Grape berry metabolism in field-grown grapevines exposed to different irrigation strategies

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1 Vitis 44 (3), () Grpe erry metolism in field-grown grpevines exposed to different irrigtion strtegies C. R. DE SOUZA 1), 2), J. P. MAROCO 1), T. P. DOS SANTOS 2), M. L. RODRIGUES 2), C. M. LOPES 2), J. S. PEREIRA 2) nd M. M. CHAVES 1), 2) 1) Lortório de Eofisiologi Moleulr, Instituto de Tenologi Quími e Biológi, Oeirs, Portugl 2) Instituto Superior de Agronomi, Liso, Portugl Summry The response of grpe erry metolism to vine wter sttus ws investigted in field grown grpevines (Vitis vinifer v. Cstelão) in southern Portugl. Wter ws supplied s: full irrigtion (), to minimum wter defiit orresponding to 1 % of rop evpotrnspirtion (Et), prtil rootzone drying () nd defiit irrigtion (), oth orresponding to n irrigtion mount of % Et, nd rinfed, i.e. no irrigtion tretment (). In, wter ws supplied to one side of the root system during eh irrigtion period, lternting sides every 1 d pproximtely. During the growing period, nd vines showed intermedite pre-dwn lef wter potentil (Ψ pd ) vlues (round -.4 MP) y the end of the growing seson, vines -.2 MP nd -.8 MP. Berry weight s well s the ontent of gluose nd frutose per erry inresed in irrigted vines (,, nd ) ompred to vines. Although oth mli nd trtri id delined in non-irrigted vines, there ws no signifint differene etween tretments t hrvest. The tivities of invertse, mlte dehydrogense nd mli enzyme were not ffeted y irrigtion throughout the ripening proess. The ontriution of other ftors involved in the redution of sugrs nd orgni ids in erries of non-irrigted vines re disussed. These results show tht defiit irrigtion, like nd, do not hve ny negtive impt on growth nd qulity of grpe erries ompred to fully irrigted vines, ut my result in improved erry qulity ompred to rinfed vines. K e y w o r d s : Berry, defiit irrigtion, invertse, mli enzyme, prtil rootzone drying, Vitis vinifer. Introdution The use of irrigtion in the Mediterrnen vitiulture hs een onsidered s stndrd prtie, minly during drought periods when it hs eome n effetive mens of regulting wter vilility to grpevines. Mintining the lne etween vegettive nd reprodutive growth is, however, one of the most diffiult prolems in irrigted vitiulture. In reent yers, different irrigtion tehniques hve een developed, suh s regulted defiit irrigtion (R), where irrigtion is redued during defined period of erry development nd prtil root-zone drying (), where wter is pplied t lternte sides of the root system to lne vegettive nd reprodutive growth (MCCARTHY 1997; BATTILA 2; LOVEYS et l. 2). Previously, we showed tht redued irrigtion (e.g. nd defiit irrigtion,, where the sme mount of wter pplied in is distriuted on oth sides of root systems) deresed vegettive vigour nd stomtl ondutne ompred to fully irrigted plnts (), without ltering signifintly yield (SANTOS et l. 23; SOUZA et l. 23). The effets of these irrigtion strtegies on grpe erry metolism need, however, to e evluted. Although some detils of grpe erry ripening hve een desried, the influene of some environmentl ftors (inluding wter vilility) on the rte of sugr import nd the tivities of relted enzymes re still poorly understood (BOSS nd DAVIES 21). Vuolr id invertse is onsidered the mjor enzyme involved in surose rekdown nd umultion of hexoses in grpe erries (HAWKER 1969; BROWN nd COOMBE 1984; DAVIES nd ROBINSON 1996). The net derese in the mli id ontent during erry ripening hs een ttriuted minly to redued mlte synthesis y phosphoenolpyruvte roxylse (PEP roxylse), to its degrdtion y NADPdependent mli enzyme (HAWKER 1969; LAKSO nd KLIEWER 197) nd to mlte dehydrogense (TAUREILLES-SAUREL et l. 199). The im