Ma

Sa

Gb

c

a

ARA

KMAEFRS

1

feiua1(bc2swriiep

0h

Postharvest Biology and Technology 75 (2013) 37–44

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

jou rna l h omepa g e: www.elsev ier .com/ locate /postharvbio

ode of action of abscisic acid in triggering ethylene biosynthesisnd softening during ripening in mango fruit

akimin S. Zaharaha, Zora Singha,∗, Gregory M. Symonsb, James B. Reidc

Curtin Horticulture Research Laboratory, Department of Environment and Agriculture, School of Science, and International Institute of Agri-Food Security (IIAFS), Curtin University,PO Box U1987, Perth 6845, WA, AustraliaAgricultural Research and Development, Tasmanian Alkaloids, PO Box 130, Westbury, Tasmania 7303, AustraliaSchool of Plant Science, University of Tasmania, Locked Bag 55, GPO Hobart, Tasmania 7001, Australia

r t i c l e i n f o

rticle history:eceived 22 May 2012ccepted 23 July 2012

eywords:ango

bscisic acid

a b s t r a c t

The role of abscisic acid (ABA) in triggering ethylene biosynthesis and ripening of mango fruitwas investigated by applying ABA [S-(+)-cis,trans-abscisic acid] and an inhibitor of its biosynthe-sis [nordihydroguaiaretic acid (NDGA)]. Application of 1 mM ABA accelerated ethylene biosynthesisthrough promoting the activities of ethylene biosynthesis enzymes (1-aminocyclopropane-1-carboxylicacid synthase, ACS; 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) and accumulation of1-aminocyclopropane-1-carboxylic acid (ACC), enhanced fruit softening and activity of endo-

thyleneruit softening enzymesipeningugars and organic acids

polygalacturonase and reduced pectin esterase activity in the pulp. The activities of ethylene biosynthesisand softening enzymes were significantly delayed and/or suppressed in the pulp of NDGA-treated fruit.The ABA-treated fruit had higher total sugars and sucrose as well as degradation of total organic acids,and citric and fumaric acids compared with NDGA treatment. These results suggest that ABA is involvedin regulating mango fruit ripening and its effects are, at least in part, mediated by changes in ethyleneproduction.

. Introduction

Abscisic acid (ABA) has been reported to play a crucial role inruit maturation and senescence (Giovannoni, 2001, 2004; Rodrigot al., 2003; Zhang et al., 2009a,b). Lower levels of endogenous ABAn unripe fruit and its substantial accumulation during fruit mat-ration suggest that ABA plays a key role in modulating ripeningnd senescence in climacteric fruit such as peach (Rudnicki et al.,968; Zhang et al., 2009a), avocado (Adato et al., 1976), tomatoButa and Spaulding, 1994; Sheng et al., 2008; Zhang et al., 2009b),anana (Lohani et al., 2004), apple (Buesa et al., 1994), and non-limacteric fruit including grape (Inaba et al., 1976; Zhang et al.,009a) and orange (Kojima, 1996). Additionally, the de-greeningtage of ABA-deficient orange mutants commenced later than in theild type consistent with a crucial role for ABA in maturation and

ipening of orange fruit (Rodrigo et al., 2003). Some earlier reportsndicated that endogenous levels of ABA increased towards harvest

n the fruit skin and pulp of ‘Nam Dok Mai’, ‘Nang Klangwan’ (Kondot al., 2004) and ‘Alphonso’ (Murti and Upreti, 1995) mangoes. Ourrevious study showed that the accumulation of endogenous ABA

∗ Corresponding author. Tel.: +61 8 9266 3138; fax: +61 8 9266 3063.E-mail address: z.singh@curtin.edu.au (Z. Singh).

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2012.07.009

© 2012 Elsevier B.V. All rights reserved.

during the climacteric rise stage might initiate climacteric ethyleneproduction during ripening of ‘Kensington Pride’ mango fruit(Zaharah et al., 2012).

Further, ABA (10−6 M) application has been reported to has-ten the ripening process and induce some structural changes in‘Alphonso’ and ‘Langra’ mangoes (Palejwala et al., 1988; Parikhet al., 1990). Application of ABA or high endogenous levels havealso been reported to stimulate ethylene production, and promoteripening in other climacteric fruit such as tomato (Sheng et al.,2008; Zhang et al., 2009b; Zhu et al., 2003), peach (Zhang et al.,2009a) and non-climacteric grape berries (Deytieux et al., 2005;Zhang et al., 2009a). Zhang et al. (2009b) reported that the applica-tion of 100 �M ABA up-regulated the expression of the Le-ACO1 andLe-ACS2 genes, which encode 1-aminocyclopropane-1-carboxylicacid oxidase (ACO, EC 1.14.17.4) and 1-aminocyclopropane-1-carboxylic acid synthase (ACS, EC 4.4.1.14), and consequentlyincreased ethylene production and accelerated tomato fruit ripen-ing. Further, the application of inhibitors of ABA biosynthesis[fluridone or nordihydroguaiaretic acid (NDGA)], inhibited theexpression of both Le-ACO1 and Le-ACS2 genes, and delayedtomato fruit ripening (Zhang et al., 2009b). The accumulation

