Environmental and Experimental Botany 64 (2008) 256–263

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Environmental and Experimental Botany

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Girdling induces oxidative damage and triggers enzymatic and non-enzymaticantioxidative defences in Citrus leaves

Fernando Rivas, Fernando Fornes ∗, Manuel AgustíInstituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, P.O. Box 22012, E46071 Valencia, Spain

a r t i c l e i n f o

Article history:Received 4 October 2007Received in revised form 8 July 2008Accepted 13 July 2008

Keywords:ProlineSoluble sugarsStarchGlutathioneAscorbic acid

a b s t r a c t

The effects of girdling on oxidative damage, antioxidant enzyme activity, antioxidant metabolites andproline (Pro) were studied in leaves arising from different shoot types of potted 2-year-old ‘Loretina’ man-darin (Citrus reticulata Blanco) trees during the spring flush period. Girdling increased malonyldialdehyde(MDA) and basal chlorophyll (Chl) a fluorescence (Fo) in young leaves 30 days after girdling but not inthe mature leaves (ML) suggesting a disruption of photosynthetic apparatus and oxidative damage inyoung leaves. This phenomenon was accompanied by increasing levels of Pro. Paralleling these changes,an increase of all antioxidant enzyme activities occurred in leaves from vegetative (VG) and multiflow-ered leafy shoots (MLY) of girdled trees. Similarly, in ML of girdled trees, ascorbate peroxidase (APX),catalase (CAT) and glutathione reductase (GR) activity also increased. However, dehydroascorbate reduc-tase (DHAR) activity decreased and superoxide dismutase (SOD) activity remained unchanged. Total leaf

Water–water cycle enzymes carbohydrate content and starch also increased as a result of girdling in all shoot types. Whilst solublesugars increased markedly in young leaves, they increased only slightly in ML. In conclusion, this studyprovides evidence that girdling gives rise to oxidative damage in Citrus during carbohydrate accumulation,

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. Introduction

Girdling is a well-known technique broadly used in horticul-ure to manage the growth and reproduction of many deciduousnd evergreen tree species. In Citrus, depending on the time oferformance, trunk girdling can be used to promote flowering,

mprove fruit set and fruit growth, and advance fruit maturity (seeoren et al., 2003 for review). However, growers sometimes refer

o girdling as a risky practice since the overall tree growth coulde retarded even though no external symptoms of damage arepparent (Winkler et al., 1974). Furthermore, girdling is not recom-ended to be applied in weak trees and during adverse growing

onditions (Goren and Monselise, 1971).The effects of girdling have been linked to the interruption of

he downward sap flux, thereby increasing carbohydrate availabil-ty (Wallerstein et al., 1974) and modifying the hormonal balancen the canopy (Goren et al., 1971). In Citrus, concomitantly with the

ccumulation of carbohydrates in leaves, damage of the thylakoidalembranes accompanied by a decline in the photosynthetic CO2

ssimilation rate have been reported (Bondada and Syvertsen,005). Traditionally, this effect has been ascribed to a profuse starch

∗ Corresponding author.E-mail address: ffornes@bvg.upv.es (F. Fornes).

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098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2008.07.006

nzymatic defence mechanisms.© 2008 Elsevier B.V. All rights reserved.

ccumulation, producing large granules and causing disassemblynd disruption of the internal membrane system (Schaffer et al.,986; Bondada and Syvertsen, 2005). However, a revised view ofhis topic holds that the accumulation of soluble sugars may inducehe overproduction of reactive oxygen species (ROS) in leaves whichan finally lead to complete cells destruction (see Couée et al., 2006or review). Furthermore, this could be linked with the severe stressymptoms evidenced by leaf chlorosis, leaf drop or even tree death,ccasionally observed several months after girdling (Noel, 1970;oren et al., 2003).