of the present study ws to enlrge our knowledge regrding the effets of wter vilility on grpe erry development nd mturtion in order to mth physiologil down-regultion y wter stress with ontrolled irrigtion progrms in field-grown grpevines. Thus, the effets of different irrigtion tehniques on the sugr nd id ontent s well s on the tivities of key enzymes involved in sugr nd orgni id metolism in ripening grpe erries were investigted. Mteril nd Methods P l n t m t e r i l n d i r r i g t i o n t r e t m e n t s : The experiments were onduted in ommeril vineyrd Correspondene to: Dr. M. CHAVES, Instituto Superior de Agronomi, Tpd d Ajud, Liso, Portugl. Fx: E-mil: mhves@is.utl.pt

2 14 C. R. DE SOUZA et l. loted 7 km est of Lison, t the Centro Experimentl de Pegões, 3º38 N nd 8º39W, during the 22 seson. The mediterrnen limte is hot nd dry in summer while winters re mild. The verge nnul preipittion nd temperture is 694 mm nd 16 ºC, respetively. The soil is derived from podzols, with sndy surfe lyer (.6-1. m) nd ly t 1 m depth. The vriety used ws Cstelão (Vitis vinifer L.), n erly ripening red wine vriety (SANTOS et l. 23), grfted on 113 Pulsen in The grpevines were sped 2. m etween rows nd 1. m within rows nd trined on vertil trellis with pir of movle folige wires for upwrd shoot positioning. The vines were spur-pruned on ilterl Royt Cordon (pproximtely 16 uds per vine). Irrigtion ws pplied with drip emitters (4 l h -1 for nd nd 2 l h -1 for ), two per vine, positioned 3 m from the vine trunk t oth sides of the rows. Wter ws supplied ording to rop evpotrnspirtion (ET) lulted from the evportion of Clss-A pn nd orreted with the rop oeffiients (K) proposed y PRICHARD (1992). The tretments were: rin-fed, non-irrigted (); prtil rootzone drying (, % of the ET ws supplied to only one side of the root system, lternting sides eh 1 d pproximtely); defiit irrigtion (, % of the ET ws supplied to oth sides of the vine, % on eh side); full irrigtion (, 1 % of the ET ws supplied to oth sides of the root system, % on eh side). Wter ws supplied twie per week from the eginning of erry development (June) until hrvest (Septemer). The totl mount of wter supplied to plnts ws 197 mm (493 l per vine). The nd vines reeived hlf of tht mount. The totl rinfll during the experimentl period (mid-june until the eginning of Septemer) ws 8.8 mm nd totl nnul rinfll in 22 ws 77 mm. W t e r s t t u s : Pre-dwn lef wter potentil ws mesured with Sholnder-type pressure hmer (Model 1; PMS Instrument Co., Corvllis, OR, USA). The mesurements were done on 6 fully expnded leves per tretment. S u g r n d o r g n i i d n l y s i s : The grpes erries used for sugr nd orgni ids nlyses were tken during the summer of 22, t 1-d-intervls pproximtely. Four replite smples of erries were hrvested from different exposed unhes t rndom nd immeditely frozen in N 2 nd stored t -8 ºC until nlysis. Ten frozen erries were weighed nd used for sugr nd orgni id determintion. After mertion of thwed erries, the smples were immeditely filtered through two lyers of heeseloth nd entrifuged t 27,2 g for 1 min. An nion exhnge resin (Bio-Rex, Bio Rd Ls) ws used to seprte the superntnt into sugr nd id frtions. A 1 ml liquot of the superntnt ws pipetted into n Eono-olumn (Bio Rd Ls) ontining Bio-Rex resin nd llowed to run freely, followed y elution with deionised wter to finl volume of 1 ml. This elute ontined solule sugrs, while orgni ids were retined on the olumn. The ltter were desored from the resin with 1 ml of 1 % H 2 SO 4, followed y deionised wter to finl volume of ml. This frtion ontins the id ompounds. Eh frtion ws filtered through.22 mm memrnes (Millipore) efore HPLC mesurements. Gluose nd frutose s well s mli nd trtri id were nlysed using Dionex