Sigm Aldrih). Proportions of flvn-3-ol suunits re presented s mole perentges to normlize dt for moleulr mss. Sttistil nlyses. The T vlues were summrized y experimentl period nd temperture-ontrol tretment in SAS (ver 9.1; SAS Institute, Cry, NC) using the MEANS proedure. Therml time ove 10 C se temperture ws omputed from 12-min verges of T nd is expressed s degree dys (DD). Sttistil nlyses of metolite nd summrized temperture dt were performed using SttGrphis Centurion softwre (Sttpoint Tehnologies, Wrrenton, VA). Signifint differenes within yers were determined using one-wy ANOVA t p < 0.05 nd mens seprted using Fisher s LSD. Two-ftor ANOVA (tretment nd yer) ws used to determine tretment nd yer effets ross ll yers of experimenttion (p < 0.05). Dt tht were not normlly distriuted were nlyzed using Kruskl Wllis ANOVA s indited in individul tles. Stepwise liner regression used kwrd seletion with n entry riterion of F > 4.0. Results Berry temperture. Dily men, mximum, nd minimum T (Figure 1) indite the effetiveness of temperture ontrol in ompressing the DTR. Dily men tempertures were similr for ll tretments; therefore vlues re shown for mient only. The dily men T of low typilly differed y less thn 0.5 C from tht of mient erries. There ws more vrition in dily men T in 2006 thn in either 2007 or 2008 nd longer periods of tempertures ove 35 C. Effetive temperture ontrol nerly eliminted tempertures >35 C or <10 C for dmp erries (Tle 1). In oth experimentl periods nd ross ll yers, the DTR for dmp lusters ws ompressed more thn those of mient nd low lusters, whih were not different from eh other. Preverison, there ws higher verge DTR in 2008 thn in either 2006 or 2007 (p < 0.001, not shown), ut less dy-to-dy vriility in men temperture. After verison, the verge DTR ws higher in 2006 (p < 0.01). Normlizing DD to the durtion of the experiment resulted in lower DD for 2008 thn either 2006 or 2007 (p < 0.05; dt not shown) in. After verison, more therml time umulted in 2006 thn 2007. The DD umulted y erries differed etween dmp nd mient only in of 2006 (Tle 1). Amient nd low lusters exeeded 25 C, 30 C, nd 35 C for the sme mount of time, while dmp lusters exeeded 25 C, 30 C, nd 35 C for less time in ll yers, exept during I of 2007. Consequently, dmp lusters were mintined etween 20 nd 25 C for greter Figure 1 Exemplry dt (n = 4) showing verge dily mximum (three upper tres), mient dily men (enter tre), nd verge dily minimum (three lower tres) tempertures during in 2006 (A), 2007 (B), nd 2008 (C), nd with theoretil minimum (10 C), mximum (35 C), nd optimum (20 25 C) tempertures for erry metolism indited.

Diurnl Temperture Rnge nd Berry Development 115 proportion of the experiment thn were mient or low lusters. The timing of exposure to higher tempertures (>35 C) in mient nd low erries differed mong yers (Figure 1). Tht is noteworthy, s potentilly detrimentl tempertures would hve ourred during slightly different stges of erry development in eh yer. Berry size nd omposition. The ompressed DTR of dmp resulted in higher erry mss t verison thn mient or low in ll yers (Tle 2). Berry volume ws proportionl to erry mss in ll ses (r 2 0.97, dt not shown). Lrger erries in omintion with higher olortion nd higher solule solids ontent t