Whenever damage or malfunction of the photochemical appa-atus occurs, the excess of absorbed light could also give riseo an overproduction of ROS such as superoxide radicals (O2

−•),ydrogen peroxide (H2O2) and hydroxyl radicals (OH•), caus-

ng damage to the cell structure (Halliwell, 1987; Mittler et al.,004). However, plants have evolved a subtle integrated systemf enzymatic and non-enzymatic antioxidants to keep ROS strictlyontrolled, thereby preserving cell integrity and functioning dur-ng oxidative stress (Noctor and Foyer, 1998; Asada, 1999; Apel andirt, 2004). The plant antioxidant system includes the enzymes

uperoxide dismutase (SOD), catalase (CAT), ascorbate peroxidaseAPX), monodehydroascorbate reductase (MDHAR), dehydroascor-ate reductase (DHAR) and glutathione reductase (GR) as well asntioxidant compounds such as ascorbate (AsA) and glutathioneGlut), most being involved in the so-called water–water cycle,

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hich serves to scavenge the O2−• produced from the reduction

f O2 by photosystem I (Noctor and Foyer, 1998; Asada, 1999).Proline (Pro) synthesis has also been proposed as a non-

nzymatic means to scavenge OH• radicals, and as an alternativeink for energy to regulate the cellular redox state (see Hare andress, 1997 for review). Pro can also protect cell membranes fromalt-induced oxidative stress by enhancing activities of variousntioxidants (Yan et al., 2000). The accumulation of this amino acidn cells has been reported for different abiotic stresses such as salin-ty (Arbona et al., 2003), drought (Hsu et al., 2003), chilling (Kushadnd Yelenosky, 1987) and UV-B radiation (Chris et al., 2006), and itas also been reported that Pro accumulates in Citrus leaves afterirdling (Fornes et al., 1990).

Based on this current knowledge, we hypothesized that if anyamage or malfunction of the photosynthetic apparatus occursuring carbohydrate accumulation brought about by girdling,xidative stress could trigger enzymatic and non-enzymatic mech-nisms that should lessen oxidative damage. To our knowledge theonitoring of antioxidant mechanisms under these specific cir-

umstances has so far not been reported. In this study we providevidence to show that girdling induces oxidative damage and trig-ers enzymatic and non-enzymatic ROS scavenging mechanisms initrus leaves during carbohydrate accumulation.

. Materials and methods

.1. Plant material, growth conditions and treatment

Ten containerized 2-year-old trees of ‘Loretina’ mandarin (Citruseticulata Blanco), grafted onto Carrizo citrange (Citrus sinensis [L.]sbeck × Poncirus trifoliata Raf.) were used in the experiment. Treesere grown outdoors in Valencia (Spain, 39◦ NL), in 10-l plastic

ontainers with a sandy-loamy soil and fertilized with nitrogen at0 g tree−1 year−1. All plants were drip irrigated daily with enoughater to maintain a minimum leaching fraction of 25%. Insects andiseases were monitored at 2-day intervals and controlled whenecessary.

Girdling was done at anthesis (when 60% of the flowers werepened) by making a 1 mm wide cut in the bark completelyncircling the trunk 10 cm above the rootstock with a sharp hooked-lade. Care was taken to avoid injuring the xylem or removingark.

At the beginning of the experiment and 30 days after girdlingDAG), when the young leaves were assumed to be a source of pho-oassimilates instead of a sink (Ruan, 1993; Rivas et al., 2007), threeypes of leaves were sampled from trees to accomplish a broadnalysis of the canopy: (1) mature leaves (ML) approximately 14onths old, from the spring flush of the previous year; (2) young

rowing leaves (2 month old) from vegetative shoots (leafy shoots;G) of the current spring flush, and (3) young growing leaves (2onths old) from mixed shoots (leafy-flowered shoots; MLY) also

rom the current spring flush. For each sample, 25 leaves of each ofhe three types were picked from every tree and were immediatelyreeze-ground with liquid N2 and stored at −70 ◦C until analysis.or carbohydrate and Pro, additional samples were taken from theame trees at 7, 15 and 60 DAG and they were lyophilized beforenalysis. All samples were taken before midday and analysed ateast three times.

