Effects of excess boron on growth, gas exchange, and boron status of four orange scion rootstock combinations

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1 J. Plnt Nutr. Soil Si. 21, 173, DOI: 1.12/jpln Effets of exess oron on growth, gs exhnge, nd oron sttus of four ornge sion rootstok omintions Ou Sheng 1,2, Gofeng Zhou 1, Qingjing Wei 1, Shung Peng 1 *, nd Xiuxin Deng 3 1 College of Hortiulture nd Forestry Sienes, Huzhong Agriulturl University, Key Lortory of Hortiulturl Plnt Biology, Ministry of Edution, Wuhn 437, Chin 2 Institute of Fruit Tree Reserh, Gung Dong Ademy of Agriulturl Sienes, Gungzhou 5164, Chin 3 Ntionl Key Lortory of Crop Geneti Improvement, Huzhong Agriulturl University, Wuhn 437, Chin Astrt Field oservtions indite tht oron (B)-toxiity symptoms my our in itrus plnts from inpproprite folir sprying or overfertilizing with B espeilly under low-rinfll onditions, where B n umulte to levels tht eome toxi to plnt growth. Previous work hs indited tht different rootstoks n gretly influene the sion s tolerne to B toxiity, however, little is known out the response of different itrus sion rootstok omintions to exess-b onditions. In the present study, we investigted the effets of exess B on plnt growth, gs exhnge, B onentrtion, nd distriution of four sion rootstok omintions, Newhll nd Skgg s Bonnz nvel ornge (Citrus sinensis Os.) sions grfted on Crrizo itrnge (C. sinensis L. Os. Ponirus trifolit L. Rf.) nd Trifolite ornge (Ponirus trifolit L. Rf.) rootstoks. One-yer-old plnts of the four sion rootstok omintions were grown for 183 d in snd perlite (1 : 1, v/v) medium under greenhouse onditions. The plnts were irrigted with hlf-strength Hoglnd s nutrient solution ontining two B onentrtions,.25 (ontrol) nd 2.5 (exess B) mg L 1. It ws found tht, prt from the omintion of Newhll grfted on Crrizo itrnge, the dry weights in vrious prts of the other three omintions were redued y the exess-b tretment. Furthermore, the plnts of Skgg s Bonnz grfted on Crrizo itrnge showed the highest growth redution mongst the four sion rootstok omintions. In most ses, the greter redutions in dry weight were found in roots s ompred to the other plnt prts under exess-b onditions, inditing tht roots were more sensitive to B toxiity thn the other tissues. In the se of Newhll plnts grfted on Crrizo itrnge, the entire plnt growth ws inresed y exess-b tretment. Boron onentrtions in ll plnts prts inresed signifintly y inresing the B supply in the nutrient solution. Leves were the dominnt sites of B umultion nd showed the gretest inrese in B onentrtion ompred to the other plnt prts, s B onentrtion in the nutrient solution inresed. Our results indite tht the omintion of Newhll grfted on Crrizo itrnge ws more tolernt to B toxiity, while the omintion of Skgg s Bonnz grfted on Crrizo itrnge ws reltively more sensitive to B toxiity, in omprison with the other sion rootstok omintions. However, Newhll plnts ontined more B in leves nd in roots thn Skgg s Bonnz plnts when they were oth grfted on Crrizo itrnge, inditing tht the mehnism underlying suh gret differentil growth responses of the two sion rootstok omintions to B toxiity my not e ssoited with B exlusion from roots or redued trnslotion of B to shoots. Furthermore, B distriution in different plnt prts implied tht the mehnism ws lso unlikely relted to ltered distriutions of umulted B in plnt tissues. However, inherent ility to tolerte exessive B onentrtion in plnt tissues my e involved in B tolerne. Key words: oron distriution / oron toxiity / Citrus sinensis / gs exhnge / grfting / sion rootstok Aepted Mrh 22, 21 1 Introdution Boron (B) toxiity is n importnt disorder tht n limit plnt growth on soils of rid nd semirid regions (Nle et l., 1997), where itrus plnts re widely grown. Also, toxiity symptoms my our in itrus plnts from inpproprite folir sprying or overfertilizing with B espeilly under low-rinfll onditions, sine under suh onditions B nnot e suffiiently lehed nd therefore my umulte to levels tht eome toxi to plnt growth. Inpproprite pplition of B fertilizer ours when nvel ornge trees re grown in southest Chin, where B toxiity indues lef hlorosis nd senesene, dereses tree vigor s well s fruit numer, size, weight, nd qulity, onsequently resulting in gret loss of yield nd ommeril vlue (Jing et l., 29). * Correspondene: Dr. S. Peng; e-mil: gnjuli_22@mil.hzu.edu.n (Shung Peng), shengou6@yhoo.n (Ou Sheng) 21 WILEY-VCH Verlg GmH & Co. KGA, Weinheim