verison indite onsiderle hstening of erry development y ompressing DTR. The mss of skin per erry t the end of ws higher in dmp erries thn in mient erries in 2006 nd 2007. Dt from onurrent experiment showed tht the rte of erry development of dmp erries ws higher thn tht from equivlent levels of dytime ooling (-8 C from mient) or nighttime heting (+8 C from mient) lone (Cohen et l. 2008). At verison there were internnul differenes in the numer of seeds per erry, in the order 2008>2006>2007 (p < 0.001, dt not shown). After I, there were no differenes in erry mss due to tretments (Tle 2). Skin dry mss ws lower nd solule solids were higher in dmp thn in mient in 2007. Internnul differenes in solule solids in mient erries (2007>2006; p < 0.01) my hve ontriuted to differenes in tretment effet on other mesured vriles. Similrly to verison, there were no differenes in numer of seeds per erry or seed fresh mss t hrvest due to tretments. Seed pronthoynidins. At verison, seed PA ontent per erry ws not signifintly ffeted y tretments (dt not shown). Among yers, per seed ontent ws highest in mient erries in 2008 (p < 0.01). Overll, seed PA ontent (p < 0.01) ws highest in 2007, ut lowest in tht yer if expressed per erry, proly euse of lower seed numers per erry. Averge polymer size (men degree of polymeriztion; mdp) of seed PA ws lower in mient thn in dmp erries in 2007 nd 2008 (Tle 3). By verison, seeds hd Tle 2 Berry mss, skin mss per erry, solule solids, nd perent erry olortion t verison () nd hrvest (I). Tretment Berry mss (g) Skin mss (mg/erry) Solule solids (Brix) Colored erries 2006 Amient 0.50 12.0 6.4 <1.0 Blow 0.52 11.1 6.7 1.0 Dmp 0.75 14.1 10.7 32.5 2007 Amient 0.69 12.0 8.6 <1.0 Blow 0.60 10.9 7.6 4.0 Dmp 0.81 14.1 10.8 40.0 2008 Amient 0.63 13.2 nd 5.8 <1.0 Blow 0.60 11.3 6.0 <1.0 Dmp 0.83 13.9 10.0 36.8 I d 2006 Amient 0.98 nd 22.3nd 22.2 nd n Blow 1.01 22.6 23.2 n Dmp 1.04 26.4 23.6 n 2007 Amient 1.07 nd 25.4 25.3 n Blow 1.02 20.6 25.1 n Dmp 0.94 18.1 26.5 n Perent olortion only relevnt t verison; n: not pplile. Letters denote differenes etween tretments within yer using Fisher s LSD, p 0.05; itlis denote Kruskl-Wllis ANOVA t p 0.05; nd = not different. d I experiment onduted in 2006 nd 2007 only. Tle 1 Tretment durtion (dys), therml time summries (DD), verge diurnl temperture rnge (DTR), nd numer of hours ove indited tempertures during eh experimentl period. Hours (n) ove indited temperture Durtion DTR Yer (dys) Tretment DD sum ( C) >40 C >35 C >30 C >25 C 2006 45 Amient 605.2 19.6 2.7 nd 58.1 256.7 501.0 Blow 596.6 18.9 1.1 46.6 235.5 481.1 Dmp 580.5 10.2 0.0 1.9 65.1 356.4 2007 44 Amient 595.4 nd 18.9 0.6 nd 36.9 236.1 504.6 Blow 597.5 18.5 0.0 37.2 235.3 503.4 Dmp 592.3 10.6 0.0 6.1 76.9 382.5 2008 46 Amient 589.9 nd 21.1 0.5 nd 38.3 237.6 502.6 Blow 589.5 20.3 0.0 29.1 226.4 495.0 Dmp 566.4 11.8 0.9 3.5 30.2 317.3 I 2006 43 Amient 431.7 nd 24.1 4.0 nd 43.4 180.9 310.3 Blow 417.6 22.3 0.8 26.0 156.7 296.2 Dmp 419.2 14.4 0.3 11.9 57.4 205.4 2007 41 Amient 391.0 nd 21.8 0.9 nd 14.6 nd 108.5 276.2 Blow 380.4 20.4 0.6 9.4 81.0 257.5 Dmp 374.3 12.7 0.0 0.0 5.5 131.7 Expressed s degree dys ( C) ove se 10 C. Letters denote differenes etween tretments within yer using Fisher s LSD, p 0.05; nd = not different.