.2. MDA analysis

The extent of lipid peroxidation was measured through the thio-arbituric acid test (TBA) which determines MDA as an end-productf lipid peroxidation according to the protocol adapted for leavesy Heath and Parker (1968), with the modifications described by

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ental Botany 64 (2008) 256–263 257

hindsa et al. (1981). Briefly, leaf tissue (250 mg FW) was homog-nized with 5 ml of 0.1% tricloracetic acid (TCA). The homogenateas centrifuged at 9000 × g at 4 ◦C for 12 min. The supernatant wasltered (Millipore, Mitex 0.45 �m) and 1 ml of the filtrate with 4 mlf a solution containing 20% TCA and 0.5% TBA was incubated inhot water bath (95 ◦C) during 30 min. The reaction was stoppedy placing the tubes in an ice bath. Samples were centrifuged, andbsorbance of the supernatant was read at 532 nm using a spec-rophotometer (Beckman DU®-70, Fullerton, USA) and corrected fornspecific turbidity by subtracting their absorbance at 600 nm. Themount of MDA was calculated by using an extinction coefficientE) of 155 mM−1 cm−1. Results were expressed as nmol MDA g−1

W.

.3. Chl a fluorescence measurements

Chl a fluorescence was assessed using a pulse amplitude mod-lated system (Junior-PAM, Gademann Instrument, Germany) asescribed previously by Rivas et al. (2007). Measurements wereerformed at girdling and 30 DAG and taken before midday08.30–10.30 h). Before the measurements, leaves were dark-dapted for at least 1 h and then basal Chl fluorescence (Fo) wasssessed by applying a far-red pulse (<0.1 �mol m−2 s−1). For allhoots, measurements were carried out on 15 leaves per tree, per-orming six readings per leaf.

.4. Carbohydrate analysis

Soluble carbohydrates were extracted with 80% ethanol, puri-ed through cation and anion columns and quantified by HPLC asescribed by Rivas et al. (2006). Starch from the remaining pelletas autoclaved and incubated with amyloglucosidase before HPLC

uantification of released glucose as indicated in Rivas et al. (2006).

.5. Proline analysis

For Pro analysis 50 mg DW was extracted at 4 ◦C with 1 ml of00 mM KH2PO4–KOH (pH 7.5). The homogenate was centrifugedt 15,000 × g at 4 ◦C for 15 min. Four hundred microliters of theupernatant were mixed with 600 �l 1% (w/v) ninhydrin in a 60%cetic acid solution (Magné and Larher, 1992). The mixture waseated at 100 ◦C for 20 min and then cooled for 5 min in an iceath. Two milliliters of toluene were added and the sample wasigorously shaken for 15 s before being placed in darkness at roomemperature for at least 4 h. The absorbance of the upper phase washen read spectrophotometrically at 520 nm. A calibration curveas used to determine Pro content and results were expressed inmol g−1 DW.

.6. Water status measurements

To measure � w 5 mm fresh leaf discs from each side of theidrib were excised with a cork borer. Discs were placed in the

ample chamber of a Wescor C-52 thermocouple psychrometerWescor Inc., Logan, UT, USA). � w measurements were performedfter ensuring initial vapor equilibrium in the chamber. � w wasetermined by measuring the voltage across the thermocoupleith a Wescor HT-33T microvoltimeter compared to voltages deter-ined with NaCl standards. Measurements were carried out before

idday on 5 leaves of every shoot type collected from each tree.To measure RWC, leaf petioles were excised and leaf fresh weight

FW) was recorded. The entire leaves were floated on distilled watert 25 ◦C in dim white light for 18 h to obtain saturated weight (SW),hen dried for 48 h in a forced-draft oven (80 ◦C) to obtain the dry

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eight (DW). The RWC was calculated from the relationship: RWC%) = [(FW − DW)/(SW − DW)] × 100.

.7. Extraction and determination of antioxidant enzyme activity

For extraction and determination of antioxidant enzyme activ-

ty, leaf tissue (250 mg FW) was homogenized in 10 ml of 100 mM2HPO4/KH2PO4 (pH 7.5) containing 2 mM EDTA and 2% solubleVP (Calatayud et al., 2002). Additionally, for APX extraction,mM of AsA was added to the medium to avoid inactivationuring extraction and assay (Asada, 1992). The extract was then

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ig. 1. Time course of starch and soluble sugar (sucrose + glucose + fructose) content in leandarin with regard to shoot type. Girdling was performed on the trunk at anthesis

ifferences at P ≤ 0.01 between treatment in the same shoot are denoted by asterisks (eaves; VG, young leaves from vegetative shoots; MLY, young leaves from multiple-flower

ental Botany 64 (2008) 256–263

entrifuged at 9000 × g during 12 min and the supernatant wasltered (Millipore, Mitex 0.45 �m) and used immediately for thenzyme activity assay. All the extraction process was carried outt 4 ◦C.