2 47 Sheng, Zhou, Wei, Peng, Deng J. Plnt Nutr. Soil Si. 21, 173, It hs een reported tht growth vritions in response to high onentrtions of B were oserved in mny rop speies nd even mong different genotypes within the sme speies (Ferrey et l., 1997; Nle et l., 1997). The mehnisms underlying suh vrines re omplited nd remin unler. However, ording to the previous studies, they re minly ssoited with B exlusion from roots, redued trnslotion to shoots nd voidne y mens of shllow root systems. The ility to restrit B uptke into the plnts n minimize the physiologil impirments used y B toxiity (Nle, 1988; Nle et l., 1997; Pull et l., 1992; Hyes nd Reid, 24). On the other hnd, n inherent ility to tolerte exessive B onentrtion in plnt tissues (Torun et l., 26) or the differentil ntioxidnt response tht my redue B-toxiity dmge in some speies (Cervill et l., 27) ws suggested. Furthermore, mny studies showed tht the primry phenotypi effet of B toxiity is poorer root growth ompred to tht of plnts grown t optiml levels of B supply (Lovtt nd Btes, 1984; Nle, 1988; Reid et l., 24; Choi et l., 26). Moreover, the results from Choi et l. (27) indite this mehnism is ssoited with omplex ontrol of surose distriution etween lef nd root tips tht ssists in mintining root growth under B toxiity. However, sine B onentrtions in roots re reltively low ompred with those in leves, even t very high levels of B supply, visile symptoms of B toxiity do not develop in roots (Nle et l., 1997). Thus, differentil tissue tolerne my ply n importnt role in plnt tolerne of suh nutritionl disorder (Pull et l., 1988). On the other hnd, it is likely tht B-toxiity effets pper to e loosely orrelted with umultion of high onentrtions of B in the shoots, whih is funtion oth of the onentrtion of B in soil nd the time of exposure (Reid, 27). Results demonstrted for whet suggest tht symptom soring for B tolerne my e more relile thn mesuring plnt B onentrtions (Klyi et l., 1998; Torun et l., 26). Therefore, omining nlysis of B sttus in vrious plnt tissues with evlution of growth responses ould ontriute to n understnding of the tolerne of plnts sujeted to B toxiity. Wheres B defiieny n e resolved y pplition of B-enrihed fertilizers, toxiity is more diffiult prolem to mnge. Previous work hs indited tht the ornge plnts re sensitive to exess of B (Ms, 199), nd different rootstoks n gretly influene sion s tolerne to B toxiity (El-Motium et l., 1994; Ppdkis et l., 24, ). Therefore, sreening of genotypes hs identified importnt rop vrieties tht n ope with reltively high soil B onentrtions (Ferrey et l., 1997; Nle et l., 1997), whih n lso e used s n pproprite mens for ddressing potentil B toxiity in nvel ornge prodution. Newhll (Citrus sinensis Osek v. Newhll) nd Skgg s Bonnz (C. sinensis Osek v. Skgg s Bonnz) nvel ornges re two importnt itrus ultivrs tht re widely grown in Chin, providing high-qulity fresh fruits for domesti onsumers. Crrizo itrnge (Citrus sinensis L. Os. Ponirus trifolit L. Rf.) is urrently one of the most importnt itrus rootstoks ll over the world, minly due to its vigorous growth nd tolerne to tristez virus (Forner-Giner et l., 23). On the other hnd, Trifolite ornge (Ponirus trifolit L. Rf.) hs een widely used s old-hrdy nd dwrfing rootstok for itrus prodution in Chin. Moreover, little is known out the dptility of the different itrus sion rootstok omintions, i.e., Newhll nd Skgg s Bonnz nvel ornge sions grfted on rootstoks of Crrizo itrng nd Trifolite ornge, to exess-b onditions. The ojetives of this work were: (1) to evlute the effets of exess B on plnt growth, gs exhnge, nd B onentrtion nd