of ABA (3000 ng g−1 fresh weight) during mango fruit ripeningalso appears to induce climacteric ethylene production whichmay be modulating its ripening process (Zaharah et al., 2012).However, the mechanism by which ABA regulates ethylene

3 ology

bd

t3e�bAfcteameidtafeis

2

2

o2Fp

ttfeabga

2

iS(LPaAtuUortAPoit

8 S.S. Zaharah et al. / Postharvest Bi

iosynthesis and mango fruit ripening has not been examined inetail.

Mango fruit softening is associated with increased activi-ies of polygalacturonase (PG), exo-polygalacturonase (exo-PG; EC.2.1.67), endo-polygalacturonase (endo-PG; EC 3.2.1.15), pectinsterase (PE; EC 3.1.1.11), pectin lyase (PL; EC 4.2.2.2) and endo-1,4--d-glucanase (EGase or cellulase; EC 3.1.1.4), which are initiatedy ethylene (Chourasia et al., 2006, 2008; Singh and Singh, 2011).n endo-�-1,4-glucanase homologue, MiCel1 from mango shows

ruit-specific and ripening-related expression which was positivelyorrelated with an increase in EGase activity particularly duringhe later stages of ripening (Chourasia et al., 2008). Previously, thexogenous application of ABA has also been reported to increase thectivity of PG, but has inconsistent effects on the activity of pectinethyl esterase (PME) during ‘Zihua’ mango fruit ripening (Zhou

t al., 1996). Overall the research on the effects of applied ABA andts inhibitors in regulating the activities of fruit softening enzymesuring mango ripening is sporadic and inconclusive. Therefore,he aim of the present study was to investigate how applied ABAnd its biosynthesis inhibitor regulate ethylene biosynthesis andruit softening, including the activities of ethylene biosyntheticnzymes such as ACS and ACO, ACC content, as well as fruit soften-ng enzymes including PE, exo-PG, endo-PG, EGase and the levels ofugars and organic acids in the pulp of the fruit during ripening.

. Materials and methods

.1. Fruit

Mangoes (Mangifera indica L. cv. ‘Kensington Pride’) werebtained from a commercial orchard located at Dongara (latitude,9.26◦15′S and longitude, 114.93◦55′E), Western Australia on 26thebruary, 2009. The fruit were firm (75.6 ± 6.71 N) and had a res-iration rate of 2.68 ± 0.40 mmol CO2 kg−1 h−1.

All the fruit were de-sapped by allowing the sap to exude fromhe end of the stalk by physical inversion on the de-sapping trayso avoid sap burn injury over the skin. The fruit were treated withungicide (Sportak 0.55 mL L−1 with Prochloraz as an active ingredi-nt, sourced from Bayer CropScience, Kwinana, Western Australia),ir-dried, packed in soft-board trays, and transported to Perth, WA,y a refrigerated truck. Fruit of uniform size and maturity (hard,reen skin and light cream pulp colour), free from visual blemishesnd symptoms of diseases were used for the experiments.

.2. Treatments and experimental design

Fruit were dipped for 5 min in an aqueous solution contain-ng 1.0 mM S-(+)-cis,trans-abscisic acid (ABA; Syntake Chemical,hanghai, China) or 0.2 mM nordihydroguaiaretic acid [NDGA; 4,4′-2,3-dimethyltetramethylene)dipyrocatechol] (Sigma–Aldrich Pty.td., Castle Hill, Australia) and 0.05% of ‘Tween 20’ (Sigma–Aldrichty. Ltd.) as a surfactant. The concentrations of applied ABAnd NDGA were selected based on our previous experiments.ir-dried fruit were packed in soft board trays, and allowed

o ripen at ambient temperature (21 ± 1 ◦C and 56 ± 11.1% RH)ntil the eating soft stage (subjective firmness rating score = 4).ntreated fruit were dipped in an aqueous solution containingnly 0.05% of ‘Tween 20’. Ethylene production and respirationate were determined daily during fruit ripening. The ACC con-ent, the activities of ethylene biosynthesis enzymes (ACS andCO) and fruit softening enzymes including exo-PG, endo-PG,

E and EGase, and the levels of individual sugars as well asrganic acids were determined from the pulp tissues at two-dayntervals during the ripening period. To determine the activi-ies of ethylene biosynthesis enzymes, fruit softening enzyme

and Technology 75 (2013) 37–44

s, levels of total and individual sugars and organic acids, pulp tissuesamples were taken and immediately immersed in liquid nitro-gen (−196 ◦C) and stored at −80 ◦C. Softness and visual skin colourof individual fruit were also recorded at two-day intervals duringthe ripening period. The experiment was laid out as a completelyrandomized design, including treatments and ripening period as afactor. Ten fruit were used as an experimental unit and replicatedthree times.