APX (EC 1.11.1.11) activity was measured by following theecrease in absorbance at 290 nm (E = 2.8 mM−1 cm−1). The assay

ixture contained 2 ml 100 mM K2HPO4/KH2PO4 (pH 7.5), 0.5 ml

.5 mM AsA, 50 �l leaf extract and 0.5 ml 4 mM H2O2. The reactionas initiated by adding H2O2 (Nakano and Asada, 1981). Spec-

rophotometrical measurements were recorded at 2 and 5 min afterncubation.

aves from girdled (closed circles) and non-girdled trees (open circles) of ‘Loretina’(60% of flowers opened). Values are the mean ± S.E. of five replicates. Significant*). Means separation by Duncan’s multiple range test. Abbreviations: ML, matureed leafy shoots.

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CAT (EC 1.11.1.6) activity was determined by following theecrease in absorbance at 240 nm (E = 39.4 mM−1 cm−1). The assayixture contained 2.5 ml 100 mM K2HPO4/KH2PO4 (pH 7.5), 0.5 ml

00 mM H2O2 and 100 �l leaf extract. The reaction was initiated bydding H2O2 (Rao et al., 1996).

SOD (EC 1.15.1.1) activity was measured as described by Beyernd Fridovich (1987). The reaction mixture contained 1.5 ml 50 mM2HPO4/KH2PO4 (pH 7.8), 0.5 ml 60 mM methionine, 0.5 ml 57 �Mitroblue tetrazolium (NBT), 0.5 ml 0.9 �M Riboflavin, 0.025% (w/v)riton X-100 and 100 �l leaf extract. The absorbance at 560 nm wasecorded after a 7-min illumination period. In this assay 1 unit (U) ofOD is defined as the amount required to inhibit the photoreductionf NBT by 50%. Results were expressed as U mg−1 Protein.

DHAR (EC 1.8.5.1) activity was measured following the increasen absorbance at 265 nm (E = 14.3 mM−1 cm−1). The assay solutionontained 2.5 ml 100 mM K2HPO4/KH2PO4 (pH 7.5), 200 �l 15 mMehydroascorbate (DHA) and 200 �l leaf extract. The reaction was

nitiated by adding 200 �l 10 mM GSH (Dalton et al., 1986).GR (EC 1.6.4.2) activity was assayed by monitoring the change

n absorbance at 340 nm resulting from the enzymatic-dependentxidation of NADPH (E = 6.2 mM−1 cm−1). The reaction mixtureonsisted of 2.5 ml 100 mM K2HPO4/KH2PO4 (pH 7.5), 200 �l.2 mM NADPH and 200 �l 3 mM GSSG (Sigma) and 100 �l leafxtract. The reaction was initiated by adding NADPH (Grace andogan, 1996).

All enzyme activities, with the exception of SOD, were expresseds nmol min−1 mg−1 Protein. Protein content in all enzyme extractsas determined following Bradford (1976).

.8. Extraction and assay of antioxidant metabolites

For analysis of AsA, 250 mg FW was homogenized with 10 ml% metaphosphoric acid and centrifuged 12 min at 6000 × g at◦C. The supernatant was filtered (Millipore, Mitex 0.45 �m)nd 1 ml of the supernatant was mixed with 2 ml of 100 mMH2PO4/K2HPO4 (pH 6.8). AsA content in the sample was assessed

ollowing the decrease in absorbance (265 nm) after adding 1 unitf AsA oxidase (E.C. 1.10.3.3 from Cucurbita sp., Sigma) (Luwe etl., 1993). The reaction was monitored until absorbance reachedconstant value. AsA content was calculated using the molar

xtinction (E = 14.3 mM−1 cm−1). DHA was determined by the same

ethod following the reduction to AsA in a reaction mixture

ontaining 1 ml supernatant, 2 ml 100 mM KH2PO4/K2HPO4 (pH.8) and 20 �l 3 M dithiothreitol (DTT). The reaction was startedy adding DTT. The increase in absorbance was monitored at65 nm until it reached a constant value and total ascorbate (AsAT)