distriution of Newhll nd Skgg s Bonnz nvel ornge sions grfted on Crrizo itrng nd Trifolite ornge rootstoks, nd (2) to ompre the differentil responses of the four sion rootstok omintions under onditions of exess B. 2 Mterils nd methods 2.1 Plnt mterils nd oron tretments Newhll (Citrus sinensis Osek v. Newhll) nd Skgg s Bonnz (C. sinensis Osek v. Bonnz) nvel ornge sions grfted on two rootstoks, either Crrizo itrnge (Citrus sinensis L. Os. Ponirus trifolit L. Rf.) or Trifolite ornge (Ponirus trifolit L. Rf.) were used in this experiment. These omintions re referred to s: N/CC, Newhll grfted on Crrizo itrnge; N/TO, Newhll grfted on Trifolite ornge; SB/CC, Skgg s Bonnz grfted on Crrizo itrnge; SB/TO, Skgg s Bonnz grfted on Trifolite ornge. The uds of two sion vrieties did not sprout efore our experiment, nd the initil shoots (out 3 4 m ove the grfting unit) of the rootstok seedlings were removed. At udding time, the seedlings of oth rootstoks were 1 yer old nd seleted sed on uniform stem dimeter, 4 5 mm for Trifolite ornge nd 5 7 mm for Crrizo itrnge, respetively. Plnt mterils (rootstok seedlings nd uds of sions) used in the present experiment ws virus-free nd hrvested from the Ntionl Indoor Conservtion Center of Virus-free Germplsms of Fruit Crops t Huzhong Agriulturl University, Wuhn, Chin. When prepring for the experiment, the roots of the plnts were severely pruned (to stimulte new root development following B tretments; Ppdkis et l., 24, ). Susequently, ll the plnts were wshed with tp wter to remove surfe ontminnts, followed y trnsplnttion to lk pots (one plnt per pot) ontining 1 L B-free medium omposed of qurtz snd nd perlite (1 : 1, v/v). Before the trnsplnttion, the medium hd een wshed with 3% hydrohlori id (HCl) overnight nd then rinsed thoroughly with deionized wter, in order to ensure the B onentrtion in the medium ws less thn.1 mg L 1 determined y n indutively oupled plsm tomi emission spetrometry (ICP- AES, IRIS-Advn type, Thermo, USA). Therefter, the plnts were set up in greenhouse, nd supplied initilly with modified B-free, 1/4 strength Hoglnd s No. 2 nutrient solution (Hoglnd nd Arnon, 195) for 4 weeks, until the sprouting shoots of the sions were out 8 1 m in length (with seven to nine leves). The tretments strted t the eginning of My in 26. The plnts were irrigted with modified Hoglnd s No WILEY-VCH Verlg GmH & Co. KGA, Weinheim

3 J. Plnt Nutr. Soil Si. 21, 173, Boron toxiity in sion rootstok omintions 471 nutrient solution (prepred with doule-deionized wter), in whih the mronutrients were supplied t hlf strength nd mironutrients t full strength, exept for B, whih ws supplied t two onentrtions, viz..25 mg B L 1 (ontrol) nd 2.5 mg B L 1 (to indue B toxiity; Ppdkis et l., 24, ; Hoglnd nd Arnon, 195). The plnts were irrigted with the nutrient solutions every 2 d (pproximtely 5 ml per plnt), llowing some dringe from the growth medium to our. To void slt nd B umultion, the plnts were irrigted with 1 L doule-deionized wter one week, followed y pplition of 3 L nutrient solution, nd the exess solution drined from the ottom of the growth medium (Bellloui nd Brown, 1998). The experiment lsted for 183 d until visile typil symptoms of B toxiity ppered. During the B tretments, the plnt-growth onditions were ontrolled s follows: 55 to 12 lmol m 2 s 1 flux density of nturl light, 33 C/25 C (dy/night) of temperture, nd 8% of reltive humidity (mesured with n Li-64 portle mesuring devie, Li-COR, Linoln, USA). 