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Environmental and Experimental Botany 71 (2011) 249–259

Contents lists available at ScienceDirect

Environmental and Experimental Botany

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xogenously applied glycinebetaine enhances seed and seed oil quality of maizeZea mays L.) under water deficit conditions

asim Alia,∗, Muhammad Ashrafa,b

Department of Botany, University of Agriculture, Faisalaabad 38040, PakistanDepartment of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 19 December 2009eceived in revised form 7 December 2010ccepted 8 December 2010

eywords:eed compositionil compositionocopherolshenolicsatty acidsntioxidant activity

a b s t r a c t

A field trial was carried out to appraise up to what extent exogenous application of a potential osmo-protectant, glycinebetaine (GB), could ameliorate the inhibitory effects of shortage of water on maizeseed and seed oil composition and oil antioxidant potential. Two maize cultivars, Agaiti-2002 (droughttolerant) and EV-1098 (drought sensitive), were exposed to drought treatments at the vegetative growthstage. Both the maize cultivars used in the present study are being widely cultivated in Pakistan andhave been an important source of developing different maize hybrids. Two levels of glycinebetaine (0 or30 mM) were foliar-applied at the vegetative stage. Water stress reduced the kernel sugar, oil, protein,moisture contents and most of the seed micro- and macro-nutrients analyzed of both maize cultivars, butit increased the contents of seed fiber and ash contents. Among different seed oil un-saturated fatty acids,water stress increased the oil oleic acid contents with a decrease in linoleic acid contents, which resultedin increased oil oleic/linoleic ratio of both maize cultivars. However, no variation was observed in oilstearic and palmitic acid contents due to water stress. A considerable increase in seed oil �-, �-, �- andtotal tocopherols and flavonoids was observed in both maize cultivars. However, oil phenolic contentand 1,1′-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity decreased. Foliar-appliedGB significantly increased the contents of seed sugar, oil, protein, moisture, fiber, ash, GB contents and

micro- and macro-nutrients of both maize cultivars under well irrigated and water deficit conditions.Furthermore, exogenous application of GB increased the oil oleic and linoleinic acid contents. All differ-ent lipophilic compounds estimated in the seed oil increased due to foliar applied GB. Furthermore, GBalso increased seed oil antioxidant activity appraised in terms of oil DPPH free radical scavenging activity.By summarizing the results, it seemed that exogenously applied GB remained in intact form until laterstages of growth and counteracted the inhibitory effects of water deficit on seed and seed oil composition

ltiva

similarly of both maize cu

. Introduction

Cereal grains supply more than 60% of the entire global foodemand (Lásztity, 1999) because they comprise a substantialmount of carbohydrates, mainly starch, along with high qualityroteins and relatively little amount of lipids, vitamins, and inor-anic nutrients (Baye et al., 2006). However, it is now evident thatoth genetic and environmental factors cause a substantial vari-tion in the quality and quantity of each of these components

Dunlap et al., 1995; Baenziger et al., 2006).

Among cereals, maize (Zea mays L.) not only has a sufficientmount of carotenoids, tocopherols, and oil but also has a reason-ble amount of starch and protein content compared with other

∗ Corresponding author. Tel.: +92 41 9200312; fax: +92 41 9200764.E-mail address: qasimbot uaf@yahoo.com (Q. Ali).

098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2010.12.009

rs.© 2011 Elsevier B.V. All rights reserved.

major food crops such as rice and wheat. Although maize is prin-cipally cultivated for carbohydrate production, in the past severalyears, it has gained great significance as a source of vegetable oilfor the food industry (Balasundram et al., 2006).

Maize oil is considered the best as a vegetable oil due to its largeamount of unsaturated fatty acids, most predominantly being oleicand linoleic acids ranging from 65 to 85% (Goffman and Böhme,2001). Despite a large amount of unsaturated fatty acids, the oilis a rich source of phenolics, flavonoids, and different types oftocopherols (Balasundram et al., 2006). Phenolic compounds beingsecondary metabolites are considered non-essential for nutrition,but the interest in the appraisal of their antioxidant and bioactive

properties has amplified due to their considerable role in humanand animal health (Schussler and Westgate, 1991; Balasundramet al., 2006).

Tocopherols exist as a family of four derivatives (�-, �-, �-and �-), which differ in the number and position of methyl sub-

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50 Q. Ali, M. Ashraf / Environmental and

titutions in the chromanol ring. Tocopherols are well recognizedntioxidants in vegetable oils, and their presence increases the sta-ility of lipids against autoxidation (Goffman and Böhme, 2001).

It is well known that genetic and environmental factors play aital function in the economic importance of a crop and can affecthe oil yield and quality of its seed/grain production. For example,nfavorable conditions especially drought have shown to alter theeed composition and related qualities such as oil physico-chemicalroperties of Moringa oleifera (Anwar et al., 2006) and sunflowerAli et al., 2009). In some earlier studies, it has been reported thatack of water during all stages of growth and development is theimiting factor for seed growth that can significantly influence itsomposition (Monotti, 2003; Ali et al., 2009).

Of different osmolytes occurring in plants in response tonvironmental stresses, glycinebetaine (GB) has been studiedxtensively (Naidu et al., 1998; Iqbal et al., 2005; Chen and Murata,008; Ashraf, 2009). It is an end-product of a metabolic processnd so is not catabolized with the plant, but readily translocatedo growing points (Hanson and Wyse, 1982; Mäkelä et al., 1996).ossible mechanisms for the GB-enhanced tolerance of plants toarious types of abiotic stresses include the protection of the pho-osynthetic machinery (Zhao et al., 2007), enhanced photosyntheticate (Yang and Lu, 2005; Ma et al., 2006; Zhao et al., 2007; Mahmoodt al., 2009), induction of specific genes involved in stress toler-nce, reductions in levels of reactive oxygen species (ROS) undertress and regulation of the activities of ion-channel proteins eitherirectly or via protection of the plasma membrane (Chen andurata, 2008). It has also destabilizing effects on photorespira-

ion (Sulpice et al., 2002). Significant advances have been maden mitigating the inhibitory effects of environmental stresses byxogenously applied glycinebetaine in different crops such as riceRahaman et al., 2002), cotton (Naidu et al., 1998), sunflower (Iqbalt al., 2005), wheat (Mahmood et al., 2009), and maize (Ali et al.,007). Despite the beneficial effects of exogenously applied GBhere are reports that show that high concentrations of exoge-ously applied GB have some inhibitory effects on plant growth andas exchange attributes (Yang and Lu, 2006). Furthermore, in somearlier studies, it has been shown that leaf burn and tip chlorosisccurred in tomato, wheat, and maize when exogenously appliedB concentration was higher than 100 mM (Allard et al., 1998;äkelä et al., 1998; Chen et al., 2000) and even exogenously appliedB more than 20 mM caused a decrease in growth of maize plantshen applied under non-stress conditions (Yang and Lu, 2006).