116 Cohen et l. umulted onsiderle quntities of flvn-3-ol monomers (dt not shown). Totl monomer ontent followed internnul ptterns similr to totl seed PA ontent. Tretment differenes t verison were limited to higher monomer ontents per seed in dmp erries in 2006. Flvn-3-ol monomer omposition in seeds differed mong tretments t verison (Tle 3). In 2006, the proportion of (+)-tehin (Cm) ws highest in dmp erries. Proportions of (-)-epitehin (ECm) were highest in dmp erries in 2007 nd 2008 nd when dt were pooled ross yers (p < 0.001). The proportion of (-)-epitethin-3-o-gllte (ECGm) ws onsistently lowest in dmp erries in ll yers. Neither DD lone nor DD omined with DTR ws signifint preditor of ECGm (p = 0.1). The dt indite tht DTR ompression results in lower gllte-esterifition of seed flvn-3-ol monomers. Averge DTR ross explined 83.2% (2006), 51.9% (2007), nd 97.2% (2008) of vrine in proportions of ECGm (p < 0.001). Pooling dt ross yers yielded liner model %ECGm = 0.0113961 + 0.00634433*DTR (dj r 2 = 0.507, SE = 0.0264, p <0.001). Reltive rnks in PA polymers due to tretment nd yer were similr to those oserved with totl PA ontent t verison, exept tht the distriution of terminl suunits showed no influene of temperture. Overll, the proportion of ECGt ws highest in 2006, followed y 2007, then 2008 (p < 0.001). The proportion of (+)-tehin extension suunits (Cx) ws higher in dmp erries in 2006 (Tle 3); vlues were highest in 2008 when pooled ross yers (p < 0.001). The proportion of (-)-epitehin s n extension suunit (ECx) did not differ due to tretment; pooled vlues were highest in 2007 (p < 0.001). Contrry to ECGm, the proportion of (-)-epitehin- 3-O-gllte extension suunits (ECGx) ws not different etween tretments. Pooled vlues followed the sme pttern s ECGm with respet to yer (2006>2008>2007; p < 0.001). After I, there were no signifint tretment effets on seed PAs, either per erry (dt not shown) or per seed (Tle 3). Per seed PA ontent ws higher in 2007 thn in 2006 (p < 0.001), similr to. The ontent of seed flvn-3-ol monomers ws higher in 2007 on oth per erry nd per seed sis (p < 0.01), ut no tretment effet ws oserved. There were no signifint differenes in seed PA mdp due to tretment; verge mdp ws higher in 2006 thn in 2007 (p < 0.001). The proportion of ECGm in dmp erries ws lower thn tht in mient erries in 2006; ECGm proportions were higher in 2006 thn in 2007 (p < 0.001). Consistent with totl PAs, flvn-3-ol monomers, nd outomes from, there were no differenes in totl polymeri seed PAs etween tretments fter I, lthough overll ontents were higher in 2007 (dt not shown, p < 0.001). The distriution of terminl suunits showed no effet of tretment; internnul differenes were onsistent with those for monomer nd extension suunits (Tle 3). The proportions of Cx nd ECx were higher in 2007, while tht of ECGx ws lower (p < 0.01). Skin pronthoynidins. Tretments hd no effet on totl skin PA ontent per erry in either period (Tle 4). Dmp resulted in the lowest PA ontent per unit erry fresh mss when dt were pooled ross yers (dt not shown, p < 0.01). The mdp ws lower in dmp erries in 2006 nd when dt were pooled ross yers (p < 0.01). There were no signifint differenes etween tretments in ontent of flvn-3-ol monomers or terminl suunits in erry skins t Tle 3 Totl pronthoynidin (PA) ontent, men degree of polymeriztion (mdp), nd suunit omposition (mole perentge) s portion of totl in seed PA t verison () nd hrvest (I). Tretment Totl PA (mg/seed) mdp PA omposition Monomers Terminl units Extension units Cm ECm ECGm Ct ECt ECGt Cx ECx ECGx EGCx 2006 Amient 2.38nd 11.7nd 55.1 27.9nd 17.0 18.9nd 3.4nd 77.7nd 6.9 70.1nd 21.5nd 1.5nd Blow 2.46 10.8 55.8 28.3 15.8 20.9 7.6 71.4 7.2 70.9 20.3 1.5 Dmp 2.73 13.7 59.8 30.1 10.1 8.3 4.0 87.7 9.1 68.4 20.6 1.9 2007 Amient 3.21nd 8.9 58.0nd 32.8 9.2 22.4nd 19.2nd 58.5nd 8.0nd 77.2nd 13.4nd 1.3nd Blow 3.01 9.4 59.0 30.7 10.2 21.7 15.6 62.8 8.4 77.0 13.3 1.4 Dmp 2.75 10.8 56.0 36.9 7.1 16.5 17.8 65.7 8.7 76.7 13.3 1.3 2008 Amient 2.51nd 6.8 53.7nd 31.4 14.9 40.2nd 17.2 42.5 9.6nd 73.3nd 14.6 2.6nd Blow 2.14 9.0 53.9 32.1 14.0 38.0 7.0 54.9 9.9 71.9 15.5 2.7 Dmp 1.94 7.9 51.7 41.3 7.0 42.2 16.0 41.8 10.5 71.6 15.9 2.0 I 2006 Amient 1.31nd 9.5nd 52.5nd 37.8nd 9.7 26.5nd 17.8nd 55.7nd 8.0nd 69.6nd 22.3nd 0.2 Blow 1.23 9.9 53.8 38.1 8.1 20.3 19.8 59.8 7.4 71.0 21.2 0.4 Dmp 1.24 9.8 52.7 40.2 7.1 25.1 18.1 56.8 7.6 68.0 21.5 2.9 2007 Amient 2.11nd 6.5 55.7nd 38.1nd 6.2nd 36.5nd 25.9 37.5nd 8.8nd 76.0nd 13.5nd 1.6nd Blow 1.87 6.4 56.1 38.2 5.8 35.2 28.5 36.4 8.5 76.8 12.9 1.8 Dmp 1.77 6.9 51.6 41.8 6.6 37.1 24.7 38.3 9.1 75.6 13.8 1.5 Arevitions: C, (+)-tehin; EC, (-)-epitehin; ECG, (-)-epitehin-3-o-gllte; EGC, (-)-epigllotehin; m, t, x: monomer, terminl, nd extension units, respetively. Letters denote differenes etween tretments within yer using Fisher s LSD, p 0.05; itlis denote Kruskl-Wllis ANOVA t p 0.05; nd = not different.

