UNIVERZA V LJUBLJANI BIOTEHNIŠKA FAKULTETA ODDELEK ZA AGRONOMIJO Mateja COLARIČ VSEBNOST IZBRANIH METABOLITOV V LISTIH IN PLODOVIH HRUŠKE (Pyrus communis L.) SORT ‘WILLIAMS’ IN ‘CONFERENCE’ GLEDE NA ARHITEKTONSKO ZGRADBO RODNE VEJE DOKTORSKA DISERTACIJA Ljubljana, 2007 UNIVERZA V LJUBLJANI BIOTEHNIŠKA FAKULTETA ODDELEK ZA AGRONOMIJO Mateja COLARIČ VSEBNOST IZBRANIH METABOLITOV V LISTIH IN PLODOVIH HRUŠKE (Pyrus communis L.) SORT ‘WILLIAMS’ IN ‘CONFERENCE’ GLEDE NA ARHITEKTONSKO ZGRADBO RODNE VEJE DOKTORSKA DISERTACIJA THE CONTENTS OF SELECTED METABOLITES IN LEAVES AND FRUITS OF PEAR (Pyrus communis L.) CVS. ‘WILLIAMS’ AND ‘CONFERENCE’ REGARDING TO ARHITECTONIC STRUCTURE OF BEARING BRANCH DOCTORAL DISSERTATION Ljubljana, 2007 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. II Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Doktorska disertacija je zaključek podiplomskega študija bioloških in biotehniških znanosti ter se nanaša na znanstveno področje agronomije. Praktični del poskusa je bil opravljen v sadovnjaku Sadjarstva Hudina v Zagaju pri Bistrici ob Sotli. Nadaljnje raziskave in analize so bile opravljene na Katedri za sadjarstvo, Oddelka za agronomijo, Biotehniške fakultete, Univerze v Ljubljani. Tema in naslov doktorske disertacije sta bila sprejeta na podlagi Statuta Univerze v Ljubljani, po sklepu Senata Biotehniške fakultete in sklepu Komisije za podiplomski študij Univerze v Ljubljani (po pooblastilu senata Univerze v Ljubljani z dne, 14. 2. 2006) dne, 28. 2. 2006. Za mentorico je bila imenovana izr. prof. dr. Metka HUDINA in za somentorja prof. dr. Franci ŠTAMPAR. Komisija za oceno in zagovor: Predsednik: prof. dr. Franc BATIČ Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo Član: izr. prof. dr. Metka HUDINA Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo Član: prof. dr. Franci ŠTAMPAR Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo Član: doc. dr. Stanislav TOJNKO Univerza v Mariboru, Fakulteta za kmetijstvo Datum zagovora: Naloga je rezultat lastnega raziskovalnega dela. Izjavljam, da so vsa vključena znanstvena dela identična objavljeni verziji. Mateja COLARIČ Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. III Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 KLJUČNA DOKUMENTACIJSKA INFORMACIJA ŠD Dd DK UDK 634.13::631.546:581.19(043) KG hruška/plodovi/listi/ogljikovi hidrati/organske kisline/fenoli/upogibanje rodnih vej KK AGRIS F08/F507F62 AV COLARIČ, Mateja, univ. dipl. inž. agr. SA HUDINA, Metka (mentorica)/ŠTAMPAR, Franci (somentor) KZ SI-1000 Ljublj ana, Jamnikarj eva 101 ZA Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo LI 2007 IN VSEBNOST IZBRANIH METABOLITOV V LISTIH IN PLODOVIH HRUŠKE (Pyrus communis L.) SORT ‘WILLIAMS’ IN ‘CONFERENCE’ GLEDE NA ARHITEKTONSKO ZGRADBO RODNE VEJE TD Doktorska disertacija OP V, 59 str., 49 vir. IJ sl JI sl/en AI V raziskavi smo ovrednotili vsebnosti metabolitov v plodovih in listih evropske žlahtne hruške (Pyrus communis L.) sort ‘Williams’ in ‘Conference’ kot odgovor na upogibanje rodne veje. Petletne rodne veje smo upognili v dveh terminih: pozno poleti 2003 in spomladi 2004. Kontrolno obravnavanje so predstavljale petletne rodne veje, ki jim nismo spremenili kota rasti. Na rodnih vejah smo v letih 2004 in 2005 vzorčili liste v rastni dobi in plodove v tehnološki zrelosti. S tekočinsko kromatografijo visoke ločljivosti (HPLC) smo izmerili vsebnosti določenih ogljikovih hidratov (saharoze, glukoze, fruktoze in sorbitola) in fenolnih snovi (klorogenske, vanilne in sinapinske kisline, epikatehina, katehina, rutina, kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida) v listih in plodovih ter organskih kislin (citronske, jabolčne, šikimske in fumarne kisline) le v plodovih. Upogibanje rodnih vej je v obeh letih mnogo bolj vplivalo na vsebnosti fenolov kot na vsebnosti ogljikovih hidratov in organskih kislin. Proučevani sorti sta se različno odzvali na agrotehnični ukrep upogibanja. Zlasti poletno upogibanje je pri sorti ‘Williams’ zmanjšalo vsebnosti določanih fenolnih snovi v listih, toda pri sorti ‘Conference’ je zlasti spomladansko upogibanje povečalo vsebnosti fenolnih snovi. V letu 2004 so bile pri sorti ‘Williams’ največje vsebnosti posameznih fenolov v plodovih, ki smo jih obrali na rodnih vejah upognjenih spomladi 2004 in pri sorti ‘Conference’ v plodovih na vejah upognjenih poleti 2003. V naslednjem letu je prišlo do preobrata - morda tudi zaradi nižjih temperatur in obilnih padavin - in so bile največje vsebnosti posameznih fenolov pri sorti ‘Williams’ v plodovih s poletnega upogibanja ali kontrole in pri sorti ‘Conference’ s spomladanskega upogibanja ali kontrole. V kontrolnem obravnavanju so bile v drugem letu pri obeh sortah značilno večje vsebnosti kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida ter sinapinske kisline in epikatehina le pri sorti ‘Conference’. V poskusu smo ugotovili razlike v vsebnosti določenih metabolitov med proučevanima sortama, med obravnavanji ter med proučevanima letoma. Na rezultate je vplival tudi čas upogibanja (pozno poletno in spomladansko upogibanje). Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. IV Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 KEY WORDS DOCUMENTATION DN Dd DC UDC 634.13::631.546:581.19(043) CX pear/fruits/leaves/carbohydrates/organic acids/phenolics/branch bending CC AGRIS F08/F507F62 AU COLARIČ, Mateja AA HUDINA, Metka (supervisor)/ŠTAMPAR, Franci (co-supervisor) PP SI-1000 Ljubljana, Jamnikarjeva 101 PB University of Ljubljana, Biotechnical Faculty, Department of Agronomy PY 2007 TI THE CONTENTS OF SELECTED METABOLITES IN LEAVES AND FRUITS OF PEAR (Pyrus communis L.) CVS. 'WILLIAMS' AND 'CONFERENCE' REGARDING TO ARHITECTONIC STRUCTURE OF BEARING BRANCH DT Doctoral Dissertation NO V, 59 p., 49 ref. LA sl AL sl/en AB The study investigated the response of ‘Williams’ and ‘Conference’ European pear (Pyrus communis L.) subjected to branch bending, according to the contents of various metabolites in their leaves and fruit. Five-year-old branches were bent in the late summer of 2003 and in spring of 2004, and the third treatment with unbent branches was the control. During both growing seasons of 2004 and 2005, leaves from bent and control branches were sampled monthly from May to October and fruit at commercial maturity. The content levels of carbohydrates (sucrose, glucose, fructose and sorbitol) and phenolic compounds (chlorogenic, vanillic and sinapic acids, epicatechin, catechin, rutin, quercetin-3-D-galactoside and quercetin-3-ß-D-glucoside) in the leaves and fruit, as well organic acids (citric, malic, shikimic and fumaric acid) in the fruit were measured by high-performance liquid chromatography (HPLC). Branch bending had a greater influence on phenolics than on carbohydrates and organic acids in ‘Williams’ and ‘Conference’ leaves and in the fruit from 2004 and 2005. Both cultivars displayed various responses to branch bending in their levels of metabolites. ‘Williams’ leaves from bent branches, especially those bent in the summer, had lower contents of some phenolic compounds; however, ‘Conference’ leaves from bent branches, especially those bent in the spring, had higher phenolics contents. In the first year, the highest content levels of most phenolics in ‘Williams’ fruit were found in the current spring treatment and in ‘Conference’ fruit in the summer treatment. However, in the next year, the opposite reaction occurred, perhaps because of lower temperatures and abundant rainy weather: the highest content levels of certain phenolics were found in ‘Williams’ fruit in the summer treatment and in the control, while in ‘Conference’ fruit the highest levels occurred in the current spring treatment and in the control. Nevertheless, in 2005 significantly higher content levels of quercetin-3-D-galactoside and quercetin-3-ß-D-glucoside were measured in both cultivars, as well as levels of sinapic acid and epicatechin in ‘Conference’ fruit in the control. The differences in content levels of selected metabolites between cultivars and among treatments were confirmed in the research; moreover, the results were affected by the time of bending (late summer and spring) and by the year-to-year. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. V Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 KAZALO VSEBINE str. Ključna dokumentacijska informacija (KDI) III Key words documentation (KWD) IV Kazalo vsebine V 1 UVOD 2 ZNANSTVENI ČLANKI 2.1 SPREMEMBE VSEBNOSTI SLADKORJEV IN FENOLNIH SNOVI V LISTIH HRUŠKE SORTE ‘WILLIAMS’ MED RASTNO DOBO 5 2.2 VPLIV UPOGIBANJA RODNE VEJE NA VSEBNOSTI SLADKORJEV, ORGANSKIH KISLIN IN FENOLNIH SNOVI V PLODOVIH HRUŠKE SORTE ‘WILLIAMS’ (Pyrus communis L.) 12 2.3 UPOGIBANJE VPLIVA NA VSEBNOST DOLOČENIH FENOLNIH SNOVI V LISTIH HRUŠKE SORTE ‘WILLIAMS’ 18 2.4 VPLIV UPOGIBANJA RODNE VEJE NA VSEBNOSTI DOLOČENIH OGLJIKOVIH HIDRATOV IN FENOLNIH SNOVI V LISTIH HRUŠKE SORTE ‘CONFERENCE’ 25 2.5 VSEBNOSTI RAZLIČNIH METABOLITOV V PLODOVIH HRUŠKE SORTE ‘CONFERENCE’ GLEDE NA UPOGIBANJE RODNE VEJE 37 3 RAZPRAVA IN SKLEPI 44 3.1 RAZPRAVA 44 3.2 SKLEPI 49 4 POVZETEK (SUMMARY) 50 4.1 POVZETEK 50 4.2 SUMMARY 53 5 LITERATURA 56 ZAHVALA Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 1 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 1 UVOD Evropska žlahtna hruška (Pyrus communis L.) je zelo razširjena sadna vrsta v zmerno toplem podnebnem pasu. Sorti ‘Williams’ in ‘Conference’ predstavljata pomemben delež med sortami, ki jih pridelujemo v Sloveniji (Hudina in Štampar, 2000a). Obe sorti se poleg splošnih značilnosti razlikujeta tako v rasti in razraščanju rodnega lesa kot tudi v rodnosti. Sansavini (2002) razvršča sorte evropskih žlahtnih hrušk po rasti in rodnosti v pet modelov: tako spada sorta ‘Williams’ v model I in sorta ‘Conference’ v model III. Med že uveljavljenimi agrotehničnimi ukrepi v nasadih sadnih dreves (gojitvena oblika, rez, prehrana, namakanje) se je upogibanje rodnih vej izkazalo za enega izmed najučinkovitejših ukrepov, s katerim uravnavamo rast in rodnost (Luckwill, 1970). Uravnavanje prekomerne vegetativne rasti in števila rodnih brstov ter s tem posledično tudi pridelka ima še toliko večji pomen zaradi gospodarskih razlogov – zmanjšanja stroškov, saj se je razmerje med stroški pridelave in tržnimi cenami pridelanega sadja v zadnjih nekaj letih povečalo. Upogibanje rodnih vej je dandanes pogost ukrep v intenzivnih nasadih in je vključen tudi v gojitveno obliko sončna os (Costes in sod., 2006). Z upogibanjem zmanjšamo vegetativno rast, vplivamo na obilnejše cvetenje in večje pridelke ter imamo s tem večje donose v obdobju polne rodnosti (Lauri in Lespinasse, 2001). Podobno ugotavljajo na jablani (Malus domestica Borkh.) tudi Grochowska in sod. (2004). Goldschmidt-Reischel (1997) je primerjal dva agrotehnična ukrepa: upogibanje in rez. Drevesa jablan in evropskih hrušk so imela večje pridelke, če so jim uravnali rast in rodnost le z upogibanjem v primerjavi le z rezjo, prvi ukrep pa je vplival tudi na zgodnejšo pridelavo plodov. Upogibanje rodnih vej našija ali azijske hruške (Pyrus pyrifolia (Burm.) Nak. je vplivalo na večjo tvorbo cvetnih brstov (Ito in sod., 1999) ter na zgodnejši zaključek vegetativne rasti poganjkov (Banno in sod., 1985). Tudi evropska žlahtna hruška sorte ‘Društvenka’ se je odzvala na upogibanje s pospešeno tvorbo cvetnih brstov in z zmanjšano rastjo poganjkov (Lawes in sod., 1997). Odziv sadnih dreves na upogibanje rodnih vej na fiziološkem in biokemičnem področju še ni dovolj raziskan. Do sedaj je bilo nekaj raziskav opravljenih na jablani in našiju. Upogibanje je ugodno vplivalo na fotosintezno aktivnost listov jablane zaradi spremembe kota osvetlitve listov (Pitushkan in Shtirbu, 1985). Ito in sod. (2004) so ugotovili, da upogibanje vej našija vpliva na vsebnost dveh pomembnih primarnih metabolitov -sorbitola in saharoze v lateralnih brstih in v internodijih poganjkov v primerjavi z neupognjenimi vejami. Na našiju povečanje kota poganjka upognjenega navzdol ni vplivalo le na razvoj lateralnih rodnih brstov, temveč tudi na zmanjšano vsebnost indol-3-ocetne kisline (oslabitev apikalne dominance) v poganjkih (Ito in sod., 2001) in v lateralnih brstih teh poganjkov (Ito in sod., 1999) v primerjavi z neupognjenimi poganjki. Z upogibanjem vej so se v le-teh vejah vsebnosti avksinov in giberelinov zmanjšale ter vsebnosti abscizinske kisline in citokininov tipa zeatin povečale. Ker je upogibanje poganjkov spremenilo hormonalno ravnovesje, so bili zato lateralni brsti veliko bolj sposobni, da tekmujejo za asimilate, zato tudi večja tvorba rodnih brstov. Podoben hormonalen odziv na upogibanje rodnih vej jablane sta ugotovila tudi Sanyal in Bangerth Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 2 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 (1998), saj je upogibanje povzročilo od 2 do 2,5-kratno zmanjšanje polarnega transporta avksinov v primerjavi s kontrolnimi - neupognjenimi vejami. Odziv drevesa na upogibanje se razlikuje med sortami proučevane vrste, pomembno pa je tudi kdaj med letom in pod kolikšnim kotom so bile rodne veje upognjene, pa tudi koliko časa so bile te veje v upognjenem položaju (Lauri in Lespinasse, 2001). Kar nekaj raziskav je bilo opravljenih s področja spremljanja vsebnosti metabolitov sadnih rastlin (tako primarnih kot tudi sekundarnih). V Sloveniji so bili do sedaj najbolj raziskani plodovi (Veberič in sod., 2005; Veberič in sod., 2007) in listi jablane (Veberič in sod., 2003; Šircelj in sod., 2005; Usenik in sod., 2004). Vsebnosti primarnih metabolitov -ogljikovih hidratov in organskih kislin v plodovih različnih sort evropske žlahtne hruške, pa tudi našija, so z različnih vidikov podrobno proučili Hudina in Štampar (1999; 2000a; 2000b; 2000c; 2004) ter Hudina in sod. (2003). Sekundarni metaboliti v hruškah do sedaj pri nas še niso bili raziskani, v tujini pa so jih proučili mnogi, zlasti v zrelih plodovih (Galvis-Sánchez in sod., 2003), manj pa v listih (Gunen in sod., 2005). Plod hruške vsebuje največ vode (84 % sveže mase), sledijo ogljikovi hidrati: fruktoza (54 %), sorbitol (18 %), saharoza (15 %) in glukoza (13 %). Zaradi ugodnega razmerja med posameznimi ogljikovimi hidrati ter zaradi precejšnje vsebnosti surovih vlaken (1,5- 2,8 %), plodove priporočajo sladkornim bolnikom namesto slaščic (Blattný, 2003). Med organskimi kislinami pri večini sort hrušk prevladuje jabolčna kislina, sledi ji citronska kislina; vsebnosti kininske, šikimske, fumarne in oksalne kisline pa so veliko manjše. Jabolčna in citronska kislina prispevata glavni delež k najbolj želeni stopnji kislosti plodov, njuno razmerje pa je povezano s senzoričnimi ocenami okusa (Colarič in sod., 2005). Sorte ‘Williams’,‘Red Williams’ in ‘Rosired’ imajo citronske kisline več kot jabolčne kisline (Hudina in Štampar, 2000a). Seveda so vsebnosti metabolitov v plodovih odvisne tako od sorte (Hudina in Štampar, 2000a) kot tudi od zrelosti (Hudina in Štampar, 2000b; Herrmann, 2001). V različnih raziskavah Hudina in Štampar (2000c) ter Hudina in sod. (2003) ugotavljajo, da so vsebnosti ogljikovih hidratov in organskih kislin v plodovih hrušk velikokrat povezane tudi z različnimi agrotehničnimi ukrepi v nasadu. V plodovih žlahtne hruške so med fenolnimi snovmi (sekundarni metaboliti) izmerili največje vsebnosti klorogenske kisline, hruška pa vsebuje tudi epikatehin, katehin, arbutin, flavonol glikozide (kvercetin in izoramnetin glikozide), procianidine, kavino kislino, p-kumarno kislino, ferulno kislino (Amiot in sod., 1995; Escarpa in González, 1999; Schieber in sod., 2001; Ferreira in sod., 2002; Leontowicz in sod., 2003; Galvis-Sánchez in sod., 2003). V plodu imajo fenolne snovi pomembno fiziološko vlogo, med drugim tudi v njegovi odpornosti na mehanski in biološki stres. Poleg tega fenolne snovi prispevajo k senzoričnim lastnostim ploda - aromi, trpkosti, grenkosti in obarvanosti (Macheix in sod., 1990). Še več, fenolne snovi skupaj s surovimi vlakni zmanjšujejo možnost pojava srčno-žilnih bolezni (Gorinstein in sod., 2002). Evropska žlahtna hruška in tudi druge sadne vrste iz družine Rosaceae imajo v listih med ogljikovimi hidrati največje vsebnosti sorbitola in saharoze, manj pa glukoze in fruktoze (Loescher in Everard, 1996). Deguchi in sod. (2002) so ugotovili, da se vsebnosti zgoraj Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 3 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 naštetih ogljikovih hidratov v listih japonske hruške povečajo, če so drevesa izpostavljena stresnim razmeram (npr. slana tla in mraz). V listih evropske žlahtne hruške med fenolnimi snovmi prevladujeta klorogenska kislina in arbutin (Gunen in sod., 2005). Slednjega je več pri sortah, ki so odpornejše na hrušev bakterijski ožig (Erwinia amylovora (Burill) Winslow et al.). Pomembne fenolne spojine najdene v listih različnih sort hrušk so še epikatehin, katehin, flavonol glikozidi (kvercetin glikozidi), procianidini, p-kumarna kislina, … (Andreotti in sod., 2006). Ogljikovi hidrati so primarni produkt fotosinteze in osnovna organska snov, iz katere se sintetizirajo še druge organske snovi. Lesnate rastline iz ogljikovih hidratov, aminokislin in maščob proizvajajo različne sekundarne metabolite, kar omogoča obrambo pred okoljskimi dejavniki, med katerimi so fenolne snovi ene najpomembnejših. Mnoge raziskave so pokazale, da fenolne snovi vplivajo na rast rastline tako, da pospešujejo ali pa zavirajo delovanje hormonov (medsebojne interakcije) (Kozlowski in Pallardy, 1997; Taiz in Zeiger, 2006). Kemična sestava različnih organov sadnih rastlin ni odvisna le od vrste, sorte in podlage (Amiot in sod., 1995; Hudina in Štampar, 2000a; Colarič in sod. 2005), temveč tudi od okoljskih dejavnikov (Hudina in Štampar, 1999; 2004) in različnih agrotehničnih ukrepov (gojitvena oblika, rez, prehrana, škropljenje, namakanje….) opravljenih v nasadu (Hudina in Štampar, 2000c; Hudina in sod., 2003). Ker je upogibanje tudi agrotehnični ukrep, smo v našem poskusu želeli ugotoviti ali z njim vplivamo na vsebnosti primarnih (ogljikovi hidrati in organske kisline) in sekundarnih metabolitov (fenolne snovi) v plodovih in listih sort ‘Williams’ in Conference’, ki sta najbolj razširjeni sorti hrušk pri nas. V svetovnem merilu do sedaj še ni poznanih raziskav evropske žlahtne hruške z biokemičnega področja (vsebnosti metabolitov v njenih organih), kjer izvajamo upogibanje rodnih vej. Kot smo omenili že na začetku, je upogibanje rodnih vej v nasadih že ustaljena praksa sadjarjev, s katerim zmanjšamo rast poganjkov in pospešimo tvorbo cvetnim brstov. Z upogibanjem vplivamo na arhitektonsko zgradbo rodne veje, saj s spremembo kota spremenimo njen položaj v krošnji. Najbolj arhitektonsko raziskano sadno drevo je jablana; manj pa oreh, breskev, marelica, češnja, oljka in hruška (Štampar in sod., 2005). Obe sorti, ki sta bili vključeni v poskus, sta cepljeni na podlago kutina MA in sta bili posajeni v letu 1987 v nasadu Sadjarstva Hudina v Zagaju pri Bistrici ob Sotli. Poskus je zajemal naslednja obravnavanja: · rodne veje upognjene 1. septembra v letu 2003 (pozno poleti 2003) pod kotom 120° od navpične lege, · rodne veje upognjene 15. maja leta 2004 (pomlad 2004) pod kotom 120° od navpične lege ter · kontrola - rodne veje, ki niso upognjene (izraščajo pod kotom 45° od navpične lege. V vsako obravnavanje je bilo vključenih 10 dreves s po 1 rodno vejo na drevo (primerljivih lastnosti), ki izraščajo iz provodnika in so imele pri meritvi prirasta v aprilu 2003 popolnoma razvit eno-, dve-, tri-, štiri- in petletni les. Rodne veje, ki so bile vključene v Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 4 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 poskus, v letih 2003, 2004 in 2005 niso bile rezane. Rodne veje, ki smo jih upognili, so pred tem izraščale pod kotom 45o, tako kot kontrolne veje. V dveh zaporednih letih (2004 in 2005) smo pri obeh sortah vzorčili razvite liste med rastno dobo (mesečno od maja do oktobra) ter plodove v tehnološki zrelosti za kemične analize vsebnosti izbranih primarnih in sekundarnih metabolitov. Postavili smo naslednje hipoteze: · med proučevanima sortama hrušk ‘Williams’ in ‘Conference’ bodo razlike v vsebnosti izbranih primarnih in sekundarnih metabolitov (posebej značilna slika za vsako sorto) v listih in plodovih, ker se proučevani sorti hrušk ‘Williams’ in ‘Conference’ razlikujeta tako v morfoloških lastnostih (po rasti in nameščenosti rodnih brstov) kot tudi v pomoloških lastnostih ploda, · med rastno dobo se bodo vsebnosti ogljikovih hidratov in fenolnih snovi v listih spreminjale, · upogibanje vej bo vplivalo na vsebnost sladkorjev in fenolnih snovi v listih v primerjavi s kontrolo, · upogibanje vej bo vplivalo na vsebnost sladkorjev, organskih kislin in fenolnih snovi v plodovih (notranjo kakovost plodov) v primerjavi s kontrolo, · proučevani sorti hrušk ‘Williams’ in ‘Conference’ se bosta na upogibanje različno odzvali v vsebnosti izbranih primarnih in sekundarnih metabolitov, tako v listih kot tudi v plodovih, · pričakujemo razlike v vsebnosti fenolnih snovi, saj so iz različnih podrazredov: fenolne kisline (hidroksicimetne in hidroksibenzojske kisline), flavonoidi (flavan-3-oli in flavonol glikozidi), · da bodo vidne razlike med dvema zaporednima letoma, saj ne smemo zanemariti vpliva okoljskih dejavnikov. Glede na to katero upogibanje bo dalo boljše rezultate (vsebnosti posameznih metabolitov v plodovih in listih v dveh zaporednih letih), pa bomo lahko predvideli za proučevani sorti ‘Williams’ in ‘Conference’ najprimernejši čas upogibanja. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 5 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 2 ZNANSTVENI ČLANKI 2.1 SPREMEMBE VSEBNOSTI SLADKORJEV IN FENOLNIH SNOVI V LISTIH HRUŠKE SORTE ‘WILLIAMS’ MED RASTNO DOBO COLARIČ Mateja, ŠTAMPAR Franci in HUDINA Metka Changes in sugars and phenolics concentrations of Williams pear leaves during the growing season. Canadian Journal of Plant Science, 2006, 86: 1203-1208. Prejeto 11. 10. 2005, sprejeto 22. 5. 2006. Ogljikovi hidrati so neposreden produkt fotosinteze v listih, glavna skladiščna snov in osnovna organska snov, iz katerih nastaja večina ostalih snovi v rastlini. Fenolne snovi imajo pomembno vlogo pri obrambni funkciji rastlin proti boleznim in škodljivcem. Precej fenolnih snovi se tvori preko vmesnega produkta fenilalanina, ki povezuje primarni in sekundarni metabolizem. Kar nekaj raziskav je bilo narejenih v povezavi s spremljanjem sezonskega gibanja ogljikovih hidratov v listih sadnih dreves iz družine Rosaceae med rastno dobo, toda spremljanje gibanja fenolnih snovi je manj pogosto. Evropska žlahtna hruška (Pyrus communis L.) je iz tega vidika še precej malo raziskana sadna vrsta, zato smo izvedli raziskavo gibanja vsebnosti določenih sladkorjev in fenolnih snovi med majem in oktobrom 2004. Analize smo opravili s tekočinsko kromatografijo visoke ločljivosti (HPLC). Podobno kot pri drugih sadnih vrstah iz družine Rosaceae je bil sorbitol (alkoholni sladkor) po vsebnosti najpomembnejši določani sladkor v listih hruške sorte ‘Williams’. Znotraj določanih fenolov smo izmerili največjo vsebnost klorogenske kisline, po vsebnosti sta ji sledila rutin in epikatehin. Med rastno dobo (6 vzorčenj) smo ugotovili statistično značilne razlike v vsebnosti sorbitola, saharoze, glukoze in v vsebnosti vseh analiziranih fenolov. Najmanjše vsebnosti posameznih snovi so bile v listih z začetka rastne dobe. Z izjemo sorbitola, so bile največje vsebnosti sladkorjev v oktobru. Med rastno dobo so vsebnosti posameznih fenolov v listih sprva naraščale, nato pa se zmanjševale: vsebnosti klorogenske kisline, rutina in kavine kisline so naraščale do julija, vanilne in sinapinske kisline do avgusta ter vsebnosti katehina, epikatehina in siringinske kisline vse do septembra. Na koncu rastne dobe je bila dinamika gibanja primarnih in sekundarnih metabolitov v listih najbolj različna: vsota merjenih sladkorjev se je še povečala od septembra do oktobra, toda vsota vsebnosti merjenih fenolov se je v oktobru razpolovila v primerjavi z mesecem prej. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 6 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Changes in sugars and phenolics concentrations of Williams pear leaves during the growing season Mateja Colaric, Franci Stampar, ard Mctka Hudina Department of Agmnomy. Brotechnicaf Faculty. University of Ljubfjzna, JamMkafjeva 101, S1-100O Ljubljana, Shventa (e-mail: maisja.cotaric@ttf.um-lj.si). Received J1 October 5005, accepted 92 May2006. Colaric. M,, Stamper, K and Hudina, M. 2CC6. Clients in suEjira and plenties t iracenlrarjuni of Williams penr Ituvt* during ihv graving season. Cur,. J. Plant Sd. 86: 1203 1203 Lcuyss of Wiiljftivs ptsr weie collected during ihe glowing season torn Ma/ lo Oclobsr and the contents of sugars and phenolic cjmpounds weie analyzed by liLgh-pcrfurmunce liquid cJltaraatag-npBy method* Sorbitol was the maj« sugar (up to 83.3 g JtL"' DW), followed by sue: ose Cuu In L2.] g kg"1 DW). Concenrratloils 0* glucose aftd fructose were as high sa L2.9 and 9.(1 $ kef1 DW, reipeclivcly. Leaves contained Up [0 29 47 1.9 ifvj kg"' DW of chforOL*nie Bid, followed in cciwenTraiion by rutin (up to0739.1 :ug kg"' DW), crncaTftc hiii (up ID 737S.O mgkg- DW), rale-Chin tup 10 38^6.5 mgks"1 DW), vanillic aciJ (up (o 1832,1 mglcj ' DW). syriajjjttacdd ftp to [ [23.5'lQgfegr1 DW). esfreicacLd (on (0 122.5 nig Jig"1 DW) and sinapie Kid (up ID 94.L mi; kg~r DW) lUe. Significant differences in concentration of soihitd, supioK, Lfuco$e, and in alt analyzed phencJics were observed during the gro^i ng aesson (sii sampling dales). The lowest ccm-cenirafioDS in die leaf were found at the hr^nriitig of Sae grewtog season j it May and June. The highest contents of iueers- were in October, with the exception ot sorhitnl. raring ihe growing season, total phenolic content Gist increased, ihen defined, Chlorogrnic. »fid, rutin and caffltJC acid coMenlS increased until July, vanillic acid tnd sinapic acid BJrtil August, aid catechin, epi-ralccfnn and lyringic acid until September. However. total phenolic Content dipped by 50$ ftojy. September tu October, Key words: P*ar leaves, sugar;, plxnotics. growing season Colaric, M„ Stampar, F. et Hudina, M. 2006. Evolution de |ji couttnlratbn de siicres et de phtinuN dans: ies Teuilles rhi poirl- er WiQnlms p.-ndunl la safeond« croissiincc. Can, J PTailE Sti, 8«: 1203-12™. I Jts aireurs On! reciieiili des tcuilles 0* poirifir Williams, pendant II periode vcg&atHe (de umi ii netočne) puis Out analyjii la concen-rstioi ce iucre.s et de ptierols par ch*0-IJiatograpJiie li-quide i haute; nerfoi maiute, Le Borbirril esr lr> jncre le pluv tbendan: {ju^n's ii,(S % pur ky de poids sec) swivi dn suciuse fjuii|u'a 22.1 j pwrlcj de polds SK). La ccncenlratinn dp glnc«c d «JJc d? rrueto« oeuvenl adei^drc resjeitivcineiH jusja'a WBti. 9,0 g parleydf pnkb ssc. Le* feuilles icnfcrrnaieiK justiu'a iU 4^1 ,iiipoyj]ds and priiKipal organic sujl^aiite-i frum whtth mast otP:er nrgsnif cotnpoiundfi fflunil in plants are. synthesized. Among s,imple licosc and fruclose art cocrimcwi and abundant in woody plants, Sh^rhjc is tunstdcrEd r.rte nnqr i rrip^rtntit nllfiris-iorharidi; in higher plants.. It has u high concentration in cells and it is the main transport form of pruXuasstmilatcs from sourer, leaves (synLheais.} la sink :tksucs. (itsc), and it is also a signifL-ani reserve; earbohyd^te (KaiioY/ski and Pal lardy 1997). In the woody R&jncevt fami:>', do wliicli pear (Pyras cntnouinln I-) bp^ngs, sugar alcohol scrtiii^ (D-glyeiiol), in parallel witli sucrose are the major photosyiithetic products in the mature leaves, (he mair dansltaTiinhlK ^nd h«iic re-seuvj carbohydraie-s. in noiipftDta.-synttiesizirig celJs. SorbitDl is also fount in [he Plctitaginacfae and may be derived from glucose aa welJ ^Lfleschwr und Everard ]$%\ Moiflg et at. 1997; Dcguclii el A 2002; Watari et al. 20041, Jxavps otlen have a liig.T content of carbohydralea; nev-urthcicsi. they cnJy coflLain a smaFI proportion of tha tetaJ omoiint pjt*crit ir) iht whole tree. Tne leavts plav a decisive rule in growth and fievelopmcin of woody plant*! bevausc they are Che basic phcuosv-nthetic organii, vtf.ich consecutively influence growdi of vegetative and rcpioductive tissues.. Leaves also stoje carbith/ilraEPis ^rd mineral nutrienTs fr.ir short periods. Leaf longevity is of much itittnML, because of its importance to plant gjtiwlli and also to plant responses (KoiEovski and PfiJIaidy 1997). nirbiiliydrjKcs pioduced In th; leaves are transported lr> ihe other parts, and then used fur the growth of various organs Sorbitol accounts for Abbreviations: KPLC, hish-performancc liquid chiti- rnulugraph; DW, dry weight 1303 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 7 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 1204 ČANADIA N JOURfiA L Of PLANT SCIENCE 60-00% ol" Ihc carbon en ported from ihe- leaf (Bie!;ski 1982). In contrast te- primary metabolites.. secondary metabolites are widely distributed in plant tissues and play a prominent role in general defense shategie« of plant*. Sevcrrii thousand plienolics have been described, and their contents have been reported to vary from fi to 50 ^ kg-1 dry weight of plani tissue« (SwanSQi 20D3). However, Uitincn el al. (200O1 rejMl«] (hut (he high incraspecific ait J interspecific v filiations in metabolite composition Pound in plants ate the con-sequsnee of evolution of plant species and may be an effective means flf adaptation in variable abiotic acid Lit nit environments. Phenolic compounds »re synthesized mostly by ihe shiki, lote and acetate pathways (Kozlowski and Pallaidy 1397). Sevejal simple phenyl propanoids {with die basi? C6-C3 carbon skeleton of phenylalanine) are produied from einnamic acid via h series of hydrosylatlon. meiiiylatinr, and dehydration reactions; these include p-cournaric, caffeic, ferulic and si napi; acids, and simple cc-um arms. Ctnnamic odd is formed from phenylalanine by the action of phenylalanine ainmonia-lyase-, the branch point enzyme between primary {shikimaie pathway) and secondary (phenylpropanoid/ metaholism {Dixon and Paiva 1W5). Pherrclirs comprise a large group of endogenous growth inhibitors and therefore can affect plants directly by Lhe-ir involvement it) metabohsm and ihiir function on growth and development, and ecological functions (KosiJtwslu and Pallflrdy 1997). As well, phcnoli:; compounds play a major role in the resi sm nee to plan* pathogens These compounds exist in many different plant parts: root, shool, flower, leaf buds, wi^nly lissurs, leaf, phloem, pollen, and stylus (Misirii et a], ]W5j, Moreover, chlnmgenic acid is induced in response to wounding and can act as defers* compouhd (Dison and Paiva LM51, Many researchers have conducted studies on (he seasonal changes of caiLohydrates in leaves of Rosttdtie trees fLcescheret ol, 1382; Bielcski and Rede.well 1935; Merio and Ptewera l $91; Hhiing el al 1997; Wang el al. IS#9; Lo Bianco et al. 2000: Dcguchi el a], 2002; Vcteric ct al. H?XV, SiresIj el al. 2005} while seasonal patterns of leaf phenolic have been investigated less (Mayi ct al. 1995; Misirii el ai. 1995; Tnuttst 3001; Uecr.ik et al. 2WW). If we focus on the caiholiydrje ami phenolic dynamics of leaves at European pear (Pynts mmntwds L.) during the erowmjj season, there ejiiMs even less inrbrmalian {Ouneji et al. 2005), and data ftro mostly restricted to ripe Ihičls. Consequently, [[-is study investigated the ihangts of carbohydrates and phenoHcA partitioning in leaves uf Williams pear from May to October. MATERIALS ANO METHODS Plant Material Williams pea.' mee; grafted on quince MA were planted in 1337 in the eastern part of Slovenia (Bistrica r.ib Sodi}. To evaluate variations in (he chemical compaction of pear leaves during the growing Heasi.]i:r the health} leaves were collected ai« times From May to October (May 15, Jan. 11, Jul. W, Aug. Qo, Sep. 02,Oct. G I). For each, sample date, 12 representative pear leaves (Pyj&j coinwuitii L.) were picked Irnm die sHmr marked 5-yr-ild branches taken from shoots of cqnul length from tlic caslerci part t?f the (ree. Eigh.t icpc-titians were prepared for each of the sis sampling dates, Leaves were placed in liquid nitrogen immediately aftcj they were eolkciet and than lyophiliasd. T.ne samples were stored at -20"^ until the chtiiiical analyses wen; performed, Extraction of Sugars and Phenolic Compounds For extraction of saga™, I g of ground leaves was weighed and bidistii.cd water was added (up lo final volume of 30 mL) and left for 45 mi it at WC. The extracted samples wert centrifuged at 12000 Kjfot? min ar HTC (Eppendorf Centrifiige 5SK) H, Hamburg. Germany) and the supernatant was used for analyses of sugars after filtration through a 0.45 u.m cellulose mined esters filter (Macherty-Nagcl, Dllrcn, Gerrnamy). To prepare samples For analyses of phejiolics, 1«! ni^ of (he ground leaves were- weighed tnto a test tube and e*lraei-cd onea wiLi 10 mL of methanol contaijiiinj 1% af hiiy?Eil-ed hydrOKytoluene (used as an antioxidative agent) in an uLuasr>E:tL bath ffjr 4? min. Afler thai, the homogenate was centrifuged (12 000 x /i far 7 min at ][)°C) and the supciv iisfajjt was fVreii ihrough a 0.45 \im Chromafil polyamide filter, (ranaferjcd into a vial and ustd for analyses of phenolic cornpobrLds. Standarda of frtctMe. glucose, ^ucrese, sorhitol. {-}-epi-catcehin, eyffcic aeid. vanillio acid, uynngie acid, f+> cate chin, ehlorogenic acid, ^itiapic acid, ami rutin were used. Bidis-tilled. water was used for the preparution of startrJands of S-ugars, and Standards of plicrtolics were dhstilved and diluted in methanol. Analyses of Sugars and Phenolic Compounds A hijth-perrnrmnnne Eiquid chfontaiojrapb [HHt-O of Thermo Separaii^n Products cquipmenr (p, iviera Ficach, FL) ivas used tor .sugar analyses (fitictose, cjuoose, tucno** and fiorbitrjl), Separa-ion of sugars was done using a Rczex RCM Monosaccharide columj) (300 x 7.S mm> from Phenomaiex (Torrance, C.A). The rolurnn temperalure was maintained a! 65^C BidistilljrJ water was used foi mobile phase witf: a flnw i^ate O.fi mf.. m'n-1. The; total fun ume was o'O mui and monitoring of cluted sngaia was carried oul nning re&scrive indei driefirjr- A Surveyor HPLC system (Thermo Finnigan, 5an Jose, CA) equipped wi'.h a photo diode array detector was performed for (he analyses of phenolic compound* (chhirogeniL acid, rutin, epicatechin, catechin, vanillic acid, ij/rin^ic acid, sinapic acid^ and caffeic acid). Scpaialion whs curied oui using a Chromsep HPLC column JiS (25Q x 4,6 mm, Hyjieu-.^l .¦> OnH) rx:iiplcd v/:th a Crn'omsepguarvJ colurrm SS (1U x J mm) from ChromsatA fMidccfburs, the WetherlHrrk) and operated it 25aC. The chromatograpihic conditions were a previously described Sehieber et al. (2001) with minor modifications- The mobile phases were 2(k acetic acid in bid i stil led water (A) and 0.5 % acetic atid in hidi^tillei waler and acetonilriie ^lio 1:1) (B). Initial gradient was 90% A, isassijiglu 4^%H»rA within 50 min, 45 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 8 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 COLARfC ETAL — SEASOHAL CHANGES IN WILLIAMS PEAR LEAVE$ W)5 Tabli l.Smjai'imiteiiti I^itIjFM, men««. ijIiipuse ail ftimrmp LH|>iv.)&rJ h? metru valu« ± St durLna: sr Mjv June Sepsinber Swfcito: fflUDTKiC Fnrtfoss Sua 7Z<) ± 7.1jJ. 7.1 ±1.8 [OS.'? - .¦ ¦ * 5?.J ±.S i 13-9 *0&i JJi 10« SJ0=! 1.3 Or;fc*(r K.S±3.ib 6.4 ± w 117-« ±5.2 7ft I +T.|fli U.I ±}.ia IJ.^ t 2.1» I2fl.l t H.I a, h VhIihm ffJIciivfrt fry i tlmtal fcHirii wiiliin cucli row aru slgnlik^irty and phenolic« (chlorogcnic actd, rutin, epieatc-chdn. catcehin, vanillic nc-id, syringic acid, caffeic acid and xnmnie acid) fluctuated in Llieir contents in a seasonal pattern from May to OcLohcr. Sugai erttiLeiilrHhoTi.tj in leaf were almost invariable in the descending order: sorbitol, sucrose, glucose and fructose (Table 1). The total sugar conceniratinn (sorbitol + sucrose ¦4-glucow: 4- fructose.) of'analyzed leav^ was between iOli.7 and 120. ] g kg"' DW, except in Jinie {06.6 g kg"] DW). The content rif toial sugars increased during the growing seas«), except in May, and the highest wis] sugar« content w^i observed aftei harvest (si |r,c last sampling date). However, Bieleski and Redgwell (1985) found thai the iolitlsiiger con-CeAtiudun of apricot Jcavea was reasonably stable between JI ind I o g kg"] of fresh weight, encepi a! the beginning of the growing season. Sorbitol was. tlie major uranslwating sssirnilale in all sampling dates (63.4-72,6% of total sugars) and a mueh luwer proportion of sucrose (13j0-18.4%> was delermined. With i-ubwquent samplings [from May to October), ihe propc'itioii iif sorbitol io ihr iot^I sugars decreased, and the proportion of snercse to (be total sugars inerea&ed. Glueo&c rcprcscnled 6.2-10.7% and fructose 5.