In maize, it has been reported that different varieties vary inheir capacity to accumulate glycinebetaine (Rhodes et al., 1989)hat is not sufficient to induce stress tolerance in maize plants. Cer-ain maize genotypes are not able to synthesize GB while somethers can synthesize it (Rhodes et al., 1989). However, the syn-hetic capacity in those that can synthesize GB is relatively low andB content is only in the range of 2–5 mmol g−1 FW (Rhodes et al.,987, 1989), which is about 5–10-fold less than that in other plantsuch as barley and sorghum (Grieve and Maas, 1984, Kishitani et al.,994). So, the transgenic maize varieties are being produced to

ncrease the internal GB contents by introducing GB synthesizingnzyme genes. Furthermore, the accumulation of GB when appliedxogenously mainly takes place due to the activity of high affinityransporters (Bae et al., 1993). The transporter so far characterizedt a molecular level is a high-affinity ATP-binding cassette (ABC)ystem described as BusA and OpuA in Lactococcus lactis subsp.remoris NCDO763 and MG1363, respectively (Obis et al., 1999;ouvier et al., 2000).

Although exogenous application of glycinebetaine as foliar sprayas been employed to enhance drought tolerance in a number ofrops (Gorham et al., 2000; Meek et al., 2003; Mahmood et al.,009), little information is available in the literature on the effectsf foliar application of this osmolyte in altering the seed compo-

rimental Botany 71 (2011) 249–259

sition, levels of different fatty acids, tocopherols, phenolics andflavonoids particularly in the edible oil of maize. In view of this, wehypothesized that GB application improves the quantity as well asquality of seed oil in maize grown under water deficit conditions.Thus, the premier objective of conducting the present study was toexplore up to what extent foliar-applied glycinebetaine could alterseed composition, oil yield, seed oil fatty acid composition, oil toco-pherol content, physico-chemical properties and oil antioxidantactivity of seed oil of maize grown under water deficit conditions.

2. Materials and methods

2.1. Procurement and planting of maize seed

The seeds of two maize varieties, Agaiti-2002 (drought toler-ant) and EV-1098 (relatively drought sensitive) used for the presentexperimentation were supplied by the Maize and Millet ResearchInstitute, Yousaf wala (Sahiwal), Pakistan. The influence of foliar-applied GB on seed and seed oil composition of both maize varietieswas assessed under field conditions at the Research Area (NewBotanical Garden) of the Department of Botany, University of Agri-culture, Faisalabad, Pakistan (latitude 30◦30N, longitude 73◦10Eand altitude 213 m).

2.2. Climatic conditions

The climatic conditions at the site calculated as means areas follow: photosynthetically available radiation measured atnoon varied from 794 to 1154 �mol m−2 s−1, and mean day/nightRH 33.1/75.1% and day and night temperatures 38.28 ± 4 ◦C and22.82 ± 3.6 ◦C, respectively.

2.3. Composition of field soil

The soil used was sandy clay (average 65% clay content, 22% sandand 13% silt). The hygrometer method was used to determine thesoil texture following Dewis and Freitas (1970). The saturation per-centage of soil was 31, organic matter 0.78% and NO3-N 6.5, NH4-N3.00, available phosphorous 5.6, potassium 187 and calcium 109(all values of nutrients in mg/kg of dry soil). The soil pH was 8.1and soil electrical conductivity (ECe) 2.1 dS/m. Electrical conduc-tance (ECe), pH and inorganic nutrients of the soil saturation extractwere appraised following Jackson (1962).

2.4. Layout of the field experiment

The whole experiment was arranged in a completely random-ized design (CRD). The main plot was divided into two sub-plotson the basis of water stress treatments. In one sub-plot, normalirrigation was applied and in the second sub-plot, drought stresswas started during the early growth stage of crop growth. Eachexperimental unit was replicated four times. First irrigation for thepreparation of soil for seed sowing to both subplots was applied 15days before sowing. When the soil was at field capacity condition,the plots were well prepared for sowing the seeds. The rate of theseed was 10 kg/ha of each maize cultivar that was hand-drilled bymaintaining inter-row distance of 75 cm and plant to plant space30 cm. Plants were thinned 15 days after germination.

2.5. Drought stress treatment

After sowing of seeds, the first irrigation to all plots was applied8 days after seedling emergence and thereafter, drought stresstreatment was initiated by controlling irrigation schedule. The irri-gations to non-stressed plants were applied regularly at 15-day

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nterval after the 1st one except the plants meant for experienc-ng drought stress; however, the irrigations to the water stressedlants were applied at 21-day intervals.

.6. Glycinebetaine application

Two levels of glycinebetaine (purchased from Sigma–Aldrich,hemie, Steinheim, Germany) used during experimentation were 0nd 30 mM, which were prepared in the surfactant Tween-20 (0.1%olution) and applied foliarly only once at the vegetative stage,hen the plants were at five-leaf stage. An aliquot of 500 mL of

0 mM GB solution was applied foliarly to plants in each repli-ate using manually operated agricultural spray equipment withL tank capacity. The equipment tank was fitted with a brass spray

ance with fine spray brass nozzle and a pump barrel made of seam-ess brass tube. The foliar application of GB was carried out in thevening just before sunset. Before harvest, ears were removed. Thears were dried for 3–4 days in air during daytime. After proper dry-ng, kernels were separated from ears and used for further analysis.ll reagents used were of analytical and HPLC grades and purchased

rom Sigma–Aldrich (Buchs, Switzerland).