2.2 Lef-gs-exhnge mesurements At the end of the experiment, the gs-exhnge prmeters, i.e., the photosyntheti rte (P n ; lmol CO 2 m 2 s 1 ), stomtl ondutne (g s ; mmol H 2 Om 2 s 1 ), interellulr CO 2 onentrtion (C i ; lmol mol 1 ), nd trnspirtion rte (E; mmol H 2 Om 2 s 1 ) of ll plnts were mesured using the Li-64 devie. Mesurements were performed on sl leves, loted etween the middle nd the se of the sion s shoots of eh plnt (nine replites per B tretment nd sion rootstok omintion). Mesurements were rried out etween 9:.m. nd 12: noon t stedy light intensity (1 lmol m 2 s 1 ) under CO 2 onentrtion of (385 ± 1) ll L 1 nd lef temperture etween 28 C nd 3 C. 2.3 Smpling, plnt nlyses, nd oron determintion When the plnts were hrvested, the sion s prts inluding leves nd stem of sion, nd rootstok s prts inluding root nd stem of rootstok, were seprtely smpled. The leves were further seprted into sl leves (spring-flush), middle leves (summer-flush), nd upper leves (utumn-flush) sed on the different phses of shoot growth. All the smples were wshed initilly with tp wter nd fterwrds with deionized wter for three times. The fresh nd dry weights were mesured. Eh dried smple ws ground to fine powder nd stored in n ir-tight glss ontiner for susequent nlyses. For mesurement of B onentrtion,.5 g of eh smple were dry-shed in muffle furne t 5 C for 6 h, followed y dissolution in.1 N HCl, nd B ws determined using ICP- AES. Boron distriution ws expressed s perentge rtios (frtion,%) of B ontent (onentrtion dry weight) in different plnt prts reltive to totl plnt B ontent (Suedi et l., 1999; Möttönen et l., 21). The ptterns of B distriution in leves, roots, nd stems (either of sion or rootstok) re presented to show the hnges in reltion to the frtions of the totl mount of B tken up y plnts upon exposure to exess B. 2.4 Experimentl design nd sttistil nlyses The experiment ws set up in ompletely rndomized 4 2 ftoril design with four sion rootstok omintions (two sions of nvel ornge grfted on two rootstoks) nd two B tretments (.25 nd 2.5 mg B L 1 ). Three replitions (three plnts in eh) were designed for eh tretment. The experimentl dt were sujeted to nlysis of vrine (ANOVA) using the SAS (SAS Institute In., 1996), nd the differenes were ompred y employing the Dunn test with signifine level of p <.5. 3 Results 3.1 Symptoms of oron toxiity nd plnt growth Visile symptoms of B toxiity developed when 2.5 mg B L 1 (exess B) were pplied to the plnts of Newhll nd Skgg s Bonnz nvel ornge sions grfted either on Crrizo itrnge or on Trifolite ornge rootstoks, using hlorosis t the tip of old leves, whih extended to the rest of the lef lde nd the middle leves. The symptoms eme more severe nd pprent in Skgg s Bonnz plnts grfted on Crrizo itrnge t the end of the experiment. The sions grfted on Crrizo itrnge exhiited reltively higher dry weights thn those grfted on Trifolite ornge, irrespetively of B tretment. Of the two sions grfted on the sme rootstok, Newhll plnts ppered to produe more iomss thn Skgg s Bonnz (T. 1). Aprt from the omintion of Newhll grfted on Crrizo itrnge, the dry weights in vrious prts of the other three sion rootstok omintions were redued y the exess-b tretment. It seemed tht the redution ws more pronouned for roots. When the plnts were treted with exess B, the totl plnt dry weights deresed y 27% in plnts of Newhll grfted on Trifolite ornge, nd y 31% or 27% in plnts of Skgg s Bonnz when they were grfted either on Crrizo itrnge or on Trifolite ornge, respetively. In the se of the omintion, Newhll grfted on Crrizo itrnge, dry weights in ll the plnt prts exept in middle leves inresed when B onentrtion in nutrient solution ws rised to 2.5 mg L 1. Within this omintion, the totl plnt dry weight inresed y 1% upon exposure to exess B, ompred with the.25 B mg L 1 tretment (T. 1). 3.2 Boron onentrtion nd distriution Boron onentrtions in ll plnt prts inresed signifintly s externl B onentrtion inresed from.25 to 2.5 mg L 1, following the order: sl leves > middle leves > upper leves > roots > stems of rootstok > stems of sion, for ll sion rootstok omintions. There were 4- to 1-fold, 2.5- to 3-fold, 1.5- to 2-fold, nd 1.2- to 1.5-fold inreses of B onentrtions of leves, roots, stems of sion s well s of rootstok stems, respetively (Fig. 1). These results suggested tht lef nd root tissues were more sensitive to 21 WILEY-VCH Verlg GmH & Co. KGA, Weinheim