2.3. Determination of ethylene production

The rates of ethylene production in mango fruit during ripen-ing were determined according to Zaharah and Singh (2011b). Fruitwere sealed in a 1 L air-tight jar, fitted with a rubber septum, for 1 hat 20 ± 1 ◦C. A headspace gas sample (1.0 mL) was then injected intoa gas chromatograph to estimate the rate of ethylene production.The concentration of ethylene produced by fruit was quantifiedusing a gas chromatograph (6890N Network GC System; AgilentTechnology, Palo Alto, CA, USA) fitted with a 2 m-long stainless steelcolumn filled with 80/100 mesh size Porapaq-Q (3.18 mm inter-nal diameter; Supelco, Bellefonte, PA, USA) and a flame ionizationdetector (FID). Ethylene in the gas was identified by comparing itsretention time and co-chromatography with authentic standards(0.9 ± 0.1 �L L−1 of ethylene in nitrogen) certified as �-standard andobtained from BOC Gases, Australia Ltd., Perth, Australia.

2.4. Determination of ACC in pulp tissue

The ACC content from pulp tissue was determined by follow-ing the method of Khan and Singh (2007). The pulp of fruit tissue(2.0 g) was homogenized with 10.0 mL dH2O and 300 mg whitequartz sand using a glass pestle and mortar at 2 ± 1 ◦C tempera-ture, followed by centrifugation at 10,000 × g for 20 min at 4 ◦C.The supernatant (0.5 mL) was mixed with 0.1 mL 50 mM HgCl2 andwith or without 0.1 mL 100 �M ACC, and the final volume 1.8 mLwas made with dH2O. The reaction tubes were then sealed with arubber septum (SubaSeal®, Sigma–Aldrich Co., St. Louis, USA). Thereaction tubes were placed on ice prior to the addition of 0.2 mL of5% NaOCl (saturated with NaOH, 2:1, v/v) and then stirred for 5 s. Allthe samples were kept on ice for 24 min, then stirred again before a1.0 mL gas sample was taken from the headspace and injected intoa GC for an estimation of ACC activity. Ethylene production was cal-culated from the peak area obtained from the tissue extract (0.5 mL)in comparison with the peak obtained for the sample without inter-nal ACC (0.1 mL) as the standard. The ACC content was expressedas pmol g−1 FW.

2.5. Determination of ACS activity in pulp tissue

The ACS activity was analysed from pulp tissue as describedby Zaharah and Singh (2011a). Pulp (2.5 g) was homogenized in5 mL of potassium phosphate buffer (400 �M, pH 8.5) containing0.01 mM pyridoxal-5-phosphate (PLP), 1 mM Na2EDTA and 0.5%2-mercaptoethanol with 200 mg of white quartz sand (−50 + 70mess, Sigma–Aldrich, Australia) at 4 ± 1 ◦C using a mortar andpestle. The extract was centrifuged at 12,000 × g for 30 min at4 ◦C. The supernatant was discarded and the pellet was resus-pended in 5 mL of 20 mM potassium phosphate (pH 8.5) containing1 mM Na2EDTA, 1 mM 2-mercaptoethanol, 10 �M PLP and 30%glycerol and incubated for 30 min at 4 ± 1 ◦C before centrifugationat 12,000 × g for 30 min. Two mL of centrifugate (crude enzyme)extract was mixed with 1 mL of 200 �M S-adenosylmethionine

(SAM), 10 �M PLP, 50 mM Hepes–KOH buffer (pH 8.5) and 1%Triton X-100 in a 10 mL glass tube with total reaction volumeof 3 mL, sealed with a rubber septum prior to incubation for30 min at 30 ◦C and then cooled on ice. A 100 �L volume of

ology

5vtsfa

2

ahts1ecm1twTwta

2

ep3Z

2p

iSwpso1oTP

c04(o

(iba1b2Law

S.S. Zaharah et al. / Postharvest Bi

0 mM HgCl2 and 300 �L of 5% NaOCl and saturated NaOH (2:1,/v) were injected into a reaction tube using a syringe. The reac-ion tube was incubated on ice for 2.5 min, and 1 mL of gasample was taken from the headspace and injected into the GCor estimation of ethylene as described earlier. The ACS enzymectivity was expressed as pmol ACC mg protein−1 h−1.

