ARTICLE IN PRESS

Journal of Plant Physiology 167 (2010) 1–9

Contents lists available at ScienceDirect

Journal of Plant Physiology

0176-16

doi:10.1

Abbre

days aft

dehydro

peroxid

MSI, me

reactive� Corr

Agronom

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journal homepage: www.elsevier.de/jplph

Nitrophenolates spray can alter boll abscission rate in cotton throughenhanced peroxidase activity and increased ascorbate and phenolics levels

M. Djanaguiraman a,�, J. Annie Sheeba a, D. Durga Devi a, U. Bangarusamy a, P.V.V. Prasad b

a Department of Crop Physiology, Tamil Nadu Agricultural University, Indiab Department of Agronomy, Throckmortan Plant Sciences Centre, Kansas State University, Manhattan KS 66502, USA

a r t i c l e i n f o

Article history:

Received 5 March 2009

Received in revised form

4 May 2009

Accepted 5 May 2009

Keywords:

Antioxidant enzymes

Ascorbate

Oxidative stress

Peroxidase

Phenolics

17/$ - see front matter & 2009 Elsevier GmbH

016/j.jplph.2009.05.018

viations: AA, ascorbate; APX, ascorbate pero

er anthesis; DAS, days after sowing; DHA, de

ascorbate reductase; GR, glutathione reducta

ase; H2O2, hydrogen peroxide; LOX, lipoxygen

mbrane stability index; O2�, superoxide radic

oxygen species; SOD, superoxide dismutase

esponding author. Present Address. Post Doc

y, Kansas State University, Manhattan 6650

ail address: djanaguiraman@yahoo.co.in (M. D

S u m m a r y

Field studies were conducted from 2002 to 2005 to evaluate foliar spray of Atonik (a plant growth

regulator (PGR) containing nitrophenolates) on cotton boll abscission rate by assessing various reactive

oxygen species (ROS) contents, antioxidant content and antioxidant enzyme activity from 1 to 9 days

after anthesis (DAA). The result indicated that the nitrophenolate spray reduced hydrogen peroxide

(H2O2), superoxide anion (O2�) accumulation, lipid peroxidation (malondialdehyde – MDA), lipoxygen-

ase (LOX) activity and membrane permeability relative to the control. Antioxidant enzyme activity

(superoxide dismutase, SOD; ascorbate peroxidase, APX; peroxidase, POX; glutathione peroxidase, GSH-

Px) was significantly increased by the nitrophenolate spray. The POX (217%) and GSH-Px (242%)

activities were enhanced compared with APX (7.7%) activity at 9 DAA. Enhanced accumulation of

ascorbate (245%), phenol (253%) and proline (150%) was observed in nitrophenolate-sprayed plants

compared with control at 9 DAA. Because ascorbate content is increased by higher dehydroascorbate

reductase (DHAR) enzyme activity, the ascorbate was able to replenish reducing equivalents to phenoxyl

radicals, resulting in an increase of phenolic compounds. The increased phenolic acid content may be

involved in scavenging the ROS produced in developing cotton boll. The role of DHAR and glutathione

reductase (GR) in keeping higher levels of reduced ascorbate and low levels of endogenous H2O2 in the

developing cotton boll may be the prerequisite for boll retention. Based on the present work, we

conclude that nitrophenolate-sprayed plants counteracted the deleterious effects of ROS by the

peroxide/phenolics/ascorbate system, which causes reduced boll abscission and increased yield.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

In India, cotton is the premier commercial fiber crop, with acultivated area of 9 million hectares. Cotton plant developmentfollows an indeterminate fruiting cycle, with excessive productionof fruiting structures by individual plants. All fruiting structuresproduced cannot mature, and excess structures abscise from theplant. Kennedy et al. (1991) found that only 24–36% of flowersproduced on a plant during a growing season mature to harvest-able bolls. Abscission is a developmentally controlled processassociated with the large-scale degradation of cellular andmacromolecular components (Rubinstein, 2000). A considerable

. All rights reserved.

xidase; CAT, catalase; DAA,

hydroascorbate; DHAR,

se; GSH-Px, glutathione

ase; MDA, malondialdehyde;

al; POX, peroxidase; ROS,

toral Fellow, Department of

2, KS, USA.

janaguiraman).

volume of research has been devoted to identify the enzymes thatbring about cell separation. The culmination of abscission is thephysical detachment of the target organ, and thus much work hasbeen focused on the phenomenon of cell wall dissolution at thesite of abscission. The contemporary view of abscission is that it ismediated through oxidative damage (Roberts et al., 2002; Mulleret al., 2007, Prochazkova and Wilhelmova, 2007). Oxidativedamage is associated with several biochemical changes, such asincreased membrane permeability, leakage of ions, lipidperoxidation, increased reactive oxygen species (ROS), nuclearfragmentation as well as expression/activation of nucleases,proteases, lipases and other cell wall hydrolases leading to lossof cell structure (Paliyath and Droillard, 1992; Rubinstein, 2000;Wagsta et al., 2002).

Plant cells need protective mechanisms through which theyrespond to oxidative stress: (a) non-enzymatic antioxidants, viz.,ascorbate (AA) and glutathione (GSH) and (b) enzymaticantioxidants such as catalase (CAT), superoxide dismutase (SOD)and ascorbate peroxidase (APX; Navabpour et al., 2003; Zimmer-mann and Zentgraf, 2005). However, during senescence, thegeneral antioxidant status diminishes and the levels of ROSare enhanced (Srivalli and Khanna-Chopra, 2004), and many

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antioxidant enzymes reduce their activity (Prochazkova et al.,2001). Antioxidant enzymes such as SOD, CAT and peroxidase (POX)are involved in the scavenging of ROS (Srivalli and Khanna-Chopra,2004). The O2

� produced in the cell will be dismutated to O2 andH2O2 by SOD (Bowler et al., 1992). Peroxidases catalyze thedehydrogenation of structurally diverse phenolic and endiolicsubstrates by H2O2 and are thus often regarded as antioxidantenzymes (Shigeoka et al., 2002). CAT removes the H2O2 produced,preventing an increase in cytosolic H2O2, which is potentiallytoxic to the plant cell, leading to oxidative stress and cell death(Prochazkova et al., 2001).