4 472 Sheng, Zhou, Wei, Peng, Deng J. Plnt Nutr. Soil Si. 21, 173, Tle 1: Effets of exess B (2.5 mg B L 1 ) on dry weight (g [plnt DW] 1 ) of different plnt prts of Newhll (N) nd Skgg's Bonnz (SB) nvel ornge sions grfted on trifolite ornge (TO) nd Crrizo itrnge (CC) rootstoks. Vlues re mens of three replites (n = 3), with three plnts per eh replite. Mens within eh olumn followed y the sme letter do not signifintly differ t p <.5. F vlues for the sion, rootstok, B tretment, nd their intertions re shown. Sion/ rootstok B /mgl 1 Bsl lef Middle lef Upper lef Stem of sion Stem of rootstok Root Sion Rootstok Totl plnt N/CC N/TO d d 44.2 d d 3.88 d 5.19 d 6.54 e 2.82 ef 11.2 e 18.3 e 14.1 f 32.4 f SB/CC d f 21.6 d 14.5 ef 36.1 e SB/TO d d 3.27 ed 13. d 21.1 d 16.3 e 37.4 e d 3.61 d 4.97 d 5.18 f 2.25 f 8.79 f 16.4 f 11.1 g 27.4 g F vlues Sion 15.3** 37.6*** 53.5*** 91.1*** 61.5*** 182.4*** 221*** 219*** 443*** Rootstok 23.7*** 72.7*** 319*** 773*** 771*** 73.*** 996*** 36*** 17*** B 6.1* 3.6*** 29.2*** 22*** 4.42ns 91.1*** 213*** 85.4*** 26*** Sion rootstok 6.27* 16.9*** 41.9***.6ns 2.3*** 21.*** 61.8*** 32.3*** 86.3*** Sion B.8ns.18ns 24.8*** 64.3*** 43.2*** 24.3*** 55.2*** 45.3*** 98.9*** Rootstok B 2.1*** 2.86ns.1ns 3.4ns 17.4*** 16.3*** 5.95* 25.8*** 32.9*** Rootstok sion B 2.5ns.82ns 2.41*** 48.7*** 38.*** 56.2*** 54.4*** 78.6*** 133*** Signifine of ANOVA: * p <.5; ** p <.1; *** p <.1; ns: nonsignifint 12 N/CC N/TO SB/CC SB/TO B onentrtion / mg (kg DW) A d.25 mg L mg L 1.25 mg L mg L 1.25 mg L mg L 1 Bsl lef Middle lef Upper lef B onentrtion / mg (kg DW) B.25 mg L mg L 1.25 mg L mg L 1.25 mg L mg L 1 Stem of sion Stem of rootstok Root Figure 1: Effets of exess B (2.5 mg B L 1 ) on B onentrtion (mg [kg dry weight] 1 ) in (A) leves (sl, middle, nd upper) nd (B) stems of sion nd rootstok, nd root of Newhll (N) nd Skgg s Bonnz (SB) nvel ornge sions grfted either on Crrizo itrnge (CC) or on Trifolite ornge (TO) rootstoks. Vlues represent the mens ± stndrd errors of three replites (n = 3), with three plnts per eh replite. Brs with the sme letter re not signifintly different t p <.5 within eh prt t 2.5 mg B L WILEY-VCH Verlg GmH & Co. KGA, Weinheim

5 J. Plnt Nutr. Soil Si. 21, 173, Boron toxiity in sion rootstok omintions 473 externl high B thn the other plnt prts. In plnts treted with 2.5 mg B L 1, it ws further found tht leves nd roots from Newhll grfted on Crrizo itrnge ontined reltively more B, while those from the omintion of Skgg s Bonnz grfted on Trifolite ornge ontined reltively less B, s ompred with the other sion rootstok omintions. Leves represented the dominnt sites of B umultion, regrdless of the omintion nd B tretment, ontining from 6% to 85% with inresing externl B onentrtion from.25 to 2.5 mg L 1, while in the other prts the rtios deresed with the inrese of externl B pplition (T. 2; Fig. 2). At 2.5 mg B L 1, the frtions of B distriuted to leves were more in the omintions of Crrizo itrnge grfted plnts ( 87%) thn in the omintions of Trifolite ornge grfted plnts ( 83%). However, the former ontined less B in roots (8.% 9.9%) thn the ltter (11.8% 12.3%) t 2.5 mg B L Effets of exess B on lef-gs-exhnge prmeters Lef-gs-exhnge prmeters were gretly influened y B tretment (Fig. 3, T. 2). After treted y 2.5 mg B L 1, lef photosyntheti rte (P n ), stomtl ondutne (g s ), nd trnspirtion rte (E) deresed y out 49% 77%, 45% 63%, nd 31% 49%, respetively, s ompred with those of plnts grown t.25 mg B L 1 (Fig. 3A, B, nd D). These prmeters were redued to greter extent in plnts of Skgg s Bonnz grfted on Trifolite ornge. Moreover, reltive higher vlues in P n nd g s were reorded in leves from the plnts of Newhll grfted on Crrizo itrnge, irrespetively of B supply. On the other hnd, interellulr CO 2 onentrtion (C i ) inresed y out 15% 25% t 2.5 mg B L 1 (Fig. 3C), whih however, did not differ signifintly upon exposure to the sme B tretment mong the four sion rootstok omintions. 