Diurnl Temperture Rnge nd Berry Development 117 the termintion of either or I. No tretment effet ws oserved for Ct, ECt, or ECGt proportions fter. The proportion of Ct ws lowest in 2006 when those of ECt nd ECGt were highest (p < 0.01). By ontrst, the proportion of Ct ws lower in 2007 thn 2006 t the end of I (p < 0.01), due to the higher ontriution of ECGt to the totl pool tht yer. In generl, skin PA extension suunit ontent ws highest in 2008 (p <0.001) s with totl PA ontent. Proportions of epigllotehin (EGCx) nd ECGx during were highest in 2007 when proportions of ECx were lowest (p < 0.001); otherwise no tretment differenes were oserved for PA extension suunit ontent or omposition t verison. During I, tretments hd little effet on totl PAs in erry skins (dt not shown), with no differenes deteted t hrvest (Tle 4). Overll ontent of skin PAs were higher in 2006, oth per erry nd y unit skin mss (p < 0.05). There were no differenes in PA mdp etween tretments or yers. The distriution of extension suunits ws not ffeted y tretment in 2006 (Tle 4); totl extension suunits were higher in mient erries in 2007. In tht yer the proportion of Cx ws higher nd tht of ECGx ws lower in dmp thn in mient erries, ut these suunits represent less thn 3% of totl extension units. Skin nthoynins nd flvonols. From visul inspetion of olor, dmp erries onsistently umulted nthoynins erlier thn other tretments. At verison, more erries per dmp luster expressed olor thn per mient luster (Tle 2). Anlysis of nthoynins onfirmed this outome: ontent ws undetetle or very low in mient nd low erries nd t 10 μg per erry in dmp erries (dt not shown). The ompositionl vrition in nthoynins ws low erly in development (i.e., up to verison) due to lk of yltion, whih inresed rpidly fter verison. At the end of I, there were ontrsting tretment differenes y yer: there ws no differene etween tretments in totl nthoynins in 2006, ut nthoynin onentrtions were lower in dmp erries in 2007 (Tle 5), refleting the outome found in seed nd skin PAs nd flvonols. Normlizing the dt y erry mss eliminted ll differenes in nthoynins. In ll irumstnes the proportion of di-sustituted nthoynins ws highest in dmp erries t the end of I. The overll proportion of di-sustituted nthoynins ws onsiderly higher in 2007; differenes were evident in higher ontents of ynidin- nd peonidin-gluosides Tle 5 Totl nthoynin ontent nd omposition (mss perent) of skins t hrvest. Tretment μg/ erry Di Anthoynin Tri Aetyl Coum 2006 Amient 900.5 nd 11.6 88.4 17.4 9.8 Blow 996.6 13.6 86.4 18.5 6.6 Dmp 1078.6 21.3 78.7 13.0 4.3 2007 Amient 928.5 30.1 69.9 14.6 4.7 Blow 888.1 29.8 70.2 14.6 3.7 Dmp 693.3 44.1 55.9 10.5 2.1 Expressed in mlvidin-3-o-gluoside equivlene (µg per erry) of di-sustituted, tri-sustituted, etyl-gluosides, nd oumroylgluosides (Di, Tri, Aetyl, nd Coum, respetively). Letters denote differenes etween tretments within yer using Fisher s LSD, p 0.05; nd = not different. Tle 4 Totl pronthoynidin (PA) ontent, men degree of polymeriztion (mdp), nd suunit ompositions (mole perentge) s portion of totl terminl nd extension units in skin PAs t verison () nd hrvest (I). Tretment Totl PAs (mg/erry) PA omposition Terminl units Extension units mdp Ct ECt ECGt Cx ECx ECGx EGCx 2006 Amient 1.16nd 26.0 78.2nd 14.5nd 7.3nd 1.5nd 53.6nd 1.9nd 43.0nd Blow 1.14 25.4 81.1 11.6 7.3 1.5 53.0 1.7 43.8 Dmp 1.04 20.6 84.6 10.1 5.3 1.5 52.6 1.6 