.6. Determination of ACO activity in pulp tissue

ACO activity was determined following the method of Gornynd Kader (1996) with some modifications. Frozen pulp (2.5 g) wasomogenized in a pestle and mortar in 5 mL extraction buffer con-aining 0.1 M Tris–HCl (pH 7.2), 10% (w/v) glycerol and 30 mModium ascorbate, plus 5% PVPP. The extract was centrifuged at2,000 × g for 30 min at 4 ◦C. The supernatant was used for thenzyme assay. The enzyme was assayed in a 2 mL reaction mixtureontaining 1.8 mL of the supernatant and 0.2 mL standard reactionixture containing 0.01 M Tris–HCl buffer (pH 7.2), 10% glycerol,

mM ACC, 0.02 mM FeSO4, 5 mM sodium ascorbate, 1 mM dithio-hreitol (DTT) and 20 mM sodium bicarbonate. The reaction tubeas sealed with a rubber septum and incubated at 30 ◦C for 1 h.

he reaction tube was stirred for 5 s before 1 mL of headspace gasas withdrawn with a syringe and injected into the GC for estima-

ion of ethylene production as described earlier. The ACO enzymectivity was expressed as nmol C2H4 mg protein−1 h−1.

.7. Fruit softness

Subjective firmness of individual fruit in each replicate wasvaluated daily by hand (nondestructive) during the ripeningeriod using a subjective 5 point rating scale (1 = hard, 2 = sprung,

= slightly soft, 4 = eating soft, and 5 = over soft) as described byaharah and Singh (2011b).

.8. Determination of the activities of fruit softening enzymes inulp tissue

Activities of exo-PG, endo-PG, PE and EGase were determinedn pulp tissues following the method as described by Khan andingh (2007) with some modification. The fruit pulp tissue (13.0 g)as homogenized with 13.0 mL cold solution, containing 12%olyethylene-glycol and 0.2% sodium bisulphite (NaHSO3). Theupernatant was immediately stored at −80 ◦C for determinationf protein content. Following centrifugation at 4 ◦C for 40 min at5,000 × g, the pellet was washed with a 13.0 mL aqueous solutionf NaHSO3 (0.2%) and re-centrifuged at 4 ◦C for 40 min at 15,000 × g.he pellet was stored at −80 ◦C for determination of exo- and endo-G, PE and EGase.

The pellet was incubated on a shaker at 4 ◦C for 1 h in 15.0 mLold 50 mM sodium acetate (CH3COONa) buffer (pH 5) containing.5 M NaCl. Following the centrifugation at 15,000 × g for 15 min at◦C, the supernatant was diluted 1:1 with 50 mM CH3COONa buffer

pH 5) and was used as a crude enzyme extract for determinationf exo- and endo-PG levels.

To determine the activity of exo-PG, the enzyme extract0.15 mL) was mixed with 0.15 mL of (0.5%) polygalacturonic acidn 50 mM CH3COONa buffer (pH 4.4), and the content was incu-ated at 30 ◦C for 18 h. To determine the amount of galacturoniccid released, 2.0 mL of 0.1 M borate buffer (pH 9.0) and 0.3 mL of% cyanoacetamide were added to the reaction mixture and thenoiled for 10 min. The absorbance of the cold solution was read at

74 nm by using a UV–vis spectrophotometer (Model 6405, Jenwaytd., Felsted, Dunmow, Essex, England) and was calculated against

standard curve of d-galacturonic acid. The exo-PG enzyme activityas expressed as �g galacturonic acid mg protein−1 h−1.

and Technology 75 (2013) 37–44 39

The activity of endo-PG was determined by measuring theviscosity using a Cannon-Fenske viscometer (Size 50, CannonInstrument Company, PA, USA). Three mL of enzyme extract asmentioned previously was mixed with a cold solution containing4.5 mL of 2% polygalacturonic acid in a 50 mM CH3COONa buffer(pH 4.4). The viscosity was measured immediately after the reac-tion mixture was incubated for 18 h at 30 ◦C. The endo-PG enzymeactivity was expressed as �viscosity (mg protein−1 h−1).