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able 1ffect of girdling on Fo of dark-adapted leaves and on MDA content in leaves from differen

arameter Treatment ML

0 DAG 30 D

DA content (nmol g FW−1) Control 68.8 ± 4.6 78.2Girdling 66.2 ± 5.2 83.5

ignificance ns ns

o (relative units) Control 425 ± 19 380 ±Girdling 431 ± 16 439 ±

ignificance ns ns

irdling was performed on the trunk at anthesis (60% of flowers opened). Leaves were samalues are the mean ± S.E. of five replicates. Fo value is the mean ± S.E. of at least 50 measre denoted by asterisks (**). Means separation by Duncan’s multiple range test.bbreviations: ML, mature leaves; VG, young leaves from vegetative shoots; MLY, young leavifferences at P ≤ 0.05.

ental Botany 64 (2008) 256–263 259

as calculated as above. DHA concentration was calculated usinghe formula: [DHA] = [AsAT] − [AsA]. Results were expressed asmol g−1 FW.

Glutathione was assayed by a slight modification of the DTNB-SSG reductase method (Griffith, 1980). Three hundred milligramsW of leaves were homogenized in 5 ml of ice-cold 5% (w/v)ulfosalicylic acid and centrifuged at 9000 × g for 10 min at 4 ◦Co sediment insoluble material. One milliliter of the supernatantas neutralized with 1.5 ml 0.5 M KH2PO4/K2HPO4 (pH 7.5). A00 �l aliquot of the neutralized sample was assayed for totallutathione (Glut). Quantification of oxidized glutathione (GSSG)as accomplished by first derivatizing reduced glutathione (GSH)f the sample with 2-vinylpiridine. To this end, 200 �l aliquot ofhe neutralized sample was incubated for 1 h at 25 ◦C with 20 �lf 2-vinylpiridine. Authentic GSSG standards were performed foruantification of Glut and GSSG. For GSSG determination, GSSGtandards were incubated with 2-vinylpiridine in the same condi-ions as the samples. The incubation mixture contained 50 �l of theample, 50 �l 6 mM 5,5′-dithiobis (2-nitrobenzoic acid), 0.25 unitsf GR (EC 1.6.4.2 from S. cereviseae) and the reaction was started bydding 50 �l 0.3 mM NADPH. Glut and GSSG were determined withhe End-Point method by measuring absorbance at 405 nm after0 min. Concentrations of the samples were calculated by compar-ng the absorbance values with the standard curve. Results werexpressed as equivalents of GSH g−1 FW.

.9. Experimental design and statistical analysis

Trials were conducted as a randomised complete block designith 2 treatments (control and girdling) and 5 replicates (trees) per

reatment. Analysis of variance was performed on the data. Meansomparison was made by Duncan’s multiple range test (DMRT).ercentages were analysed after arcsine transformation of theata.

. Results

.1. Soluble sugars and starch contents

Girdling caused an accumulation of carbohydrates whicheached a maximum above the non-girdled control at 30 DAG, in

ll leaves from every shoot type (Fig. 1). Starch accumulated morehan sugars in ML (116% over control) and VG (147% over control)han in MLY (17% over control). Soluble sugars accumulated signif-cantly in young leaves from VG (68% over control) and MLY (43%ver control) but not in ML.

t shoot types of ‘Loretina’ mandarin

VG MLY

AG 0 DAG 30 DAG 0 DAG 30 DAG

± 2.1 29.2 ± 1.6 35.7 ± 0.7 29.5 ± 0.9 33.4 ± 2.3± 3.0 30.8 ± 2.5 52.4 ± 2.6 28.7 ± 1.1 61.4 ± 1.1

ns ** ns **

42 235 ± 72 209 ± 31 568 ± 24 418 ± 2623 253 ± 41 387 ± 67 622 ± 79 607 ± 67

ns ** ns **

pled 0 and 30 days after girdling.urements. Significant differences at P ≤ 0.01 between treatments in the same shoot

es from multiple-flowered leafy shoots; DAG, days after girdling; ns, non-significant