.7. Characterization of maize kernel and kernel oil

.7.1. Proximate analysisMaize kernels were evaluated for crude protein, moisture, ash,

rude fat, starch and crude fiber by using appropriate protocolss depicted in AACC (Anonymous, 2000), i.e., Method No. 44-15A,ethod No. 08-01, Method No. 46-30, Method No. 30-25, Method

2-10, respectively.

.8. Oil extraction

After proper drying, the seeds (200 g) of each treatment wererushed into 80 mesh of particle size. Then the properly weighedrushed seed material was packed in paper thimbles and placedhem in a soxhlet apparatus fitted with 500 mL volumetric flask.he extraction was carried out using n-hexane as a solvent for 8 h.

.9. Chemical parameters of oil

Determinations of kernel oil iodine value, saponification value,nsaponifiabale matter, peroxide value, density and contents ofree fatty acids (FFAs) of the extracted oil were performed follow-ng the established AOCS methods (Cd 1-25, Cd 3-25, Ca 61-40,d 8-53, Cc 10a-25 and F9a-44, respectively; AOCS, 1997). Specificxtinctions [ε1% 1 cm (�)] at 232 and 270 nm were appraised follow-ng the IUPAC method II. D. 23 (IUPAC, 1987). Oil samples diluted

ith iso-octane were read at 232 and 270 nm on a spectropho-ometer (model, U-2001, Hitachi, Japan). The amount of p-anisidineas determined following an IUPAC method II. D. 26 (IUPAC, 1987).oloured complexes of the oil samples were developed by readinghe oil samples p-anisidine for 10 min and absorbance was read at50 nm � using a spectrophotometer (U-2001, Hitachi Instrumentsnc., Tokyo, Japan).

.10. Fatty acid composition of seed oil

Fatty acid methyl esters (FAMEs) were prepared following theUPAC standard method (2.301) and quantified using a gas chro-

atograph (Perkin-Elmer, model Clarus 500) fitted with an Rt-2340

B (RESTEK, Corp., 800-356-1638, USA) methyl-lignocerate-coated

film thickness 0.20 �m) polar capillary column (60 m × 0.25 mM)nd an FID detector. Analytical grade nitrogen gas with a flow ratef 5 mL min−1 was used as a mobile phase. Other conditions main-ained were: oven initial temperature, 80 ◦C; ramp rate, 3 ◦C/min;

rimental Botany 71 (2011) 249–259 251

final temperature, 210 ◦C; detector temperature 220 ◦C; and injec-tor temperature, 210 ◦C. The quantification of FAMEs was based ontheir absolute retention times after comparing with appropriatestandards purchased from Sigma–Aldrich (Buchs, Switzerland).

2.11. Tocopherol content

The tocopherol content in the kernel oil was analyzed by HPLC(Sykam GmbH, Kleinostheim, and Germany) following Lee et al.(2003). The HPLC system was equipped with an S-1122 dual pistonsolvent delivery system, an S-3210 UV/VIS diode array detector.Twenty �L of the extract was injected into Hypersil ODS reversephase (C18) column (5 �m particle size, 250 mM × 4.6 ID Thermo-hypersil GmbH, Germany) fitted with a C18 guard column andmethanol:acetonitrile:methylene chloride (50:44:6, v/v) mobilephase at 1 mL min−1 flow rate. The peak areas were recorded andcalculated by a computer with SRI peak simple chromatographydata acquisition and integration software (SRI instrument, Tor-rance, CA) at 295 nm. The quantification of tocopherols was doneby comparing the samples with pure standards purchased fromSigma–Aldrich (Buchs, Switzerland).

2.12. Total phenolic contents

The amount of total phenolics was assessed using Folin-Ciocalteu reagent (Chaovanalikit and Wrolstad, 2004). Briefly 10 goil was mixed with 60 mL n-hexane and extracted with 60%methanol. The solvent was evaporated to dryness (40 ◦C) usinga rotary evaporator (Heidolph, model, Laborota 4001, Germany)to obtain the crude extract. Then the crude extract (50 mg) wasmixed with 0.5 mL of Folin-Ciocalteu reagent and 7.5 mL of deion-ized water. The mixture was kept at room temperature for 10 min,and then 1.5 mL of 20% sodium carbonate (w/v) was added. The mix-ture was heated in a water bath at 40 ◦C for 20 min and then cooledin an ice bath; absorbance was measured at 755 nm using a spec-trophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan).Amounts of TP were calculated using a gallic acid calibration curvewithin the range of 10–100 mg L−1. The results were expressed asgallic acid equivalents (GAE) g/100 g of dry matter.

2.13. Total flavonoids

Total flavonoids were appraised following the procedure ofDewanto et al. (2002). Two mL of aqueous extract of dry matterwas placed in a 10 cm3 volumetric flask, and then was added 5 mLof distilled water followed by 0.6 mL of 5% sodium nitrite solution.After 5 min, 0.6 mL of AlCl3 solution (10%) was added. After another5 min, 2 mL of 1 M NaOH was added and the volume made up to10 mL with distilled H2O. The solution was mixed thoroughly andabsorbance noted at 510 nm. TF concentrations were expressed ascatechin equivalents g/100 g of dry matter.

2.14. DPPH radical scavenging activity

The free DPPH radical scavenging activity of the oil extracts wasestimated following the Sultana et al. (2007) and Iqbal and Bhanger(2007). Five cm3 of a freshly prepared solution of 1,1′-diphenyl-2-picryl-hydrazyl (DPPH) at concentration 0.025 g/L was added to1.0 mL of the extract of dry matter in methanol. The mixture wasincubated in at room temperature in the dark for 30 min. Then thedecrease in absorbance was measured against a blank at 515 nm

(the change in colour from deep-violet to light-yellow). The follow-ing formula was used to calculate the radical scavenging activity:

DPPH radical scavenging (%) = (AB − AA)AB

× 100

2 Experimental Botany 71 (2011) 249–259

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52 Q. Ali, M. Ashraf / Environmental and

here AB – absorbance of blank sample (t = 0 min); AA – absorbancef tested extract solution after 15 min of incubation (t = 30 min).