(]-9.2l34 Dfrnul uajraiS, Moreover, several authors reported thai throughout the jjnuwtng season sorbitol was jiIjh* di= major sugar found in leaves of many species. t>f the woody ficmiene family including die economically imponant genus htaion (apple). T/ytv-rst from Elstar apple trees hid a slightly lower sorbitol to total sugars content (up io 4S.7%) fSircclj ct ah 2005), while s'jeose had a higbrr pnjpfirticn to total sugars (up to tfjtffo) than our findings in pear, Merlo and Passero (1501) described the levels of leaf sugars- of peach [fr;wu.i prrsicti (L-) Batsch.) during (he growing season, when iheir con-tents, especially of sorbitol, increased. In veiy young leaves only 20% nf toia) stfgans were present as sorbitol. However, at the last sampling (In October) soibtto] reached 40% of total sugars-, Sucroac and glucose were predominated (40 arid 30% of total sugars, -respectively) in very young leaves ar.d Lieu decreased in TT.aiurc leaves (up to no and Zfflft at the. last sampling, respectively J. The percentile <>r fructose-wanreqsoniibly stable throughout tbc growing seasuti (atoul 10% of toial sugsrs>. Similar results were also ocutined by Lo B i once et al, {200C'>. In their strtLf.y, sc-Tbitol nad suercse wen: the iwo main sugars found in mature peach leaves, although the suciose content wsis from four- to eightfold lower tliau tlie «tnibia>l ut>n[Kni, Biclcski ajid Redgwell (14JS5) have sJiown that throughout each stage ofapricot leaf dcveloprrierit. all leaves conlauicd sorbitol as the majior s-jgar (between 60 and 70% of total sugars), and a much lower proportion of sucrose (from 24 to 16%). Sorbitol was aLso found as u major phtocm oompancr.i in rosaceous fruit Lrees and can re-present a major portion of the wtrbon molecules in the phloem sap. Tu a study with peach leaves (Mying ctal, I9y7)h sorbitc-i accorintcd for 60 to 90% of the carbon exported from the source leaves, Tn our study, t'^e lowest sorbitol content in pear leaf was oa&erved in June, and the penk of sorbitol content was reached in September. The a^ow-mcnlioncd samplings significantly differed in sorbitol contents (Duncan's test, P <0-05), Sorbitol was found to allow continuation nf phorosynik:tic activity and carfcor: metabolism under adverse environmental condi-lions such as water stress (aJvmuiLC trf sorbttoi). Sonbitol accumulation in plants is considered an adaptive reaetiai to drought s'.rcss (Itudina and Stampar 1999). Moder-att drought influenced higher soitttol concentratiori in apple leaven, while severe drought caused a Jecreuse- in sorbitol concern rat i on (Sircelj et al. 2002). Degcchi ct al, (2002) reported sugar contents [sorbitol, sucrose, fructose and sucrose) increased in fajjancse pear lenves responding to salinity and chilling stresses. Zhou and Quebedeau* (2003> Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 9 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 10 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 COLAPtC ETAL — SEASONAL CHANGES IN MLLlAMS PEAR LEAVES 1207 Among sampling dales, the highest contents uT epicaie-ehin and oaiwhin were rsaehed in September (7:373.A and 3546-5 mg kg"1 DV, respectively), followed by H decrease in October. The highest proportions of bvth epitalKtiin (I7.fi*) umicateehin (Q.?^) tototal pbenolics weta noticed in September. Similar seasonal changes weft also wnfumed forcpicatcchin and caitcchin in apple Jeaves fMayr et a.]. 1995). Although the coKienLs of van I'lie ncid in pear leaves reached the minimum values in May, Us proporion to total ohenolies wis the highest (ll.*&). Vanillic acid 'tallied h ;.jcalt in August, declined in Sepfervibcr snd increased once again iti October, The content of syringic acid constantly incensed until September and (lien declined. Caffejc acid rcHched its peak in. July, and did not signiflcanriy decrease unfil October. The content of sinapic acid gradually increased until August, and decreased significantly thereafter. Arbutin ischsracterisliL uf jusy IPiivcistnxJ web also identified. but difficulties in iti separation ai the twginnin* of each analysis occurred; therefore, the data arc not shown here, A-nhulin. has also been found in strRwoeiry, bearherry, wlieat and in wheat products (Clifford 2O00t.However, others have also had difficulties with arbtilin in phenolics extractions (Seliicbcr et al. 2 concentrations, would have an influence on increasing primary and secondary leaf metabolites, was refuted, CONCLUSION The chnnges in sjgar ihd phenolic contents tn leaves of Williams pear were confirmed during the growing season. Leave« from the early sample dates m spring had lower levels of ca.rbohydfates, direct products of photosynthesis, and secondary metabolites, which play a pmrnlneiti role in the general defense strategies ot" plants. The highest eontenls of sugars were observed in October, except for sorbitol. The lowest cements of phenol i cs were in May, then increased quickly, reaching a peak sometime in summer and decreasing thereafter jntil October. The patterns of primary and scconrisry membolites from September to October were quite different, (he content of total sugars continued to increase, while the total phenolks contents crapped by 50%. Similar 10 other rosaceous trees, sorbitol was always lire major sugar followed by sucrose, Chlorogcnic acid was identified as a nlajor phenolie in pear leaf, followed by rutin, epicatechin. eatechin, vanillic acid and syringic acid, ACKNOWLEDGMENTS This work is part of Ihe program Horticulture P4-G(JIj-0481, giiintcd by the Slovenian Ministry of Higher Education, Science and Technology. BiBkski, ft, L. and Jterljwell, R, J, 1PBS, Sirbilcl \craus sucrose us photosynthesis and (Tanslocatijn products in developing ipricot tcav«. .Aurt;r. J, Plant Pliysiol. 12; 057-668. Hieleshi Rr L. 19&2. Ha^ic ultotiols. Pages 15?U1?2 tn P. A. Lcevvus and w. Tanner, eds. Plant carhnhydrateg. I. Intrscellular Carbohydrates. Wcw Seiias, Vo], ]3A- Springer-VfcrlaLh Berlin, fjcrmany. OlifTord, M, N, 2(00. MiacclliiriErnui p.TcnoIs in roods and beverages - Njture, occurrencs. niui dietary boidejl J. Sci. Food A^iic. S0l H^liS7, fTtilarir, M., Vftherir, R., Solar, A., HudJrtu, M. und Slamp:ir, P. 2005, Phenol 1; acids, synr.gaidcKydc, and jugiure in fruits of djffenmt ti>lLivLxs of Jufiam Kgw L. ;, Agric, Food Cbem, 53: Cui, Th) Nakaniurai K,, Ma, L,, Li, J.-Z. und Kayahura, II, 2D#5, Analyses of achutin jjiJ chtunogenit; acid, [tie najor phenr^ lie constiiuenrs ia Grienial pear. J. Ajric, Food Caem j;li SXAl-HVil. Decucfii, M.. VV,-i|,ii! rd)«, M, and Kblruiyum«, Y, 2M)2. Increase iit sorbitol ?iusyntJicsis in stressed Japanjsf pear leaves, Acta Hnrtic. 5«?: 511-517, Oivun, E, A, and PaJva, N. L. IW5. Siress-iiidiiccri phenyl- p-firran™d merarmtism. The Pliknl C"eiJ 7: lt*5-ltHI7. Crimen, V^ MUirli, A. and fJuJcan. It. 2UU5. Uaf phenolic content wf pear cjllivars resis-tant or iUSMptthl* 10 fife blight. 5d. Hoiric. IIS: 2J3-23I, Kudilta. M, and Stampur, F. 1999. Infl jsnte of water stress aid .ritiluhiljuu urea un the -KugiT content and nrganic Sicid riurinpr the »nowti-. period in the ^car fruits (Pvpu-.t rommuitrS L) cv. ¦WiltnmisMJhytenW: I 6?-] II. tto, A., Hayama, H. amd Knsllinilirn, V. 2002. Sorbitol meLubo-JiRm in h'^ds during fio'Aier bud formation; a corrparisan of Iwn Jiipynese p**r (J*ynis pyiifolia {Bum:.) Nat.) caltivais pOSSKSiil^ diftcrail fluiverine dabits. Sci, flOJtJC, 96; 1IS3-I7J. KEtin, iw. A_, norp, J, A. and Benlnger, C. w. 2005. aieet of elevated COj levels and leaf area removal on sorbitol, suerrM«. nnd phluridun content in 'GalaTMalling 9 l?ple leavea, J. Am, Sue Hortie. SO. 130: 326-330. Kozloirfki, T. T.and Pallartiy, S. (J. 1^*7. P1)ysiolfjLy of ^tiody pla;ils. 2nd ed. Atjufcmic prsss^ San Dipgo. CA. 411 pp. T.aitinen, M.-T.., Jtilltunen-TlltlO, R. and KOuSi, M. 2UDU. Variation in phenolii uompnonds within a hinch [Semlf *i«tt4utii) population. I. Chcni. Ecol. 26: 163(>-I622. Lo Blanco, R., Rtfhger, M. and Sung, S. S. 2000, liffcct of dtiouajlt vii SOtuitu] ajid sucrua; rnelubulism in .^ints lad sourqei of peach Physio!. Plant. lOfi: 71-78. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 11 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 1208 CA NADtA N JOURNAL OF PLAHT SCIENCE Lwschtr, W. II„ MbiW, (i. O. and Kennedy, R. A. 19B2. S^rtiiDpE metabolismar.d iinlt-suiipce iinsti.iiiiverann;; in develop. Iftg apple leaves. Plant Phj,siol. 70: 3"jtS—i3tS. loewlierh v\i, ji, stno h^verarrf. J. D. IWfr, Sugar almjhd metabolism in jinks anet sources, Pa^ea L85-20r7 in B, Zamiki ud A. k, &: liitfcr, cd j, PhOtoaSsimi kte distribution ir. p] a lili . 1111J ciuijs Snurire-sinJ; relationship, Mattel Dtktcr, New York, NY. .Yluyr, Uhl Treutter, D., SMUos-Buelga, C«, Timor, H. nnd ' V-i. ¦it, W, [995. De-velopmenfiiL changes V ttic phenol COtlMn (ratiiMH of 'tiotdeii De I ici OTIS' ipplt fmitF. and leaves Phylocheniisliy 3Sr L15 3-1 'S^-Matic, L. and russtn, C. 1*91. Clianses in tarbutr/dniUKt sod ciuyme Isvels uuiiug development scirhfbnl syithesiring .speci«. Physiol. Piani. 83: o.2 l-Wtj. Misirli, A,, Gulcan, R. and ve Tjtnr.jistver, A. lVWt A relation ship betwaen 8» phenclif coinpCfiJnds smd the resistance 10 Stkycnima [mtmiiia) iaui {Adectl e[ rabi.) in some apricot ¦varieties. Acta liii-rk. 384; 209-211. Mviiiid, A., <.'iirLn.u]uL-. V., 7irtpi*rlin, R., SynneJlih L. and flmirii ll$re, J, p. lft)7, Phloem loading in peaelir symplncric or anaplastic Physiol Plant, 1W: 480-1*6. Schioher, A, Keller, P. Dnd Cork-, R. KKN, Dercnminitli™ or plicnolic aciils and fiivunoids of apple a:i;l pear by hlgrl-perfoc-man« liquidchnorotograjihy. J. n-mrnntngr. A fli.fi: Ki-2Tx Sfcttlj, H,, Tatisz, M, Grill, f>. and Bstit, J^2UU5.Eicclterniea] responses in Leaves or' two apple Iree erillivars subjected to pn> greasing (franjln. J. Pla.it Physiol, ItVJ: E306-L31C. Siwk, Y, Taikuda, M,, Jlatann, S., Kansi.TiUiiii, \.L Vurinidii. It, Slm'altik«, K. and 'tainjikijS- 3)1X5. Changes in die acridity and gene fnpnfHion of SOCbiloJ- and sucrae-related enjymes wiltl leaf ccvrl-opmejit oi U Hrtmo:' pear. J. Jon. Snc, HoiTie. Sfi. 75; 45-Sff Swiinsnn, R. fi. 2003, Tannins and polyphenols. Pages 5729-573? fri 3. fJaballcro, L, C. TfUgO, and P. M. Finglas, cds. Einryelopediii of Food sciences and nutti:ioilh 2nd ed, Acudrraic Picas, London, UK. Trenti«', D. 2*81, BL'jfiynLhesiii of phenolic compounds 3tvi Its TrjjulaCion iuappte, Plain(JnVWIh EieguJ. 34: 7l-sq. Slwntli, V., Mikiilir.CtlkCu-wjk, M, Solar, A. and Slumpar, F. 31)84. Hiavrjicli of leaves m De]a;ion ki apple wab resistance. Z PfliTiienkr. Pflanycnscliulz lit: 137-144. Vebtric, R., Vodnik, D. uijud Stain par, F, 2IM3, CaTbOTl partt- [luiiing iimJ seasonal dynamics of car&otiydraies in the burtc, leaves and fniiis of apple (JWaJhj rfvmttftca Borkh.^ vv. 'tjuititn DciiCkms'. Eur, J. Norhe. ScL 6H: 2Z1-226. Wang, ZM Pan, Q, and Qucbcdcaux, JJ. 1S9P. Carbon pajlitroa- irig into sorbitol, lUCrose, a^d itartb in joun;e 31(1 lint apple Jcaiies at, effected l)y elenled COj. Lnvimn. Ei-p. Vict. 41: S9^lr>. VVuturi, J« Kohio, V.H Vamakl, S,, '¦> -j «'.:u\i. k., ToyDfEUtu, K., TnbtlChE, T. and Shiratake, K. ^004, IdeniilicatLm of so:bitoL ifaitEpcrtert exprcsied in ihe phloem c-f apple source leaver. M . Cell Physiot. 4S: 1032-104J. Zhou, R. flftd OaebedcauK, fl. HIOJ. Cbatlgea i:i plitUuiv j[lies:s Bnd<;ai'lJC(ty[J[aLe.Liit:lLbr?li.HminmaturrH}inJE leaves i n FpponW K> whrtlfi pEanl source-sink manipuktion. J. Am. Soc. Honic. Sti. 12K: 113-119. Zhfluh R„ Siclter R. C. and QnthtdtHui, B, 20IR Apple kaf sucrosc-T)h) (md Cflffeic acid (»,72-1.04). Signincam difftrences in content Of fructose, sorbitol, total sugars, catechol, epitaLcchin, ainapic ado, syringic ocid, sad » sum of determined phenolic compounds wcro observed armmr the treatments. Fruits from summer bending branches had die lowest content of individual sugars, citrit ucid and phenolic compound* and the highest content of malic, shikiiuic and fumaric acid. "ITie highest content of fructose, sorbitol, sucfuse, cutal rogarn, caflfeic acid, catcchin, epicatechln and syHnglc add were determined in tho fruits from iht &prinS treatment, In tlie control treatment the highest content of glucose, cEfcrie acid, cidnrajnmic acid, sinapic acid, vanillic acid, sb well a sum of determined ptlMloLiCs, were observed, The lowest content of fumaric acid was in the spring treatment and of malic and smkimic acid in the control. ® 2006 Society of Chemical Industry Keywords; psar fruit; sugars; organic adds; phcnolks; bending INTRODUCTION Chemical composition of pear fruit, which determines its quality, has been mvestisaced to a lesser extent in comparison with, apples o? peaches, However, for :te dfitermiuaTiori various chromatographic procedur« exist, among which high-performance liquid chromatography (HPLC) has been used most frequently. Tn i his way many researchers' * have reported that pear fruits of the cttitivar (cv) 'Williams' contain fructose, glucose, sorbitol and suirmse. Among organic acid's, 'Williams' pear fruit* l.i?h ain citriCj malic, shikimic, fumaric and also ojuime add.2 ¦5 Chlorogenic add was the ma;or pheimlit in French pears cv 'Williams1, followed by epicatediin and ±en eatechin.' Separately from pear pulp of cv 'Decana' only chiorogenic acid and cpicatcchtn vvere isolated by Eacarpa and Gonzaieii,15 whereas in the pear peel arbutin, eatechin, cafieie acid, rutin and some glycosides, a& we3 as chlorogenic acid and catechin were jdenulied,13 In Portuguese peaf fruits the highesi content of chlorogeriic acid and flavonoids was also found in the peel, whereas in ths pear pulp only clihimgwric acid was determined,^ Leonto^ica ^r fU,'* liettcied siynifit;antly higher content of phenolic acids in Isiaeli pear peels than in pear pulpSj and Spanish pear peels and pulps vfw 'T5lanquifla* wefe an even better source of phentjlin acids." Similar findings have been obtained in other studies, emphasising th-e importance of consuming the whole fruit Jwidi peel), because phenolic* are a natural sour« of antioxidants. However, the fear of pesticide residues tsftea leads people co peel 9 fruit before eating it, Consequently, f rganic fruit is more desired for consumption.: i-'l Sugars and on;an:c aad composition of pear fruits grown in Slovenia have already been investigated^"1 Nevertheless, there i&no known information available on the individual phenolic content of pears ejrown in Slovenia Each species1"1 and each cuJtivar?,l:; of fiu i i 1ih.s its uwu significant chemical composition l hnw/evet, differences in compound amounts may occur dcpenditig oa ihe tnaturity stage,7,16 environmental tac:ors and iKizhnoloiical measures (i.e., training system, priipijjgi tree nutrition, irrijaijon) applied in an orchard.-' Consequently, bending treatment may aflfect chemtcal composition. Ico ei id.IS reported the influence of bending shoots on (lowering und au.L?ir mctHbo'ism15 of lateral buds in Japanese pear (fynu pyrifolia {Burni,) Nak.1. Bending is ' ConHBOCriefica to: Mar^a Cm^ De^srtTienl afigmunv/ Eiot»ctink:al Fae^ly, Unlraratycl LJubl^n« Airmikarie-^a Id, 91-1005 \±Maa. Sbranla F-mH f irBTe^a.colarlSftirju^- \L\ Cantrac-J$iaM (pMSWGlMrln MtnfetryrfHk*)« Eduoatloa Sciertci fry) "(Khndosj, GUiHWumt nuTitJEf P4-IX'ia-04A1 (fllKtrnitf 7 September L(HJs; rsri&sd vi sorbitol, citric acid, ihJkimic add, fumade acid, (->epicateehin, cafMc add aid venilliL at:i equipped wide u jshtito diode array (PDA) detector and controlled by the ChrtimQuefl 4.0 Chroinacojrraphy workstation software system. Separitions were carried out using a Chremsep HPLC cohimn S3 '2^0 k 4-. 6 mm, Hypereil t> ODS) coupled wiih a Chromsep guard column SS ("0 x 3mm) from CliLoiiipads (Middelbu^g! The Netherlands). Column temperature was maintained atSS'O. The chromatographic conditions were similar to iliose previously described by Schieber i?r til.,"'' Willi .-ninor modjficaljons. The mobile phase A ivaa 2% acetic acid prepared in bidistUec water; solvent B was. 0,5% acetic add in bidistilled water and Dce:onitri]e (ratio 1:1). The gradient was as follows; 90 to 45% of J Sd Prmd A&ts. S6r246 J-2467 (2000) DOL 10.1002/jifa Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 15 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 16 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 M Cofcric a at. TaMsS. AvHra«e conlan! of pTermllc compound? in IMNarns' acaiS atBr different treatments Phmoic canprxino CiHturfim Chbrogcnc acid FplcaftKhln Einapic add Bangle sfna VflrSic scki Sum Summer 2003 0.7S ±0.11 26:67 ± 1,746 2L>,S6 X L3.45 46.55 ±L.T7a 0.83 i O.GSa 35.46 ± 4.30a 1.87 ± 0.03 451.95 = ol ,50a Bending ^reafmant Sprinc 2D0fl 1.04 ±0.12 44.81 =5.H/tj 313.17 ±LL.62 a3.09± 10.56b l.18±0.nab 131.32= 10.33b ^52 ±0.65 5EL 132 = 4G ,48b Central Q.30±0.1L 36.60±4.6Gab 357.34 ± 2Q.ee 50.82 ±10-3Seb ¦ .72 ± 0.26b 12C.65±9.S7ab 3.43 ± 073 5SC.3St*18iflb Valuta are urtniw itmJ as 1he mean ± SE In mg kg n FW. Vsluos 'oHcwad by different letters within o»ch iw am signrf tartly different actefding to 1he LSD mulliple-'Si*** tal at P -^ 0.O5. Content of phenolic compounds As shown in Tabic 3, cb.lonoeen.tc acid was the major phenolic compound found in pear fruits h Thai is in accordance with C'iffotd,^ who reported quite siir.il ar ch I erogenic acid content in pear fruits (up to 2S0mgkg' ] FW). I.ower content of chlorogenic a;id was reported for 'Williams' peats (up to 10C tug kg"1 FW) by Herrmann* and Atniot m tzl.7 arid cvun lower content for cv 'Red Williams* (21 ing kg ' FW) .*¦ Comparing Our European pear fruits (Fyrus f.nmniu-ttii L.) Cui « a/J4 observed that European pears were a much better source of chlorogcnic acid (309 mfk^'') than were Oriental piara rJV&« breisthnrideii) (163 nig fee.-1}. Juice made from 'Williams' pears contained I74mgL~" of chlorogenic acid, as shown by the findings, by Tannoven and Frksj,3' Syringic acid showed nearly one third the con-ctiitraiiiiii of ^Monogenic arid content in -Jiis study, Epicatechin cojiteini was very cltoe tu that shewn by Tsanova-Savova fit iil.,?s wtio reported iha; prat fruits contained 2Q.9-8L.2uigkg~L FW, Herrmann* found 'Williams' pears had up to 30rag kg-1 FW of epicatechin, Amiot st alJ cited 21 retake;-' FW of epicatechin snd Tannoven and Eksi37 determined that 'Williams' juice contained 34.9 mg L ' of epicatechin. However, Schieber siAr.H reported that 'Red Williams' pears contained only up to Gmgkg ' FW of epicatechin. Cftiechin valuta were about one half of epicatechin iji all t ten tin en is (up to 44. Si mgkg_] FW in pears from spring treatment). HerniianTi' reported that 'Williams' fruits exhibited up to lOtnykg-1 FW of catechin, Tsanova-Savova a al'j:i detenriincd lower mean values (3.7 mg kg-1 FW) and Aiuiot a ai? also cited similar values o I' catechin (5rngkg_I FW). Vanillic, sinapic and cafleiL- acids were minor phenolic com.puu.nds found in pear fruits. According to the findings of Tannoven and Ek^i,27 Williams" pear juice contained 11.0mgL_J Dfeaffeic add. Much higher ennrenr of caff tie acid was found in Israeli pear pulps (19Buujkg~ ' FW),"* and Spanish pear pulps of Cv 'Blanquilla1 were an even better suurL-e of cafleic add C?01 mglce;-1 FW).