To determine the activity of PE, the pellet as described earlierwas re-suspended in 15 mL cold solution containing NaCl (7.5%,w/v) + EDTA (0.75%, w/v) at pH 6.5 and incubated at 4 ◦C for 10 min.Following centrifugation at 15,000 × g for 15 min, a 20 mL of cit-rus pectin solution (1%, w/v) at pH 7.5 was mixed with 5 mLenzyme extract solution. The reaction mixture was titrated against0.01 N NaOH and maintained at pH 7.4, while incubating at 30 ◦Cfor 10 min. During the titration and incubation time, the reac-tion mixtures were continuously and slowly shaken by hand. Thetotal amount of 0.01 N NaOH to maintain pH 7.4 was used to cal-culate the PE activity. The PE enzyme activity was expressed asmM NaOH mg protein−1 h−1.

Determination of EGase activity was performed by stirring theextraction pellet with 15.0 mL of 0.1 M citrate–phosphate buffer(pH 6.0) containing 1.0 M of NaCl for 1 h. Following centrifugation at15,000 × g for 15 min, 3.0 mL of supernatant was mixed with 6.0 mLof 0.2% carboxymethyl cellulose in citrate–phosphate buffer (pH6.0). The viscosity changes were measured immediately and after18 h incubation at 30 ◦C using a viscosity meter. The EGase enzymeactivity was expressed as �viscosity (mg protein−1 h−1).

2.9. Protein determination

Protein content in fruit pulp was determined followingthe Bradford (1976) method using bovine serum albumin(Sigma–Aldrich Pty. Ltd., Castle Hill, Australia) as a standard.

2.10. Determination of individual sugars and organic acids

To determine the individual sugar and organic acid levels, frozenpulp (15 g each) was pooled from the inner and outer mesocarpfrom the central part of ten fully ripe fruit. The frozen pulp sampleswere thawed and homogenized in 25 mL Milli-Q water (Millipore,Bedford, MA, USA), using a mini-mixer (DIAX 900, Heidolph Co.,Ltd., Schwabach, Germany) for 1 min to extract individual sug-ars and organic acids. A subsample (equivalent to 1 g pulp) wascentrifuged (Eppendorf 5810 R, Hamburg, Germany) at 10,000 × gfor 15 min at 15 ◦C, the supernatant collected and diluted withMilli-Q water to make up the volume to 50 mL. It was filteredthrough a syringe using a 0.2 �m nylon filter (Alltech AssociatesLtd., Baulkham Hills, Australia) and loaded into a 1.0 mL glass vial.The concentration of sugars and acids were quantified using areverse-phase high performance liquid chromatography system(RP-HPLC; Waters, Milford, MA, USA) and conditions of analy-sis have been reported in detail earlier by Zaharah and Singh(2011b). The concentrations of sucrose, glucose, fructose, and citricacid were expressed in g 100 g−1 fresh weight (FW), whilst tar-taric, malic, shikimic, and fumaric acids content were expressedin mg 100 g−1 FW.

2.11. Statistical analysis

The experimental data were subjected to two-way (treat-ment × ripening period) analysis of variance (ANOVA) using SAS

(release 9.1.3, SAS Institute Inc., Cary, NC, USA). Fisher’s least sig-nificant differences (LSD) were calculated following a significant(P ≤ 0.05) F-test. All the assumptions of ANOVA were checked toensure validity of statistical analysis. Pearson correlations were

40 S.S. Zaharah et al. / Postharvest Biology and Technology 75 (2013) 37–44

Ripening period (d)

109876543210

Eth

yle

ne

(nm

ol

C2H

4 k

g-1

h-1

)

0

2

4

6

8

10

Control

ABA

NDGA

Fig. 1. Changes in ethylene as influenced by ABA and its biosynthesis inhibitor(NDGA) treatments (T) during mango ripening (RP) at ambient temperature. Verticalb(

peif

3

3

ofmcmfc

3r

aa0pspd

pwi2tf

ttcaahwlA

(C)

Ripening period (d)

1086420

AC

O

(C2H

4n

mo

l m

g p

rote

in-1

h-1

0.0

0.5

1.0

1.5

2.0

(B)

AC

S

(pm

ol

AC

C m

g p

rote

in-1

h-1

))

0

20

40

60

80

(A)

AC

C

(pm

ol

g-1

FW

)

0.0

0.4

0.8

1.2

1.6Control

ABA

NDGA

Fig. 2. ACC content (A), ACS (B) and ACO enzyme activities (C) in pulp tissuesas influenced by ABA and its biosynthesis inhibitor (NDGA) treatments (T) dur-

ars represent SE, and invisible when the values are smaller than the symbol. LSDP ≤ 0.05) for T = 0.31, RP = 0.59, T × RP = 0.51.

erformed between ethylene production and ethylene biosynthesisnzymes, ethylene production and fruit softening enzyme activ-ties, fruit firmness and ethylene biosynthesis, fruit firmness andruit softening enzyme activities using the same program.