.15. Determination of inorganic micro- and macro-elements inernels

Well dried ground seed material (0.2 g) was digested with 2 cm3

f H2SO4 + H2O2 following Wolf (1982). Potassium and calciumn the digested samples were appraised using a Jenway flamehotometer (model PFP-7). Kjeldahl method was employed foretermining N and P following Allen et al. (1986). The remaininglements Fe, Mg, Mn, Zn, Cu, Ni, Cd, Cr and Pb were determinedsing an atomic absorption spectrophotometer (A Analyst-300)erkin-Elmer, USA.

.16. Glycinebetaine determination in leaves and seeds

For the estimation of GB, leaves were detached from the droughtreated and non-treated plants and dried for 72 h at 70 ◦C in an elec-ric oven for further analysis. For the estimation of GB one g of driedeaves or seeds was ground in 2 mL ethanol and kept in a water batht 90 ◦C until complete evaporation of ethanol. Then 2 mL of deion-zed distilled water was added and shaken vigorously for 2 min.he corresponding crude extract was centrifuged at 14,000 × g formin so as to remove insoluble particles.

GB was measured by high performance liquid chromatog-aphy (HPLC) following Gibon et al. (1997). The crude extractbtained was purified with an AG1-X8 anion exchanger. To theurified crude extract, 50 �g g−1 analytical grade GB (purchasedrom Sigma–Aldrich, Chemie, Steinheim, Germany) was added asn internal standard to reduce spectral interferences and for accu-ate and precise quantification of GB in the leaves and seeds. ThePLC system was equipped with an S-1122 dual piston solventelivery system, S-3210 UV/VIS diode array detector. Twenty �L ofhe extract was injected into Hypersil ODS reverse phase (C18) col-mn (5 �m particle sizes, 250 mM × 4.6 ID Themohypersil GmbH,ermany). The GB separation in the samples was done using iso-ratic condition of elution with a mobile phase of pH 3.7 comprisingf 13 mmol/L sodium 1-heptanesuphonate solutions added withmmol/L Na2SO4. The peak areas were recorded and calculatedy a computer with SRI peak simple chromatography data acqui-ition and integration software (SRI instrument, Torrance, CA) at00 nm. The quantification of GB was done by comparing the sam-les with pure standards purchased from Sigma–Aldrich (Buchs,witzerland).

.17. Statistical analysis

Data for each attribute were subjected to CoStat Computerrogram (version 6.303, PMB 320, Monterey, CA, 93940 USA)or calculating ANOVA. Mean values were compared with a LSDorked out following Steel and Torrie (1986).

. Results

Imposition of drought stress significantly reduced the seedugar and oil content of both maize cultivars; however, drought-nduced reduction in seed oil contents was more in cv. Agaiti-2002han that in EV-1098. The latter cultivar had higher seed oilontent than the former one under non-stress conditions. Seed

ugar contents did not vary significantly in the two cultivarsnder water deficit conditions (Table 1). Exogenous applicationf GB as a foliar spray significantly increased the seed oil con-ents in both cultivars under stress and non-stress conditions,ut this GB-applied increasing effect in relation to seed sugar Ta

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Experimental Botany 71 (2011) 249–259 253

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70.

038

0.08

3

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ean

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Q. Ali, M. Ashraf / Environmental and

ontents in both cultivars was observed only under non-stress con-itions.

A significant reduction in seed protein and moisture contentsf both maize cultivars was observed due to imposition of droughttress. No significant difference between the cultivars was observedn relation to these parameters both under well watered and under

ater deficit conditions. However, seed starch contents remainednchanged due to imposition of drought stress both maize cultivarsTable 1). Foliar applied GB significantly increased the seed protein,

oisture and starch contents of both maize cultivars under bothtressed and non-stressed conditions.

Seed fiber and ash contents in both maize cultivars increasedignificantly due to desiccation stress. Higher contents of seed ashnd fiber were observed in cv. EV-1098 than those in Agaiti-2002nder stressed and non-stressed conditions (Table 1). This increase

n seed fiber and ash contents was further enhanced due to exoge-ous application of GB under both water stressed and well wateredonditions.

Drought stress significantly altered the levels of all macro-inerals (K, Mg, P, N and Ca) and micro-minerals (Mn, Cu, Fe, Zn,o, Cr and Cd) analyzed in the seeds of both maize cultivars excepto, Cr and Cd which remained unaffected due to water stress.ata presented in Tables 2 and 3 show that contents of all theseinerals were reduced significantly in both maize cultivars underater stress. Externally applied GB mitigated the inhibitory effects

f drought stress on seed nutrients in both maize cultivars.GB contents in leaves and seeds of both maize cultivars

ncreased significantly due to imposition of water stress (Fig. 1).he cultivars did not differ significantly in leaf GB contents bothnder water stress and non-stress conditions however, in case ofeed slightly higher GB contents were observed in cv. Agaiti-2002han those of EV-1098 both under water stress and non-stress con-itions. Foliar-applied GB significantly increased the endogenous

evels of GB in both leaves and seeds of both maize cultivars. Muchncrease in leaf and seed GB contents due to foliar application of GB

as observed in cv. Agaiti-2002 as compared to that in cv. EV-1098.Data for seed oil palmitic acid and stearic acid show that impo-

ition of water stress had no significant effect on the levels of theseatty acids in both maize cultivars. However, both maize cultivarsesponded differently to exogenous application of GB in relationo oil stearic acid contents and this response was only under non-tress conditions. In cultivar Agaiti-2002 there was a slight decreasen stearic acid contents due to foliar application of GB while thepposite was true for cv. EV-1098.

Oleic acid and linoleinic acids increased while linoleic acidecreased in both maize cultivars due to drought stress. Oilleic/linoleic ratio of both maize varieties also increased due toater shortage. Foliary-applied GB significantly affected the con-

ents of these unsaturated fatty acids in the seed oil of bothaize cultivars. Oil oleic acid, linoleinic acid and oleic/linoleic ratio

ncreased, while linoleic acid decreased in both maize cultivars.owever no effect of water stress or exogenously applied GB wasbserved on oil total saturated and unsaturated fatty acids of bothaize cultivars (Table 4).Oil saponification value was significantly reduced in both maize

ultivars due to drought stress. However, oil un-saponifiable matterncreased significantly in kernel oil of both maize cultivars due to

ater stress, but oil iodine value remained unchanged due to impo-ition of water stress. Foliar GB application significantly increasedhe oil saponification and iodine value of both cultivars, but the oiln-saponifiable matter decreased significantly (Table 5).