Plant growth regulators (PGRs) have been used to improveyield. One such PGR is Atonik (Asahi Co., Ltd.; Nara Prefecture,Japan), a commercially available product containing the activeingredients 1.25 g L�1sodium 5-nitroguaiacolate (NaC7H6NO4),2.5 g L�1 sodium ortho-nitrophenolate (NaC6H4NO3) and 3.75 gL�1sodium para-nitrophenolate (NaC6H4NO3). These active ingre-dients, termed nitrophenolates, can stimulate plant growth byaltering the activity of specific antioxidant enzymes such as SOD,CAT and POX (Djanaguiraman et al., 2004; Djanaguiraman et al.,2005a, b). Atonik has been used on various crops in more than 20countries and was registered for pesticide use in cotton(Gossypium hirsutum), rice (Oryza sativa) and soybeans (Glycine

max) in 1995 as ARYSTA-Exp-NP321 (Asahi Co., Ltd.; NaraPrefecture, Japan). ARYSTA-Exp-NP321 has the trade nameChaperone, which was registered by the Environmental ProtectionAgency (EPA) with the patent pending in 2000. Chaperone wasintroduced into the cotton market in 2004 as a protein transportenhancer and is currently the only agrochemical containing thesenitrophenolates registered for cotton (Bynum et al., 2007).Phenolics are able to act as radical scavengers or radical-chainbreakers, strongly extinguishing oxidative free radicals such as thehydroxyl-radical-yielding products with much lower oxidativecapacities compared with the parent compounds (Grassman et al.,2002). Thus, the ability of phenolics to ameliorate or lessen theimpact of ROS has been explored.

With this background, the present study was conducted toexamine whether applied nitrophenolates can alleviate oxidativedamage by reduced ROS accumulation in the developing boll byenhancing the antioxidants and antioxidant enzymes; we alsoexamined consequent yield improvement.

Materials and methods

Crop management

Cotton (G. hirsutum) variety MCU 12 was sown in a clay loamtextured soil with a pH of 7.6 and electrical conductivity (EC) of0.31 dS m�1 from 2002 to 2005 in the experimental field of TamilNadu Agricultural University, Coimbatore (111N; 771E; 426.7 mamsl), India. At the experimental site, the maximum andminimum temperature and relative humidity ranged between33 and 20 1C and 80% and 60%, respectively. The minimum andmaximum irradiances were 800–1100mM m�2 s�1 (PAR). The soilof the experimental field was low in available N (180 kg ha�1),medium in available P (6 kg ha�1) and high in available K(376 kg ha�1). The experiment was designed to evaluate the foliarspray of nitrophenolates on yield improvement in cotton byassessing the production of various oxidants, antioxidants,antioxidant enzymes and membrane damage. The experimentutilized two treatments :(i) foliar spray of nitrophenolates at0.8 g ai ha�1 at square formation stage [35 days after sowing(DAS)] and initiation of flowering stage (58 DAS) and (ii) anuntreated control with six replications. Twelve plots with a size of25 m2 were prepared. The crop was sown during August with a

spacing of 75 cm�30 cm. Pendimethalin @ 3.3 L ha�1 was applied3 DAS, using a hand-operated sprayer fitted with a deflecting-typenozzle. Sufficient moisture was present in the soil at the time ofherbicide application and one hand weeding was performed 45DAS. Gap filling was performed on the 10th day of sowing andthinning of the seedlings was done on the 15th day of sowing. 50fifty per cent of N and K and a full dose of P2O5 was appliedas basal in the form of urea, single super phosphate and KCl.The remaining 50% of the recommended doses of N and Kwere applied as top dressing 40–45 DAS. Recommended plantprotection measures were followed. At early stages of squareformation, Endosulfan 35 EC was sprayed @ 2 L ha�1 using agooseneck nozzle to cover the under surface of the foliage to getgood control of sucking pests. During the boll formation andmaturation stage, Phosalone 35 EC was sprayed @ 2.5 L ha�1 tocontrol bollworms.

The variation in the production of oxidants, viz., hydrogenperoxide (H2O2) and superoxide radical (O2

�), lipoxygenase (LOX)enzyme activity, malondialdehyde (MDA) content, membranestability index (MSI) and antioxidant enzymes namely SOD, CAT,APX, POX and glutathione peroxidase (GSH-Px) activitieswere recorded from 61 to 69 DAS in the developing bolls.Likewise, various metabolites, viz., ascorbate, dehydroascorbate(DHA), soluble phenol and proline contents and activities ofenzymes such as dehydroascorbate reductase (DHAR) andglutathione reductase (GR) were recorded 61 DAS (1 day afteranthesis – DAA), 63 DAS (3 DAA), 65 DAS (5 DAA), 67 DAS (7 DAA)and 69 DAS (9 DAA) in the developing bolls. The tenth main stemnode was chosen as a representative fruiting branch to quantifyvarious oxidants, antioxidants and antioxidant enzyme activity,since it typically possesses the greatest leaf area and contributesto the majority of the yield on a per node basis (Oosterhuis andWullschleger, 1988).