. Results

.1. Ethylene production

Fruit treated with 1.0 mM ABA exhibited a climacteric peakf ethylene production that was 36% higher than in the controlruit (Fig. 1). The NDGA-treated (0.2 mM) fruit showed a cli-

acteric peak of ethylene production that was 43% lower thanontrol fruit (Fig. 1). When averaged over the ripening period, theean of ethylene production was 9% higher in the ABA-treated

ruit and 30% lower in NDGA-treated fruit, compared with theontrol.

.2. ACC content and activities of ACO and ACS during fruitipening

The ABA-treated fruit exhibited higher ACC contents (1.28nd 1.06 pmol g−1 FW, respectively), than NDGA-treated (0.54nd 0.30 pmol g−1 FW respectively) and control fruit (0.96 and.21 pmol g−1 FW respectively) on days 2 and 4 of the ripeningeriod (Fig. 2A). The application of NDGA significantly (P ≤ 0.05)uppressed the levels of ACC in the pulp on day 2 of the ripeningeriod as compared to control, but this effect had diminished byay 4 (Fig. 2A).

The ABA-treated fruit exhibited higher activities of ACS in theulp tissue on days 4, 6 and 10 of the ripening period comparedith the control (Fig. 2B). By contrast, ACS activity was suppressed

n NDGA-treated fruit as compared with the control fruit on day of the ripening period. However, on day 4 to day 10 of ripeninghe ACS activity did not differ significantly between NDGA-treatedruit and the control (Fig. 2B).

ACO activity was reduced in NDGA-treated fruit during day 2o day 10 of ripening as compared with the control (Fig. 2C). ABA-reated fruit exhibited higher ACO activity as compared with theontrol fruit on days 2, 4, 6 and 10 of ripening (Fig. 2C). Whenveraged over ripening days, the mean of ACC content, and the

ctivities of the ACS and ACO enzymes in the pulp tissues wereigher (55, 58 and 26%, respectively) in ABA-treated fruit comparedith the control. There were significant (P ≤ 0.001) positive corre-

ations (r = 0.74, r = 0.62 and r = 0.75) between ACC content, ACS andCO activities and ethylene production, respectively.

ing mango ripening (RP) at ambient temperature. Vertical bars represent SE, andinvisible when the values are smaller than the symbol. LSD (P ≤ 0.05) for ACC con-tent: T = 0.1, RP = 0.16, T × RP = 0.28; ACS: T = 7.19, RP = 10.17, T × RP = 17.62 and ACO:T = 0.11, RP = 0.14, T × RP = 0.26.

3.3. Fruit softness and the activities of fruit softening enzymes infruit pulp

The exogenous application of ABA promoted fruit softening onday 2 of ripening compared with the control (Fig. 3A). The NDGA-treated fruit exhibited delayed fruit softening during the ripeningperiod, compared with the control and ABA-treated fruit. Averagedover all treatments used, the mean fruit softness was 7% higher inABA-treated fruit, and 17% lower in NDGA-treated fruit, comparedwith the control.

ABA or NDGA treatment did not significantly affect exo-PG activ-ity in pulp during the fruit ripening period compared with controlfruit (data not shown). When averaged over the ripening period, themean activity of exo-PG increased in all treatments during ripeningfrom day 0 to day 4.

In contrast to exo-PG, the ABA treatment did increase the activ-ity of endo-PG in the pulp (by 81% compared to day 0) on day 2 ofthe ripening period, resulting in a peak in activity of this enzymethat was two days earlier than the peak seen in control and NDGA-treated fruit (Fig. 3B). After this peak, the activity of endo-PG in thepulp of ABA-treated fruit declined rapidly and was in fact lowerthan the control level by day 4 to day 8. In contrast to ABA, NDGA-treatment reduced the activity of endo-PG in the pulp from day 2 today 6 of ripening compared with the control (Fig. 3B). Averaged ofall over treatments used, the mean endo-PG activity was 13% higher

in ABA-treated fruit than in the control, whilst NDGA-treated fruitshowed 16% lower activity as compared with the control. Therewas a significant (P ≤ 0.01) positive linear relationship (r = 0.46)between the activity of endo-PG and fruit softening during ripening.