HPLC system (Dionex Corportion, Sunnyvle, CA) equipped with n Aminex HPX-87H olumn (Bio-Rd, Rihmond, CA) nd differentil refrtometer detetor (LKB, Bromm, Sweden). The olumn ws operted t 6 ºC nd mm H 2 SO 4 ws used s the moile phse t.6 ml min -1 flow rte. The ompounds were eluted in 2 min. E n z y m e s o f s u g r n d i d m e t o l i s m i n g r p e e r r i e s : Five frozen erries (without seeds), tken from the smples used for sugr nd orgni id nlyses just efore verison (July) nd t the end of the ripening period (August), were ground in mortr ontining liquid N 2 nd.-.6 g of frozen powder ws mixed with ml of 1 mm Hepes-KOH (ph 8.), 1 mm MgCl 2, 1 mm β-merptoethnol, 2 mm dithiothreitol (DTT),.1 % triton X-1, 4 % (v/v) Complete-protese inhiitor oktil with EDTA, 1 % polyethyleneglyol (PEG) 4, nd 1 % glyerol. The extrt ws entrifuged t 27,2 g for 1 min nd the superntnt ws used for determintion of enzymti tivities of id invertse (EC ), mli enzyme (EC ) nd mlte dehydrogense (EC ). The tivity of invertse ws determined s desried y HUNTER et l. (1994) with some modifitions. The extrt ws inuted in mixture ontining 1 mm NOA ph (4.), 1 mm surose for 1 min t 3 ºC. The retion ws stopped y ddition of DNSA-regent (1 % 3,-dinitrosliyli id (w/v) in. M KOH nd 1 M potssium sodium trtrte, thus shifting the ph fr into the lkline rnge). The mixture ws kept in oiling wter for 1 min, ooled to room temperture nd olour intensity ws red t 4 nm ginst lnk with zero s retion time. The tivity of mli enzyme ws mesured y following redution of NADP t 34 nm using modified method of LAKSO nd KLIEWER (197). The ssy mixture ontined 1 mm Hepes-KOH (ph 8.), 1 mm MgCl 2 nd.13 mm NADP. The retion ws strted y ddition of 4 mm of L-mlte nd the tivity ws mesured y following the redution of NADP t 34 nm. The tivity of mlte dehydrogense ws ssyed using modified method from LOPEZ-MILLÁN et l. (2). The ssy mixture ontined mm Hepes-KOH (ph 8.),.1 mm NADH nd.4 mm oxlette. The retion ws strted y ddition of oxlette nd the tivity ws mesured y following the oxidtion of NADH t 34 nm. S t t i s t i l n l y s i s : Sttistilly signifint differenes etween ftor groups were evluted with Tukey s HSD for =. using the Sttisti softwre (ver. SttSoft, Tuls, OK, USA). All mesurements shown re mens ± SE. Liner regressions were otined using Sigm Plot softwre (vers. 7., SPSS Siene, Chigo). Results The sesonl evolution of pre-dwn lef wter potentil (Ψ pd ) showed signifint differenes mong tretments

3 Metolism in field-grown grpevines exposed to different irrigtion strtegies 1 (Fig.1). As expeted, the full-irrigted tretment () mintined the highest vlues of Ψ pd (out -.2 MP) throughout the seson. In ontrst, Ψ pd ws signifintly lower in non-irrigted vines () s ompred to other tretments nd t hrvest rehed vlues t out -.8 MP. gve vlues higher thn throughout most of the seson, ut oth tretments showed similr vlues t hrvest. Ψ Ψpd (MP) pd (Mp) Verison /6 1/7 1/7 29/7 12/8 26/8 Fig. 1: Sesonl evolution of pre-dwn lef wter potentil (Ψ pd ). Vlues of Ψ pd re mens ± SE. : non-irrigted, : prtil rootzone drying, : défiit irrigtion nd : full irrigted. Berry weight of the irrigted tretments (, nd ) were signifintly higher (3 %) s ompred to t hrvest (Fig. 2). The onentrtion of gluose nd frutose in the erries inresed during the growing seson (Fig. 3), wheres mli nd trtri id deresed (Fig. 4). In generl, erry omposition ws not signifintly ffeted y the irrigtion tehniques when the results were expressed on onentrtion sis (g l -1 ) (Fig. 3, ). However, in order to eliminte the dilution effet due to hnges of erry size, sugrs expressed on per erry sis (mg erry -1 ) showed sttistilly signifint differenes mong tretments Berry weight (g.erry -1 ) 3, 3, 2, 2, 1, 1,, 8/7 22/7 /8 