Oxidant production and lipid peroxidation

Lipid peroxidation was determined by malondialdehyde con-tent produced by thiobarbituric acid (TBA) as described by Behraet al. (1999). A 200 mg boll sample was homogenized in 5 mL of0.1% trichloroacetic acid (TCA). The homogenate was centrifugedat 10,000g for 5 min at 4 1C. An aliquot of 0.3 mL supernatant wasmixed with 1.2 mL of 0.5% TBA prepared in 20%, TCA andincubated at 95 1C for 30 min. After stopping the reaction in anice bath for 5 min, samples were centrifuged at 10,000g for 10 minat 25 1C. The absorbance was measured at 532 nm. Aftersubtracting the non-specific absorbance at 600 nm, MDAconcentration was determined using on extinction coefficient of155 mM–1 cm–1. Lipid peroxidation was expressed as MDA contentin nM g�1 FW. The H2O2 level was colorimetrically measured asdescribed by Okuda et al. (1991). H2O2 was extracted byhomogenizing 0.5 g of tissue with 4 mL of perchloric acid(200 nM). The homogenate was centrifuged at 12,000 g for10 min. Oxidation of ferrous ions to ferric ions by H2O2 in acidicpH was monitored and a stable complex of ferric ions with Xylenolorange dye at 560 nm was measured. H2O2 was expressed innM g�1 FW. Superoxide anion was estimated according toChaitanya and Naithani (1994) and expressed as change in ODmin�1 g�1 FW. Weighed bolls were homogenized in ice-coldsodium phosphate buffer (0.2 M, pH 7.2) containing diethyldithio-carbamate (10�3 M). The homogenate was immediatelycentrifuged for 1 min at 3000 rpm. In the supernatant, superoxideanion was measured by its capacity to reduce nitrobluetetrazolium (NBT, 2.5�10�4 M). The absorbance of the endproduct was measured at 540 nm.

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Lipoxygenase (LOX EC 1.13.11.12) and membrane stability index (MSI)

LOX (1.13.11.12) was measured spectrophotometrically andexpressed as mM min�1 mg�1 protein (Gallego et al., 1996). Thebolls were macerated with a mortar and pestle, and the enzymeswere extracted at 4 1C in 10 mL of 0.1 M sodium phosphate buffer,pH 6.5, containing 1% Tween 20. The slurry was centrifuged at10,000g for 15 min at 4 1C, and the supernatant was used for theassay of LOX. LOX activity was calculated following the rise inthe extinction at 234 nm using an extinction coefficient of25,000 M–1 cm–1. The membrane stability index was determinedby recording the EC of leachates in double distilled water at 40and 100 1C (Chaudhuri and Choudhuri, 1992) and expressed as apercentage.

Enzyme assays

For SOD, CAT, APX and POX enzyme assays, frozen tissue washomogenized in ice-cold 0.1 M Tris–HCl buffer at pH 7.8 contain-ing 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithio-treitol and 5 mL of 4% polyvinyl pyrrolidone per gram freshweight. The homogenate was filtered through a nylon mesh andcentrifuged at 20,000g at 4 1C. The supernatant was used formeasuring enzyme activity.

SOD (EC 1.15.1.1) was determined by the nitroblue tetrazoliummethod by measuring the photoreduction of NBT at 560 nm(Beyer and Fridovich, 1987). One unit of SOD activity is equivalentto the amount required to inhibit photoreduction of NBT by 50%.Catalase (EC 1.11.3.6) was estimated according to Samantary(2002) and expressed as mM H2O2 destroyed min�1 mg protein�1.CAT activity was assayed in a reaction mixture containing 50 mMphosphate buffer (pH 7.0), 10 mM H2O2 and enzyme aliquot. Thedecomposition of H2O2 was followed at 240 nm (e ¼ 39.4 mMcm�1). APX (EC 1.11.1.11) enzyme activity was measured accordingto Gerbling et al. (1984), following the oxidation of AA to DHAspectrophotometrically. The enzyme activity was calculated fromthe observed rates of decrease in absorbance and the millimolarextinction coefficient for AA, which is 0.80 and 0.055 mM�1 cm�1

at 298 and 310, respectively.Peroxidase (EC 1.11.1.7) activity was measured as the rate of

decomposition of hydrogen peroxide by POX, with guaiacol ashydrogen donor, by measuring the rate of color developmentspectrophotometrically at 436 nm due to guaiacol oxidation(e ¼ 26.6 mM cm�1).

GSH-Px (EC. 1.11.1.9) activity was measured by a modificationof Flohe and Gunzler’s method (1984) by using H2O2 as substrate.The enzyme was extracted by a pre-chilled KNaHPO4 buffer at pH7.0, with a homogenizer. The supernatant obtained by centrifuga-tion (10 min at 3000 rpm) was used as an enzyme crude extract.For the enzyme reaction, 0.2 mL of the supernatant was placed ina test tube and mixed with 0.4 mL reduced glutathione (0.1 mM)and 0.2 mL KNaHPO4 (0.067 M). The above reagents withoutenzyme extract were used for the non-enzyme reaction. After pre-heating the mixtures in a water bath at 25 1C for 5 min, 0.2 mLH2O2 (1.3 mM) was added to initiate the reaction. The reactionlasted for 10 min and was terminated by adding 1 mL 1% trichloroacetic acid, and the mixture was put in an ice bath for 30 min. Themixture was then centrifuged for 10 min at 3000 rpm: 0.48 mL ofthe supernatant was placed in a cuvette, and 2.2 mL of 0.32 MNa2HPO4 and 0.32 mL of 1.0 mM 5,50-dithiobis (2-nitrobenzoicacid) (DTNB) were added for color development. The absorbancewas measured with a Beckman spectrophotometer at a wave-length of 412 nm within 5 min. GSH-Px activity was calculatedusing the extinction coefficient of 6.22 mM�1 cm�1 and expressedas mM min�1 mg�1 protein.