44.3 2007 Amient 0.96nd 24.9 90.0nd 8.8nd 1.2nd 1.3 47.4nd 2.2nd 49.2nd Blow 1.18 29.0 88.1 10.2 1.7 1.1 44.5 1.9 52.4 Dmp 1.03 22.4 91.4 7.1 1.5 1.2 46.8 2.1 49.9 2008 Amient 1.41nd 27.1nd 89.3nd 8.6nd 2.1nd 1.4nd 54.4nd 1.6nd 42.5nd Blow 1.49 28.0 88.2 9.9 1.9 1.4 52.4 1.5 44.8 Dmp 1.77 27.5 90.7 7.5 1.8 1.3 52.4 1.6 44.7 I 2006 Amient 1.34nd 26.2nd 89nd 11nd 0.0nd 1.4nd 54.4nd 1.4nd 42.8nd Blow 1.10 26.4 85.1 11.8 3.2 1.5 53.5 1.3 43.6 Dmp 1.31 24.5 84.6 13.0 2.4 1.4 52.8 1.2 44.6 2007 Amient 1.17nd 26.3nd 76.5 16.6nd 6.8nd 1.4 47.6nd 2.0 49nd Blow 0.94 23.0 72.3 20.9 6.9 1.5 49.2 2.6 46.7 Dmp 0.90 23.4 77.8 15.5 6.7 1.7 49.9 2.7 45.6 Arevitions: C, (+)-tehin; EC, (-)-epitehin; ECG, (-)-epitehin-3-o-gllte; EGC, (-)-epigllotehin; t nd x, totl terminl nd extension units, respetively. Letters denote differenes etween tretments within yer using Fisher s LSD, p 0.05; nd = not different.

118 Cohen et l. onomitnt with lower mlvidin-3-o-gluoside nd ylted derivtives (dt not shown). The proportion of nthoynins in the form of etyl-gluosides ws lower in dmp erries in oth yers nd lowest in 2007 when dt were pooled (p < 0.001). Similrly, oumroyl-gluosides were lso lower in dmp thn mient, nd higher in 2006 thn 2007 (p < 0.001). In generl, dmp resulted in lower proportions of trisustituted nthoynins or in etyl- nd oumroyl-gluoside forms, whih grees with work onduted elsewhere relted to redued dytime temperture nd in ool yers in Merlot nd Shirz erries (Downey et l. 2004, Trr et l. 2008). By yer, tretments ppered to hve no effet on flvonol ontent per erry t verison or hrvest (Tle 6). When dt were pooled ross yers, mient erries hd lower totl flvonol ontent (p < 0.05). Aross yers on mss sis, whih shows the effet of erry size, low erries hd the highest ontent (dt not shown, p < 0.01). Similr to nthoynins, hemil diversity in flvonols ws more limited during. Initilly, queretin derivtives were dominnt; hene higher proportion of di-hydroxylted (di-oh) flvonols in mient nd low erries. Dmp erries umulted higher proportions of mono-hydroxylted (mono-oh) flvonols y verison only in 2006. The proportion of di-oh flvonols t hrvest ws higher in dmp erries in 2007 nd when dt were pooled ross yers (p < 0.01). By yer, the proportion in the di-oh form ws highest in 2007 (p < 0.001), similr to tht of nthoynins nd opposite tht of skin PAs. Disussion The DTR of field-grown grpe erries ws ompressed y delivery of old ir during the dy nd wrm ir t night. Tle 6 Totl flvonol ontent nd omposition (mss perent) of skins t verison () nd hrvest (I). Tretment μg/ erry Mono Flvonol Di Tri 2006 Amient 19.8 nd 3.2 96.0 0.9 nd Blow 22.4 3.3 95.3 1.4 Dmp 21.4 4.6 94.1 1.3 2007 Amient 27.1 nd 3.4 95.0 1.6 nd Blow 39.0 2.9 95.5 1.6 Dmp 36.8 4.1 93.7 2.2 2008 Amient 32.4 nd 1.9 nd 96.5 nd 1.6 nd Blow 39.1 1.7 97.0 1.3 Dmp 44.4 2.0 95.8 2.2 I 2006 Amient 78.3 nd 4.6 nd 67.5 nd 27.8nd Blow 66.5 3.9 63.7 32.4 Dmp 73.1 3.7 72.6 23.8 2007 Amient 71.4 nd 5.7 nd 77.1 17.3 Blow 66.2 6.5 77.4 16.1 Dmp 47.2 5.3 82.3 12.3 Expressed in queretin equivlene (µg per erry) of mono-, di-, nd tri-hydroxylted flvonols (Mono, Di, nd Tri, respetively). Letters denote differenes etween tretments within yer using Fisher s LSD, p 0.05; nd = not different. These re the first suh dt to e olleted outside ontrolled environments (Koyshi et l. 1967, Kliewer nd Torres 1972, Kliewer 1973). The tretments ltered the durtion