|J Atburin was also identified, hut otiring to difficulties in its separation at the befiinning of each HPLC analysis the data ars not s?iown here. Schieber et ?i,-2 also had problem* with arbutin interpretation; it* content in cv 'Red Williams.1 was only 0 4mgkg-1 FW, Gal vis-Sanchez u cd? analysed pear pulp ant: peel separately, and arbutin, was found only in the pear peel U 158 mg kg"1 FW). Frui:s (rem summer bending branchci (! September 2003^ had the lowest con;ent of all individually analysed phenolic compojnds. Content of catechin. epicatechin: sinapic acid; syringic acid and a sum of tujalyned phentjlics differed siguif'cantly among Lie treatments (ANOVA, ISTI lesi ar P'< 0.05). The hiehest conccnuations of caSctc acid, catectitiij epicat-cchin and syringic add were detected in the fruits from spring bending brojichea. Chlorceenac acid, sinapic add atrd vanillic add, as well as total analysed pheno-¦ics, eahibited the highes: values in control treatments, We propose thai the bending of branches altered i"hr branch mttabolism and consequently infliiL-nccs uri rrt]f: composition. D-j« to lower content of most analysed compounds in th" fruits ¦from the summer treatment and higher content from the spring treatment; it is suppoicd that spring bent branches Saved the assimilates i'or lrui"&, but summer bent branches spent assimilates for other tissues before the fruit required them for development and growth. CONCLUSION This investiracion demonstrated the sugar, organic acid and phenolic composition of 'Wiiliams' pears. Fruici! wer; harvested from branches on trees; involved in three different trc-atir.ents (branch was bent in summer 2003; branch wan bent in spring 2004; arid nu Iwni branch -control). Statistical analysis showed significantly lewer content of fruuttise, sorbri.il, tum] sugar?.. cate;hins epicatediiti, sinapic acid, syringic acid, and sum of determined phenolic compounds in fruits from the summer bending branches. Significantly, the highest Content of fructose, sorbitol, total sugars, catechin, epicatechin and syringic acid w?r? in fruits from the spring bending branches, art(! i he highest content of sinapic acre from the control ireaim;r,t. It has been shown that differences J 3ei Fff^Agric B&2463-34G7 (.2006) DOI: 10.1002Jjsfo Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 17 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Jnfiuerhic uf bending en sugar > urgainic acid arid jiliertolic content in jwars among treatmisite regarding content qfanfare, organic acids and plisnolics could be :hje rem.t nf branch reorientation. Further E-cudiej ate needed to confirm and clarify lio-w flicse differences occur. AKNOWLEDGEMENT Tins work is pan of the pn>grHim Horticulture P4-0013-0481, granted by die Slovenian Ministry of Higher Education, Scicnzc and Technology. 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L.lj J-Z and Ka/s!ii*A H^ Analyse of nrhi.ilin urn: ch'orogCTiif a^idj [hs U)ljtirphsn^li;^i^nptitVltnti in Onen;s,l pear. J /Igric Ftvd Ghent Sl&lB&l ¦ 38 B7 (jKJOS). 15 CwLaritM, Vebenc R, SiamnarF arnl Hudina M, Evaluation o:' peatli sjid r.ecLaiine fruit iuaii;j snd eorrclaiL-otu bctwrtu. atniocj and thrmiat Jttr.butrs. 7 Sri Fmu ^s* j;:Jt]J-Jfc!b (3005). 15 Siuim K, K«rvn D and Stimpar F, Tltc coOWOsJlioti uf fruit ofOllffioSfLl mrawbErTj'viricrits dcrondtns or muftinty ststpr. IvmMJ/itm 83:417-122 fW03J, n IludiraM. and SutmpurF, Influcnji,!; uf water Fticsa snd atamilflrio-n arcs, o.^ EhK sugur onnt^n- R^d organic acid du^in^ th* growth period in the peir fi-uit> {^vw «|j|flr«t!tj L,^ tv. "Williams'. Pft>mnM:H)7-]l I (HMji, L 8 ItoA, Yacgaki H, Hnyumt Fl, Yflmngv.chi 1, KuaabaS ajid VoEhloki H, ReiudLnE stifuis aumulaiEs dowering anil indntrces liurmtirii; 1?it1^ in lateral buds *>f fipau^ffi flsar. HatiSiaiir.ui %4:l224 IJ18 (LWD). J* [to A, YListiiuLa H, Hjjjfarri!i H and Kashimura T, RconcmaTJon of shoois to rtu? horisonul pHxih'or. influenoEE [he fii^ar nvj[ihi>liim t>? Literal buds and shoot iotcrnojd« in Ja].'Ln«e psnf (f^Tu-i fyrifeiic (Bunti.) Nat.). J Han Sn Bistmii 75:416 422 {2004). 20 LuokwiLLC, Tlic rtralrw] cf xruwlti mnd fruitlu]n:sa nf spplc irc*s, ji PAymrfuigy o/ jnw O^j, ed. by LuclmiD LC uid Cutiinj CV. Addemk frrss, London, pp. 237-234 fWO)- 21 Dolenc K end Srampnr P, An inv«dga[itin of die appJitiiLSn urji '^u^ditioni tjfauolyafsof HFLCnuahyds fgT dtrcrmanhs tujMrs and Hjrsank aiids b fhitrt. Jfj-j ffr^j Biat Foe UL 6ft«-ICfi flWTJn 23 Sebi;i;r Aj KcLkr I* and Carle R, DeterniinariiMi of ¦¦!.:¦:.¦:. ^cid^ and ll.^viirkiiiils ^rappl^arid p^'arbv hijtb-ptrfirfmartL'^ Hqnid fhitmutosiaphy. J Cftvinwirqgr/f 910:26! -J,Ti 0€C1), 23 LlhtsuIicj^'H and Everaid JD, S-uear akohot mcrarjollsin da rinks ajid aoutcijF, in Wpicwjj'miter* itjidrrhvijijr m finMj d«f Cmpr/ Sc«Jte-5.>jft EtiJa:i}uifii, 24 TengY, Tamuis Fj Ts,nalx;K and NakaiT, Parndajiing rartcrns of pliutosynttwtcs from ddlcrcnt ehiooi Eypes in rSiiivitiki' prjr fP>FTii f/\-n~lo!i-i NaJcsi). J Hvn Set Biuiecjt 77?7^Jl-7n5 (2002). 25 Diie;nV Jt>, Aerda, in Etxyclupmdi&zfFaxi$&*(& mdNtiriuim f2nd *dn), ed hj1 (TlaEialJein R; Tnieo LC ?nd FbiBisa ?M Academic Pjcss, I»ndnn: pp. 'J 17 (2003J. 26 Clifford MM) Chl^roserdc atids ar.d oiLk: drLuauiaLta: icatyre, occij?renes onri dfetsr?brrntcai, JSerFmiiAl^c TV-:ih2-ni H;ii D and Ehai A, Piicnolkc cnni|«uiid\ in pe*r iukr Irom diBTerent ^uldvais. PmiJCWr «S:9j? y J (3005^. 2B TsBjiova-Sa-fora S, RLbatvvn F cud Gciuvu. M, <+>Caircbin and (-J-cpictrechiQ m B-jIgstisn. Ehiitc. /Fuod Camp«-Jaa,' MtlWl-Ma.fjMOS). DOI: 10,1032/JEia J1S7 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 18 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 2.3 UPOGIBANJE VPLIVA NA VSEBNOST DOLOČENIH FENOLNIH SNOVI V LISTIH HRUŠKE SORTE ‘WILLIAMS’ COLARIČ Mateja, ŠTAMPAR Franci in HUDINA Metka Bending affects phenolic content of William pear leaves. Acta Agriculturae Scandinavica, Section B, Soil and Plant. Science, 2007, 57: 187-192. Prejeto 31. 1. 2006, sprejeto 24. 5. 2006. Upogibanje vej je pri sadnih drevesih uveljavljen tehnološki ukrep in zelo razširjen v intenzivnih nasadih. Učinek upogibanja rodnih vej se odraža tako na zgodnejšem in večjem pridelku kot tudi na zmanjšani vegetativni rasti. Na biokemičnem nivoju je bilo ugotovljeno, da vpliva upogibanje na vsebnost hormonov in njihovo premeščanje ter na metabolizem sladkorjev. Učinek upogibanja rodnih vej hruške na fenolno sestavo listov do sedaj še ni bil poznan. Fenolne snovi so pomembna skupina endogenih rastnih inhibitorjev, ki imajo v rastlini pomembno vlogo v odpornosti na bolezni in škodljivce. V poskusu smo naključno razporedili 30 proučevanih dreves hruške sorte ‘Williams’ v tri obravnavanja, kjer smo upognili po eno petletno rodno vejo na drevo, in sicer 1. septembra 2003 in 15. maja 2004. Tretjo obravnavanje so predstavljale označene petletne rodne veje, ki niso bile nikoli upognjene - kontrolne veje. Z upognjenih in kontrolnih vej smo vzorčili liste od maja do oktobra 2004. Določili smo naslednje fenolne snovi: rutin, epikatehin in katehin, kavino, klorogensko, sinapinsko, siringinsko in vanilno kislino, katerih vsebnost je bila najmanjša v maju, nato so vsebnosti posameznih fenolov naraščale, dosegle največjo vrednosti v juliju, avgustu ali septembru ter se zatem do oktobra zmanjševale. Med posameznimi obravnavanji smo najmanjše vsebnosti pri večini določanih fenolnih snovi izmerili v listih na upognjenih rodnih vejah, zlasti na vejah upognjenih pozno poleti 2003. Značilno največje vsebnosti posameznih fenolov so bile v listih iz kontrolnega obravnavanja. Domnevamo, da je sprememba kota rodne veje hruške sorte ‘Williams’ sprožila fiziološki odgovor z različnimi vsebnostmi fenolnih snovi v listih z upognjenih in neupognjenih vej. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 19 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Aeia AgrkultitKe Scaxdmavica Section E^Soil and Piani Science, 2007; 57: 187-192 ORIGINAL ARTICLE Bending affects phenolic content of William pear leaves MATEJA COIARIC, FRANCI STAMPAR & METKA HUDDStA Depawneni of Agronomy, Biutechtarai Fondly, Ihasitraity ojI juh^aaa., F.jub^ana, Skvenia Abstract ITl« *tt*et of branch bending (in th« phenolic content in pear leaves was invesflgated. Leaves were sampled from the tit* bran-ches which were bent in summer 5003 '\ September), in tpr,ng 2314 (\5 -Miy), and from unbent ire«, Samplings vine noade djrinj- the- growing season May to October 2004, din die following dat«; 1st sampling, L S Maj; 2nd sampling, 11 JuJKj 3rd sampling, 9 July; 4th Mmpiing, i August; 5 th Wnpiine., 2 September; and f)dt jampLing, i October. The leaves contained caffcic acidj chlrrOEffnic acid, sinapic add, syringic acid, vanillic acid, ruliu, epiuaieelli.i and catecliin. The Lowes: foments Qfplienolies 'Vf.rc. ffmnd on Che firer neensmn. Arrerlhat, in Ihe ieujuence of Samp ling dicei, an increase was noticed at fitac, but aftet fetching their highest point, the cor.tents of pf.eruolies decreased. In most taies fewer contents of phenolic suHiipOTindi were fcufid in leaves from bent branches (especially from branch« bent in suinnur), and the highest ori0-7), Likev^&e, Grochowska, Htsdun and Mifea (2UU4) showed chat branch bending increased fruiting of apple trees,, but it 1: :u: no effect on pluoi trees, I:t ihe lateral buds of Japanese pear* branch bending Slimuiflted flowering ai well as influencing hormone levels, rheir distribution and transport (Ito^ Yaegdki, Hayama, Kusaba, Yamaguchi & Yoahioka, 109.9), snd sugar roetabolwni (tto, Yoshiokn, Hayama & Kashimura, 2004). Sanyal and Btiigerth (1993) found that branch bending had an influence on the increase in flower budi number, higher internal ethylene production* and on reduction in polar auxin transport and cyioldnin levela in the branches. Since r»o icnowri literature pravices informatiori about che effect of branch bending on the phennR: eantcnt of fruit ttee leaves, its effect was examined in thin study. In general, only few studies on the ron;ent of phenolic compounds in pear leaves are available; neverdiekss, phenolic compounds are widely distributed in plant tissues where they play a signif.cant mle in general defence sii'ategtes of plants (Swansea 20031. K07kiwski and Pallardy (1507) explained that phenolic compounds belong- to a large fironp of ecidogenous growdi inhibitorii and can therefore CMYEn«nd«m: M. dlHr.C, DcOflfCDlTir nf Ajr'flnnmy, EimMtinkn: Ficully. trTTivcreity ntT LjjhljzttB, Hatotiiteqat IUI, SJ-IUIK) Liublpns, Sbivtnia. Ty: 4-?Jfi L «1 II L]. Feu: -HJS6 I tS [<1 *S. F-nnail: miir:s mlnri^Hh-.imi-li.si rssNJi(;iw-4Tin [wiWtsstJ ibsj-jsis aniirte^i st;o7 m&t & vtxsb iwii' ki iLiMM':ynrj;njfinuhmiTH'h Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 20 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 I S3 AfL Golaric ei al. affect plains directly by influencing their metabolism, growth and development. The aim of our research was to evaluate tiie effect of the change in branch angle on content levels of phenolic compounds in its leaws. Therttore, William pear branches were bent in Late summer and in Iste spring; ^r.d then phenolic variations among bending and control treatments were determined at different sampling dat;s. Materials and methods Plant tnausrial The experiment was implemented on pear trees {Pyrin consmtutii L.J, cv. Williairij grafted on quince MA. The trees were planted in the eastern part of Slovenia (Bistrita ub Sotli) in 19B7 and were divided into three different treatments (10 trees per treatment). One five-year-old branch pet tree on the tree's eastern side was bent a L an angle ufčO'1 from a vertical 10 a horizontal position: 1) in late summer 2003 (1 September - the summer treatment), or 2) in late spring 2004 (15 May - the spring ;rearrnent)j or 3) was not bent at all — the control group. From those branches, healthy leaves were sainted six times through the growing season from May to October 2004 to evaluate variations in rhe leaf phenolic composition among treatments. The samplings took place en the following dates: 1st sampling, 15 May; 2nd sampling, 11 June^ 3rd sampling, 9 Julyj 4-th sampling, & August; 5 th sampling, 2 September; and 6th sampling, 1 October. On each sampling da:c, 12 representative pear leaves were picked from the same marktd five~ycar-old branch and frcm the equally long shoots to mtke up one sample. For each treatment, eight replications were mads. Leaves were iiiiineUJaielv frozen iti liquid nitrogen and then lyophilized. Sample preparation Edch sample was ground in a mortar. 10 achieve a fine texture. Precisely 100 rng of taidi sample was weighed into a icsi tube and extracted with 10 ml of methanol containing 1% of antioxidant agent BHT (butylu:ed hydroxytolucnc or 2,^i-Mrr-butyl-4-methylphenol) tor 45 min in an ultrasonic bath cooled with ice. The extracted sample was csntri-fuged at J 2,000 xg flor 7 mir. at 10" C (Eppendorf centrifuge 5810 R, Hamburg, Germany), The supernatant was filtered through a Chromafil pclyarnide filter with a 0.45 11 m pore diameter {Machcrcy-NageS, Diiren, Gertnany), transferred into a vial and used for HPLC analyses of phenolic compounds. Atialyiei cf phenolic compounds An Hl'l.C analysis was performed using a Surveyor HIJ1.(' system and a diode array detector, controlled by a C ]hromQuesr 4.0 chromatography workstation software system (Ihermo h'innigan, Nan Jase, (TA). Separation of phenolic compounds was carried our using a Chromsep HPLC column SS 0250* 4.6 mm, Hypersit 5 ODS) coupled with a Chromsep guard column SS (10*3 mm) froin Chrampack (Middelburg, The Netherlands), and the column temperature maintained at 25CC Two per cent acetic acid was prepared in bidistilled water a& solvent A. Solvent B was 0.5% acetic acid in bidistilled water and acetonitrile (ratio 1:1). The gradient used for the phenolics analysis was as follows: from initial 90% A to 45% A (50 min), from 43% A to 0% A HO min), from 0% A to 90% A (5 min) (Schicbcr, Keller & Carle, 2001). The analysis deration was 65 min, with 15 min of cquilibtiation treatment (90% A) between each analysis. The flow rate was 1 ml min- , The injection volume was 20 til. lotriiijictiiioti and quantification of pfienolic compounds Identifica-jon was carried out by monitoring absoi> baoce at 280 nm for catechin, rutin, vanillic acid end epicHLediii), and a L 320 inn for chiorogenic acid, syringii; acid* uaffeit; add and sitmpic add, Quantification of analysed compounds was carried out as previously described (Coiaric, Yeberic, Sniar, Hudna & Stampar, 2005). Also, standards of phenol i cs, which were dissolved and diluted in methanol; were uses for identification and quantify cation. Dam (tnafytts Dara were analysed using the Statgrapiiics plus 4.0 ptogrnrnme (Manugisries Inc., Rockville, MD, USA). The bending effect on rhe content level of individual phenolic compounds was tested by oneway analysis of variance (ANOVA) for each sampling date. Means were separated by the test tor least significant difference (LSD) at a significance level of 0.05. Results and discussion Tn William pear leaves, the levels of caffeic, chloro-gsni;, sinapic, syringic and vanillic aeid, as well as rutin, catechin and cpicstcchin were defined. Generally, chlorogcnsc acid was found to have the highest conren: in all treatments compared to the other compounds analysed - the opposite of sina pic acid; Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 21 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 22 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 23 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Phenolic čDntcrtl nf William pear Uaves 191 very similar crmtems, and after reaching- 3 peak no earlier than the fifth sampling date (Figure 1), thai contents declined, Mayr, Tre utter, Santos-Euelga, Bauer nud Feucht (1995) tlso reported epicstechin as exhibiring a maximum value in older apple 1 eaves. It is- evident froc[] die results above that branch bendinz and The lime when ii is carried out do influence :he contents of phenolic compounds^ which are important endogenous growth inhibitors that can affect plants directly through their involvement in mctabolianij growth and development (Ko-zlOwski & Pa! lardy; 1997}^ the reason being the existence of phenolic compounds in various pans of the plant, such as root, branch, flower and leaf hud, woody tissue;, leaf, phloem^ pollen, and stylus (Mis-irli, Gulcnn & ve Tanrysever, 1005). Apart ftoni technological growth, regulation measures, such as bending, with dje aim of reducing vegetative growth and improving flower bud quality, alternative solutions were soughr. During tin lastfcv years, the chemica! growth regulator prohcKadicuie-calcium (P C a) has proven successful as one of these, Furthcnrtorcj, this solution controls fire blight infections and PiyUa fiyri multiplication, an-d i L llms considerably reduces the required application of fungicides (Deckers & School^ 2004). It is being recommended because prohexadione-calcium alters f]avonoi;¦•.;,-.:¦ -v: Scxnti Sr BioiecJwelvgy, 79, S52-25V, fJuntn, If., MiiLrli, A.h & Gultan, R. faOO1!). I^af fihanAlk CJftle-nr of ixar Culli\ftrS reLiilllnHjrtijSCcii:Jble tu fire Might. Scamiu HorUCullKrazi JW, 2 13-221. lie, f^t Yoshiotiij H„ Haynmt, H., & KashLmmra, Y (L004). WivriuiwTwn of iboo4J to the huiiauinal poHttcm influi.-3i.Li rh# sii^nf meraravreiT] (jflstcrjil biidsjiid ^.oot mwrn/>dc?in Japantst pear \P$nts pynfolvx tjtotm.) NilkO. J'JurrMj v/ Horpcutwret 5crmof Or Sioieclmeftgy-. 79> 41d-422. Ice-. A., Yaegaki, H., Hayamaj H., Kuaata^ S., VimaguL-Li, L, i ISxhiuka, H. (tyfJ1?). Rend ing ^rjvfi sljm-ulut« fi(iwip"inn and infliijnci« hnrmont Imi in lutrrsl one.? of Japanrae p:sr. HonStiesKS, ii-, I214-i22-i. KoikfinM, T. T. & PmUnly, S, G. iliVT) WbyAAoff at wiauV plflLita. Atatitrnit Prt^t-. Sail Diego, 41! pa. Lsuri., P. E., Si Lespinasse, J. M. (SfVJl). GtmQtyp? vL iippl^ tr=& zfft.ax trriiwlh arr| fn.lit]nj Ji^pon*"* CO iJinot bfTElnf af vnriou-i timei al" year. Journal af tan Amttiam Satiety far HurtlL-aliural S^'fti \2§, l(j^-|7il, Ljclnvili, L. C. (I°70). The Munirel Dl gjcuvtli and friLtfijuiara of applf ir«s. InLt Ijittwill, & CV CjnLvj (ErLs.^ Pftysivaigy u} ins Umps (pp. 237-254). Academic lJresnh Lnn(fnnh New To* LundegBndh, B., tk MartensWPj f<, (^003)- Organically produced planl ;'pocIe; evirifti« cf liealrti benefitE. An* A^ri\,Kliain\i SjanJinEii«!, šfcitott B, Sei! ceid Plant Sctttcf-. 5J, 3 15. Mxyj> U.>Titirtncr^ D.j Sintoa-Buclsa. C.j Bauer, H..& Fcucln, W. (JW51. Etvelopmenial ;hapg!ts in rte ptetiol :i>nc(Jntfa-Liuils ul" 'Gulden Delidyna.' aipte Jruirs and [rarea. Pky.o-~J.wiilxry, Jfl, 1151-1153. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 24 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 102 M. Colark et al. MisiiK; A.sGuLmus R., &veTanry5ev?r, A. :19P5), A relationship between [he phenolic compounds smd [he tesistarue to ScJtnrtiitKt frjigijjJjgJ fcra (Adech tt niti,) Lil ;omt aprtiH Tardeties. Aaa Homtuhurat-, 184, 205-211. Itftnattli, S., XtelUItti JI, Sptukmen, H- B„ ft Kacfcrnichcr, W. (ItUJti), Tvffrr.tt nf prohrxadionr-Ca »n the ftavo-ncdj iijotLi-boliso of applr wiiti jcipect ro plane rcslsTan« agjiita fiic blight Act:: tfWtttVbnM, 4&>, 351)-34 J, Sbt.ysJ, D.j, a Bangrrttij F. (i«S) Stresa induced ethylene evnluti^n and it!.; posdMc r?ljifi pn-ship to HUltitl tJSin^por:, CTpiobimn levclsh and Hcnvrx bud induction ir. shows of Jpftk =*ll:J btii^ u.id bearing applt lrcu». Ptant fjrwth RtguitlfiWi > 24, 137-1 3-4. Sthkhcr, A., Kdl«, P., tV Cark K (2tf01>, Deteimitudtm of pbienulic acids and flavrttttitls of apple Drvi pwf hy high-ptrfun \vnvx Ikguiii ttrummltajaplij. Juifntrf of Giirommogn-pfy-A , 919, 265-27J. Swamnn., B. G. (IQQJ) Tftftnins and poJyphenols. la B Cabillsn*. LC Ttugn, & PM 1'inpla« (lids.), Cttiythpoetlia a/ ftitfn1 iScj'fjimj cpicatechin, vanillic acid, quercetin-3-D^galactoside and quercetin~3-[i-D-glucoside were purchased from Fluka Chemie GmbH (Buchs, Switzerland). Malic acid was obtained from Merck KgaA (Darmstadt, Germany) and (+)-cateehin from Carl Roth (Karlsruhe, Germany). Chlorogenic acid, sinapic acid, rutin (quercetin-3-ruunoside) and butylated hydroxytoluene (BHT) [2,6-di-ter/-buty]-4-metuylphenol], used as an antioxidative agent in the extraction solution, were purchased from Sigma Chemical Co, (St. Louis, MO, USA). Methanol, acetonirrile and bidi stil led water were of IIPLC grade. Methanol was obtained from Riedel-de-Haen (Seelze, Germany) and acetonitrile from Sigma-Akfrich Chemi e GmbH (Steinheim, Germany). B i distilled water purified in a MiJli-Q water purification system by Millipore (Bedford, MA, USA) was used- Sugar standards were prepared in bid i stilled water and phenolic standards were dissolved and diluted in methanol. 