The GR (EC 1.6.4.2) estimation method is based on the increasein absorbance at 412 nm when 5, 50-dithiobis (2-nitrobenzoicacid) is reduced by GSH (Smith et al., 1988) and expressed asmM min�1 mg�1 protein. Briefly, the boll was homogenized in0.1 M NaK phosphate buffer, pH 7.5 containing 0.2 mM diethyle-netriamine pentaacetic acid and 4% (w/v) polyvinylpolypyrroli-done in an ice-cold glass homogenizer and centrifuged at 30,000g

for 10 min at 4 1C. The supernatant was used to measure GRactivity. The assay mixture contained 100 mM, pH 7.5 NaKphosphate, 0.2 mM diethylenetriamine pentaacetic acid,0.75 mM 5, 50 DTNB, 0.1 mM NADPH, 0.5 mM GSSG and 50mLextract in a total volume of 1 mL. DHAR (EC 1.8.5.1) enzymeactivity was measured by monitoring the reduction of DHA at265 nm according to Foyer et al. (1989) and expressed asmM min�1 mg�1 protein. Briefly, 1 gr of boll was homogenized in2 mL of 50 mM phosphate buffer (pH 7.0) that included 1 mMethylenediaminetetraacetic acid, 2 mM 2-mercaptoethanol and 2%(w/v) polyvinylpolypyrrolidone. The homogenate was centrifugedat 13,000g for 40 min at 4 1C. The supernatant was used to assayDHAR activity. The molar extinction coefficient used to calculatethe activity of this enzyme was 7.0 mM�1 cm�1.

Metabolites

Ascorbate (AA) and dehydroascorbate (DHA) contents

Ascorbate and dehydroascorbate were assayed as described byLeipner et al. (1997). They were assayed photometrically by thereduction of 2,6-dichlorophenolindophenol (DCPIP). Two hundredmilligrams of bolls were homogenized in 5 mL ice-cold 2% (w/v)metaphosphoric acid in the presence of 1 g NaCl. The homogenatewas filtered through a paper filter. An aliquot of 300ml was mixedwith 200ml 45% (w/v) and 100mL 0.1% (w/v) homocysteine toreduce DHA to AA for the determination of the total AA pool. Todetermine AA, the homocysteine solution was replaced by thesame volume of distilled water. After 15-min incubation at 25 1C,1 mL 2 mM citrate–phosphate buffer (pH 2.3) and 1 mL 0.003%(w/v) DCPIP were added. The absorbance at 524 nm was measuredimmediately. The content of AA was calculated by reference to astandard curve. The amount of DHA was obtained as thedifference between the total AA pool and the AA and expressedin nM g–1 fresh weight.

Soluble phenol and proline contents

Soluble phenol was assayed spectrophotometrically afterextraction with 80% ethanol. The amount of soluble phenolicswas determined according to the Folin–Ciocalteu’s procedure(Singleton and Rossi, 1965). Phenol content was expressed as mg(gallic acid equivalents) g�1 fresh weight. Proline was extracted in3% sulfosalicylic acid and estimated by using acid ninhydrinreagent and measuring the absorbency of the toluene chromo-phore at 520 nm (Bates et al., 1973) and expressed as mg g�1 FW.

Shoot length, yield and yield components

The shoot length, boll weight and seed cotton yield wererecorded at harvest. The shoot length measurement was takenfrom the soil surface to the apex of the plant at harvest. Ten plantsper plot were tagged at the square formation stage and initiationof the flowering stage to document the number of abscisedsquares and bolls. The fertility coefficient was calculated as thenumber of flowers produced to the number of bolls retained in aplant, and expressed in percentage. In all years of the study, 10plants were randomly chosen from the center ridge of each plot todetermine boll weight (grams of seed cotton per boll). Boll mass

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was determined by dividing the weight of seed cotton by thenumber of bolls harvested.

Statistical analysis

Three replicates were performed. Oxidants, antioxidants,antioxidant enzyme assays, membrane damage observations andLOX enzyme activity were taken from each replication (n ¼ 18).The mean values with S.E. are provided in all tables. The data wereanalyzed statistically using a general linear model for analysis ofvariance (Wilkinson et al., 1996). Significance between control andtreatments was compared at the 0.05 probability level.

Results

Oxidants production, lipid peroxidation and membrane damage

In order to test the hypothesis that abscission of cotton bollsoccurs through oxidative damage, measurements were made onH2O2 content, O2

� content, MDA content, LOX enzyme activity andmembrane permeability. The concentrations of H2O2 and O2

� arepresented in Table 1. The concentration of H2O2 was significantlyincreased from the first DAA to the ninth DAA in both control andnitrophenolate-sprayed plants. However, the concentration ofH2O2 decreased from 298 to 147 nM g�1 by the nitropheno-late spray when compared with the control at the ninth DAA.In the case of superoxide anion production, a significant increasewas observed from the first DAA to the ninth DAA, and thedifference between control and nitrophenolate spray was1.68DOD min�1 g�1 FW at the ninth DAA. MDA content inplants was significantly higher from the fifth DAA to the ninthDAA, with the increase being 82% and 91% more that plantssprayed with nitrophenolates during the seventh DAA and theninth DAA, respectively (Table 1). LOX enzyme activity of bollswas 18.1 and 22.2mM min�1 mg�1 protein at the seventh DAA andthe ninth DAA, respectively, in control plants. However, in thenitrophenolate-sprayed plants, LOX activity was 9.3 and 11.9

Table 1Changes in hydrogen peroxide concentration, superoxide anion concentration and lipid

Days after anthesis Hydrogen peroxide (H2O2) concentration

(nM g�1 FW)

Superoxide anio

(DOD min�1 g�1

Control Nitrophenolates-sprayed plant Control

1 7772.1a 4972.7b 1.0270.11a

3 18474.4a 8375.2b 1.8470.16a

5 26278.5a 11475.8b 2.3470.23a

7 27876.7a 12276.3b 2.6970.27a

9 29877.6a 14776.8b 3.5470.35a

Means7SE within a row with different letters are significantly different (Po0.05); n ¼