19/8 Fig. 2: Sesonl trends of erry weight for vrious irrigtion tretments. Vlues re mens ± SE. (For revitions see Fig. 1). (Fig. 4, d). Gluose nd frutose umultion ws signifintly higher in irrigted tretments thn in vines. No signifint differenes were oserved mong tretments with respet to the evolution of orgni id onentrtion (Fig. 4, ). However, on per erry sis mli id ws signifintly higher in vines ompred to vines (Fig. 4 ). nd vines showed no signifint differenes ompred to or vines. Trtri id remined high during most of the experimentl period in irrigted tretments, the differenes eing only sttistilly signifint etween nd erries (Fig. 4 d). At the lst smpling dte, there were no signifint differenes in trtri id etween tretments. The gluose/frutose rtio ws high (out ) t the eginning of July, however fter verison the rtio deresed to 1.1 in ll tretments (Fig. ). There were no signifint differenes etween tretments in the evolution of gluose/ frutose rtio. The trtrte/mlte rtio, inresed from verison until hrvest, the only signifint differenes etween tretments ourring y the end of August, when erries showed signifintly higher rtios thn nd Gluose Frutose /7/2 22/7/2 verison /8/2 19/8/2 d 8/7/2 22/7/2 /8/2 19/8/ Gluose Frutose Fig. 3: Sugr umultion in erries sujeted to different irrigtion tretments. Vlues re mens ± SE. (For revitions see Fig. 1).

4 16 C. R. DE SOUZA et l. Mli id Trtri Aid verison d Mli id Trtri Aid 8/7/2 22/7/2 /8/2 19/8/2 8/7/2 22/7/2 /8/2 19/8/2 Fig. 4: Orgni id pttern in erries sujeted to different irrigtion tretments. Vlues re mens ± SE. (For revitions see Fig. 1). Gluose/Frutose /7/22 22/7/22 /8/22 19/8/22 8/7/22 22/7/22 /8/22 19/8/ Trtrte/Mlte Fig. : Effets of irrigtion tretments on the gluose/frutose rtio () nd trtrte/mlte rtio (). Vlues re mens ± SE. (For revitions see Fig. 1). (Fig. ). However, there were no differenes etween irrigted tretments. Fig. 6 shows the impt of soil wter vilility on enzymes involved in sugr nd id metolism of grpe erries nlysed efore verison (July) nd t the end of the ripening period (August). When the enzyme tivities were expressed on per grm fresh weight sis, only invertse tivity ws redued in irrigted tretments y the end of August (Fig. 6,, ). However, there were only sttistilly signifint differenes etween nd vines. This suggests tht the deline in tivity per grm fresh weight in vines ws the result of inresed erry weight. The tivity of invertse, mlte dehydrogense nd mli enzyme ws not ffeted y irrigtion tretments when the tivities were expressed on per erry sis (Fig. 6 d, e, f). However, the tivities of invertse nd mli enzyme were signifintly higher in August thn in July. Moreover, invertse tivity ws positively relted to the onentrtion of gluose nd frutose during ripening (Fig. 7, ) while the redution of mli id ws negtively relted with the inrese in tivity of mli enzyme (Fig.7 ). Disussion Vine wter sttus ws lerly ffeted y the different tretments s shown y the sesonl evolution of Ψ pd (Fig.1). The defiit irrigtion tretments, nd, resulted in intermedite vlues of Ψ pd etween nd, lthough hd higher vlues thn on most smpling dtes. The redued lef re nd/or deresed stomtl ondutne of s ompred to oserved previously (SANTOS et l. 23; SOUZA et l. 23) my explin the high vlues of pre-dwn lef wter potentil oserved in vines throughout the seson. Severl studies hve shown tht irrigtion delys sugr umultion nd inreses erry size, oth generlly onsidered detrimentl to wine qulity (FREEMAN nd KLIEWER 1983; JACKSON nd LOMBARD 1993). In our study, lthough erry weight inresed in the irrigted tretments (Fig. 2), the mount of gluose nd frutose per erry ws lso inresed y irrigtion (Fig 3, d) inditing proportionl inrese in erry sink strength nd/or inresed sugr vilility.