4 Disussion In our experiment, the mjor effets of B toxiity on itrus plnts were the deline in dry mtter of vrious plnt prts, inhiition of root growth, lef hlorosis developing to rown spots t the tips nd the edges of the leves leding to nerosis. Symptoms were more severe in exess-b-treted Skgg s Bonnz plnts grfted on Crrizo itrnge (T. 1). The symptoms of B toxiity ppered on old (sl) leves, t the end of the trnspirtion strem (Brown nd Shelp, 1997), in greement with other oservtions desried for itrus plnts when they were treted with exess B (Ppdkis et l., 24, ). The toxiity symptoms my e used y the metoli disruption due to high internl onentrtions of B inding to the riose moieties of ATP, NADH, or NADPH from the metoli pool, whih inhiit Tle 2: F vlues nd levels of signifine for sion, rootstok, nd B tretment nd their intertions from ANOVA on B onentrtion nd B distriution (in lef, stem of sion, stem of rootstok s well s in root) nd on gs-exhnge prmeters, viz. the photosyntheti rte (P n ), stomtl ondutne (g s ), interellulr CO 2 onentrtion (C i ), nd trnspirtion rte (E). B onentrtion B distriution Gs exhnge lef stem of sion stem of rootstok root lef stem of sion stem of rootstok root P n g s C i E Sion 135*** 39.7***.96ns 85.7*** 2.14ns 94.2*** 8.21* 7.28* 8.42** 16.6** 1.86ns 5.81* Rootstok 196*** 54.9*** 139*** 43.8*** 12.7** 1.96ns 28.8*** 42.4*** 1.4** 7.28* 1.64* 7.15* B 94*** 3785*** 24*** 6596*** 1431*** 1471*** 495.4*** 752*** 243*** 353*** 3.94*** 27*** Sion rootstok 6.5* 3.89ns 2.54ns 11.2** 5.17* 28.2*** 1.39ns.42ns 1.91ns 12.7** 1.49ns 17.8*** Sion B 22.7*** 2.14ns 2.22ns 39.8***.9ns 11.4**.64ns 1.32ns 1.43ns.69ns.26ns.14ns Rootstok B 49.2*** 26.2*** 13.** 96.4*** 8.44* 12.2** 25.4***.26ns 18.6*** 11.**.2ns 4.23* Rootstok sion B 5.1***.6ns 16.8*** 6.56* 9.75** 7.19*.6ns 11.** 2.23ns 1.ns.5ns.36ns Signifine of ANOVA: * p <.5; * *p <.1; *** p <.1; ns: nonsignifint 1% 8% Lef B ontent rtio 6% 4% 2% % N/CC N/TO SB/CC SB/TO N/CC N/TO SB/CC SB/TO.25 mg L mg L 1 Stem of sion Stem of rootstok Root Figure 2: Effets of exess B (2.5 mg B L 1 ) on perentge (%) of totl B ontent prtitioned to lef, stem of sion, stem of rootstok, nd root of Newhll (N) nd Skgg s Bonnz (SB) nvel ornge sions grfted either on Crrizo itrnge (CC) or on Trifolite ornge (TO) rootstoks. Vlues represent the men ± SE of three replites (n = 3), with three plnts per eh replite. 21 WILEY-VCH Verlg GmH & Co. KGA, Weinheim

6 474 Sheng, Zhou, Wei, Peng, Deng J. Plnt Nutr. Soil Si. 21, 173, Interellulr CO 2 onentrtion / μmol mol 1 Photosynthesis rte / μmol CO 2 m 2 s A N/CC N/TO 6 SB/CC SB/TO C.25 mg L mg L 1.25 mg L mg L 1 B onentrtion in nutrient solution d d Stomtl ondutne / mmol H 2 O m 2 s 1 Trnspirtion rte / mmol H 2 O m 2 s B D B onentrtion in nutrient solution d e d d Figure 3: Effets of exess B (2.5 mg B L 1 ) on (A) photosynthesis rte (lmol CO 2 m 2 s 1 ), (B) stomtl ondutne (mmol H 2 O m 2 s 1 ), (C) interellulr CO 2 onentrtion (lmol mol 1 ), (D) trnspirtion rte (mmol H 2 Om 2 s 1 )of Newhll (N) nd Skgg s Bonnz (SB) nvel ornge sions grfted either on trifolite ornge (TO) or on Crrizo itrnge (CC) rootstoks. Vlues represent mens ± stndrd errors of nine replites (n = 9). Brs with the sme letter re not signifintly different t p <.5. norml iosyntheti tivities or energy trnsdution (Reid et l., 24; Reid, 27), onsequently leding to impired growth nd development. On the other hnd, exess B indues oxidtive dmge y lipid peroxidtion nd hydrogen peroxide umultion, whih my lso ontriute to the expression of the B- toxiity symptoms (Gunes et l., 26; Cervill et l., 27). Roots were more sensitive to externl B thn the other plnt prts, sine in most ses, the inhiitory effets of exess B on growth were more pronouned in roots (T. 1). The inresed inidene of norml mitosis in root meristem, whih influenes ell division nd ultimtely dereses the rte of root elongtion (Klein nd Brown, 1981; Liu et l., 2), ws proly involved in deresed root growth, whih would limit the ility of the plnts to tke up essentil minerl nutrients nd wter, nd onsequently ffet the growth of oveground of the plnts (Reid, 27). Otherwise, root tips re lso site of B toxiity, nd nlysis of physiologil nd morphologil hnges of root tips ould predit the potentil ourrene of B toxiity, sine the primry effet of toxi B might e on root tips when high B ws pplied to the rooting medium (Reid et l., 24). Aording to the growth performne of the plnts fter longterm exposure to exess B, only the sion rootstok omintion of Newhll grfted on Crrizo itrnge exhiited etter root growth, produed more new young leves (upper leves) nd onsequently hd signifint inreses (1%) in totl plnt dry weight under exess-b onditions (T. 1). In ontrst, the other omintions showed redution ( 3%) in entire plnt dry weight upon exposure to exess B (T. 1). When Skgg s Bonnz plnts were grfted on Crrizo itrnge, this omintion showed the gretest growth redutions in root tissues (43%) nd produed less upper young leves (T. 1). These results indite tht Newhll grfted on Crrizo itrnge ws the most tolernt omintion while Skgg s Bonnz grfted on Crrizo itrnge my e the most sensitive omintion onerning B toxiity, mongst the four sion rootstok omintions tht were evluted in our study. From our dt of plnt dry weight nd B onentrtion, it n e esily lulted tht Crrizo itrnge grfted plnts umulted more B thn Trifolite ornge grfted plnts (dt not shown), implying tht the roots of Crrizo itrnge ould tke up more B thn the roots of Trifolite ornge. However, t 2.5 mg B L 1 Newhll plnts ontined more B in leves thn Skgg s Bonnz plnts, irrespetively of the rootstok used (Fig. 1). These results indite tht the mehnism underlying the gret differentil growth responses etween the two sion rootstok omintions (N/CC nd SB/ CC) to B toxiity nnot e explined in terms of B exlusion from roots or redued trnslotion of B to shoots. However, the mehnism my e ssoited with the inherent ility to tolerte exessive B onentrtion in plnt tissues. It ws suggested y severl studies tht the internl omprtmenttion of B in vuoles is plusile explntion for B tolerne 21 WILEY-VCH Verlg GmH & Co. KGA, Weinheim

7 J. Plnt Nutr. Soil Si. 21, 173, Boron toxiity in sion rootstok omintions 475 (Torun et l., 23, 26; Mhlkshmi et l., 1995). Torun et l. (26) evluted genotypi vrition in tolerne to B toxiity in 7 durum whet (Tritium durum) genotypes nd found tht there ws no reltionship etween shoot B onentrtions nd reltive dereses in shoot dry weight used y B toxiity, ut the most B-sensitive genotypes generlly hd muh lower mount of B in shoot thn the genotypes showing higher tolerne to B toxiity. Similr results were lso shown in red whet (Tritium estivum; Klyi et l., 1998) nd in rley (Hordeum vulgre L; Mhlkshmi et l., 1995; Torun et l., 23): The tolernt ultivrs mintined high levels of B in shoots or/nd roots. It ws further speulted y Kur et l. (26) tht formtion of B-polyol omplexes in the vuoles would e n effetive wy of storing B without dmge to ellulr metolism. The B-sensitive genotype my lk this mehnism to limit toxi B onentrtion in the ytoplsm, with onsequent dereses in tissue iomss (Kur et l., 26). Previous work hs proven tht different rootstoks n gretly influene sion s tolerne to B toxiity (El-Motium et l., 1994; Ppdkis et l., 24, ), whih lso n e onluded from our results tht oth sion nd rootstok influenes determine growth responses (Ts. 1 nd 2). It ws further oserved in our experiment tht omplited sion rootstok intertions exist in the four sion rootstok omintions, showing different growth responses to exess-b stress (Ts. 1 nd 2). There were few reports on the omintions of sion rootstok upon exposure to exess B in itrus plnts. However, previous studies hve suggested tht root rhiteture ould diretly ffet exploittion effiieny of nutrition nd wter required for plnt growth (Berntson, 1994; Nielsen et l., 1994), while rohydrte derived from the lef photosynthesis nd phytohormones synthesized in the growing shoots n ffet the rhiteture of the root system nd its funtion (Thler nd Pgès, 1996; Wightmn nd Thimnn, 198). In our experiment, exess-b tretment my hve used signifint derese in net photosynthesis (Fig. 3), leding to negtive effets on growth nd the ility to supply photosynthtes, whih n redue the trnslotion of rohydrtes to root, inhiiting growth nd funtion of the root. Our results show tht B onentrtions in the leves of Newhll or Skgg s Bonnz plnts inresed s B