S.S. Zaharah et al. / Postharvest Biology and Technology 75 (2013) 37–44 41

(C)

Ripening period (d)

86420

0.05

0.10

0.15

0.20

0.25

(B)

0

1

2

3

4

5

6

(A)

Fru

it S

oft

ness

(Sco

re 1

-5)

0

1

2

3

4

5

6

Control

ABA

NDGA

Endo

-PG

[∆ v

isco

sity

(m

g p

rote

in-1

h-1

)]

PE

[mM

NaO

H m

g p

rote

in-1

h-1

]

Fig. 3. Fruit softening as well as the changes in endo-PG [�viscosity(mg protein−1 h−1)] and PE (mM NaOH mg protein−1 h−1) in the pulp as influ-enced by ABA and its biosynthesis inhibitor (NDGA) treatments (T) during mangoripening (RP) at ambient temperature. Vertical bars represent SE and invisiblewfT

rTptcdaW(artP

twid

3

at

(B)

Ripening perio d (d )

1086420

Su

cro

se (

g 1

00

g-1

FW

)

8

12

16

20

(A)

To

tal

sug

ar

(g 1

00

g-1

FW

)

12

16

20

24

Control

ABA

NDGA

Fig. 4. Changes in the concentration of total sugar (A) and sucrose (B) (g 100 g−1 FW)as influenced by ABA and its biosynthesis inhibitor (NDGA) treatments (T)during the fruit ripening period (RP) at ambient temperature. Vertical barsrepresent SE. LSD (P ≤ 0.05) for the concentration of total sugar: T = 1.83,RP = 2.58, T × RP = NS and the concentration of sucrose: T = 0.48, RP = 2.10, T ×RP = NS.

hen the values are smaller than the symbol. LSD (P ≤ 0.05) and NS = not significantor fruit softening: T = 0.11, RP = 0.14, T × RP = 0.24; endo-PG: T = 0.51, RP = 2.72,

× RP = 1.24; PE: T = 0.01, RP = 0.02, T × RP = NS.

The activity of PE in the pulp tissue declined over theipening period irrespective of the treatment applied (Fig. 3C).he ABA-treated fruit exhibited lower PE activity in theulp (10%, 31%, 25% and 22%) on days 2, 4, 6 and 8 ofhe ripening period, respectively, when compared with theontrol. The NDGA-treated fruit exhibited 24% higher PE activity onay 4 of ripening compared with the control fruit, but did not showny difference to the control on day 6 to day 8 of ripening (Fig. 3C).hen averaged over all treatments, the mean PE activity was lower

0.14 mM NaOH mg protein−1 h−1) in ABA-treated than in controlnd NDGA-treated fruit (0.17 and 0.18 mM NaOH mg protein−1 h−1,espectively). There was a significant (P ≤ 0.001) negative correla-ion (r = −0.82) between mango fruit softening and the activity ofE during the ripening period.

The activity of EGase in pulp was not affected by ABA or NDGAreatment during fruit ripening period (data not shown). However,hen averaged over the ripening period, the mean activity of EGase

n all treatments increased on days 2 and 4, stabilised on day 6 andeclined at the ripe stage.

.4. Changes in concentration of total and individual sugars

ABA-treatment increased the accumulation of both total sug-rs and sucrose on day 2 of the ripening period compared withhe control fruit (Fig. 4A and B). In contrast, NDGA-treatment

delayed the accumulation of total sugars and sucrose comparedto both ABA-treated and untreated fruit. Despite this delay, theconcentration of total sugars and sucrose in the NDGA-treatedfruit eventually surpassed that in the ABA-treated fruit, but stilldid not reach the level recorded in control fruit. Neither ABA norNDGA treatment had an affect on the concentration of glucoseand fructose during fruit ripening period (data not shown). Whenaveraged over the ripening period, the mean concentrations ofglucose and fructose in all treatments increased up to day 6 ofthe ripening period and then declined with the advancement ofripening.

3.5. Changes in concentration of total and individual organicacids

The concentrations of total organic acids, citric acid, malic acid,shikimic acid and fumaric acid in the pulp tissue declined withthe advancement of fruit ripening irrespective of the treatmentapplied (Fig. 5A–C and some data not shown). The exogenous appli-cation of ABA and NDGA only had an effect on the concentrationsof total organic acids and citric acid (Fig. 5A and B). The concentra-tions of total organic acids and citric acid in the pulp declined ata faster rate from days 2 to 4 of ripening in ABA-treated fruit thanin the control. In NDGA-treated fruit, the concentrations of totalorganic acids and citric acid in the pulp declined at a slower rateuntil day 4 of ripening than in the control. At the ripe stage (days8 and 10 of ripening), the concentrations of total organic acids andcitric acid did not differ among treatments. The concentrations oftrace organic acids (tartaric acid, malic acid, and shikimic acid) didnot vary among treatments during the ripening period (data not

shown).