Peroxide and p-anisidine values and seed oil free fatty acidsncreased in both maize cultivars due to desiccation stress. No sig-ificant difference between the cultivars was observed with respecto oil peroxide value and free fatty acids. However, p-anisidinealue was significantly higher in cv. Agiti-2002 as compared to that Ta

ble

2Se

edm

acro

-min

Cu

ltiv

ar

Aga

iti-

2002

EV-1

098

LSD

at5%

leve

Dat

aar

ep

rese

n

254Q

.Ali,M

.Ashraf/Environm

entalandExperim

entalBotany71 (2011) 249–259

Table 3Seed micro-minerals as influenced by exogenous application of GB when sprayed at the vegetative stage to the water stressed and non-stressed plants of two maize cultivars.

Cultivar Drought treatment GB level (mM) Mn Cu Fe Zn Mo Cr Cd Pb

mg/100 g dry wt. �g/g dry wt.

Agaiti-2002Control

0 0.71 ± 0.04 b 0.18 ± 0.01 b 2.18 ± 0.04 c 2.33 ± 0.07 b 0.33 ± 0.01 a 0.015 ± 0.001 a 0.013 ± 0.001 a

30 0.69 ± 0.02 b 0.18 ± 0.01 b 2.20 ± 0.08 c 2.36 ± 0.08 b 0.35 ± 0.01 a 0.014 ± 0.001 a 0.014 ± 0.002 a

Drought0 0.52 ± 0.03 a 0.16 ± 0.01 a 1.95 ± 0.08 a 2.09 ± 0.07 a 0.28 ± 0.01 a 0.013 ± 0.001 a 0.014 ± 0.001 a

30 0.56 ± 0.03 a 0.16 ± 0.01 a 2.06 ± 0.04 b 2.17 ± 0.08 a 0.30 ± 0.07 a 0.014 ± 0.002 a 0.013 ± 0.001 a

EV-1098Control

0 0.67 ± 0.05 c 0.19 ± 0.01 c 2.17 ± 0.03 c 2.14 ± 0.06 a 0.34 ± 0.01 a 0.013 ± 0.001 a 0.014 ± 0.001 a Was not indetection limit30 0.73 ± 0.06 c 0.19 ± 0.01 c 2.23 ± 0.06 c 2.26 ± 0.07 b 0.37 ± 0.01 a 0.015 ± 0.002 a 0.014 ± 0.001 a

Drought0 0.48 ± 0.04 a 0.18 ± 0.01 b 1.87 ± 0.04 a 1.87 ± 0.04 c 0.28 ± 0.01 a 0.014 ± 0.002 a 0.013 ± 0.001 a

30 0.58 ± 0.04 b 0.17 ± 0.01 a 1.98 ± 0.09 b 1.93 ± 0.10 c 0.30 ± 0.01 a 0.014 ± 0.001 a 0.013 ± 0.001 a

LSD at 5% level 0.062 0.009 0.091 0.11 0.013 0.002 0.001

Data are presented as means ± standard error. Means within a same column sharing the same superscript letters do not differ significantly at the 5% level.

Table 4Composition of seed oil fatty acids as influenced by exogenous application of GB when sprayed at the vegetative stage to the water stressed and non-stressed plants of two maize cultivars.

Cultivar Drought treatment GB level (mM) Pamitic acid Stearic acid Oleic acid Linoleic acid Linoleinic acid Oleic/linoleic Saturatedfatty acids

Un-saturatedfatty acids

Percentages

Agaiti-2002Control

0 10.19 ± 0.74 a 2.06 ± 0.08 ab 31.02 ± 2.21 c 55.33 ± 2.91 c 0.29 ± 0.02 c 0.57 ± 0.07 c 12.24 ± 0.69 a 86.64 ± 0.82 a

30 10.93 ± 1.17 a 1.94 ± 0.09 b 32.76 ± 2.41 c 53.00 ± 1.15 d 0.32 ± 0.01 d 0.62 ± 0.06 c 12.86 ± 1.11 a 86.07 ± 1.40 a

Drought0 10.65 ± 0.43 a 2.13 ± 0.09 a 38.35 ± 1.56 a 46.19 ± 1.74 a 0.36 ± 0.01 a 0.84 ± 0.07 a 12.78 ± 0.52 a 84.89 ± 0.68 a

30 11.52 ± 1.27 a 2.14 ± 0.11 a 44.98 ± 1.67 b 39.08 ± 2.63 b 0.42 ± 0.02 b 1.17 ± 0.12 b 13.66 ± 1.18 a 84.48 ± 0.97 a

EV-1098Control

0 10.28 ± 0.92 a 1.94 ± 0.10 b 32.70 ± 0.82 c 54.30 ± 1.90 c 0.30 ± 0.01 c 0.61 ± 0.04 c 12.22 ± 0.87 b 87.30 ± 1.09 ab

30 10.51 ± 0.47 a 2.10 ± 0.09 a 37.82 ± 1.70 a 48.45 ± 2.31 a 0.35 ± 0.02 a 0.79 ± 0.07 a 12.61 ± 0.57 a 86.62 ± 0.60 a

Drought0 10.45 ± 0.62 a 2.09 ± 0.12 ab 37.62 ± 2.24 a 47.36 ± 2.77 a 0.35 ± 0.02 a 0.81 ± 0.10 a 12.54 ± 0.75 a 85.32 ± 0.51 a

30 11.54 ± 1.49 a 2.08 ± 0.05 ab 45.62 ± 1.29 b 39.13 ± 2.58 b 0.42 ± 0.01 b 1.18 ± 0.10 b 13.62 ± 1.49 a 85.18 ± 1.28 a

LSD at 5% level 1.44 0.14 2.71 3.48 0.022 0.12 1.42 1.44

Data are presented as means ± standard error. Means within a same column sharing the same superscript letters do not differ significantly at the 5% level.