to whih erries were exposed to purported optimum tissue tempertures, tht is, ~20 to 25 C with respet to photosynthesis nd primry metolism (Koyshi et l. 1967, Buttrose nd Hle 1973). High ( 35 C) nd low ( 10 C) ir tempertures hve een shown to lter erry development nd umultion of sugrs, ids, nd nthoynins in grpes; however, responses n e ultivr dependent nd vry temporlly (Buttrose et l. 1971, Kliewer 1973, Mori et l. 2007, Trr et l. 2008). In this study DTR ompression hstened erry development, s evident in greter erry mss nd lrger volume, skin olortion, higher skin weight per erry, nd higher solule solids ontent t verison. While it is unler wht mgnitude of DTR ompression is required to hieve n effet, DTR ompression of one-hlf the mgnitude desried in this study showed similr results onerning morphology t verison (Cohen et l. 2008). The dt suggest tht ompressing the DTR of erries grown in res with short ripening period ould e n effetive wy to elerte erry development. Compressing DTRs resulted in derese in ECGm in seeds, likely driven y lower dy tempertures (Cohen et l. 2008). Tht the proportion of ECG s extension nd terminl suunits ws not ffeted y DTR suggests tht the mehnisms regulting monomeri nd polymeri flvn-3-ols in grpe seeds re independent. In other work (Cortell nd Kennedy 2006), exposure to sunlight resulted in derese in ECGx nd ECGt in Pinot noir seeds, lthough ECG ws not reported s monomeri suunit. Reserh with te (Cmeli sinensis L.) did show lower levels of gllte esterifition of flvn-3-ols ssoited with leves hrvested during ooler months (Yo et l. 2005). The overll redution in seed PAs etween verison nd hrvest is likely relted to oxidtion, oservle y rowning of the seed ot leding to n inility to extrt nd quntify the PAs (Kennedy et l. 2002). Differenes due to DTR were most evident s lower proportions of ECG monomeri nd terminl suunits etween smples olleted t verison nd t hrvest in 2006 nd 2007. Tht my reflet hnges due to seed mturtion, development of the seed ot, nd differenes in rte of polymeriztion ttriuted to flvnol glloyltion, demonstrted elsewhere in model solutions (Cheynier nd Rirdo-d-Silv 1991). Although reserh with purified PAs suggests tht those originting from seed will ffet stringeny nd my e orse or itter, pplition to wine prodution nd qulity remins unertin (Gmuti et l. 2006). Seed dditions during wine prodution hve resulted in improvements in wine olor nd potentil rom hrter (Lee et l. 2008). Aumultion of totl skin flvonols t verison did not pper to e ffeted y DTR in ny single yer, when expressed per erry. Previous work hs demonstrted the effet of solr rdition exposure on flvonol umultion in grpes (Downey et l. 2004, Trr et l. 2008). In the urrent study,

Diurnl Temperture Rnge nd Berry Development 119 differenes in flvonol ontents were limited to prtitioning sed on flvonoid B-ring hydroxyltion, with ompressed DTRs resulting in higher proportions of di-oh flvonols t hrvest. The dt presented here onfirm tht the regultion of grpe flvonoids differs y stge of erry development. Shifts in flvonoid prtitioning due to ompressed DTRs were different t verison nd t ommeril mturity, whih my reflet differenes ssoited with vrious MYB-type regultory genes expressed throughout erry development nd differenes in the retivity or loliztion of metolites (Czemmel et l. 2009, Terrier et l. 2009). Unlike nthoynins, PAs re sujet to polymeriztion