Table 2Changes in lipoxygenase enzyme activity and cell membrane stability in bolls of contr

Days after anthesis Lipoxygenase (LOX) enzyme activity (mM min�1 mg�1 pr

Control Nitrophenolates-spraye

1 5.670.40a 5.170.24a

3 8.270.51a 7.770.34a

5 15.270.68a 8.070.53b

7 18.170.81a 9.370.72b

9 22.270.92a 11.970.81b

Means7SE within a row with different letters are significantly different (Po0.05); n ¼

during the same period (Table 2). Membrane injury recorded fromthe first DAA to the ninth DAA in bolls revealed that maximummembrane injury occurred in control compared withnitrophenolate-sprayed plants. Control plants had an injury rateof 55.4% compared with 39.3% in nitrophenolate-sprayed plants atthe ninth DAA (Table 2). MDA, LOX and membrane damageshowed significant differences from the fifth DAA to the ninthDAA.

Antioxidants and antioxidant enzyme activity

All antioxidant enzyme activity was higher in nitrophenolate-sprayed plants compared to control plants. A significant increasein the activity of antioxidant enzymes, viz., SOD, POX and GSH-Px,was observed from the first DAA to the ninth DAA in bolls(Table 3). An increase in SOD activity by 24.4 enzyme unit mg�1

protein was observed from the first DAA to the ninth DAA incontrols compared with a much higher increase of 38.9 enzymeunit mg�1 protein in nitrophenolate-sprayed plants. GSH-Pxactivity increased from 5.1 to 14.2mM min�1 mg�1 protein fromthe first DAA to the ninth DAA in control plants. In contrast, in theplants sprayed with nitrophenolates, the increase was from 12.2to 34.5mM min�1 mg�1 protein from the first DAA to the ninthDAA. CAT activity did not show significant changes from the firstDAA to the ninth DAA (Table 3). Observation of the influence ofnitrophenolate spray on APX and GR activity revealed that APXactivities increased by 23.6% and 16.0% over controls at theseventh DAA and the ninth DAA, respectively. However, GRactivity increased to 55% and 49% at the seventh DAA and theninth DAA, respectively with nitrophenolate spray (Table 4). Theincrease in APX and GR activity by nitrophenolates spray wassignificant from the first DAA to the ninth DAA. POX activities ofbolls was higher in nitrophenolate-sprayed plants compared withcontrol plants. At the seventh DAA and the ninth DAA,nitrophenolate spray increased POX activity by 2.5 and 2 times,respectively, over controls (Table 4). On the ninth DAA,nitrophenolate spray increased the AA content by 2.5 times overunsprayed control plants (Table 5). However, the DHA content was

peroxidation in bolls of control and nitrophenolate-sprayed plants.

n (O2�) concentration

FW)

Malondialdehyde (MDA) content

(nM g�1 FW)

Nitrophenolates-sprayed plant Control Nitrophenolates-sprayed plant

0.5370.16b 9.270.48a 8.970.21a

0.9870.21b 11.170.48a 10.870.38a

1.2670.28b 21.470.71a 11.770.64b

1.4670.31b 24.770.97a 13.570.82b

1.8670.36b 28.370.82a 14.870.91b

18.

ol and nitrophenolate-sprayed plants.

otein) Cell membrane stability (MSI) damage (%)

d plant Control Nitrophenolates-sprayed plant

19.371.1a 18.471.2a

20.571.2a 18.971.6a

31.472.5a 25.771.7b

45.873.8a 31.372.1b

55.4374.8a 39.372.7b

18.

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Table 4Changes in peroxidase enzyme, glutathione peroxidase enzyme and glutathione reductase enzyme activities in bolls of control and nitrophenolate-sprayed plants.

Days after anthesis Peroxidase (POX) enzyme activity

(mM min�1 mg�1 protein)

Glutathione reductase (GR) enzyme activity

(mM min�1 mg�1 protein)

Glutathione peroxidase (GSH-Px) enzyme

activity (mM min�1 mg�1 protein)

Control Nitrophenolates-sprayed plant Control Nitrophenolates-sprayed plant Control Nitrophenolates-sprayed plant

1 0.03970.0071a 0.13170.0081b 0.05770.0015a 0.09170.0013b 5.170.74a 12.270.81b

3 0.04570.0082a 0.16470.0092b 0.13270.0167a 0.35270.0147b 6.570.82a 17.470.90b

5 0.06970.0062a 0.19370.0094b 0.17470.0178a 0.36970.0232b 9.470.86a 22.970.75b

7 0.09170.0083a 0.22570.0089b 0.26570.0183a 0.41270.0247b 12.170.91a 28.070.96b

9 0.12370.0091a 0.26870.0084b 0.29670.0231a 0.44370.0379b 14.270.94a 34.571.1b

Means7SE within a row with different letters are significantly different (Po0.05); n ¼ 18.

Table 5Changes in ascorbate content, dehydroascorbate reductase enzyme activity and dehydroascorbate content in bolls of control and nitrophenolates-sprayed plants.

Days after anthesis Ascorbate (AA) content (nM g�1 FW) Dehydroascorbate (DHA) content

(nM g�1 FW)

Dehydroascorbate reductase (DHAR) activity

(mM min�1 mg�1 protein)

Control Nitrophenolate-sprayed plant Control Nitrophenolate-sprayed plant Control Nitrophenolate-sprayed plant

1 67711a 202718b 711721a 912734b 1.2470.13a 1.7570.11b

3 102712a 243717b 777723a 1020735b 1.4170.14a 1.9770.17b

5 108716a 280716b 861728a 1105738b 1.7170.12a 2.3670.17b

7 135712a 354714b 926730a 1230746b 1.9770.18a 2.5270.19b

9 170714a 417712b 1113732a 1430749b 2.3170.19a 2.9870.22b

Means7SE within a row with different letters are significantly different (Po0.05); n ¼ 18.