2 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 28 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Plant material and treatment The experiment was performed on pear trees - 'Conference'/Quince MA, planted in 1987 in the eastern part of SI oven i a. The peaT orchard was maintained according to standard commercial practice for integrated fruit production (orchard management and cultural practice, i.e. spraying, irrigation, etc). Three different management treatments were applied to the trees, which were chosen randomly, and each single treatment was repeated on ten trees, One 5-year-old branch per tree [on the tree's eastern side and of comparable properties) was bent down to an angle 120° from the vertical position in late summer 2003 (summer treatment - 1 September) or in late spring 2004 (spring treatment - 15 May), and the third treatment was a control (branches were not bent). All bent and control branches prior to the experiment were grown at an angle 45° from the vertical position. The bent branches were tied down, vising a cord and thus remained at the stated angle throughout the study. All bent and control branches were allowed to develop without pruning from 2003 through 2005. Branches fruited and were harvested on 9 September 2004 and 1 September 2005, The weather conditions (temperature, sunshine duration and precipitation) during the growing season 2004 were comparable to the long-term averages (1961 to 1990), but in 2005, 174 and 183% more rainfall was measured in July and August, respectively (Table I), than the long-term averages for these two months. In 2004 and 2005, healthy and mature spur leaves were sampled monthly from May to October (on a sunny day, at midday, in the middle of each month), immediately frozen in liquid nitrogen, then lyophilised and stored at -20°C, awaiting carbohydrate and phenolic extraction. Extraction from leaves The dried leaves were ground in a mortar with pestle. For carbohydrate extraction, bidistilled water tip to a final volume of 50 ml was added to 1 g of leaf tissue and left for 45 min at 20°C, with occasional stirring. Afterwards, samples were eentrifuged at 12000 g for 7 min, at 10°C (Eppendorf Centrifuge 5S10 R, Hamburg, Germany). Then the supernatant was filtered through a 0,45 urn cellulose mixed Esters filter (Macheiey-Nagel, Diiren, Germany) and used for the carbohydrate analyses. For phenolic analysis, 100 mg of the weighed sample was transferred to a test tube and extracted once with 10 ml methanol in an ultrasonic- bath for 45 min. The mediano! contained 1% of the antioxidative agent BUT. Afterwards, the extracted sample was centrlfiiged at 12000 L for 7 min, at 1GCC. The supernatant was then filtered through a 0.45 um Chromafil poiyamide filter, transferred into a vial and used for analyses of phenolic compounds. Carbohydrates and phenol ics analyses Carbohydrates (sucrose, glucose, fructose and sorbitol) were determined with high-performance liquid chromatography (HPLCj from Thermo Separation Products (Riviera Beach, FL, USA), equipped with a Rezex RCM Monosaccharide column (300 * 7.8 mm; Phenomenex, Torrance, C A, USA) operated at 65 "C and a refractive index (Rl) detector fur monitoring eluted carbohydrates. Bidistilled water was used as the mobile phase for the column at a flow rate of 0,6 ml min'. Total run time was 60 min. Chromatographic separaliori of phenolic compounds was performed using a Surveyor HPLC system (Thermo Pinnigan, San Jose, C A, USA) consisting of a vacuum degasser, a quaternary pump, a thermostatted autosampler (10°C) and a photo diode array detector. A Chromsep HPLC column (250 «4.6 mm, Hypcrsil 5 ODS) operated at 25°C, coupled with a Chromsep guard column SS (10 * 3 mm) from Clirompack (Middelburg, The Netherlands) was used. The HPLC system was controlled with ChmmQuest 40 Chromatography workstation software (Thermo Finnigan, San Jose, CA, USA). The chromatographic conditions were according to Schieber et ai (2001), Acetic acid solution in bidistilled water (2%, v/v) was used as the mobile phase A, and 0,5% acetic acid in bidistilled water and Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 29 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 acetonitrile (ratio 1:1, Wv) was B. The elution gradient was: 0-50 min, from 10% to 55% B; 50-60 min, from 55% to 100% B; and 60-65 min, from 100% to 10% B. The total run time was 65 min, then washing and reconditioning of the column was performed for 15 min between each analysis (10% B). The How rate was 1 ml min~\ and the injection volume was 20 pi. Phenolics were monitored at 280 and 320 nm. Identification and quantification of phono lies was done according to Colaric et ol, (2005). Dora analyses Data were analyzed using the Statgraphics Plus 4.0 program (Manugistics, Inc., Rockville, MD, USA), and the effects of bending on the content of individual carbohydrates and phenolic compounds was tested by one-way analysis of variance (AN OVA; general linear model) for each sampling date and year, separately. Means between treatments were separated by Tukey's test (J* = 0.05). Darn are presented as mean t standard error, Contents of carbohydrates in 'Conference* leaves are given in g 100 g" and contents of phenolics in mg 100 g of dry weight (DW) through each growing season and separately for each compound analysed (Figures 1 and 2). RESULTS AMD DISCUSSION [n both growing seasons, sorbitol was the primary carbohydrate, and chlorogenic acid was the primary phenolic (Figures 1 and 2). According to Ito el ai. (2004), sorbitol was the most abundant sugar in both the bud and internode segment of Japanese pear, Our results also showed a seasonal dynamic involving individual carbohydrates and phcnolics; moreover, it was found that the pattern among phenolics was rather similar. Generally, in the first year, Ehe lowest values of analysed phcnolics were observed in May, and then the levels increased to a maximum in July; ailerwhieh their contents decreased slowly, but not below the May values. An exception was epicatechin, whose contents increased till September but afterwards decreased. However, in the second year, the phcnolics dynamics were not as explicit as in the year before: the maximum was reached earlier -mostly in June, and after decreasing until August, anollier small increase appeared from August to October, It is suspected that phenolic dynamics could be influenced by weather conditions (Table I). Warmer and sunny weather with less rain in May and June 2005 could have accelerated the phcnolics peak. Afterwards, very rainy and less sunny conditions eould have caused a decline in phenol i cs until August, and then a little warmer and sunny September and October may have resulted in anotheT small increase of phcnolics. In 2004, large differences emerged between the lowest and the highest value of each phenolic among sampling dates. The phenolic dynamic was similar to what has been reported for 'Williams' leaves (Colaric el at, 2007). buL Lhii contents of some phenolics were lower in that study. Moreover, phenolics in apple leaves showed a similar seasonal pattern: their contenls increased until it peak in July or August, and afier that decreased (Usenik el al, 2004). Carbohydrates did not change through the growing season in an understandable progression, as did phenolics. In the first year, the highest contents of sucrose, glucose and fructose were noticed in October and of sorbitol in May, The lowest, contents of sucrose, glucose and fructose occurred in June and of sorbitol in September, In the next year the lowest values of sucrose and glucose were again attained in June and of fructose and sorbitol in August. Maximum values of analysed sugars occurred in October, except for sucrose, where higher values occurred in July. Generally, some higher values of carbohydrates were noted in the second year in comparison with the levels in the first year. These carbohydrate levels, especially in the First year, arc comparable to levels found in 'Williams' leaves (Colaric el ait 2006). Additionally, the highest carbohydrate content in 'Williams' ieaves was noticed in October, except for sorbitol, which peaked in September. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 30 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Many studies have tracked the seasonal changes of carbohydrates in the leaves of rosaceous trees, mostly in apples. In the study of Heruter and Studer Feusi (1997), glucose and sorbitol decreased, while fructose and sucrose increased during the growing season. Our results, however, are more in accordance with Veberic et a}. (2003), who found fructose, glucose and sucrose contents slightly increased from August to the end of the growing season. Branch bending is a conventional cultural practice that improves the balance between vegetative growth and fruiting in apple trees (Lauri and Lespinasse, 2001); however, on the physiological level, the response to bending has not been well understood. ItO et al. (1999; 2004) reported that changes in carbohydrates in buds are affected by bending treatments. Sorbitol and sucrose contents decreased significantly in the lateral buds 3 days after bending, but increased in the shoot intemode 30 days after bending {Ito ct al, 2004). In our experiment, carbohydrate changes in 'Conference' leaves after the bending treatment showed no clem-tendency in 2004 or 2005 (Figures 1 and 2). In 2004, significantly higher sucrose content was observed unce in leaves from the control treatment (in .lune), but then no significant differences were observed for sucrose the next year. For glucose and fhictose, no significant changes among treatments were observed in the first year. However, in the second year, significant but unclear differences wen; indicated in fructose content in August and in glucose content in July. .September and October. Tn the first year, the highest sorbitol content occurred in October, in leaves from the summer treatment, a phenomenon which was repeated next June; however, in the next October, sorbitol significantly increased in the control as well. According to Sanyal and Bangerth {1998), mechanically induced stress is created by branch bending, and sorbitol, which is, besides sucrose, the major photosynthetic product in the mature rosaceous leaves, increased under various stress conditions (Hudina and Stampar, 1999; Sircelj et al, 2005). However, according to our results above, there seemed to be no apparent stress induced by branch bending. Phenolic compounds are important endogenous growth inhibitors that can affect plants directly by influencing their meLabolism, growth and development (Kozlowski and Pallardy, 1997).'Branch bending and the time of bending had a greater influence on phenolics than on carbohydrates. In the first sampling year, significantly higher contents of some phenolics were measured in leaves from the spring treatment. Specifically, higher values of dilorogenic, vanillic, and sinapic acid and rutin were found in September. However, in August a significantly higher content of epicatcchin occurred in leaves from the summer treatment and a higher content of catechin in leaves from the summer treatment and the control Moreover, in June the epicatechin content was significantly higher in the control. The phenolic content in 'Williams' pear leaves was investigated in the first year after branch bending bv Colarie et al. (2007), but this revealed responses different from those for 'Conference' leaves. 'Williams1 leaves from branches bent in the summer had significantly lower contents (where control leaves had the highest contents at the same time) of the following phenolics: chlorogenic acid and rutin In July, sinapic acid in June, August and September, catechin in September and epicatcchin in July and September (Colarie et erf., 2007). In the second yeaT, 'Conference* leaves from the spring treatment exhibited significantly higher levels of chlorogenic and vanillic acid in September and October, as well as significantly higher catechin levels in October. No significant differences in rutin content among treatments were evident. However, in June, the lowest content of sinapic acid was observed in leaves from the spring treatment, and in October in leaves from the summer treatment, whereas the highest contents were in the control leaves. In addition, the dynamic of the two quercetms, quercetin-3-U-galactoside and quercefin-3-fl-D-gIucoside, was observed in the second growing year (but not measured in 2004). In May, the content of quencet.in-^-D-galactoside was significantly higher in leaves from the control, but in September it was higher Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 31 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 in leaves from the spring treatment. The content of quercetin^-p-D-glucoside was significantly higher in leaves from the control at four samplings. In the rainy summer 2005 (Table I) it was seen in leaves from [he control some lower (but not significantly lower) contents of measured phenolic acids and rutin and scime higher contents of both flavan-3-ols and quercetin-3-D-galactoside and quetcetin-3-fS-D-giucoside (Figure 2). Moreover, the biosynthesis of phenolic compounds is induced by various stress situations (cultural practise, wounding, pathogen attack, environmental conditions, etc.). According to Sanyal and Bangerth (1998), branch bending inflicts mild stress. It was shown by Cohen el ai (1994) thai water stress induced the accumulation of phenolics and suppressed vegetative growth. According to Ruhmann et al. (2002), phenolics suppressed shoot growth, but nitrogen negatively affected phenolic content in apple leaves and then increased the susceptibility of trees to apple scab infections. Liffl-Endrich et ai. (2000) reported that the quantitative and qualitative composition of hydroxy-crnnami;; acids and llavan-3-ols, and the quantitative composition of quercetins-glycosides were enhanced by sucrose content. Higher leaf chlorogenie acid content was detected in pear genotypes resistant to fire blight ^Gunen et a!., 2005). According to Treuttcr (2006), flavonoids have a significant role in plant resistance, e.g. flavan-3-ols affected resistance of apple to scab, catcchin acted as an allclochemical and quercetin-3-rutinoside acted as a defensive compound. Leaf wounding affected the accumulation of flavonol glycosides and flavan-3-ols in pear leaves (Andreoiti et al.., 2006). Colarec et ai (2007) suggested that no stress occurred in bent branches since the reaction of most measured phenolics was the lowest in leaves from bent branches and the highest in the control leaves, allhough the study of 'Williams leaves lasted for one year only. Surprisingly, 'Conference' leaves do not show so clear a tendency, hut when significant differences were noticed for chlorogenie and vanillic acid, Iheir contents were highest in leaves from the spring treatment in bolh growing seasons. As well, at one sampling, the highest contents of sinapic acid and rutin were in leaves from the spring treatment in 2004, and of catcchin and queredin-3-D-ga!actc-side in 2005. Al one sampling, both measured flavan-3-ols (in 2004), as well as sinapic acid and quercetin-3-D-galactoside (in 2005) exhibited significantly higher values in the control, Moreover, the flavonol glycoside, quercetin-j-p-D-glucoside, was highest in the control leaves, significantly greater than the bending treatments at four samplings. Al just one sampling, significantly higher content of cpicatechin was found in leaves from the summer treatment (in the first year after bending), Bending is a cultural practice to balance vegetative growth and fruiting. However, it is also difficult to conclude if those abovementioned and inconsistent variations in 'Conference' leaves among bending treatments are solely the result of bending or whether they are indirectly affected by more general physiological processes at the level of the tec as a whole, and by the year to year variations, genotype properties, time of bending, etc. This investigation was part of Programme Horticulture No. P4-0013-0481, and the authors would like to thank, the Slovenian Ministry of Higher Education, Science and Technology for financial support. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 32 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 REFERENCES AndrEOTTI, C, Costa, G. AMD TREUTTBR, D. (2006). Composition of phenolic compounds in pear leaves as affected by genetics, ontogenesis and the environment. Scientia Horticulture 109,130437. Beruter, J. and STUDER Feltsi, M, E. (1997). The effect of girdling on carbohydrate partitioning in the growing apple fruit. Journal of Plant Physiology, 151, 331-341. Cohen, Y., Treutter, D. and Feucht, W. (1994). Water stress induces changes in phenol composition of leaves and phloem ofPrvnus avium L. Acta Ho?ticuliuraet 381, 494-497. COLARic, M.> StamPAR, F, and IJudtna, M (2006). Changes in sugars and phenolies concentrations of Williams pear leaves during the growing season. Canadian Journal of Plant Science, 86, 1203-1208. Colaric M, Stampar, F. and Rudina, M. (2007). Beading affects phenolic content of William pear leaves. Acta Agricultural Scmdinayica, Section B - Plant Sot! Science, 57. 187-192. Colaric, M„ Veberic, R,, Solar, A., Hudina, M, and Sta m par, F, (2005). Phenolic acids, syringaldehyde, and juglone in fruits of different cullivars utJvghms regia L, Journal of Agricultural and Food Chemistry, 53,6390-639(5, ENWONMENTAL Auency Of THE REPUBLIC OF SLOVENIA. Monthly meteorological reports. [Online], Available; http://www.arso.gov.si/o_agenciji/knji-znica/publikiicijt;/billtn.htm [accessed 3 March 200?]. Goldschmidt-Rrischeu K (1997), Regulating trees of apple and pear by pruning and bending, Swedish Journal of Agricultural Research, 27, 45-52, Grdchowska, M. J., HiODUK, M. ANn MKA, A. (2004). Improving productivity of four fruit species by growth regulators applied nnce in ultra-low doses to the collar. Journal of Horticultural Science tS- Biotechnology, 79n 252-259. Gunen, Y.h Misirli, A. AND GULCAN, R, (2005). Leaf phenolic content of pear cultivars resistant or susceptible to fire blight. Scientia Horticultural?, 105, 213-221. HUDINA, M. AND STAMPAR, F. (1999). Influence of water stress and assimilation area on the sugar cement and organic acid during the growth period in the pear fruits {Pyrux communis L.) cv. 