nd, therefore, ompositionl differenes my e the result of suunit ondenstion nd ssoited differenes in extrtility, whih ffets downstrem nlysis. Differenes in the hemil struture of flvonoids diretly ffet their retivity or stility, mking our understnding of metolite prtitioning nd modifitions importnt for wine prodution. Conlusion Results suggest tht typil vintge vriility is more likely due to the timing of events suh s high or low tempertures thn to n integrted sesonl vlue. Compressing the DTR ffeted the rte of erry development efore verison ut hd little influene on umultion of PAs in erry skins or seeds. Flvonol nd nthoynin ompositions fvored di-sustitution with ompressed DTRs, while totl ontent ws not onsistently ffeted. Additionl work onerning the effet of trnsient temperture exposure is neessry to determine the existene of sensitive developmentl stges orresponding to vrious metolites. Literture Cited Amerine, M.A., nd A.J. Winkler. 1944. Composition nd qulity of musts nd wines of Cliforni grpes. Hilgrdi 15:493-673. Boulton, R. 2001. The opigmenttion of nthoynins nd its role in the olor of red wine: A ritil review. Am. J. Enol. Viti. 52:67-87. Buttrose, M.S., nd C.R. Hle. 1973. Effet of temperture on development of grpevine infloresene fter ud urst. Am. J. Enol. Viti. 24:14-16. Buttrose, M.S., C.R. Hle, nd M.W. Kliewer. 1971. Effet of temperture on omposition of Cernet Suvignon erries. Am. J. Enol. Viti. 22:71-75. Cheynier, V., nd J.M. Rirdo-d-Silv. 1991. Oxidtion of grpe proynidins in model solutions ontining trns-ffeoyltrtri id nd polyphenol oxidse. J. Agri. Food Chem. 39:1047-1049. Cohen, S.D., J.M. Trr, nd J.A. Kennedy. 2008. Assessing the impt of temperture on grpe phenoli metolism. Anl. Chim. At 621:57-67. Coome, B.G. 1995. Growth stges of the grpevine: Adoption of system for identifying grpevine growth stges. Aust. J. Grpe Wine Res. 1:104-110. Cortell, J.M., nd J.A. Kennedy. 2006. Effet of shding on umultion of flvonoid ompounds in (Vitis vinifer L.) Pinot noir fruit nd extrtion in model system. J. Agri. Food Chem. 54:8510-8520. Czemmel, S., R. Strke, B. Weisshr, N. Cordon, N.N. Hrris, A.R. Wlker, S.P. Roinson, nd J. Bogs. 2009. The grpevine R2R3- MYB trnsription ftor VvMYBF1 regultes flvonol synthesis in developing grpe erries. Plnt Physiol. 151:1513-1530. Downey, M.O., J.S. Hrvey, nd S.P. Roinson. 2003. Anlysis of tnnins in seeds nd skins of Shirz grpes throughout erry development. Aust. J. Grpe Wine Res. 9:15-27. Downey, M.O., J.S. Hrvey, nd S.P. Roinson. 2003. Synthesis of flvonols nd expression of flvonol synthse genes in the developing grpe erries of Shirz nd Chrdonny (Vitis vinifer L.). Aust. J. Grpe Wine Res. 9:110-121. Downey, M.O., J.S. Hrvey, nd S.P. Roinson. 2004. The effet of unh shding on erry development nd flvonoid umultion in Shirz grpes. Aust. J. Grpe Wine Res. 10:55-73. Gmuti, A., A. Rinldi, R. Pessin, nd L. Moio. 2006. Evlution of Aglinio grpe skin nd seed polyphenol stringeny y SDS- PAGE eletrophoresis of slivry proteins fter the inding retion. Food Chem. 97:614-620. Hendrikson, L., M.C. Bll, J.T. Wood, W.S. Chow, nd R.T. Furnk. 2004. Low temperture effets on photosynthesis nd growth of grpevine. Plnt Cell Environ. 27:795-809. Kennedy, J.A., nd G.P. Jones. 2001. Anlysis of pronthoynidin levge produts following id-tlysis in the presene of exess phlorogluinol. J. Agri. Food Chem. 49:1740-1746. Kennedy, J.A., nd A.W. Tylor. 