onentrtions in nutrient solution inresed, nd tht leves were the dominnt sites of B umultion (Figs. 1, 2), whih oinided with the previous studies (Ppdkis et l., 24, ). This ould e explined y the fts tht B trnsported through the xylem is gretly influened y trnspirtion (Rven, 198; Brown nd Shelp, 1997). Moreover, effets of exess B on perentge of totl B ontent prtitioned to different plnt prts showed similr hnges in the distriution pttern mongst the four sion rootstok omintions, despite the gret differenes in totl mounts of umulted B found mong them (dt not shown). This suggests tht the reltive B tolerne in the four sion rootstok omintions ws lso unlikely to e ssoited with ltered distriutions of umulted B in plnt tissues, in ordne with the previous results in erels (Nle et l., 1997). Our dt of gs-exhnge prmeters refleted lose reltionships with high B supply, whih deresed lef photosyntheti rte nd stomtl ondutne, ut inresed interellulr CO 2 onentrtion of the Newhll nd Skgg s Bonnz plnts grfted either on Trifolite ornge or on Crrizo itrnge (Fig. 3), inditing tht oth stomtl nd nonstomtl ftors were involved in the dereses of P n under exess-b onditions. Similr responses were reported in other itrus speies (Ppdkis et l., 24, ) nd kiwifruit (Sotiropoulos et l., 22) when they were grown under B toxiity. Boron toxiity n use derese in hlorophyll ontent nd struturl dmge of hloroplsts, whih led to derese in CO 2 -ssimiltory pity of the mesophyll ells (Ppdkis et l., 24, ), ultimtely ffeting plnt growth. Furthermore, lef-gs-exhnge prmeters reorded in Skgg s Bonnz plnts grfted on Crrizo itrnge, were gretly ffeted y exess-b tretments, showing the lowest vlues in P n, g s nd E fter exposure to exess B (Fig. 3, T. 2), whih my e linked to the more severe negtive effets of exess B on this sion rootstok omintion. 5 Conlusion Amongst the four sion rootstok omintions evluted in our experiment, Skgg s Bonnz plnts grfted on Crrizo itrnge showed the highest growth redution under exess B, wheres the plnt growth of Newhll plnts grfted on Crrizo itrnge ws inresed y the tretment of exess B. These results indite tht the omintion of Newhll grfted on Crrizo itrnge ws more tolernt to B toxiity, while the omintion of Skgg s Bonnz grfted on Crrizo itrnge ws more sensitive to B toxiity. The mehnism underlying suh gret differentil growth responses etween the two sion rootstok omintions to B toxiity ws not explined in terms of B exlusion from roots or redued trnslotion of B to shoots, ut n inherent ility to tolerte exessive B onentrtion in plnt tissues of Newhll nvel ornge. Aknowledgment This study ws supported y Ntionl Nturl Siene Foundtion of Chin (No , ). The uthors re grteful to Dr. Ionnis E. Ppdkis (Aristotle University, Greee) for his vlule tehnil ssistne nd omments on this work. Referenes Bellloui, B., Brown, P. H. (1998): Cultivr differenes in oron uptke nd distriution in elery (Apium grveolens), tomto (Lyopersion esulentum) nd whet (Tritium estivum). Plnt Soil 198, Berntson, G. M. (1994): Modelling root rhiteture: Are they trdeoffs etween effiieny nd potentil of resoure quisition? New Phytol. 127, Brown, P. H., Shelp, B. J. (1997): Boron moility in plnts. Plnt Soil 193, WILEY-VCH Verlg GmH & Co. KGA, Weinheim

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(1996): Periodiity in the development of the root system of young ruer trees (Heve rsiliensis Müll. Arg.): Reltionship with shoot development. Plnt Cell Environ. 19, Torun, A. A., Yzii, A., Erdem, H., Çkmk, I. (26): Genotypi vrition in tolerne to oron toxiity in 7 durum whet genotypes. Turk. J. Agri. For. 3, Torun, B., Klyi, M., Ozturk, L., Torun, A., Aydin, M., Ckmk, I. (23): Differenes in shoot oron onentrtions, lef symptoms, nd yield of Turkish rley ultivrs grown on oron-toxi soil in field. J. Plnt Nutr. 26, Wightmn, F., Thimnn, K. V. (198): Hormonl ftors ontrolling the initition nd development of lterl roots. I. Soures of primordi-induing sustnes in the primry root of pe seedlings. Physiol. Plnt 49, WILEY-VCH Verlg GmH & Co. KGA, Weinheim