'Williams', Phyton, 39, 107-111. Ito* A„ Yaegakl H., Hayama, H„ Kusaba, S,, YaJviaglchi, I. and Yoshjoka, H. (1999). Bending shoots stimulates flowering and influences hormone levels in lateral buds of Japanese pear. HorlSciencs,M, 1224-1228. ITO, A., Yoshioka, H., Hayama, H, and Kasuimura Y. (2004). Reorientation of shoots to the horizontal position influences the sugar metabolism of lateral buds and shoot internodes in Japanese pear {Pyrus pyrifolia (Burm.) Nak,), Journal of Horticultural Science ugji die iimplin^ dates in Use year 20O5 (the mean values and standard error bars)- Msdisr * statistically sigmficnm at P -value below 0.05 and ** bcLow 0,01 (one-way ANOVA), Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 36 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 37 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 2.5 VSEBNOSTI RAZLIČNIH METABOLITOV V PLODOVIH HRUŠKE SORTE ‘CONFERENCE’ GLEDE NA UPOGIBANJE RODNE VEJE COLARIČ Mateja, ŠTAMPAR Franci in HUDINA Metka Content levels of various fruit metabolites in the ‘Conference’ pear response to branch bending. Scientia Horticulturae, 2007, x: 000-00. Prejeto 14. 3. 2007, sprejeto 22. 3. 2007. Sorta ‘Conference’ predstavlja pomemben delež med sortami hrušk, ki jih pridelujemo v Sloveniji. Rodi redno in obilno, in to kar 80 - 90 % iz brstičev na dveletnih ali triletnih vejah kot tudi na starejših. V poskusu smo upognili petletne rodne veje pozno poleti 2003 in spomladi 2004, kontrolno obravnavanje pa so predstavljale neupognjene rodne veje. V tehnološki zrelosti smo v letih 2004 in 2005 obrali plodove ter primerjali vsebnosti analiziranih ogljikovih hidratov, organskih kislin in fenolnih snovi med obravnavanji. V letu 2004 nismo ugotovili značilnih razlik v vsebnosti določanih ogljikovih hidratov, toda v letu 2005 sta bili v kontrolnih plodovih značilno večji vsebnosti glukoze in fruktoze in najmanjša vsebnost saharoze. V kontrolnih plodovih smo v prvem letu izmerili značilno večje vsebnosti jabolčne in fumarne kisline in manjše vsebnosti klorogenske in vanilne kisline ter v naslednjem letu najmanjše vsebnosti jabolčne kisline in največje vsebnosti nekaterih fenolnih snovi (epikatehina, kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida). Med fenoli je bila v letu 2005 le vsebnost klorogenske kisline značilno večja v plodovih na vejah upognjenih spomladi 2004. Vsebnosti posameznih fenolnih snovi so bile v letu 2004 nekoliko večje v plodovih na vejah upognjenih poleti kot v plodovih na vejah upognjenih spomladi. V vsebnosti sorbitola, citronske kisline, katehina in sinapinske kisline v nobenem letu ni bilo razlik med obravnavanji. Razlike v vsebnosti posameznih metabolitov v plodovih sorte ‘Conference’ so bile posledica upogibanja rodnih vej, na le-te razlike je vplival tudi čas upogibanja (pozno poleti in spomladi). Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 38 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 ARTICLE IN PRESS ELSFVTFR Available cn-Jne al wvvw.KUH-ieeclirEtt-MHTi %* ScienceDirect Srienlia HonJcukiEae y\x <2(fl7) kii-iki SWNTIA HQRTICULTURAE aw -¦¦¦jt: lsLvl±rf:MT.,li:LJi.E',-^Lhon j Content levels of various fruit metabolites in the 'Conference' pear response to branch bending M. Colaric *, R Stamper, M. Hudina Untwrsrtf uf Ljrtbt/tiAj. BMcckmc-c! faculty. t'CJMranwtff a/ Afrtraiir.y, Ckairftw Fnrit (TmiWjij. Jmrnibiirjevu !C1, St-{00t) !.jnMje.Tp, S!"\VHi« RcMh'ed 14 Mirfi 2007; rraiwii in revised form J] Mutch 3007; accepted Z2 Mtwh 20T? A (Mined The icsponsf of 'Cojtfcienut.' peu subjected tu bra.iucji tending In the content levels cif various uistabohtes tn its fruit wiis investigated. 1'hc fruits in eommeiicial malmitj w«rfc sauijilfJ in 20O4 and 100,1 fnom branch« bent ill the late surrmei of 201B frtjfl summer LrciHmentl. from hr.inches belt in the Jate spring. Dt' 20O4 (the spting huntuieiitj icid frnoi LonmVl branches. Tie »Went levels ol'-jnnnhydTiKcs. Off suit acids and phenolic compounds vic^rf. fiotnpji-rd among rreatrueiiti in lv,o smxessiw ysars, The fruit revealed various responses i it cunlcnt levels of imlubDlitea. In 1hc firs* year after Vnrlir.j>, no Signi h'cstnt dJHejerces. i*cre found imlietuitKpiluf e;nih caTtKthydraie. but in 2g]u«)Kide. The cuinpariiOTi u:* the two bending treatment; alnne in 2004 shewed lha.[ I Ik summer Lreitrmerl often produced b slightly hiP.tar vjiiuc ofejdh phenolic incooipaLJSjfjiL tu llje ipqiiig HmttmaiL. Huwewr. in 2(K)5 tin? sijnii fican'Iy highest content of chlojogenic acid wa; in fruit from the spri« p. treatment. "sorbitol, as we]] as citric acjd, catecliiji and iiirapc Lid J showed no clear t-sndencv among ireaumeots, witter in2004 rucrns2UU5.1! Is sugjeskd i:iir ihe*p>ariiiticns of 'Conference' frail subjected k different bend: no HeaimeniE could ItOI bi lLtertSuLl of bending plonc, but that Ihcy could be iiK?ttefitlj aFftcl *1 h? sioSoeictil Ik ftuitlrce. Hoivevcr, it seemed that varialiC'rtsnfSiu'fiettedbydtctimcof henrii ng *r<1 by the veaj'to-Trear, tuidsLch [¦espouses can hi mliibuled lu tlK 'Cofifurerie«' genmype onjy. Y; 2007 Elsevkr B V. All rf^Hj reserml, Kt/V'uitl:: ITti; 1'ywi.i canu\ami!.; TlTjinirli hwntiirj; Pivi«., CuibiiijdraJfci; Oijj^ic Kt\ik\ Phai(j|]i; i fnlroduction Ptar (Pynu ty^mniiirfiy L.) is » poptrlai frllit itl The T-Biivpftratc legions, and Ln Addition i-o "Wllltams", 'Confcrance." feprcssnis an ijiijjottam shanr of the culttvws grov/n m Slovenia ^HLtiiiui and iilaiiipar, 20fl0). 'Conference' pear is a very t'er.iie, high yielding culiivar Ehat timps SO-W35; on ^utri trom brindies Df 1- and 3-year of age as weJl as on older ernes (Smijsavini, 3002'h. 'ITic necessity to i^^ulate e^i^iKive ve^stiittvic- (braneh) y:L»wlb and to increase flowering artd fniitin| btoornc: ^ven moteEignifiiant fftecofuoiriicrtHiicirs, i.e.. mst reduction, lints the ratio bel'^sen ptodiicitLin costs unci niirkct prices foi' ftuii hai; increased in recent years. Among iraditLymtl mithoda of * CoircLj>cmiIiiL aiiihoc. T*l.. +5S& ] .Cj 11 31; fi«r ^36 I 423 10 it. i:-n\n!l aritbm: nBi^jaroliiricShf.iinl-lj.a (M. -CcJaiie). njtM-'fSai - nm fioiit maucr © 'MOT F.ImIh R.V, All righls rtwr,-«J. *ii:!u.iDi!iij.Mi5nti.2u:j7,L>3..;;jh oichard mcnagnvieTn and ctiltufil piatttutiK applied, in tin ojvhiinJ lu wTHruI growth, and fruitiiig, branch henKfint} haa proved the ts\asx suetUwifpl. Kranoh bending is- a Jon/; cstablislied and widely u^d c^lturg] practice in high-density orcnards, and its conospi bus nowilikys ^ccn integrated into the ReiLnxc: training system (Combs et j|„ itifitij, Jjuvesiet aJ. (1997} report thai bejidijij* resulted in higher flontl precKiiy and in red.ucwl slioot vjgour of the :Doysnnt du f !i:miL-c" pear. Apple and peflr trees yielrL-vf mom; fnhi( a.od prodiiced fruit enjlter :f n?I!iilared! orjjy by bending than, those fegulLted Jjy prunint; Lilcmc- (CoJdiv;[iiiiidt-RjeiM;he!, Jši^>7). Howevef, Laisri and Lfcfipinasse -(200]) have, shown thut ih4 iricc.'.i ™ction to bending ahn varic; with tlie geno'ype. aiid Hie lime uf hcudlnij, as well its rtitli the ani^ie of bendinj", the duralion ofbendinE tirnem elv. Fruit sneh as pcar.H arc an excellent source of carbohvdrntea for a diet. Despite the fact (hat pcans cxmlaln a good quantity of : [his njticJe in press .is: Ce>\r,rit., M. $t j| Coniejit levels cf *>aaiwja iiui( metut-jlites i^ th; 'CotifMIKe' p2ar rarxmne iu brjoidi beJLdtLg, Sci. HrtrticjU. CJMIHTj. da\:lft.\f\]t>(i ^ienla.aOOrfJJ.OLfi Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 39 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 HOHTI-!W;N.ii-fPJSiwL ARTICLE I *i. Gofotic fr ot fSot'.lm IfyfSiaiXaittg XXX \10Q7', vx XXX sugar; (cm average 54% fruclifce, ISIS sorbitol, and only lS'jfc sucrose- find 13% glucose;} and dieta:? tlher r J ?—2S g/kg FW) (B'.uilny, 200?), they ant n rrcorr.menc.able GobStilUM for diabtMitS and the ob«e; inmeover, dietary liher together with phenollcs helps (0 reduce the risk ot'develofji.ig didjo^scular disease(Gonnsiein et.il.. 2002). In fflfist pear genotypes, rasli« acid predciminatej; among organic at ids. However, Hudbta and Stamp«! (20001 reported that 'WHlfenitt*, 'Red Wiliianu and 'Rosined' peb'.fi contain a higher percentage of citric acio. Both acid* mentioned; err rnajtir ton I ri baton m th= opii inal degree of acidity, and their ratio (malic acid/ciiric. acid ratio) co-re'iaicH with flhe sensory evaluation of las-js (Culaiič et al., 2C05). Phenolic compounds have n grBai physiologic JI Rile in Bruit, as well in i la resistance to mechanic;! I unrl biological stress. Phenolic compounds in find are of great imerest 1* the consumer. hecaus* they tre an important factor in trj.;: qu&iny; tlwy contribute in Hie.ir sensory qwili'.ies (aroma, asttinsency. bitternes.;; and colour), some of them have pharmacological pnipertiei, too (ami-inflammatory, antita-mur, antiallergic, etc.; (Machei* et a: . l^lt). In Lighl of the positive findings for biding, Ihe aim of this study i« io de:eimine the response of 'Conference' psar to branch bending with respect id Ihe cuntant ul" vafjoits coetahoBtes m its fruit. The corner levels of carbohydrates. organi: acids and pheno'it compounds, which determine ihe nuirilicnal value of firuit, ore compared according to Treatments in two successive years. Apart from seosoty evaluation and physical mewiromeate of tlie fruit, ihe paraneters analysed ncpreseni rhe main indicator of fruit quality, 2, Material and methods 2.1. Chemicals Carbohydrates (sucrose, glucose. &ucto»Rnd vnihiiol). citric add, shLkittiicncid, fumaric acid, (—)-epKaleebJtL, vanillic acid, 4L.ertetin-J'P-galai:toside and ¦qntrCetift-S-P-D-glucoiiide were obtained from Fink* Cheraie GmbH {Bachi, Switzerifmd) The mnlie acid cairie froju Merck KyaA (Daimsiadt, Germany) and (+J-cfl.tochin from Car! RotJi (K urlsruhe, Germany), Chkuflptftfc acid, smapic Kid, rutin (o^eTcelia-j-rutinw^Lri) and hutylated hydrnn.yiolu.eKe (BHT) wetcotrtsined from Sigma Oteniieal'Or}. (Si- Louis, MO, USA). Methanol was purchased Tiur:: Riedel-de-Haen (Seelie, Germany) and aceton irrile fnfnti Sigma-Aldrich ChemJe G mhH (SleinhKi 111, G urmatiy) B idisr died ivalcr p* iri hed m a Miili-Q -xah-.T punfli;JLJO" S,y!itcn-i by MiUiiJui« (Bedfojd, MA, USAiwi^ P****! Mfth.innl. acetitnilrile tnd bidih-ti!1c«J ¦»'aiet were oft^ie HFLC giade, Su^ai tnd oiganic flcui startrlards, were prepared in IMniisiJIIed water and phenolic ilandards ju methanol, 2.2. Plant melerial 'i'he study ifivolwd pear uess 'ConfereiKc'/Qui^ue MA planted in 19B7, traced to a spindle sysirsm and grew« in the eaa-ern pan of Slovenia (hi tandy loam soil. Lieea were S|JE.ccd 3H m h 1.4 m, and the row orientLtlion wbs souih-nunL. Ule pew: cirtkaitl was mainlined LiL-cordirgto st;:nd:iTd 60*nmenda] praeiiue for i nlegrated fruil ptioduction (ore hwi j i is lafienrtrt inj tuliui j] praetice, -..e. .sprayLeg, iirigarion. etc.). Those differed maniigieme™ treamients were appherftftlhelrecs, andcueh single treatment ivas repeatrd tvukndy an ten trses, 'Jbeatments were the followjjsg.: (i) the summer treatment, where one 5-year-old branch' per tree (cm the tree's cA^iem side and of comparable propen ies> was bent tu un aritjle of I 'MX' from t he vertical po^i tion in laic summer 20(H (Is'. September 2003^ (li) the spring irealnicnt, where one 5-year-old biaiich per tree was hent in laic soririi- 2004 {LSlh May 2004}; (iii) (he control ire.Tjmeni, where labeled, branches were nut ben; (but remained 45' from ihe vertical}. Before hending. the bent br;tnche^ weie erown like ihe coii^ro; branches—at an angle, of 45J frnm the venicai position. All ben I brandies, as Well control braflches, were allowed to develop without pruning from 2003 to 2005. The undamped pear liuit werf hn^ienied fmm thoattrancr-es-fthiee repressnta-dvfl &uit finracftdlbtanch, »pealed cm ten irsespcrtrcutmentl at commeroitll maturity on 9th Se-piemLa 2'JCid and lsi September 20(15.Immediately ,nf;erharvest, the fruns w«R«KE«da( -20 "C. 2.3. Extraction pnxmiare The samples w;r? homogenized to a puree with tlie T25 basic ULuaTurrax hr>mfij^eniser([Kl^ T.nhtirtcchTiik. Jarke and KunkEl GmbH, Slaufcn. CJeftuanyl Ihcn the puree was prepared separately for the carbohydrate and organic acid ansiJyaes, and for the- phenolic compound analyses. For fljgars ar^d, ciryamc adds extraction, bddisrillcct wafer up to ft iinai volume of 50 ml was add-d 10 10 g of puree and leTt for 45 ~.in it room tctripcratiin:, with cccasiotial stirrittg. After eMwcuori. the puree w;is,(xnirihiued m 1 Z.flOfj x g (Pppcndorf flentrifuge 5810 R, Hamburg, Germany) fw 7 aia at id "C, Tlie aufjeriiataiit wns ihrn tiltertid through a 0,45 p,m cellulose mivsd esletE filter (Mdcherey-Nagel, ULien, Germany) inw a visi and used for the carbohydrate i-,nd organic and analysis, For phenolic: analyses, 1 g. of fhe homtijjenized sample *ss trausfeired lo a test !.uae. and ejctMKlftd tmce with 10 ml t>f me:hanol fm45 rain in ;m u.^fitsonic buh cooled with ice. The me^hano' con mined 1% of the antioxldaiiive a^ent BHl; which had no influence on :he. extraction pracess and ths HPLC-analysis. The exu-acted sample wai centrifuge^ 12JXQ x a for 7 min at 1(1 "C. Than the nupcrrialbijjl was fihered Ihrough B 0.45 u.m Chromsfil (Machcruy-Nagel) polyaniide filter and used fur the phenolic compound analysis. 1A. (ii'LV iinalyxex Chromatographic scparatkm of the carbohydrates and rr^anic aicida was pcrforrr.ed using a Thennu Separation Produtls HPLC system (Rivier?. Besirh. PL, USA). Tlie HPLC-analys« of varbohydratej ^ecc made M&inL a Rere^ RGM-monosacchiiride eoluinn (3 00 m m k 7.8 mm) operated ar 55 °C from Hienomcncn (TorranCt, CA, USA), The mobile phase w;ik hidiitilled w^tcr and Ihi flow rate wr.s O.fi nlh'ttlin, i"he duratieu of the analysis was 6D m in, and * refractive iodev Jftl) drterror (Sbodex Bt-"?1, Shcj-wi Denko K.K.. Kawasaki, Japan) was used Pot monitoring eluled carbohj-drotef.. Tlie a die lhls arlicle m prets tu; Oslarws; M tt «3s of , Sci. BprtlcHlt, (20C), doi:m.H]LiiJja-]jina.J reported ibaL 'Oojifcrcncc' differed fram 'Williams' in jtrvwLti and fniitjng habits. 'Conference: has lhc advantage that iT is regarded as regular beaiiiift cultim, which bean 80-W* nit spurs (mode] 3 of five frjiting modeli), mostly fram branches of 2-3 y-ar* in age, nut h never stops cropping <¦"' tlie SP1"^ of the oldest branches, while 'W!liiam&" cruns less than 50& on the spur buds. The decision about Lite tuniflg or branch bending stil L remains open for 'Conference' pears, which stiowedaii unclear tendency hrtw&ert hoih bending tre-Htiipe-niK. jlihuuuh some cSifltrerjces were found among Lhc fruit invo'ved in bendingand those in the control ircatncn^ (Tables L -6). But fcr the :WilliJIIlii, ptais ilesu be intern«! that branch rending in spring :s more recommended (Culuricetal., ZUOoj However, it seems from the above results thai branch bendinji hmi u positive effect on fhc. l^el at nhenniics in 'Conference' fruit in 2(104, d. Cundusion The fecus of the rcseerch was ths effect of branch angle change on 'Conference' fruit in twrj suwessivt: year* at the chemical level. The fruit revealed various responses in carbohydrates, organic a~id& and phenolLcs. content levels, Mcvertlisks«, it was. sJiowij that fruit from the osnlno! reached significant^ hir/hei contem levels of maLii; neid and lower iionreiu lev-ils of certain ptoenoliui! in ihe first y-ati af'ei bsndjng, but in the next year lhc c*.w?t in 2005. I: can be inferred ;hat thuse variations of 'Uofifericnre' frill [ among bending trattmoiLs i*>u!d not be the iesult of bending alone, but thai lhej could be indirectly affwted by other physiological responses of ^he. fruit tree, However, it seems (hat variation? ire iiffec.ted by bending time and by the year-ro-i'ear variation!;, and such responses can he attributed to the 'Conference' yjpntiiyjje only. AckPiOwttdKtmeiiti This investigation was pari uf the Hoiticuirune programme N-o. P4-0013-Q^tH, and the authors would like tu t'r,ank die Slovenian. Ministry of Higher Education, Sdenpe and TeeJnLOkigy tor financial supptnl. References Anc'retfii, C, riKii, ("I., T:tuii-r, D., 2X16. tiunpv«iLian ot phenolic com- pL'iniJ; in piar I«™* »i nfTcH«! by jcucLf t, OWOJ^nf "is nnd Ifaz JfV:cO(1-mnit Zr.i. Tlnrt. UN, ]L*1)-IJV. Elanrf.t. MOJ. Ptin. In; Cstalltru, fl-.Tnifc. LC,, Pnjb.-.I'.M. [Bb^, Cticycl(if»Hi= uf KMd fcicnctj mid NU[ri[irtrt. Anemic Prc^, ljni:dui!, pp. a43S-iil3J. CoL*ic.M.r3[ui-(ipnr. R.HtltliM, M, JU0rB;i-Jinc4irftciiptiariciUf 4joiu:M of ¦ : I .,i. pear Leu^'5. QjtiirtC. M., btDlnpnr, F„ Sfllnr- A., HuJi la. M., 2W1S. InfiiiHiLC uP Liaifn hendins ur vu^ai, Oi gall if nfiJ nrd phennlic lciiMliiC 111 thlLla of ' Williiim!' ptBHi L^rtJY Mammy LJ, J. Sii FiumI Aerie, 86. H63-1J6-: C^laric, M.. Vrtimu. K., ;šiaii.|t.. R, EDudi.iu, M„ 211*5. livalml^uof peocn nnJ nccdirliefejLl quality ani comelmiii« t*mfefi scikij1 *nJ thmiii ^1 aftrioWW ) Sfi. fn.Yl Agric. S^, 2» L L-2616. C^tes, E-, I JJri, PE., E*pnaj4l. Jl. , 300*. Analy-jjii|f JjuLl Uct afchi(fl!liiiK: iilifllLSllSMtiforlrTKmnrajcnieTil jjcJ hut pr-SdlKUCfl. lir J*nptH, J. (Ed.), Htirt. Rev. i2, 1-41. ariUrflunidl-fidKltel. C . 19!"- He^ularing Uet5fll*pple4iiKt ptafbv pnialng and briuJinj. Swcd. I, Agric, Rk;, 2^, 4J-J2. Goiiiiitf in. S.. MartiTltfHow, mjftjf.ipaii!^ 1^1 tv. "WilUama' Pny1on3Y, :07-t]t. R, 2nflO. Sajii-mid orianic atHs mirtoTts of Eun^nw (/V«' *iMHi™a LI Jud Asisn (P.urai- wmfifla Rrhd.) pe« r.il i vai s A.luj AlilTUfUl. 79. ,Si7-2.1Cl. Ho. A„ YlniMfto. H.. Hny.»inaiH„ Kswhiniiin, If, 100*. Rmrientpiiiniiiifshs^i?; K- 1Lw Isifimnml pnsilinn inlluCrHJDS Lie aujjni incl*t^|iiar ti! laKfa. bodi anr- ahooi ir.iefliatej In JajKintw p?ur (PjjrKi ^ynru^u (Bunm.) hf.*.). I. Hmi. ,^1. Biotwli. 73,416-J21. Lauri, P.-E., LcioipiauBi,J^M.,2001. LViutKypr^rappli' itkks;iVLLtigrowLiami iniitiri-eipftnsaj lo aluot tendine =¦¦ jjrious :iiiitt of ve«. J, AfL Sot. Hon.icl. IS«, IW-174. Laivct, *3.^., .ip=™:= t-B-. 'I'umiii, U.S.. Mjik. S.M., I9?7. Tuo« a.uali(]f nnd tarnpj mair^cmcni sfftcf; w \be jiywih auJ Sofn":l ffr^rKj- ?hyuunj ¦Do/tnLK Ju Ojit«^' pear ncca. rt. Z, J. Cnip Hun. Scl- 2J, IT7- 134. MacticLii. J,I„ Plcutiet. ft,, BlMl.1, J-, t^Ml Fruit FMnlxs. CRC Ptr«, B-rrs Rhwti. ^Ltiichof, A., roller. P.. Carle. R„ 2001- Dacrmiruiuor uf ptiemil'L aj.LJJi jlv) lIllMTKflii uripplt LillJ pi^l Ljf llijll-JJCrfi^inaiK= lii[jid iUuflialOgnipriy. J- Chnjimlri^. A 510, 1oS-2hJ. Sunu^Lni, j„ 2[XK. fcn JutiJi^iaach nwnVlj nckiicd u yield control ord prunini- Aiia HOLl. M6i S27-6J3, Samysl, D.. Bunptrth, P., !*93. Saeii induced idiylciii: tvolJtion nnd its prsfjhlt ret*l.