2003. Anlysis of pronthoynidins y high-performne gel permetion hromtogrphy. J. Chromtogr., A 995:99-107. Kennedy, J.A., M.A. Mtthews, nd A.L. Wterhouse. 2002. Effet of mturity nd vine wter sttus on grpe skin nd wine flvonoids. Am. J. Enol. Viti. 53:268-274. Kliewer, W.M. 1964. Influene of environment on metolism of orgni ids nd rohydrtes in Vitis vinifer. I. Temperture. Plnt Physiol. 39:869-880. Kliewer, W.M. 1965. Chnges in the onentrtion of mltes, trtrtes, nd totl free ids in flowers nd erries of Vitis vinifer. Am. J. Enol. Viti. 16:92-100. Kliewer, W.M. 1973. Berry omposition of Vitis vinifer ultivrs s influened y photo- nd nyto-tempertures during mturtion. J. Am. So. Hort. Si. 98:153-159. Kliewer, W.M. 1977. Influene of temperture, solr rdition nd nitrogen on olortion nd omposition of Emperor grpes. Am. J. Enol. Viti. 28:96-103. Kliewer, W.M., nd R.E. Torres. 1972. Effet of ontrolled dy nd night tempertures on grpe olortion. Am. J. Enol. Viti. 23:71-77. Koyshi, A., T. Fukushim, N. Nu, nd K. Hrd. 1967. Studies on the therml onditions of grpes. VI. Effets of dy nd night tempertures on yield nd qulity of Delwre grpes. J. Jp. So. Hort. Si. 36:1-7. Koym, K., nd N. Goto-Ymmoto. 2008. Bunh shding during different developmentl stges ffets the phenoli iosynthesis in erry skins of Cernet Suvignon grpes. J. Am. So. Hort. Si. 133:743-753. Lmuel-Rventós, R.M., nd A.L. Wterhouse. 1994. A diret HPLC seprtion of wine phenolis. Am. J. Enol. Viti. 45:1-5. Lee, J.M., J.A. Kennedy, C. Devlin, M. Redhed, nd C. Rennker. 2008. Effet of erly seed removl during fermenttion on pronthoynidin extrtion in red wine: A ommeril prodution exmple. Food Chem. 107:1270-1273. Mori, K., N. Goto-Ymmoto, M. Kitym, nd K. Hshizume. 2007. Loss of nthoynins in red-wine grpe under high temperture. J. Exp. Bot. 58:1935-1945. Mori, K., S. Sugy, nd H. Gemm. 2005. Deresed nthoynin iosynthesis in grpe erries grown under elevted night temperture ondition. Si. Hort. 105:319-330. Pirie, A.J.G., nd M.G. Mullins. 1980. Conentrtion of phenolis in the skin of grpe erries during fruit development nd ripening. Am. J. Enol. Viti. 31:34-36.

120 Cohen et l. Smrt, R.E., nd T.R. Sinlir. 1976. Solr heting of grpe erries nd other spheril fruits. Agri. Meteor. 17:241-259. Spyd, S.E., J.M. Trr, D.L. Mee, nd J.C. Ferguson. 2002. Seprtion of sunlight nd temperture effets on the omposition of Vitis vinifer v. Merlot erries. Am. J. Enol. Viti. 53:171-182. Trr, J.M., J.C. Ferguson, nd S.E. Spyd. 2000. A hmer-free method of heting nd ooling grpe lusters in the vineyrd. Am. J. Enol. Viti. 51:182-188. Trr, J.M., J. Lee, S.E. Spyd, nd C.F. Sgel. 2008. Berry temperture nd solr rdition lter yltion, proportion, nd onentrtion of nthoynin in Merlot grpes. Am. J. Enol. Viti. 59:235-247. Terrier, N., L. Torregros, A. Ageorges, S. Vilet, C. Verries, V. Cheynier, nd C. Romieu. 2009. Etopi expression of VvMyPA2 promotes pronthoynidin iosynthesis in grpevine nd suggests dditionl trgets in the pthwy. Plnt Physiol. 149:1028-1041. Ymne, T., S.T. Jeong, N. Goto-Ymmoto, Y. Koshit, nd S. Koyshi. 2006. Effets of temperture on nthoynin iosynthesis in grpe erry skins. Am. J. Enol. Viti. 57:54-59. Yo, L.H., N. Cffin, B. D Ary, Y.M. Jing, J. Shi, R. Singnusong, X. Liu, N. Dtt, Y. Kkud, nd Y. Xu. 2005. Sesonl vritions of phenoli ompounds in Austrli-grown te (Cmelli sinensis). J. Agri. Food Chem. 53:6477-6483.