2 xperimental Botany 64 (2008) 256–263

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Fig. 2. Effect of girdling on antioxidant enzymatic activity in leaves from differ-ent shoot types of ‘Loretina’ mandarin. Leaves were sampled 30 days after girdling.Data are the means ± S.E. of five replicates. Significant differences between treat-ment and shoot in each enzyme activity at P ≤ 0.05 and P ≤ 0.01 are denoted by (*)and (**), respectively. Means separation by Duncan’s multiple range test. Abbrevi-alor

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60 F. Rivas et al. / Environmental and E

.2. Changes in Fo and MDA content

The Chl a fluorescence analysis revealed that girdling increasedhe basal Chl a fluorescence emission of dark-adapted leaves (Fo)f VG and MLY but not in ML (Table 1). Similarly, MDA increasedignificantly in VG and MLY as a result of girdling, whereas MDAas not modified in ML. MDA content in ML of control and girdled

rees remained significantly higher than in VG and MLY.

.3. Enzymatic antioxidant activity and metabolites

Activity of APX, SOD, CAT, GR and DHAR increased in VG andLY leaves of girdled trees in comparison with control trees (Fig. 2).

omparatively, ML showed a smaller increase in APX, CAT and GRctivities, whereas DHAR activity was reduced and SOD activity wasot modified by girdling in these leaves.

Girdling caused a decrease in foliar AsA and DHA content in VGnd MLY (Fig. 3). In VG of girdled trees, AsA and DHA content wereeduced by 23% and 16.4%, respectively, whereas for MLY, the reduc-ion was 34% and 11.8%, respectively. By contrast, in ML, girdlingaised both AsA and DHA content by 23%. ML from control treeshowed lower values (P ≤ 0.01) of AsA and DHA than those found inG and MLY. The leaf ratio AsA/DHA was not modified by girdling

n any of the leaf types.Glut pool size (GSH + GSSG) increased in leaves of all shoots

fter girdling. The increase was related to the rise of both GSHnd GSSG (Fig. 3). These increases were more acute in the youngparticularly in MLY) than in the mature leaves. After girdling, theatio GSH/GSSG increased significantly in both ML and MLY andecreased significantly in VG.

.4. Proline content

Pro content followed a similar pattern in all shoots up to 30AG (Fig. 4). At 15 and 30 DAG, girdling caused a significant accu-ulation of Pro in all leaf types. However, from 30 to 60 DAG, the

hanges in Pro concentration depended on the type of shoot. MLYro leaf content remained significantly higher at 60 DAG in girdledrees, whereas Pro content of VG became significantly lower and

L remained unchanged after girdling in comparison with controlrees.

. Discussion

In this study, we hypothesize that girdling gives rise to a stressue to metabolic changes derived from the interruption of phloemransport. Evidence for this stress condition could be cellularamage, photosystems malfunction symptoms or even changes innti-stress defence mechanisms triggered by the plant.

In fact, our results demonstrated that girdling increases lipoox-dation (expressed as MDA content) and Fo emission (Table 1) inoung leaves (VG and MLY), revealing the occurrence of oxida-ive damage and malfunction of the photochemical apparatus inhese shoots (Gilmore et al., 1996). On the other hand, ML experi-nced no changes in these parameters after girdling (Table 1). Thencrease in the Fo emission may be induced by the release of free Chlrom protein–pigment complexes (Wingler et al., 2004). Under thisircumstance, unbound Chl molecules can readily produce highlyeactive singlet oxygen species in the presence of light and oxygeneading to lipid peroxidation (Merzlyak and Hendry, 1994; Matile et

l., 1999). This phenomenon could explain the concurrent increasef Fo and lipid peroxidation observed in young leaves of girdledrees (Table 1).