5 Metolism in field-grown grpevines exposed to different irrigtion strtegies 17 Invertse (µmol surose.g FW -1 h -1 ) MDH (µmol NADH.g FW -1 h -1 ) Mli Enzyme (µmolnadh.g FW -1 h -1 ) /7/2 28/8/2 Irrigtion Wter tretment tretment d e f Invertse (µmol surose.erry -1 h -1 ) MDH (µmol NADH.erry -1 h -1 ) Mli Enzyme (µmol NADH.erry -1 h -1 ) Fig. 6: Effets of different irrigtion tretments on the tivity of invertse, mlte dehydrogense (MDH) nd mli enzyme. Vlues re mens ± SE. Gluose Frutose Mli id R 2 =.7 P<. R 2 =.79 P< Invertse (µmol surose erry -1 ) R 2 =.8 P= Mli enzyme (µmol NADH erry -1 ) Fig. 7: Reltion etween gluose nd frutose with invertse tivity (, ) nd mli id with mli enzyme tivity (). On two dtes ll tretments were inluded. Vlues re mens ± SE. The higher sugr levels per erry in the irrigted tretments reltive to were not ompnied y n inrese in invertse tivity per erry, whih ould ount for inresed sink strength. Most of the sugr in the erries is synthesized in leves y photosynthesis nd trnsported to the erries vi the phloem, minly s surose (RUFFNER et l. 199; DAVIS nd ROBINSON 1996). The high umultion of sugrs per erry in irrigted vines ould result from inresed photosynthesis mesured in these tretments s ompred to non-irrigted tretment in the sme experiment (SOUZA et l. 24). Although the invertse tivity ws not ffeted y the wtering regimes, there ws signifint inrese in enzyme tivity t the end of the ripening period, in proportion to the inrese in erry size (Fig. 6 d). The high levels of hexoses umulting in the erries were strongly ssoited with the rise in invertse tivity s ws lso shown y HAWKER (1969) nd PEREZ nd GOMEZ (2). The sugr umultion proess in grpe erries is still not well understood, nd lthough solule invertse my e importnt in the umultion of hexoses in the vuole, their synthesis does not seem to e the trigger for its umultion (DAVIS nd ROBINSON 1996; DREIER et l. 1998). Other mehnisms must e involved in the regultion of sugr umultion, suh s differenes in wter potentil etween soure nd sink, whih fvours the movement of phloem sp into the erries (LANG nd DÜRING 1991). Moreover, phloem unloding of sugr ould hve een ffeted y wter stress, s ws reently demonstrted y WANG et l. (23). Before verison, there ws derese in the orgni id ontent in ompred to vines, when the results were expressed on per erry sis (Fig 4, d). Mlte, the

6 18 C. R. DE SOUZA et l. mjor orgni id in grpe erries, hs een proposed to e degrded y NADP-mli enzyme nd lso y mlte dehydrogense (MDH) (HAWKER 1969; LAKSO nd KLIEWER 197). The redution of mli id in non-irrigted vines ould not e explined y hnges in the tivity of these enzymes euse there were no signifint differenes etween irrigtion tretments in the tivity of oth enzymes, either efore verison or t the end of ripening. The inresed temperture of sun-exposed erries of non-irrigted vines, ould lso ontriute to redue mlte ontent due to redued synthesis y PEP roxylse whih hs lower therml stility ompred to mli enzyme (LAKSO nd KLIEWER 197). The derese in mli id in ll tretments throughout the seson ould e ttriuted, in prt, to n inrese in tivity of the mli enzyme (Fig., 6 f, 7 ), unlike other studies where no diret orreltion ws found etween the hnge in mli id onentrtion nd the tivity of mli enzyme in the erry tissue during ripening (HAWKER 1969; RUFFNER 1982; GUTIÉRREZ-GRANDA nd MORRISON 1992). The intrellulr omprtmenttion, rther thn enzyme tivity, hs lso een invoked to regulte the mli id metolism during the ripening proess (RUFFNER 1982; KANELLIS nd ROUBELAKIS-ANGELAKIS 1993; FAMIA et l. 21). In