Table 6Changes in proline content and total phenol content in bolls of control and nitrophenolate-sprayed plants.

Days after anthesis Proline content (mg g�1FW) Total phenol content (mg g�1 FW)

Control Nitrophenolates-sprayed plant Control Nitrophenolate-sprayed plant

1 295714a 365711b 0.09770.012a 0.21570.014b

3 379712a 473712b 0.12670.015a 0.29470.012b

5 456715a 646713b 0.15170.017a 0.34170.013b

7 589713a 807716b 0.17270.018a 0.39370.016b

9 693716a 1040715b 0.20170.016a 0.51270.017b

Means7SE within a row with different letters are significantly different (Po0.05); n ¼ 18.

Table 3Changes in superoxide dismutase, catalase and ascorbate peroxidase enzyme activity in bolls of control and nitrophenolate-sprayed plants.

Days after anthesis Superoxide dismutase (SOD) enzyme activity

(enzyme units mg�1 protein)

Catalase (CAT) enzyme activity

(mM H2O2 min�1 mg�1 protein)

Ascorbate peroxidase (APX) enzyme activity

(mM min�1 mg�1 protein)

Control Nitrophenolate-sprayed plant Control Nitrophenolate-sprayed plant Control Nitrophenolate-sprayed plant

1 14.971.1a 23.670.8b 17.771.1a 19.670.9a 4.370.28a 5.670.27b

3 23.271.8a 35.771.2b 23.171.2a 25.271.2a 4.970.21a 6.170.20b

5 31.572.4a 48.671.8b 28.471.6a 29.571.6a 5.370.29a 6.970.29b

7 37.072.3a 55.872.4b 36.271.8a 39.471.7a 7.270.26a 8.970.25b

9 39.371.1a 62.572.5b 38.471.9a 42.172.0a 8.170.24a 9.470.39b

Means7SE within a row with different letters are significantly different (Po0.05); n ¼ 18.

M. Djanaguiraman et al. / Journal of Plant Physiology 167 (2010) 1–9 5

increased to 28% over controls on the same day. The DHAR activitywas increased significantly over control plants by nitrophenolatespray from the first DAA to the ninth DAA (Table 5). The maximumincrease was recorded at the first DAA (41.1%), followed by thethird DAA (39.7%). In both nitrophenolate-sprayed and controlplants, phenolics and proline contents increased from the firstDAA to the ninth DAA (Table 6). However, the magnitude ofincrease was higher in nitrophenolate-sprayed plants. In controlplants, at the ninth DAA the phenolics and proline contentsshowed 16% and 17% increases as compared with values at the

seventh DAA. In nitrophenolate-sprayed plants, increases of 30%and 28% were recorded during the same period.

Shoot length, yield and yield components

The influence of nitrophenolate spray on shoot length did notshow significant difference in any of the study years. However, asignificant difference was observed between control and nitro-phenolate-sprayed plants for the number of abscised squares and

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Table 7Effect of foliar spray of nitrophenolates during square formation (35 DAS) and initiation of flowering stage (58 DAS) stage on shoot length (cm), number of squares

abscised plant�1, number of bolls abscised plant�1, fertility co-efficient (%), boll weight (g seed cotton boll�1) and seed cotton yield (kg ha�1).

Parameters Year

2003 2004 2005

Control Treated Control Treated Control Treated

Shoot length (cm) 78.178.1a 84.578.8a 76.878.0a 70.877.6a 76.878.5a 83.879.5a

Number of squares abscised plant�1 28.472.1a 20.472.3b 20.671.8a 16.971.5b 21.872.5a 16.871.8b

Number of bolls abscised plant�1 8.271.1a 5.471.2b 9.571.1a 6.171.0b 8.970.9a 6.470.8b

Fertility Co-efficient (%) 53.575.9a 62.576.1b 53.875.8a 66.475.9b 55.476.1a 64.875.8b

Boll weight (g seed cotton boll�1) 4.6970.5a 4.8070.6a 4.6670.4a 4.7270.5a 4.6570.6a 4.7170.6a

Seed cotton yield (kg ha�1) 16857180a 19807191b 21687241a 26147260b 19867220a 25057268b

Means (7SE) for each year followed by same letter are not significantly (P ¼ 0.05); n ¼ 20.

Means7SE within a row with different letters are significantly different (Po0.05); n ¼ 18.

M. Djanaguiraman et al. / Journal of Plant Physiology 167 (2010) 1–96

bolls plant�1, fertility coefficient and seed cotton yield between2003 and 2005 (Table 7). Foliar spray of nitrophenolates reducedthe number of abscised squares plant�1 to 28.1%, 17.9% and 22.9%during 2003, 2004 and 2005, respectively, compared withcontrols. Likewise, nitrophenolate spray reduced the number ofabscised bolls plant�1 34.1%, 35.7% and 28.0% during 2003, 2004and 2005, respectively, compared with controls (Table 7). Theaverage boll weight was not significantly influenced by foliarspray of nitrophenolate. Nevertheless, the fertility coefficient waspositively influenced by nitrophenolate spray (Table 7). The lowyield in control compared with nitrophenolate-sprayed plants wasdue to higher abscission rate of squares and bolls, since the bollweight was not improved by nitrophenolate spray. The increase inyield by nitrophenolate spray was 17.5%, 20.5% and 26.1% overcontrols during 2003, 2004 and 2005, respectively.