Q. Ali, M. Ashraf / Environmental and Experimental Botany 71 (2011) 249–259 255

ays L.

inobfpesn

wcaHdmtat

tvispAonE

acTuweilid

isAac

Fig. 1. Effects of foliar applied GB on leaf and seed GB content of two maize (Zea m

n cv. EV-1098 under stressed and non-stressed conditions. Exoge-ous application of GB slightly increased the oil p-anisidine valuef both maize cultivars under stressed and non-stress conditions,ut, oil peroxide value and free fatty acids decreased slightly due tooliar application of glycinebetaine. Similarly, like other oil physicalroperties, oil density was also affected due to water stress, but thisffect was observed only in cv. Agaiti-2002, in which it increasedignificantly under drought conditions (Table 5), however, no sig-ificant effect of GB was observed on seed oil density.

Maize kernel oil dienes and trienes, a measure of oil oxidation,ere affected due to water limited conditions, but this effect was

ultivar specific. In cv. Agaiti-2002, the seed oil dienes remained un-ffected, while trienes increased due to imposition of water stress.owever, in cultivar EV-1098 both oil dienes and trienes increasedue to desiccation stress (Table 5). Similarly, the response of bothaize cultivars to foliar applied GB in relation to oil dienes and

rienes was cultivars specific. In cv. Agaiti-2002, both oil dienesnd trienes decreased due to foliar applied GB but the reverse wasrue in cv. EV-1098.

Imposition of water stress significantly increased �-, �-, �- andotal tocopherol contents in the kernel oil of both maize culti-ars. Of different tocopherols, �-tocopherol occurred abundantlyn the seed oil of both maize cultivars under non-stress and watertress conditions. Significantly higher values of different toco-herols were observed in cv. EV-1098 as compared to those in cv.gaiti-2002 (Fig. 2). Foliar application of GB further increased theil tocopherol contents in both maize cultivars under stress andon-stress conditions, but the much increase was observed in cv.V-1098 as compared with that in cv. Agati-2002.

Seed oil phenolics and flavonoids in both maize cultivars werelso affected significantly due to drought stress. The oil phenolicontent decreased significantly while that of flavonoids increased.he cultivars differed significantly only in oil flavonoid contentsnder non-stress conditions, because higher values of oil flavonoidsere observed in cv. EV-1098 than those in cv. Agaiti-2002. How-

ver, exogenous application of GB as foliar spray significantlyncreased the contents of oil phenolics and flavonoids of bothines under stress and non-stress regimes, but much increase asnfluenced by exogenous GB was observed under water deficit con-itions in both cultivars (Fig. 2).

Seed oil antioxidant activity measured as DPPH radical scaveng-

ng activity was reduced in both maize cultivars due to droughttress. This reduction was more in cv. EV-1098 than that in cv.gaiti-2002. Seed oil of cv. Agaiti-2002 showed higher antioxidantctivity as compared with that in cv. EV-1098. Exogenous appli-ation of GB increased the seed oil antioxidant activity of both

) cultivars when grown under water stress and non-stress conditions (n = 4 ± S.E.).

maize cultivars both under stressed and non-stressed conditions.Oil antioxidant activity in both cultivars was positively related tooil phenolic, flavonoids, tocopherol and oil oleic acid contents onlywhen GB was applied as foliar spray under both stressed and non-stressed conditions (Fig. 2).

4. Discussion

Low supply of water during different phases of plant growthparticularly at the reproductive stage is very harmful for seed devel-opment (Boutraa and Sanders, 2001). Grain yield is governed bythe speed and period of grain filling stage which is a post-anthesisphenomenon (Nel, 2001). Availability of assimilates, particularly,carbohydrates can affect this phenomenon substantially. Assim-ilation and partitioning of assimilates during grain developmentare, undoubtedly of vital value (Carvalho et al., 2004). Some earlierreports have shown that unfavorable conditions such as droughtcan alter the seed composition and related qualities (Nel, 2001;Anwar et al., 2006). In view of a number of reports it is now evidentthat glycinebetaine GB can alleviate some of the drought-inducedinhibitory effects on plants (Ashraf and Foolad, 2007; Mahmoodet al., 2009). For example, in sunflower, it was observed that exoge-nously applied GB mitigated the detrimental effects of drought onachene yield, whereas oil content remained unaffected (Iqbal et al.,2005; Hussain et al., 2008). However, it was not assessed whethercomposition and quality of seed and seed oil are affected due toexogenous application of GB as foliar spray under water deficit con-ditions. So, in the present study for the assessment of the effects offoliar-applied GB on seed oil quality, two maize cultivars, Agaiti-2002 and EV-1098 were used. While appraising the relative droughttolerance of a number of available maize cultivars, Ashraf et al.(2007) ranked cv. Agaiti-2002 as drought tolerant and EV-1098 asdrought sensitive.

Proximate analysis of corn kernels in the present study revealedthat imposition of water stress reduced the kernel sugar, oil, proteinand moisture contents with a subsequent increase in the seed fiberand ash contents in both maize cultivars; however, no effect wasobserved on seed starch contents of both maize cultivars. Exoge-nous application of GB ameliorated the adverse effects of waterstress with a subsequent increase in all these seed parameters bothunder stress and under non-stress conditions. Some earlier stud-

ies show that accumulation of GB protects the activity of enzymesand upholds the structure of cellular membranes for their normalperformance under water stress conditions (Sakamoto and Murata,2002; Ashraf and Foolad, 2007) and in the presence of absorbed GB,translocation of assimilates might be improved from source to sink

256 Q. Ali, M. Ashraf / Environmental and Expe

Tab

le5

Phys

icoc

hem

ical

pro

per

ties

ofth

eke

rnel

oila

sin

flu

ence

dby

exog

enou

sap

pli

cati

onof

GB

wh

ensp

raye

dat

the

vege

tati

vest

age

toth

ew

ater

stre

ssed

and

non

-str

esse

dp

lan

tsof

two

mai

zecu

ltiv

ars.

Cu

ltiv

arD

rou

ght

trea

tmen

tG

Ble

vel

(mM

)Sp

onifi

cati

onva

lue

(mg

ofK

OH

/gof

oil)

Un

-sap

onifi

able

mat

ter

(%)

Die

nes

[ε1

cm(�

232

nm

)]Tr

ien

es[ε

1

cm(�

268

nm

)]p-

An

isid

ine

valu

eIo

din

eva

lue

Free

fatt

yac

ids

(mg

ofK

OH

/gof

oil)

Pero

xid

eva

lue

(meq

/kg)

Den

sity

(25

◦ C)

(g/c

m3)

Aga

iti-

2002

Con

trol

019

1.07

±8.