ontrst to mli id, the ontent of trtri id per erry ws lmost onstnt during the growing seson, s hs een oserved in other studies with grpe erries (KANELLIS nd ROUBELAKIS-ANGELAKIS 1993). However, like mli id the gretest redutions over time were oserved in erries. In generl, the low orgni id onentrtions in erries of vines my e relted to the effet of wter stress on ron ssimiltion in leves nd/or trnslotion of photossimiltes to the erries. It is widely epted tht most orgni ids re formed t the expense of surose imported from the leves ( MARCO et l. 1977; TERRIER nd ROMIEU 21) nd in this experiment photosynthesis ws depressed in non-irrigted vines reltive to irrigted tretments (SOUZA et l. 24). The rtios of gluose/frutose nd trtrte/mlte my e used s qulity indies to define the optiml hrvest dte. There ws no effet of vrious irrigtion tehniques on the pttern of the gluose/frutose rtio during the seson (Fig. ). The highest rtio mesured t the eginning of July ws due to the low levels of frutose present in the green erries. During the erly stges of erry development, gluose ounts for 8 % of sugr ontent, ut in ripe erries the rtio of gluose to frutose is lmost equl, s hs een reported y KLIEWER (1967) nd KANELLIS nd ROUBELAKIS-ANGELAKIS (1993). The irrigtion tretments hd more pronouned effet on the trtrte/mlte rtio (Fig. 6 ), whih inresed in non-irrigted vines ompred to irrigted tretments. The vlues of this rtio were out of the rnge onsidered optiml ( ) ording y RIBÉREAU-GAYON (197). This inrese my hve een the result of mli id degrdtion ssoited with the rise in temperture tht usully ours in erries of sun-exposed lusters. In ft, higher perentge of exposed lusters were oserved on vines ompred to the other tretments (SANTOS et l. 23), nd this ws ompnied y higher erry tempertures (SANTOS et l., unpul.). In onlusion, the defiit irrigtion tretments, nd, hd no negtive impt on growth nd qulity of grpe erries s ompred to the tretment, ut resulted in n improvement of erry qulity ompred to vines. In vitiulture, defiit irrigtion my help to regulte yield nd qulity in ddition to its role in optimizing of rop wter use effiieny. The lower sugr ontent in erries of non-irrigted vines my hve resulted from less ron ssimilted y folige wheres higher luster exposure in plnts my hve resulted in higher trtrte/mlte rtio. Aknowledgements We grtefully knowledge the use of n HPLC equipment nd the tehnil ssistne in sugr nd orgni id nlyses enled y Professor Isel Spener nd Césr Fonse (Universidde Nov de Liso). The finnil support from the EU projet IRRISPLIT (IAC3-CT ) is knowledged. C.R.S reeived dotorl fellowship y Fundção pr Ciêni e Tenologi (PRAXIS XXI/BD/2186/99). We lso thnk A. Rodrigues nd E. Brei for tehnil support in lortory nd field experiments. Referenes BATTILA, A.; 2: Applition of the regulted defiit or irrigtion to grpevines (Vitis vinifer) in su-humid re. Pro. 3th Int. Symp. Irrig. Horti. Crops. At Horti. 37, BOSS, P. K.; DAVIES, C.; 21: Moleulr iology of sugr nd nthoynin umultion in grpe erries. In: K.A. ROUBELAKIS-ANGELAKIS (Ed.): Moleulr Biology nd Biotehnology of the Grpevine, Kluwer Ademi Pulishers, The Netherlnds). BROWN, S. C.; COOMBE, G.; 1984: Proposl for hexose group trnsport t the tonoplst of grpe perirp ells. Physiol. Veg. 22, DAVIS, C.; ROBINSON, S. P.; 1996: Sugr umultion in grpe erries. Plnt Physiol. 111, MARCO, G.; GREGO, S.; TRICOLI, D.; TURI, B.: 1977: Cron isotope rtios ( 13 C/ 12 C) in frtions of field-grown grpe. Physiol. Plnt. 41, DREIER, L. P.; HUNTER, J. J.; RUFFNER, H. 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