Discussion

Abscission refers to separation events, and it is the final phaseof the senescence process (Prochazkova and Wilhelmova, 2007).Hormonal signals and competition for photoassimilates remainfactors of prime importance among those influencing abscission(Gomez-Cadenas et al., 2000). However, our previous experimentin tomato showed abscission of tomato fruit occurs throughoxidative damage (Djanaguiraman et al., 2004). Oxidative stressarises from an imbalance in the generation and metabolism ofROS, with more ROSs (such as H2O2, OH� and O2

�) being producedthan are metabolized (Dhindsa et al., 1981). The response of theplant and its components to ROS depends on the ROS in questionas well as on its concentration. It also depends on the develop-mental stage of the plant part. It is well established that cottonbolls abscise within 10–15 days of anthesis (Constable, 1991;Crozat et al., 1999). In this condition, ROS detoxification mechan-isms must be activated early (start of anthesis stage) to avoidoxidative damage. The results of the present study indicated thatthe ROS (O2

� and H2O2) started to accumulate from the first DAA tothe ninth DAA in both control and nitrophenolate-sprayed plants.However, nitrophenolate-sprayed plants showed comparativelylow ROS content. The same trend was also observed for LOXenzyme activity, MDA content and cell membrane damage. Theincreased level of ROS in control plants may be due to elevatedproduction of free radicals. Lipid peroxidation can be initiated byROS or by the action of LOX (Halliwell and Gutteridge, 2000), andseverely affects functionality and integrity of cell membranes. Thecontrol plants showed enhanced MDA content, which is an indexof lipid peroxidation. The decrease in MDA content in nitrophe-nolate-sprayed plants could be attributed to a general protectiveeffect of the antioxidant system.

According to Mayak et al. (1981), superoxide anions that areproduced in increasing quantities during senescence of carnationpetal induce the degradation of phospholipids and the fatty acidsreleased by this breakdown are then peroxidized, which in turnaffects membrane permeability (Simon, 1974). The comparativelylow contents of O2

� and H2O2 in nitrophenolate-sprayed plantsmay be due to trapping of free radicals by phenolics. Phenolicsinhibit the oxidation of lipids, fats and proteins by donation of aphenolic hydrogen atom to the free radical (Aruoma et al., 1993;Halliwell et al., 1995). The stereo-electronic effects of phenolicsand phenoxyl radicals are largely responsible for their reactivitywith the radicals (Burton et al., 1985). The reaction mechanismsby which the hydrogen atom of phenolics is transferred to aradical can occur in two distinct pathways: hydrogen atomtransfer and proton-coupled electron transfer (Mayer et al.,2002). Frankel et al. (1996) and Jang et al. (2007) also documentedthe antioxidant property of phenolics. Thus, phenolics are able toact as radical scavengers or radical-chain breakers, extinguishingstrongly oxidative free radicals (Stadler et al., 1995; Moran et al.,1997).

Plants possess a well-defined enzymatic and non-enzymaticantioxidant defense system to protect themselves against thesedeleterious effects by scavenging ROS. The combined action ofSOD and CAT/APX/GSH-Px/POX is critical in mitigating the effectsof oxidative stress, as the former merely acts on the superoxideanion, converting it to another reactive intermediate (H2O2) andthe latter acts on H2O2, converting it to water and oxygen (Mates,2000). The various scavenging enzymes encoded by the ROSnetwork can be found in almost every subcellular compartment.In addition, more than one enzymatic scavenging activities for aparticular ROS can usually be found in each of the differentcompartments. When the functions of the different enzymes inthe different cellular compartments are considered, it is importantto note that H2O2 can diffuse between different compartments.The water–water cycle detoxifies O2

� and H2O2, and alternativeoxidase reduces the production rate of O2

� in thylakoids; in someplants, Fe-SOD might replace Cu/Zn-SOD in the chloroplast. ROSsthat escape this cycle and/or are produced in the stroma undergodetoxification by SOD and the stromal AA–GSH cycle. Peroxir-edoxin and GSH-Px are also involved in H2O2 removal in thestroma. ROSs produced in peroxisomes during photorespiration,fatty acid oxidation or other reactions are decomposed by SOD,CAT and APX. SOD and other components of the AA–GSH cycle arealso present in mitochondria (Mittler et al., 2004). In the presentstudy, we found that CAT did not participate in active H2O2

reduction, although SOD was active in scavenging the superoxideproduced in the developing bolls. Differential CAT and SODactivities under abiotic stress, inducing oxidative damage, havebeen reported by Jung (2003). Replacement of CAT by GSH-Px and

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POX in senescing tissue was previously shown by Barber (1980).Moreover, induction of specific isozymes of POX in many plantshas been recognized as one of the biomarkers of senescence(Kukavica and Jovanovic, 2004). The combined action of SOD andGSH-Px is significant in diminishing the effects of oxidative stress.In the present study, we observed that CAT did not participatein active H2O2 reduction irrespective of the date of sampling,although SOD was active in scavenging the superoxide radicals, asevidenced by significant differences between the treatments. Thiscould be because CAT is present only in the peroxisome and haslow substrate affinity, as it requires simultaneous access of twomolecules of H2O2 (Djanaguiraman et al., 2005a, b). GSH-Pxenzyme, acting in concert with other antioxidants and free-radicalscavengers in the cell, reduces hydrogen peroxide and organicperoxides to water and corresponding alcohols, in the presence ofreduced GSH. GSH-Px was more efficient in destroying H2O2 thanCAT. This could be because GSH-Px is present throughout the celland has higher substrate affinity in the presence of GSH as areductant (Noctor and Foyer, 1998). That the increase in GSH-Px,which is a scavenger of H2O2 and lipid hydroperoxides, resulted inreduced formation of superoxide anion radicals is confirmed inthe present study by a lower value of superoxide anion contentand membrane damage in nitrophenolic-treated plants comparedwith control plants.

It has been proposed that POX may play an important role inthe rapid defense responses of plant cells to oxidative stress(Zabalza et al., 2007). The greater inhibition of all the antioxidantenzymes in control plants relative to nitrophenolate-treated plantindicates more inactivation of all the antioxidant enzymes. Thismight be due to the toxic effects of the high turnover rate of H2O2

or its harmful ROSs, which impair enzyme activities (Noctor andFoyer, 1998). The results of the present study strongly suggest thatnitrophenolate-sprayed plants have greater ability to eliminateROS through higher POX activity because of higher substrate(guaiacol) availability. This substrate is one of the activeingredients of Atonik. It has been suggested that POX could actas an efficient H2O2-scavenging system in plant vacuoles in thepresence of phenolics and reduced AA (Zancani and Nagy, 2000).