60b

2.11

±0.

13bc

1.87

±0.

008

a1.

79±

0.00

4c

2.08

±0.

07bc

113.

49±

2.60

a1.

35±

0.05

c6.

15±

0.28

a0.

92±

0.00

4a

3020

5.00

±7.

64c

2.03

±0.

07c

1.64

±0.

007

b1.

54±

0.00

4b

2.17

±0.

08c

124.

05±

4.45

b1.

35±

0.02

c5.

39±

0.33

b0.

92±

0.00

3a

Dro

ugh

t0

175.

38±

8.41

a2.

47±

0.11

a1.

87±

0.00

7a

1.87

±0.

004

a2.

33±

0.07

a11

6.60

±4.

13a

1.64

±0.

06a

6.58

±0.

30a

0.92

±0.

004

a

3018

5.31

±6.

49ab

2.24

±0.

06b

1.64

±0.

006

b1.

54±

0.00

6b

2.36

±0.

08a

125.

97±

2.82

b1.

47±

0.10

b6.

16±

0.49

a0.

92±

0.00

2b

EV-1

098

Con

trol

019

6.12

±8.

22b

2.11

±0.

18bc

1.63

±0.

003

b1.

54±

0.00

7b

1.87

±0.

04d

115.

16±

4.22

a1.

41±

0.06

c6.

30±

0.42

a0.

92±

0.00

3a

3019

7.00

±6.

66b

2.05

±0.

06c

1.85

±0.

004

d1.

85±

0.00

6e

1.93

±0.

10d

124.

05±

4.45

b1.

30±

0.06

cd5.

31±

0.41

b0.

92±

0.00

3c

Dro

ugh

t0

178.

55±

6.97

a2.

35±

0.14

ab1.

71±

0.00

6c

1.61

±0.

004

d2.

14±

0.05

bc11

5.34

±2.

97a

1.70

±0.

08a

7.60

±0.

28a

0.93

±0.

001

a

3018

9.73

±9.

32b

2.24

±0.

09b

1.87

±0.

006

a1.

79±

0.00

3c

2.26

±0.

07ac

122.

98±

3.72

b1.

52±

0.04

b6.

37±

0.40

a0.

92±

0.00

2a

LSD

at5%

leve

l11

.76

0.17

11.7

60.

007

0.11

5.60

0.10

0.56

0.00

5

Dat

aar

ep

rese

nte

das

mea

ns±

stan

dar

der

ror.

Mea

ns

wit

hin

asa

me

colu

mn

shar

ing

the

sam

esu

per

scri

pt

lett

ers

do

not

dif

fer

sign

ifica

ntl

yat

the

5%le

vel.

rimental Botany 71 (2011) 249–259

(Nayyar et al., 2005). Similarly, Quan et al. (2004) found that higheraccumulation of GB in GB-accumulating maize plants safeguardedcell membranes and improved the activity of different enzymescompared with those in non-accumulator plants under stress con-ditions. The exogenously applied GB might contribute to restrictcytoplasmic dehydration and maintain turgor in plants subjectedto water deficit conditions (Iqbal et al., 2008) thereby maintaininghigh photosynthetic efficiency. Thus, increased net photosyntheticcapacity could then lead to improved capability of the plant to allo-cate more assimilates to developing fruits and seeds (Mäkelä et al.,1998).

Glycinebetaine has been reported to be taken up readily by leaftissues when applied exogenously (Park et al., 2006). In this study,the authors applied GB exogenously on leaves of tomato plantsand reported that foliar-applied GB was translocated to the meris-tem containing tissues including floral buds and shoot apices. Inanother study, it was demonstrated that GB was translocated toactively growing and expanding portions of plants, and this long-distance translocation of GB was reported to be mediated by thephloem (Mäkelä et al., 1996). Similarly, in the present study, leafand seed GB contents of both maize cultivars increased due to foliarapplication of glycinebetaine that shows that GB might have beentransported from leaf to the seed during the developmental stagefrom the vegetative stage to seed development. This is parallel towhat has been observed in an earlier study (Mäkelä et al., 1996)in which it was shown that large amount of foliar-applied GB wastransported to meristem-containing tissues including floral budsand shoot apices.

The fatty acid composition of corn oil is considered vital in deter-mining nutritional quality and possible uses of oil for industrialapplications. The results for different saturated and un-saturatedfatty acids of corn seed oil analyzed in the present study showthat the contents of oleic acid and linoleinic acid in the kerneloil increased due to water stress with a subsequent decrease inlinoleic acid. This drought-induced increase in oleic acid resultedin an increased oleic/linoleic ratio of kernel oil of the droughtstressed plants, however, saturated fatty acids remained unaf-fected. Exogenous application of GB under stress and non-stressconditions further increased the contents of oil oleic and linoleinicacids with a subsequent decrease in linoleic acid and an increase inoleic/linoleic ratio. However, the total saturated and unsaturatedfatty acids remained unchanged in kernel oil due to water stressand exogenous GB. The changes in oleic/linoleic acid ratio due toexogenous application of GB in water stressed plants as observed inthe present study confirm a direct or indirect protective effect of GBon the enzymes involved in the biosynthesis of oleic and linoleinicacids. These results indicate that it is likely that GB had a protectiveeffect rather than a direct involvement in fatty oil biosynthesis andstorage, which occurs in liposomes or oleosomes in seeds duringseed filling stage (Taize and Zeiger, 2006).