Whenever ROS are produced, an activation of the enzymatic andon-enzymatic antioxidant system can reduce oxidative stress in

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tions: ML, mature leaves; VG, young leaves from vegetative shoots; MLY, youngeaves from multiple-flowered leafy shoots; APX, ascorbate peroxidase; SOD, super-xide dismutase; CAT, catalase; GR, glutathione reductase; DHAR, dehydroascorbateeductase.

he tissues. Our results showed that in young leaves (VG and MLY)irdling increased the antioxidant activity of all enzymes of the

ater–water cycle, the ascorbate-glutathione ROS scavenging cycle

SOD, APX, GR, DHAR) and CAT (Fig. 2), and raised the content oflutathione-related compounds (Fig. 3). Noteworthy was that theotal pool of AsA was reduced in young leaves of trees subjected toirdling, suggesting an increased AsA consumption or a rapid catab-

F. Rivas et al. / Environmental and Experimental Botany 64 (2008) 256–263 261

Fig. 3. Changes in total ascorbate pool (AsA + DHA), reduced ascorbate (AsA) and dehydroascorbate (DHA) content, redox ratio (AsA/DHA), total glutathione (GSSG + GSH)p ox ratD ent as , youns ts.

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ool, oxidized glutathione (GSSG) and reduced glutathione (GSH) content, and redata are the means ± S.E. of five replicates. Significant differences between treatm

eparation by Duncan’s multiple range test. Abbreviations: ML, mature leaves; VGhoots; ns, non-significant differences at P ≤ 0.05 in any of the shoots and treatmen

lization of DHA to two- and four-carbon products such as oxalatend tartrate, which can accumulate to relative high levels (Washkot al., 1992). In fact, we observed by microscopy a high quantityf crystals (possibly calcium oxalate) in squeezed leaves from gir-led trees that were not present in the control trees (data nothown). However, far from becoming a limitation for the enzymaticsA-dependent reaction, AsA content was found to be sufficient

o maintain a high enzymatic and non-enzymatic ROS scavengingctivity, as suggested previously by Asada (1999). In ML the changesaused by girdling in these enzyme activities and metabolites weref less magnitude than in young leaves. The antioxidants AsA andlutathione are crucial for plant defence against oxidative stressNoctor and Foyer, 1998). It is believed that maintaining a high ratiof reduced to oxidized forms of these metabolites is essential forhe proper scavenging of ROS in cells. This ratio is maintained by thenzymes GR and DHAR whose activities, as stated before, increasedy girdling more in young than in mature leaves (Fig. 2).

One of the most evident effects of girdling in Citrus is the accu-ulation of carbohydrates above the girdle (canopy) and their

epletion below it (root system) (Li et al., 2003). The changes in the

ynthesis and transport of the different carbohydrates caused byirdling depend on the presence of developing fruits on the tree.i et al. (2003) demonstrated that genes encoding the enzymesnvolved in starch synthesis were expressed in girdled Citrus treesnly when fruits were absent. Our experiment agrees with these

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io (GSH/GSSG) induced by girdling in different shoot types of ‘Loretina’ mandarin.nd shoot are denoted by (*) and (**) for P ≤ 0.05 and P ≤ 0.01, respectively. Meansg leaves from vegetative shoots; MLY, young leaves from multiple-flowered leafy

esults. After girdling, leaves from every shoot type of girdled treeshowed increased carbohydrate, reaching maximum differences at0 DAG (Fig. 1). Afterwards, differences vanished probably due tohe reestablishment of the phloem transport given that cambiumctivity produces callus which heals the wound in approximately 4eeks (Wallerstein et al., 1974; Williams et al., 2000). Physiological

nd metabolically recovery from the stress induced by girdling haseen reported in roots by Wallerstein et al. (1978). Nevertheless,he quality of this accumulation was a function of shoot type. Onlyhe leaves from shoots which do not support growing fruits (MLnd VG) accumulated an important quantity of starch in responseo girdling, whereas in MLY leaves the accumulation of starch overhe control was small (Fig. 1). On the other hand, girdling caused aignificant accumulation of soluble sugars in young (VG and MLY)ut not in mature leaves. The lack of starch accumulation in Cit-us leaves in response to developing fruits on the shoots, evenhen girdling is performed, has been previously reported by other

uthors (Schaffer et al., 1986; García-Luis et al., 1995; Li et al., 2003).nterestingly, in control trees only MLY leaves increased the starchontent throughout the experimental period, suggesting an over-

roduction of carbohydrates in response to the high demand fromhe growing fruits (Syvertsen et al., 2003; Rivas et al., 2007).