The role of GR in H2O2 scavenging in plant cells has been wellestablished in the AA–GSH pathway (Noctor and Foyer, 1998). Thelower levels of GR activity in control plants make them stresssensitive (Aono et al., 1995). The induction of GR activity innitrophenolate-treated plants is not a response of the AA–GSHcycle, as APX activity was less influenced by nitrophenolate spray.The GR activity increase may be related to the enhancement ofGSH content. Foyer et al. (1995) reported that GSH content levelsare more strongly influenced by GR activity than by enzymaticactivities of the biosynthetic pathway.

Lesser oxidative damage in the nitrophenolate-sprayed plantshas been attributed to higher AA content and induction of AA-GSHcycle enzymes. The susceptibilities of an Arabidopsis mutant withlow ascorbic acid and transgenic Arabidopsis plants with less than5% wild-type leaf GSH content to oxidative damage furtherhighlight their importance in oxidative stress (Xiang et al.,2001). It has been widely accepted that the capacity of plants toreduce oxidized AA decreases as plants mature. In addition, theenzyme galactono–g-lactone dehydrogenase, which catalyzes thelast step in AA biosynthesis, also decreases with age. This may bethe reason for low levels of AA in control plants (Bartoli et al.,2000). In the present study, higher abscission rates in controlplants may be due to failure in H2O2 management and to drasticoxidation of the AA–GSH pool. The high H2O2 level and/oroxidation of the AA pool might have an inhibitory effect onantioxidant enzymes.

The increased AA level in the nitrophenolate-sprayed plantsmight be involved in reduction of superoxide, quenching of singlet

oxygen or regeneration of reduced a-tocopherol (Bartoli et al., 1999;Smirnoff and Wheeler, 2000). Further, ascorbic acid reduces lipidperoxidation (Shalata and Neumann, 2001). The enhanced AA levelsin nitrophenolate-sprayed plants may be due to de novo AAbiosynthesis and recycling of the oxidized forms of AA, MDA andDHA via MDAR and DHAR, respectively (Conklin and Barth, 2004).

Possible positive roles of proline under oxidative stress havebeen proposed, including stabilization of proteins, regulation ofthe cytosolic pH and regulation of the NAD/NADH ratio by singlet-oxygen quencher and scavenger of OH� radicals (Matysik et al.,2002). Smirnoff and Cumbes (1989) reported that proline reactswith OH� under hydrogen abstraction by forming the most stableradical. Enhanced proline content under oxidative stress was alsoobserved by Gunes et al. (2007). It can therefore be inferred thatproline is an effective scavenger of ROS.

Nitrophenolates applied at a rate of 0.8 g ai ha�1 significantlyincreased cotton seed yield in all of the study years. Earlier reportsclaimed that nitrophenolates increased cotton lint yield bysignificantly increasing individual boll weight (Fernandez et al.,2003), increasing the number of harvested bolls per plant(Townsend, 2004) and increasing the number of seeds per boll(Fernandez and Correa, 2004). Nitrophenolates have also beenshown to decrease membrane leakage by 34.5% (Guo andOosterhuis, 1995). Fernandez et al. (2002) reported an increasein the percentage of fruit retention and lint turnout of cottontreated with nitrophenolates. The increase in the seed cotton yieldby nitrophenolate spray in the present study is in accordance withthese findings. The increased yield by nitrophenolate spray in thepresent study is due to reduced abscission of squares and bolls.

This study indicates that oxidative damage comprises a highlyregulated series of biochemical events. At the terminal phase,plants lose antioxidant capacity and the release of ROS increases.At this stage, cells undergo lipid peroxidation and membranedegradation. ROSs seem to be involved in ethylene synthesis,which causes abscission. However, plants possess an array ofantioxidant defense systems, which includes ROS-detoxifyingenzymes (SOD, GSH-Px and POX) and metabolites (AA, phenolicsand proline) to remove potentially harmful ROS.

Under conditions of high AA levels and low APX activity, asseen in nitrophenolate-sprayed plants, POX participated in thescavenging of H2O2 through the POX/phenolics/ascorbic acidsystem. Higher GR activity observed in this study indicates amechanism of antioxidant defense occurring through enhancedoxidation of GSH to GSSH by DHAR, yielding AA. This AA, inaddition to the AA produced by non-enzymatic disproportionationof MDHA, may be used in the peroxide/ phenolics/AA system todetoxify H2O2 (Sgherri et al., 2003). We can hypothesize a cyclewhere H2O2 is scavenged by phenolics through a POX; phenolicsare oxidized to phenoxyl radicals, which can be reduced by AA.This cycle can occur in both the apoplast and in the vacuole,where phenolics are particularly concentrated (Santiago et al.,2000). This suggests that DHA produced from AA oxidation inthese compartments must be transported back into the cytosol forregeneration to AA. In the present experiment, the increased AAcontent with nitrophenolate spray may be sufficient to replenishreducing equivalents to phenoxyl radicals, and thus explaining theincrease in the phenolic compounds. Further studies are requiredto reveal the role of ionically bound isoforms of POX in theformation of polymerized phenolics and the production of H2O2

for lignification. However, in control plants, some sort of signalresults in down regulation of GSH-Px, POX activity and AA leveland an increase in endogenous H2O2 level. This H2O2 in turn mayact as a signaling molecule, inducing the programmed cell deathpathway as well as triggering membrane lipid peroxidation,leading to membrane deterioration and ultimately resulting inboll abscission.

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