Different abiotic stresses including desiccation stress cause thegeneration of reactive oxygen species (ROS) in different plantorganelles such as peroxisomes, mitochondria, and chloroplasts(Smirnoff, 2005). Various hydrophilic (ascorbic acid and salicylicacid) and lipophilic (tocopherols) compounds play a significant rolein reducing the ROS in various plant parts. Because desiccation isthe final phase of seed development in most crop species (Taizeand Zeiger, 2006), seeds have low water contents and low levels offew water-soluble antioxidants and antioxidant enzymes. This sug-gests that they are inadequately protected from oxidative stress atthis critical seed filling stage. Thus, it is likely that during the seed

filling stage, the amount of lipophilic antioxidant compounds suchas tocopherols is more important in scavenging the ROS therebymaintaining seed oil quality. In the present study, �-, �-, �- andtotal tocopherol contents increased due to foliar application of GBto water stressed plants of both maize cultivars. These results are

Q. Ali, M. Ashraf / Environmental and Experimental Botany 71 (2011) 249–259 257

F idants

swbimeaps

ig. 2. Effects of foliar applied GB on tocopherols, phenolics, flavonoids and antioxtress and non-stress conditions (n = 4 ± S.E.).

imilar to those of Steven and Diane (2002) in which drought stressas reported to cause a 2–3-fold increase in �-tocopherols in soy-

ean. Likewise, drought stress caused an increase in tocopherolsn an Arabidopsis mutant (npq1) (Havaux et al., 2000) and Ros-

arinus officinalis (Munné-Bosch and Alegre, 2000). As describedarlier (Collakova and DellaPenna, 2003) tocopherols are lipophilicntioxidants and they protect the polyunsaturated fatty acids fromeroxidation (Kamal-Eldin and Appelqvist, 1996). In the presenttudy, a positive association has been found between un-saturated

activity of seed oil of two maize (Zea mays L.) cultivars when grown under water

fatty acids such as oleic, linoleic and linoleinic acids and each of�-, �-, �- and total tocopherol contents. These findings are analo-gous to those of Kriese et al. (2004) who found a positive correlationbetween some fatty acids, and �-tocopherols and total tocopherols.

Unfavorable conditions, especially drought, might alter the seedcomposition and its quality (Nel, 2001). In the present study, it wasobserved that water stress caused a marked reduction in maizekernel macro-minerals (K, Mg, P, N and Ca) and micro-minerals(Mn, Cu, Fe, Zn and Cr) except Mo, Cd and Pb that remained unaf-

2 Expe

ftdstcitwer

epfiGmmta(i

ipwCdoetctptTtac

tKihmpflisdew

ataicnbi2ip

a

58 Q. Ali, M. Ashraf / Environmental and

ected. Exogenous application of GB caused an increase in most ofhe seed minerals analyzed under both stress and non-stress con-itions. This increase in mineral accumulation in seeds of droughttressed plants due to foliar application of GB could have been dueo the reason that under water deficit conditions GB exerts benefi-ial effects on translocation and redistribution of different nutrientsn plants (Nayyar et al., 2005). Furthermore, in the present study,he increase in kernel nutrient contents was positively correlatedith kernel protein contents. These findings can be related to the

arlier study of Welch and Graham (2004) who reported similaresults about maize kernel nutrient contents.

The results of different physico-chemical parameters of thextracted maize kernel oils from drought stressed and well irrigatedlants show that drought caused a substantial decline in oil saponi-cation value and increased the un-saponifiable matter. ExogenousB improved the quality of oil by decreasing the un-saponifiableatter and also increased oil saponification and iodine values, theeasure of unsaturation. In the present work, seed oil unsatura-

ion increased in terms of increased oil linoleinic acid. These resultsre similar to those of some earlier reports with Moringa oleiferaAnwar et al., 2006) and sunflower (Ali et al., 2009). They found anncrease in oil iodine value with an increase in oil un-saturation.

Seed oil total phenolic contents are indicative of the total antiox-dative activity (Kumar et al., 2009) due to the availability of thehenolic hydrogens (as hydrogen-donating radical scavengers),hich predict their antioxidant activity (Rice-Evans et al., 1996).ereal grains have the highest level of phenolics and antioxi-ant activity (Dykes and Rooney, 2000). In some earlier studiesn olive oil (Greven et al., 2009) and rape-seed oil (Bouchereaut al., 1996), it was observed that low rate of irrigation was foundo be significant in decreasing the concentration of oil phenolicompounds. All these reports support our results for phenolic con-ents that shortage of irrigation decreased the concentration ofhenolic compounds, but GB applied exogenously at the vegeta-ive stage enhanced the seed oil phenolics of both maize cultivars.his GB-induced increase in oil phenolic contents can be related tohe findings of Karjalainen et al. (2002), who reported that foliar-pplied glycinebetaine enhanced the levels of several phenolicompounds in strawberry under water deficit conditions.

In an earlier study it was observed that total flavonoid con-ents increased throughout the seed development in two soybeanorean cultivars (Kumar et al., 2009). Similarly, the drought-

nduced increase in flavonoid contents in maize kernel oil mayave been due to their enhanced synthesis during seed develop-ent and seed maturity. Kumar et al. (2009) reported that seeds

icked at early reproductive stages possessed low concentrations ofavonoids as compared to those picked at maturity. In the present

nvestigation, the increase in flavonoids in kernel oil of droughttressed plants due to exogenous application of GB could have beenue to the role of GB in delaying the maturity of plants (Agbomat al., 1997), that will delay the seed maturity and ultimately thereould be more accumulation of flavonoids in the seeds.

The DPPH radical scavenging activity, a measure of oil totalntioxidant activity, in maize kernel oil decreased significantly dueo water stress, but increased due to GB application under stressnd non-stress conditions. Furthermore, this increase in oil antiox-dant activity due to foliar spray of GB under stress and non-stressonditions was positively correlated with the contents of oil phe-olics, flavonoids and tocopherols. This strong positive correlationetween total phenolics and antioxidant activity was also observed

n cereals (Dykes and Rooney, 2000) and soybean (Kumar et al.,

009), which suggests that this increase in oil antioxidant activity

s contributed by the presence of high amount of phenolic com-ounds.

From the results of the present comprehensive maize kernelnd kernel oil analyses, it could be concluded that drought might

rimental Botany 71 (2011) 249–259

be considered as one of the most visible factors which affectedthe chemical composition of maize kernel and kernel oils, but theexogenous application of GB considerably counteracted the harm-ful effects of drought on seed and seed oil composition. Oil yield,degree of un-saturation, fatty acid composition, oxidative stability,oil phenolics, and flavonoids are the parameters which are mostvulnerable to drought and were positively affected due to exoge-nous application of GB.

Acknowledgements

The authors gratefully acknowledge the funding from thePakistan Academy of Sciences (PAS) (Grant No. ACS/HEC/2006/PAS/2989). The results presented in this paper are a part of Ph.D.studies of Mr. Qasim Ali.

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