Based on published data, there are some explanations whichould link the increase of ROS with the carbohydrate accumulationfter girdling. One proposal can be related with the physical damage

262 F. Rivas et al. / Environmental and Experim

Fig. 4. Time course of proline content in leaves from girdled (closed circles) andnon-girdled (open circles) trees of ‘Loretina’ mandarin with regard to shoot type.Girdling was performed on the trunk at anthesis (60% of flowers opened). Each datapoint is a mean ± S.E. of five replicates. Significant differences (P ≤ 0.05) betweentrM

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A

A

A

B

B

B

C

C

C

D

D

decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101.

reatment for each date are denoted by (*). Means separation by Duncan’s multipleange test. Abbreviation: ML, mature leaf; VG, young leaf from a vegetative shoot;LY, young leaf from a multiple-flowered leafy shoot.

o the thylakoid structure induced by big starch granules formationSchaffer et al., 1986; Bondada and Syvertsen, 2005). Another pos-ibility could be related with the accumulation of soluble sugars,imicking the effect of excessive light and then rising ROS content

n leaves (see Couée et al., 2006 for review). This last view is in accor-ance with our results since oxidative damage symptoms werebserved only in leaves that had accumulated a significant con-entration of soluble sugars (VG and MLY) in response to girdlingFig. 1; Table 1). On the other hand, soluble sugars also providearbon skeletons for the synthesis of numerous compounds thatre involved in antioxidative protection such as AsA (Smirnoff etl., 2001), and for the amino acids Cys, Glu and Gly which are theuilding blocks of glutathione (Noctor and Foyer, 1998). Further-ore, it has been shown that glucose induces the expression of

enes involved in the synthesis of enzymes such as chalcone syn-hase and SOD (Koch, 1996) implicated in plant response to abiotictress.

It is well known that Pro levels rise in Citrus leaves in response to

ertain stresses such as chilling or drought (Kushad and Yelenosky,987). Changes in Pro content due to chilling or drought stresses inur experimental conditions were unlikely since trees were grownnder identical temperatures and we confirmed that neither RWC

F

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ental Botany 64 (2008) 256–263

or � w in any of the shoots was modified as a result of girdling (dataot shown). In our experiments Pro content increased in all shootsfter girdling (Fig. 4). When a malfunction of the photosyntheticpparatus exists, the increase in Pro content could play a role inrresting further photooxidative damage by preserving membranentegrity, reducing the imbalance between the light absorbed byhotosystems and that used in the intersystem electron transportHare and Cress, 1997), and/or enhancing the activities of vari-us antioxidants (Yan et al., 2000). However, further experimentalupport will be required before a sound conclusion can be drawnut these possibilities may serve working hypothesis for futureesearch.

In conclusion, our data show that girdling causes oxidativeamage in Citrus leaves, increasing the level of lipooxidation and

nducing malfunction of the photosynthetic apparatus. A relationetween the changes in soluble sugars (but not starch) and thentioxidant metabolites and antioxidative enzymes activity afterirdling was found. The contribution of each of these factors to thexidative damage triggered by girdling remains unclear. Importantspects regarding ROS, the antioxidative pathway and their sig-alling function were not addressed in this study. Future studies onhe role of ROS and antioxidant metabolites as signalling moleculesould clarify potential mechanisms whereby girdling, resemblinghilling or water stress, might modify physiological process likeud dormancy or flowering in Citrus trees.

cknowledgments

We are grateful to M. Fuster for her assistance with thexperiments. Thanks to Dr Debra Westall and Brande Wulff forheir linguistic assistance during the manuscript writing. We alsocknowledge A. Calatayud for her critical review and improvementf the article.

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