-onEti.p to ausin (rjnitiojn, cymkinim Irwrls, ami liiwer bud i ndMion i n ^™m« rt appl"1 i^dluijs *nd lwoiriTig eppl= biaach Landing; S^i- Hunzteh. <.:007:,, ilM^Li.liliriJj.TCienlJi.ZIJin.UJ.Olb______________________________________________________________________ Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 44 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 3 RAZPRAVA IN SKLEPI 3.1 RAZPRAVA V dveh zaporednih letih 2004 in 2005 smo spremljali vsebnosti primarnih in sekundarnih metabolitov v plodovih in listih evropske žlahtne hruške sort ‘Williams’ in ‘Conference’, ki so bili vzorčeni na petletnih rodnih vejah. Rodne veje so bile vključene v različna obravnavanja: rodne veje, ki smo jim spremenili kot rasti iz 45° na 120° od navpične lege (i) pozno poleti 2003 in (ii) spomladi 2004 ter (iii) rodne veje, ki niso bile upognjene, temveč so vseskozi rasle pod kotom 45° od navpične lege. V listih, kjer smo spremljali vsebnosti metabolitov od maja do oktobra, smo določili ogljikove hidrate - saharozo, glukozo, fruktozo in sorbitol ter fenolne snovi - klorogensko, vanilno in sinapinsko kislino ter (-)-epikatehin, (+)-katehin in rutin ter v naslednjem letu dodatno še dva flavonola: kvercetin-3-D-galaktozid in kvercetin-3-ß-D-glukozid (Colarič in sod., 2006a; 2007a; 2007b). Ogljikove hidrate so spremljali tudi v listih drugih sadnih rastlin iz družine rožnic (Loescher in Everard, 1996; Deguchi in sod., 2002; Veberič in sod. , 2003; Šircelj in sod., 2005). V našem poskusu je v obeh letih pri obeh sortah pa tudi v vseh vzorčenjih v listih prevladoval sorbitol nad ostalimi določanimi snovmi (Colarič in sod., 2006a; 2007b). Hudina in Štampar (1999) sta pri evropski žlahtni hruški ugotovila, da se je v sušnih razmerah vsebnost sorbitola povečala. Podobno so Šircelj in sod. (2005) ugotovili pri jablani: sorbitol se je tvoril kljub neugodnim razmeram kot je zmeren sušni stres, toda dolgotrajna suša je zmanjšala kopičenje sorbitola. V letu 2004 smo izmerili največje vrednosti sorbitola v listih pri sorti ‘Williams’ v septembru in pri sorti ‘Conference’ v maju; največje vrednosti saharoze, glukoze in fruktoze pa so bile pri obeh sortah oktobra (Colarič in sod., 2006a; 2007b). Sezonsko gibanje ogljikovih hidratov v listih obeh proučevanih sort proti koncu prvega leta je bilo podobno kot navajajo Veberič in sod. (2003) za liste jablane: vsebnosti fruktoze, glukoze in saharoze so se od konca avgusta pa do oktobra povečale in vsebnosti sorbitola zmanjšale. Naslednje leto smo pri sorti ‘Williams’ v oktobru izmerili le največje vrednosti sorbitola, fruktoze je bilo največ v septembru ter saharoze in glukoze največ že v maju. Pri sorti ‘Conference’ smo v letu 2005 izmerili največje vsebnosti treh ogljikovih hidratov (fruktoze, sorbitola in glukoze) v oktobru, z izjemo saharoze, ki je dosegla največjo vrednost v juliju (Colarič in sod., 2007b). Med vsemi proučevanimi fenolnimi snovmi, smo izmerili največje vsebnosti klorogenske kisline. Le-ta je zelo pomembna pri odpornosti na hrušev ožig (Gunen in sod., 2005). V prvem letu smo pri obeh sortah zasledili najmanjše vsebnosti posameznih fenolnih snovi v maju, nato so se pri sorti ‘Conference’ vsebnosti povečevale do julija, zatem pa počasi zmanjševale do oktobra; katehin pa je dosegel največjo vrednost šele v septembru (Colarič in sod., 2007b). Sezonska dinamika sorte ‘Williams’ je bila nekoliko drugačna: v juliju sta največje vrednosti dosegli le klorogenska kislina in rutin ter šele avgusta vanilna in Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 45 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 sinapinska kislina in septembra katehin in epikatehin (Colarič in sod., 2006a). Podobno sezonsko gibanje fenolnih snovi so zabeležili tudi Usenik in sod. (2004) v listih jablane: vsebnosti posameznih fenolov so naraščale vse do julija ali avgusta, nato pa so se zmanjšale. V letu 2005 smo pri obeh sortah zabeležili največje vrednosti večine fenolov že v juniju, nato so se le-te zmanjšale do avgusta in nato ponovno povečale do konca sezone. Domnevamo, da so na tak potek vplivale tudi ekstremne vremenske razmere (deževje in nižje temperature), še posebej v juliju in avgustu 2005 (Colarič in sod., 2006a; 2007b). V plodovih smo v tehnološki zrelosti poleg ogljikovih hidratov ter fenolnih snovi, ki smo jih določali tudi v listih, izmerili vsebnosti organskih kislin: citronske, jabolčne, šikimske in fumarne kisline. Sorta ‘Williams’ je po času zorenja še poletna hruška (Blattný, 2003), v letu 2004 smo jo obrali 1. septembra in v letu 2005 19. avgusta. Sorto ‘Conference’ uvrščamo med jesenske hruške (Blattný, 2003) in smo jo v letu 2004 obrali 9. septembra in v letu 2005 1. septembra. Razmerje med posameznimi ogljikovimi hidrati je bilo pri obeh sortah hrušk podobno kot navaja Blattný (2003) in vsebnost fruktoze je prevladala nad sorbitolom. Močno pa se sorti ‘Williams’ in ‘Conference’ razlikujeta po vsebnosti citronske in jabolčne kisline (Hudina in Štampar, 2000a), podobno smo ugotovili tudi v našem poskusu (Colarič in sod., 2006b; 2007c). Hudina in Štampar (2000a) sta poleg sorte ‘Williams’ izmerila večje vsebnosti citronske kisline tudi pri sortah ‘Red Williams’ in ‘Rosired’, ostale sorte pa so imele največje vsebnosti jabolčne kisline. Tudi v plodovih obeh sort hrušk smo izmerili največje vsebnosti klorogenske kisline, sledila sta epikatehin in katehin. Vsebnosti omenjenih fenolov so nekoliko večje kot poročajo Amiot in sod. (1995) ter Herrmann (2001). V naslednjem letu nam je uspelo določiti tri flavonol glikozide (rutin, kvercetin-3-D-galaktozid in kvercetin-3-ß-D-glukozid), katerih vsebnost je podobna kot navajajo Schieber in sod. (2001), po čigar metodi smo tudi ločili fenole v plodovih in listih. Zgoraj smo opisali vsebnosti različnih metabolitov v listih (med rastno dobo) in v plodovih (v tehnološki zrelosti) proučevanih sort hrušk, saj do sedaj pri nas še niso bili tako raziskani, z izjemo plodov, kjer sta Hudina in Štampar (2000a; 2000b; 2004) podrobno proučila vsebnosti sladkorjev in organskih kislin. Glavni namen naših raziskav pa je bil ugotoviti ali upogibanje, kot eden izmed učinkovitih agrotehničnih ukrepov, s katerim v sadovnjakih uravnavamo rast in rodnost (Luckwill, 1970; Lauri in Lespinasse, 2001; Costes in sod., 2006), vpliva na vsebnost že omenjenih primarnih in sekundarnih metabolitov, saj do sedaj ta del še ni bil predmet raziskav. Vsebnost metabolitov je pomembna tudi z vidika kakovosti plodov, saj le-ti prispevajo k senzoričnim lastnostim (sladkost, kislost, vonj, okus, trpkost, grenkost, obarvanost), mnogi med njimi imajo zdravilne lastnosti kot npr. nekatere fenolne snovi, ki zavirajo razna vnetja, rast tumorjev, pojav alergij in srčno-žilnih bolezni (Macheix in sod., 1990; Gorinstein in sod., 2002). Ogljikovi hidrati so glavna skladiščna snov in osnovna organska snov, iz katere se tvorijo še druge organske snovi, tudi fenoli (Kozlowski in Pallardy, 1997). Fenolne snovi imajo v rastlini pomembno fiziološko vlogo, saj vplivajo tako na njeno rast kot tudi razvoj, in vlogo v odpornosti na mehanski in biološki stres (Macheix in sod., 1990). V prvem letu našega poskusa smo pri sorti ‘Williams’ zasledili, da se listi, ki smo jih vzorčili na upognjenih in na neupognjenih vejah, niso značilno razlikovali med seboj v Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 46 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 vsebnosti ogljikovih hidratov, razlikovali pa so se v vsebnosti fenolov. Kadar so bile statistično značilne razlike v vsebnosti posameznih fenolov, so bile vsebnosti le-teh največje v listih iz kontrolnega obravnavanja ter najmanjše v listih, ki so bili vzorčeni na rodnih vejah upognjenih poleti 2003 (Colarič in sod., 2007a). V vsebnosti klorogenske kisline in rutina so bile razlike v mesecu juliju, sinapinske kisline v avgustu in septembru, katehina le v septembru ter epikatehina v juliju in septembru. V listih sorte ‘Williams’ smo v letu 2005 zabeležili razlike v vsebnosti fruktoze in sorbitola, oba ogljikova hidrata sta imela v avgustu najmanjšo vrednost v kontrolnem obravnavanju, sorbitol pa še v juniju. Toda v septembru je prišlo do preobrata in sorbitol je prav pri kontroli dosegel največjo vsebnost. Tudi v drugem letu smo izmerili večje vsebnosti nekaterih fenolov v listih s kontrolnega obravnavanja, še posebej vseh treh določanih flavonol glikozidov. Statistično značilne razlike v vsebnosti vanilne kisline, rutina in kvercetin-3-D-galaktozida smo zabeležili v mesecu juniju, katehina v juliju in kvercetin-3-ß-D-glukozida v avgustu. Epikatehin je bil izjema, saj je bila njegova vsebnost v oktobru značilno najmanjša prav v listih z neupognjenih vej. Andreotti in sod. (2006) so izmerili večje vsebnosti flavonol glikozidov in flavan-3-olov v poškodovanih listih, za liste analizirane v našem poskusu, pa tega ne moremo trditi, saj smo v vseh obravnavanjih vzorčili zdrave liste. Sanyal in Bangerth (1998) trdita, da upogibanje rodnih vej povzroči v sadnih rastlinah zmeren stres, nastajanje fenolnih snovi pa se običajno poveča ob stresnih situacijah (Treutter, 2001), kar je v neskladju z našimi dvoletnimi rezultati, saj so bile vsebnosti fenolov sorte ‘Williams’ največkrat značilno večje v listih z neupognjenih vej -kontrole. Upogibanje rodnih vej tudi v listih sorte ‘Conference’ ni pokazalo jasnih razlik v vsebnosti posameznih ogljikovih hidratov, niti v letu 2004 niti ne v letu 2005 (Colarič in sod., 2007b). V prvem letu je bila v kontrolnih listih vsebnost saharoze značilno večja v juniju in sorbitola značilno manjša v oktobru. Upogibanje je mnogo bolj vplivalo na vsebnosti fenolov, saj smo v prvem letu izmerili značilno večje vsebnosti klorogenske, vanilne in sinapinske kisline ter rutina v listih na rodnih vejah upognjenih spomladi 2004. Izjema sta bila oba določana flavan-3-ola, katerih vsebnost je bila vsakega po enkrat največja v kontrolnih listihin le epikatehina enkrat v listih na rodnih vejah upognjenih poleti 2003. V naslednjem letu je bila slika sladkorjev v listih sorte ‘Conference’ še bolj nejasna: pri glukozi smo tako zasledili značilno največje vrednosti po enkrat v vsakem obravnavanju, sorbitol je junija dosegel največjo vsebnost v listih na rodnih vejah upognjenih poleti 2003 in oktobra v kontrolnih listih, največje vrednosti fruktoze pa smo izmerili avgusta v listih na rodnih vejah upognjenih spomladi 2004 (Colarič in sod., 2007b). Ito in sod. (2004), ki so proučevali odgovor lateralnih brstov in poganjkov našija na upogibanje vej, so ugotovili, da so se z upogibanjem vsebnosti sorbitola in saharoze v lateralnih brstih zmanjšale, toda v internodijih poganjkov povečale. Tudi v letu 2005 smo izmerili večje vsebnosti klorogenske in vanilne kisline ter rutina v listih na rodnih vejah upognjenih spomladi 2004, v tem obravnavanju sta imela največjo vsebnost tudi katehin v oktobru in kvercetin-3-D-galaktozida v avgustu. Sicer pa so bile največje vsebnosti sinapinske kisline (junija in oktobra), kvercetin-3-D-galaktozida (maja) in kvercetin-3-ß-D-glukozida (maja, junija, julija in oktobra) v kontrolnih listih. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 47 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 Če povzamemo dvoletne rezultate obeh sort, vidimo, da se vsebnosti ogljikovih hidratov v listih na upogibanje ne spremenijo v nekem sosledju, vsebnosti fenolnih snovi pa. Proučevani sorti sta se na upogibanje različno odzvali: upogibanje, še posebej poletno, je povzročilo pri sorti ‘Williams’ največkrat zmanjšanje vsebnosti posameznih fenolov. Pri sorti ‘Conference’ pa je upogibanje, še posebej spomladansko, povečalo vsebnosti nekaterih fenolov. Iz zgoraj opisanega lahko sklepamo, da se odziv drevesa na upogibanje razlikuje med sortami, pomembno pa je tudi kdaj so bile rodne veje upognjene, s tem pa lahko povežemo dognanja Lauri-ja in Lespinasse-ja (2001), ki sta ugotovila, da se jablane na upogibanje različno odzovejo v rasti in rodnosti, saj v veliki meri vplivata tako sorta kot tudi čas upogibanja. Proučevani sorti sta imeli podobne vrednosti posameznih fenolov, le obeh flavan-3-olov je bilo več v listih sorte ‘Williams’ ter kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida manj. Gunen in sod. (2005) so izmerili večje vsebnosti fenolov v listih na hrušev bakterijski ožig odpornih sortah hrušk. V plodovih sorte ‘Williams’, ki smo jih obrali na rodnih vejah upognjenih poleti 2003, so bile v prvem letu vsebnosti ogljikovih hidratov najmanjše, največje pa so bile v plodovih na vejah upognjenih spomladi 2004 (z izjemo glukoze), kar pa je v naslednjem letu obveljalo le za saharozo (Colarič in sod., 2006b). V vsebnosti organskih kislin v nobenem letu ni bilo značilnih razlik, čeprav so bile vsebnosti posameznih organskih kislin z izjemo citronske, v prvem letu nekoliko večje prav v obravnavanju, kjer so bile vsebnosti ogljikovih hidratov najmanjše. V prvem letu smo v vsebnosti posameznih fenolnih snovi zabeležili enako kot pri sladkorjih, z izjemo sinapinske kisline, katere vsebnost je bila sicer tudi najmanjša v plodovih na vejah upognjenih poleti 2003, toda največja vsebnost le-te pa je bila izmerjena v kontrolnih plodovih. V naslednjem letu sta bili vsebnosti glukoze in fruktoze najmanjši pri kontroli, vsebnost sorbitola pa je bila nekoliko večja v plodovih na vejah upognjenih poleti 2003. Fenoli so nam v drugem letu podali drugačno sliko v primerjavi s prejšnjim letom: največje vsebnosti smo izmerili v plodovih obranih na vejah, ki smo jih upognili poleti 2003 in najmanjše v kontrolnih plodovih. Izjema sta bila kvercetin-3-D-galaktozid in kvercetin-3-ß-D-glukozid, katerih vsebnost je bila ponovno večja pri kontroli. Vsebnost sinapinske kisline je bila najmanjša v plodovih na vejah upognjenih spomladi 2004, ostali obravnavanji pa se v vsebnosti nista razlikovali med sabo. V plodovih sorte ‘Conference’ v prvem letu nismo ugotovili nobenih značilnih razlik v vsebnosti ogljikovih hidratov med obravnavanji (Colarič in sod., 2007c). Vsebnosti jabolčne in fumarne kisline sta bili značilno največji pri kontroli. Vsebnosti citronske kisline so bile podobne med obravnavanji, a majhne, tako v letu 2004 kot tudi v letu 2005. V prvem letu so bile vsebnosti posameznih fenolov, z izjemo katehina, najmanjše v kontrolnih plodovih ter največje v plodovih z upognjenih rodnih vej, še posebej poleti 2003. V letu 2005 smo v kontrolnih plodovih izmerili značilno večje vsebnosti glukoze in fruktoze ter manjše vsebnosti saharoze. Tudi pri tej sorti so nam fenoli v drugem letu pokazali drugačno sliko. Največje vsebnosti epikatehina, sinapinske kisline ter določanih flavonol glikozidov smo izmerili v kontrolnih plodovih ter najmanjše vsebnosti vseh posameznih fenolov v plodovih na upognjenih vejah, še posebej upognjenih poleti 2003. Toda vsebnosti klorogenske in vanilne kisline so bile nekoliko večje v plodovih na vejah upognjenih spomladi 2004, kar je zanimivo, saj sta imela oba fenola največje vrednosti v enakem obravnavanju tudi v listih, in to kar obe leti (Colarič in sod., 2007b). Katehin je bil Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 48 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 še večja izjema, saj je imel najmanjšo vsebnost v plodovih na vejah upognjenih spomladi 2004 ter največjo na vejah upognjenih poleti 2003. S pomočjo zgoraj opisanih dognanj lahko povežemo rezultate dvoletnih meritev vsebnosti metabolitov v listih in plodovih posamezne sorte. Pri sorti ‘Williams’ so bile prvo leto najmanjše vsebnosti posameznih fenolov, tako v listih kot tudi v plodovih, ki smo jih vzorčili na rodnih vejah upognjenih poleti 2003, enako smo opazili le v listih tudi v naslednjem letu. V obeh letih smo izmerili največje vsebnosti fenolov v listih iz kontrolnega obravnavanja, kjer pa so bile v letu 2005 najmanjše vsebnosti v plodovih. V letu 2004 smo izmerili največje vsebnosti fenolov v plodovih na rodnih vejah upognjenih spomladi 2004. V naslednjem letu pa je prišlo do preobrata in smo v plodovih, ki smo jih vzorčili na vejah upognjenih poleti 2003, izmerili največje vsebnosti fenolov (izjema so bili flavonol glikozidi z največjo vsebnostjo pri kontroli). Pri sorti ‘Conference’ so se vsebnosti fenolnih snovi v listih in plodovih precej izključevale: v obeh letih so bile največje vsebnosti v listih na vejah upognjenih spomladi 2004 ter le v prvem letu v plodovih na vejah upognjenih poleti 2003, naslednje leto pa v kontrolnih plodovih. Izjeme so bile klorogenska in vanilna kislina, katerih vsebnost je bila največja v plodovih na vejah upognjenih spomladi 2004 in katehin z največjo vsebnostjo v plodovih na vejah upognjenih poleti 2003. V dvoletnem poskusu smo potrdili razlike med proučevanima sortama, ki smo jih tudi pričakovali, saj se sorti razlikujeta v rasti in rodnosti ter v pomoloških lastnostih ploda (Sansavini, 2002). Glede na rezultate vsebnosti metabolitov v listih in plodovih, lahko domnevamo, da je za sorto ‘Williams’ primernejše upogibanje vej spomladi ter za sorto ‘Conference’ pozno poleti. Seveda pa bilo potrebno v prihodnje poskus zastaviti še na večjemu številu sort. Za leto 2005 velja omeniti, da je bilo poletje precej deževno in hladnejše od dolgoletnega povprečja (Colarič in sod., 2007b), kar bi lahko vplivalo na preobrat v vsebnosti metabolitov, saj je vsebnost fenolov močno odvisna tudi od mnogih zunanjih ali okoljskih (stres, temperatura, svetloba) in notranjih dejavnikov (hranila, hormoni) (Macheix in sod., 1990). Vsebnosti fenolnih snovi: (hidroksicimetne kisline, flavan-3-oli in flavonol glikozidi) lahko povečamo s hranili in ogljikovimi hidrati kot je npr. saharoza (Lux-Endrich in sod., 2000). Glede na večje vsebnosti saharoze in fenolov v plodovih na rodnih vejah upognjenih spomladi 2004 pri sorti ‘Williams’, lahko povežemo ugotovitve Lux-Endrich-a in sod. (2000) z našimi, vendar pa to ne moremo trditi za sorto ‘Conference’. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 49 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 3.2 SKLEPI V dvoletnem poskusu smo podrobno proučili kemično sestavo listov in plodov v Sloveniji razširjenih sort hrušk ‘Williams’ in ‘Conference’. S tekočinsko kromatografijo visoke ločljivosti smo v listih med rastno dobo in plodovih v tehnološki zrelosti izmerili vsebnosti ogljikovih hidratov (saharoze, glukoze, fruktoze in sorbitola) in fenolnih snovi (klorogenske, vanilne in sinapinske kisline, (-)-epikatehina, (+)-katehina, rutina, kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida) ter v plodovih tudi vsebnosti organskih kislin (citronske, jabolčne, šikimske in fumarne kisline). Med ogljikovimi hidrati smo v plodovih izmerili največje vsebnosti fruktoze in v listih sorbitola, med fenolnimi snovmi pa je bilo v plodovih in listih največ klorogenske kisline. V plodovih sorte ‘Williams’ smo izmerili večje vsebnosti citronske kisline in v plodovih sorte ‘Conference’ večje vsebnosti jabolčne kisline. Vsebnosti metabolitov v listih, ki smo jih vzorčili med rastno dobo od maja do oktobra, so se med vzorčenji razlikovale, najbolj značilne razlike pa so bile v vsebnosti fenolov. Vsebnosti le-teh so bile običajno najmanjše na začetku rastne dobe, nato so naraščale do junija, julija ali vse do avgusta in se zmanjševale do oktobra. Opazili smo razlike tudi med obema letoma, saj so fenoli v drugem letu dosegli dva vrha, na kar so morda vplivale tudi vremenske razmere (okoljski dejavniki). Proučevani sorti sta se med rastno dobo razlikovali v vsebnosti metabolitov tudi med seboj, primer: v prvem letu smo pri sorti ‘Williams’ izmerili največje vsebnosti sorbitola in katehina septembra ter pri sorti ‘Conference’ največje vsebnosti sorbitola maja in katehina julija. Upogibanje rodnih vej ni pokazalo jasnega vpliva na vsebnosti ogljikovih hidratov v listih. Upogibanje (še posebej poletno upogibanje) je pri sorti ‘Williams’ povzročilo v listih manjšo vsebnost fenolnih snovi, pri sorti ‘Conference’ pa je upogibanje (še posebej spomladansko) povečalo vsebnosti nekaterih fenolov, zato menimo, da imata vpliv na učinek upogibanja tudi sorta in čas, ko smo izvedli upogibanje. Tudi v plodovih sta se proučevani sorti različno odzvali na agrotehnični ukrep upogibanje rodnih vej. Pri sorti ‘Williams’ so bile v prvem letu največje vsebnosti fenolnih snovi (pa tudi ogljikovih hidratov) v plodovih, ki smo jih obrali na rodnih vejah upognjenih spomladi 2004, zato domnevamo, da je upogibanje rodnih vej v pomladnem času za to sorto bolj primerno, tudi z vidika notranje kakovosti plodov. Pri sorti ‘Conference’ menimo, da je primernejši čas upogibanja pozno poleti, saj smo v prvem letu zabeležili največje vsebnosti fenolov v plodovih na upognjenih rodnih vejah poleti 2003. Znotraj fenolnih podrazredov so se fenoli v plodovih in listih različno odzvali na upogibanje: vsebnosti flavonol glikozidov - kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida so bile pogosto večje pri kontroli, fenolnih kislin - klorogenske in vanilne kisline pa v plodovih in listih, ki so bili vzorčeni na upognjenih vejah. V raziskavi smo potrdili razlike v vsebnosti metabolitov v listih in plodovih, ki smo jih vzorčili na upognjenih (spremenjen kot rasti) in neupognjenih vejah, razlike v času upogibanja (poletno in spomladansko), med letoma ter seveda med proučevanima sortama. Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 50 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 4 POVZETEK (SUMMARY) 4.1 POVZETEK Evropska žlahtna hruška (Pyrus communis L.) je zelo razširjena sadna vrsta v zmerno toplem podnebnem pasu. Proučevani sorti ‘Williams’ in ‘Conference’ predstavljata pomemben delež med sortami, ki jih pridelujemo v Sloveniji. Sorti se med sabo razlikujeta po splošnih značilnostih, v rasti in razraščanju rodnega lesa, rodnosti ter tudi v pomoloških lastnostih ploda. Med uveljavljenimi agrotehničnimi ukrepi v intenzivnih nasadih sadnih dreves se je upogibanje rodnih vej izkazalo za enega izmed učinkovitih, s katerim uravnavamo rast in rodnost. Upogibanje je vključeno tudi v gojitveno obliko sončna os in je celo boljši ukrep kot sama rez. Upogibanje rodnih vej vpliva tudi na manjšo rast poganjkov in njihov zgodnejši zaključek vegetativne rasti ter na pospešeno tvorbo cvetnih brstov. Odziv sadnih dreves na upogibanje rodnih vej na biokemičnem področju še ni dovolj raziskan, do sedaj pa tudi ni poznanih raziskav evropske žlahtne hruške z vidika vsebnosti metabolitov v njenih organih kot odziv na upogibanje rodnih vej, zato smo slednje poskušali ovrednotiti v našem dvoletnem poskusu. Obe sorti sta bili cepljeni na podlago kutina MA in bili posajeni v letu 1987 v nasadu Sadjarstva Hudina v Zagaju pri Bistrici ob Sotli. Poskus je vključeval tri obravnavanja: petletne rodne veje, ki smo jih spremenili kot rasti iz 45° na 120° od navpične lege (i) pozno poleti 2003 in (ii) spomladi 2004 ter (iii) petletne rodne veje, ki niso bile upognjene, temveč so vseskozi rasle pod kotom 45° od navpične lege. Na teh rodnih vejah smo v letih 2004 in 2005 v rastni dobi vzorčili liste in v tehnološki zrelosti plodove ter jim s tekočinsko kromatografijo visoke ločljivosti določili in izmerili vsebnosti ogljikovih hidratov (saharoze, glukoze, fruktoze in sorbitola), fenolnih snovi (klorogenske, vanilne in sinapinske kisline ter (-)-epikatehina, (+)-katehina, rutina, kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida) ter v plodovih tudi vsebnosti organskih kislin (citronske, jabolčne, šikimske in fumarne kisline). Med ogljikovimi hidrati smo v plodovih izmerili največje vsebnosti fruktoze in v listih sorbitola. V plodovih sorte ‘Williams’ smo izmerili večje vsebnosti citronske kisline in v plodovih sorte ‘Conference’ večje vsebnosti jabolčne kisline. Med fenolnimi snovmi je bilo v plodovih in listih največ klorogenske kisline. V listih, kjer smo merili vsebnosti metabolitov med rastno dobo od maja do oktobra, so se vsebnosti le-teh med vzorčenji razlikovale, najbolj značilne razlike pa so bile v vsebnosti fenolov. Obe sorti sta imeli v prvem letu najmanjše vsebnosti posameznih fenolnih snovi v maju, nato so se vsebnosti fenolov pri sorti ‘Conference’ povečevale do julija (le vsebnosti katehina vse do septembra), pri sorti ‘Williams’ pa do julija (klorogenska kislina in rutin), avgusta (vanilna in sinapinska kislina) ali septembra (katehin in epikatehin), zatem pa počasi zmanjševale do oktobra. V letu 2005 smo pri obeh sortah zabeležili največje vrednosti večine fenolov že v juniju, nato so se le-te zmanjšale do avgusta in nato ponovno Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 51 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 povečale do konca sezone. Domnevamo, da so na potek fenolov v letu 2005 vplivali tudi okoljski dejavniki, predvsem ekstremne vremenske razmere v juliju in avgustu. Upogibanje rodnih vej v listih ni pokazalo jasnega vpliva na vsebnosti ogljikovih hidratov pri nobeni sorti. Pri sorti ‘Williams’ v prvem letu našega poskusa v vsebnosti ogljikovih hidratov v listih sploh nismo zasledili značilnih razlik, v drugem letu pa sta imela fruktoza in sorbitol najmanjšo vsebnost v avgustu pri kontrolnem obravnavanju, sorbitol pa še v juniju. Toda v septembru je prišlo do preobrata in sorbitol je prav pri kontroli dosegel največjo vsebnost. V listih sorte ‘Conference’ je bila v prvem letu v kontrolnih listih vsebnost saharoze značilno večja v juniju in sorbitola značilno manjša v oktobru, v letu 2005 pa je bila slika sladkorjev še bolj nejasna, saj smo pri glukozi zasledili značilno največje vrednosti po enkrat v vsakem obravnavanju, sorbitol je junija dosegel največjo vsebnost v listih na vejah upognjenih poleti 2003 in oktobra v kontrolnih listih, največje vrednosti fruktoze pa smo izmerili avgusta v listih na vejah upognjenih spomladi 2004. Upogibanje rodnih vej je v listih mnogo bolj vplivalo na vsebnosti fenolov, saj je še posebej poletno upogibanje pri sorti ‘Williams’ v prvem letu povzročilo manjše vsebnosti posameznih fenolnih snovi, največje vsebnosti le-teh pa smo izmerili v listih s kontrolnega obravnavanja. Tudi v naslednjem letu smo izmerili večje vsebnosti nekaterih fenolov v listih z neupognjenih rodnih vej, še posebej vseh treh določanih flavonol glikozidov. Pri sorti ‘Conference’ pa je v prvem letu upogibanje rodnih vej (še posebej spomladansko) povečalo vsebnosti nekaterih fenolov v listih, z izjemo obeh flavan-3-olov. Tudi v letu 2005 smo izmerili večje vsebnosti klorogenske in vanilne kisline ter rutina v listih na vejah upognjenih spomladi 2004, kjer sta imela največjo vsebnost tudi katehin v oktobru in kvercetin-3-D-galaktozida v avgustu. Sicer pa so bile največje vsebnosti sinapinske kisline (junija in oktobra), kvercetin-3-D-galaktozida (maja) in kvercetin-3-ß-D-glukozida (maja, junija, julija in oktobra) v kontrolnih listih. Na podlagi navedenih rezultatov menimo, da sta imela vpliv na učinek upogibanja tudi sorta in čas, ko smo izvedli upogibanje. Proučevani sorti sta se v plodovih različno odzvali na agrotehnični ukrep upogibanje rodnih vej. Pri sorti ‘Williams’ so bile v prvem letu najmanjše vsebnosti ogljikovih hidratov in fenolnih snovi v plodovih, ki smo jih obrali na vejah upognjenih poleti 2003 ter največje vsebnosti omenjenih metabolitov v plodovih na vejah upognjenih spomladi 2004, zato domnevamo, da je upogibanje rodnih vej v pomladnem času za to sorto bolj primerno, tudi z vidika notranje kakovosti plodov. V vsebnosti organskih kislin v nobenem letu ni bilo značilnih razlik. V naslednjem letu so bile vsebnosti glukoze, fruktoze in fenolnih snovi najmanjše pri kontroli; vsebnost sorbitola pa je bila nekoliko večja v plodovih na vejah upognjenih poleti 2003, kjer smo izmerili tudi značilno večje vsebnosti fenolov (z izjemo kvercetin-3-D-galaktozida in kvercetin-3-ß-D-glukozida, katerih vsebnost je bila večja pri kontroli). Pri sorti ‘Conference’ v prvem letu nismo ugotovili značilnih razlik v vsebnosti ogljikovih hidratov med obravnavanji, čeprav so bile vsebnosti glukoze in fruktoze nekoliko večje v plodovih na rodnih vejah upognjenih pozno poleti 2003. Toda v naslednjem letu so bile ravno vsebnosti glukoze in fruktoze značilno večje v kontrolnih plodovih, kjer pa je bila vsebnost saharoze najmanjša. V letu 2004 sta bili vsebnosti jabolčne in fumarne kisline značilno največji pri kontroli, kjer smo hkrati zabeležili najmanjše vsebnosti posameznih Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 52 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 fenolov (z izjemo katehina). Največje vsebnosti fenolov smo v prvem letu izmerili v plodovih na upognjenih rodnih vejah, zlasti upognjenih pozno poleti 2003, zato menimo, da je primernejši čas upogibanja konec poletja. V naslednjem letu smo v kontrolnih plodovih izmerili najmanjšo vsebnost jabolčne kisline ter največje vsebnosti šikimske kisline, epikatehina, sinapinske kisline ter določanih flavonol glikozidov. Toda vsebnosti klorogenske in vanilne kisline so bile nekoliko večje v plodovih na vejah upognjenih spomladi 2004. Najmanjše vsebnosti posameznih fenolov so bile v drugem letu v plodovih na upognjenih vejah: bodisi upognjenih poleti 2003 ali spomladi 2004. V dvoletnem poskusu smo potrdili razlike med proučevanima sortama, pa tudi razlike v času upogibanja - pozno poletno in spomladansko upogibanje - ki smo jih tudi predvidevali. Menimo, da so bistveni rezultati iz prvega poskusnega leta, saj so bile okoljske razmere v mejah dolgoletnih povprečij, v drugem poskusnem letu pa je bilo poletje precej deževno in hladnejše, kar bi lahko vplivalo na preobrat v vsebnosti metabolitov. Vsebnost fenolov je namreč močno odvisna tudi od mnogih okoljskih dejavnikov (stres, temperatura, svetloba), nikakor pa ne smemo zanemariti tudi možnega vpliva notranjih dejavnikov (hranila, hormoni). Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 53 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 4.2 SUMMARY European pear (Pyrus communis L.) is a widespread fruit species in the temperate regions. The investigated cultivars, ‘Williams’ and ‘Conference’, represent an important share of the cultivars grown in Slovenia. They differ in general properties, in growth, branching and fruiting habit; and their fruit differ pomologically. Among the traditional methods of orchard management and cultural practice applied in high-density orchards, branch bending has proven the most successful measure for regulating vegetative growth and fruiting. Its concept has now been integrated into the Solaxe training system, and bending has proven a better measure than pruning alone. Branch bending resulted in reduced shoot vigour, an earlier end to vegetative growth in autumn, and higher floral precocity. The response of fruit trees to branch bending has not been sufficiently researched on the biochemical level. Moreover, no known investigation has been made to date into the content levels of metabolites in the tissues of the European pear as a response to branch bending. Consequently, the bending measure was evaluated in our two-year research. Pear trees ‘Williams’ and ‘Conference’ grafted onto the MA quince were planted in 1987 in the orchard owned by Sadjarstvo Hudina from Zagaj near Bistrica ob Sotli. The research encompassed three treatments: five-year-old branches were bent to an angle of 120° from the vertical position (i) in the late summer of 2003 (the summer treatment), (ii) in spring of 2004 (the spring treatment), and (iii) the control, where five-year-old branches were not bent but remained at a 45° angle from the vertical. Before bending, the bent branches were grown like control branches at an angle of 45°. During the growing seasons of 2004 and 2005, leaves were sampled monthly from May to October, and fruit from those branches were sampled at commercial maturity. The content levels of carbohydrates (sucrose, glucose, fructose and sorbitol), phenolic compounds (chlorogenic, vanillic and sinapic acid, as well as (-)-epicatechin, (+)-catechin, rutin, quercetin-3-D-galactoside and quercetin-3-ß-D-glucoside) in leaves and fruit, as well as organic acids (citric, malic, shikimic and fumaric acid) in the fruit were measured using high-performance liquid chromatography. Among carbohydrates, fructose predominated in the fruit and sorbitol in the leaves in both years. ‘Williams’ fruit had higher content levels of citric acid, and ‘Conference’ fruit higher levels of malic acid. Chlorogenic acid was the major phenolic found in pear leaves and fruit. In the leaves, where the content levels of metabolites were measured during both growing seasons from May to October, these levels differed among sampling dates, mostly in terms of phenolic compounds. In the first year, both cultivars had the lowest content levels of analysed phenolics in May, and then in ‘Conference’ pear the content levels increased to a maximum in July (catechin until September) and in ‘Williams’ to a maximum in July (chlorogenic acid and rutin), August (vanillic and sinapic acid) or September (catechin and epicatechin); afterwards, their content levels decreased slowly until October. In the next year, the maximum in ‘Williams’ and ‘Conference’ leaves was reached earlier – mostly in June, and after decreasing until August, another small increase appeared until the end of Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 54 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 the growing season. It is supposed that the dynamics of phenolics in 2005 could have been influenced by environmental factors, especially by the extreme weather conditions in July and August. Carbohydrate changes in pear leaves after the bending showed no clear tendency for either sampling date, in 2004 or 2005. For selected carbohydrates, no significant changes in their content levels among treatments were observed in ‘Williams’ leaves in the first year. However, in the second year, the control leaves had the lowest levels of fructose in August and of sorbitol in June and August. But in September, the highest sorbitol content was in the control leaves. In 2004, significantly higher sucrose content was observed in ‘Conference’ leaves from the control, where a lower sorbitol content was observed in October. In the second year, unclear differences in carbohydrate content levels emerged, as the glucose content was the highest once in each treatment; then the highest level of sorbitol occurred in June in leaves from the summer treatment and in October in the control leaves; the highest fructose content was found in leaves from the spring treatment. Branch bending had a greater influence on leaf phenolics than on carbohydrates. In 2004, ‘Williams’ leaves from bent branches, especially those bent in summer, had lower content levels of some phenolics, and the highest ones were found in the control leaves. In the second year, significantly higher content levels of some phenolics were measured in the control leaves, particularly of all determined flavonol glycosides. In ‘Conference’ leaves, branch bending (particularly bending in the current spring) influenced higher content levels of some phenolics, with the exception of both flavan-3-ols. Also in the second year, ‘Conference’ leaves from the spring treatment exhibited higher content levels of rutin, chlorogenic and vanillic acid, as well as catechin (in October) and quercetin-3-D-galactoside (in August). Otherwise, the highest content levels of sinapic acid (in June and October), quercetin-3-D-galactoside (in May) and quercetin-3-ß-D-glucoside (in May, June, July and October) were observed in the control leaves. Based on these results, it is suggested that the bending effect is also dependent on cultivar and time of bending (i. e. season). ‘Williams’ and ‘Conference’ fruit responded differently to the branch bending technique. In the year 2004, the content levels of most analysed carbohydrates and phenolics were in ‘Williams’ fruit among the lowest in the summer treatment and among the highest in the current spring treatments, it is therefore presumed that branch bending in spring is more recommended for ‘Williams’ pears, also from the point of view of internal fruit quality. There were no significant differences in content levels of individual organic acids among the treatments, neither in 2004 nor in 2005. In 2005, the content levels of glucose, fructose and phenolic compounds were among the lowest in the control fruit, and the sorbitol content was slightly higher in fruit involved in the summer treatment, where significantly higher content levels of phenolics were measured. The two exceptions were quercetin-3-D-galactoside and quercetin-3-ß-D-glucoside, the content levels of which were highest in the control fruit. In the first year after bending, no significant differences were found in carbohydrate content levels in ‘Conference’ fruit among treatments, although slightly higher glucose and fructose content levels occurred in fruit from the summer treatment. However, in the Colarič M. Vsebnost metabolitov v… hruški (Pyrus communis L.) glede na arhitektonsko zgradbo rodne veje. 55 Dokt. disertacija. Ljubljana, Univ. v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, 2007 following year, only the glucose and fructose levels were significantly higher in the control fruit, where the lowest sucrose content was observed. In 2004, the control fruit showed significantly higher content levels of malic and fumaric acid and lower levels of certain phenolics, except for catechin. In 2004, the highest levels of individual phenolics were measured in ‘Conference’ fruit grown on bent branches, particularly those bent in the late summer of 2003; therefore, a more appropriate time for branch bending is late summer. In 2005, the lowest content of malic acid was in the control fruit. Shikimic acid, epicatechin, sinapic acid and flavonol glycosides appeared in higher content levels in the control fruits. However, chlorogenic and vanillic acid content levels were higher in the fruit involved in the spring treatment. In 2005, the lowest content levels of individual phenolics were found in the fruit from bent branches: either bent in the late summer of 2003 or in the spring of 2004. In the two-year research, the expected differences in content levels of selected metabolites between cultivars and among bending treatments (bent in late summer and in spring) were confirmed. 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Zahvalila bi se tudi Sadjarstvu Hudina iz Zagaja pri Bistrici ob Sotli, da so mi omogočili izvedbo praktičnega dela poskusa v njihovem nasadu hrušk. Posebna hvala staršem, da so mi privzgojili delovne navade, da sem lahko prišla do tega cilja in sestrama Marjeti in Simoni, ki sta mi tudi pomagali pri izvedbi praktičnega dela poskusa. Hvala Edi za ljubečo podporo, razumevanje in vse lepe stvari, ki si jih naredil zame v tem kratkem času. Zahvala tudi vsem, ki so mi kakorkoli pomagali med podiplomskim študijem in pri nastajanju doktorske disertacije, pa jih nisem posebej omenila.