42 S.S. Zaharah et al. / Postharvest Biology

(C)

Ripening period (d)

1086420

Fu

ma

ric

acid

(m

g 1

00

g-1

FW

)

0.00

0.05

0.10

0.15

0.20

Cit

ric

acid

(g

10

0 g

-1 F

W)

0.0

0.4

0.8

1.2

Tota

l organ

ic a

cid

s (g

100 g

-1 F

W)

0.0

0.4

0.8

1.2

1.6

Control

ABA

NDGA

(A)

(B)

Fig. 5. Changes in the concentration of total organic acid (A), citric (B) (g 100 g−1 FW)and fumaric acid (C) (mg 100 g−1 FW) as influenced by ABA and its biosynthesisinhibitor (NDGA) treatments (T) during the fruit ripening period (RP) at ambienttemperature. Vertical bars represent SE. LSD (P ≤ 0.05) for the concentration of totaloRT

4

4e

ccaAmS(Aomtgtpfseto

rganic acid: T = 0.07, RP = 0.10, T × RP = 0.17; the concentration of citric acid: T = 0.07,P = 0.10, T × RP = 0.18; and the concentration of fumaric acid: T = 0.08, RP = 0.01,

× RP = NS.

. Discussion

.1. Ethylene production and activities of ethylene biosynthesisnzymes

Higher levels of endogenous ethylene production and/or appli-ation of exogenous ethylene promote ripening in a range oflimacteric fruit, including mango, even when applied prior to theccumulation of ABA (Singh and Janes, 2001; Lalel et al., 2003).ccumulation of endogenous ABA has also been implicated in cli-acteric ripening in avocado (Adato et al., 1976), tomato (Buta and

paulding, 1994; Sheng et al., 2008; Zhang et al., 2009b) and bananaLohani et al., 2004). Recently, we have reported accumulation ofBA (>3000 ng g−1 fresh pulp weight) prior to the climacteric peakf ethylene production during fruit ripening in ‘Kensington Pride’ango fruit (Zaharah et al., 2012). Results presented here suggest

hat ABA is critical for the onset of ripening through its role in trig-ering the climacteric peak of ethylene production. Ethylene mayhen be playing a role in mango fruit ripening at climacteric andost climacteric stages. The application of ABA promoted mangoruit ripening, whilst treatment with an inhibitor of ABA biosynthe-

is (NDGA), delayed ripening in mango (Fig. 3A). Similarly, Zhangt al. (2009b) reported the acceleration and retardation of climac-eric fruit ripening in tomato fruit with the exogenous applicationf ABA and fluridone (an ABA inhibitor), respectively. Previously,

and Technology 75 (2013) 37–44

the application of ABA has been shown to increase endogenousethylene levels during development and ripening in cherry (Kondoand Inoue, 1997), litchi (Wang et al., 2007) and tomato (Buta andSpaulding, 1994; Zhang et al., 2009b).

In the current study, the application of 1.0 mM ABA increasedethylene production during ripening at ambient temperature(21 ± 1 ◦C), whilst the application of NDGA, suppressed produc-tion of ethylene (Fig. 1). The increase in ethylene biosynthesis inABA-treated fruit during ripening can be attributed to the increasedactivities of ACO and ACS enzymes (Fig. 2B and C) as well as higherACC content in the fruit pulp (Fig. 2A). The reduction in ethylenebiosynthesis observed in NDGA-treated fruit may be ascribed tothe suppression of ACO activity (Fig. 2C), reduced ACS activity andlowered ACC content in the pulp during ripening (Fig. 2A and B).The ACO activity in NDGA-treated fruit did increase slightly on day2 compared to day 0 and this increase may have been enough toallow the limited conversion of ACC to ethylene (Figs. 1 and 2A, C).Earlier, Suzuki et al. (2005) also reported that similar lower ACOactivity is adequate to convert some ACC to ethylene in broccolifollowing high temperature treatment.

ABA application to fruit was effective in enhancing ethylenebiosynthesis (relative to the control) at the climacteric stage ofripening (Figs. 1 and 2A–C). The effect of exogenous ABA on the cli-macteric peak in ethylene production occurred just two days aftertreatment, which is consistent with a direct effect of ABA on ethy-lene synthesis (as outlined above). On the other hand, treatment ofmango fruit with NDGA caused a suppression of ethylene biosyn-thesis at the climacteric stage of ripening (Figs. 1 and 2A–C). NDGAis a known inhibitor of ABA biosynthesis in plants and effectivelyacts within a short period of time after its application (Zhang et al.,2009b), leading to lower levels of ABA, and in turn, a suppressionof ethylene synthesis.

4.2. Fruit softening and activities of fruit softening enzymes

Fruit softening during ripening occurs due to the disintegra-tion of cell walls as well as transformation and solubilization ofcell wall polymers such as pectin, cellulose and hemicelluloses(Singh and Singh, 2011). Increased fruit softening in ABA-treatedfruit has previously been reported in kiwifruit (Chen et al., 1999,2005), and strawberry during ripening (Jiang and Joyce, 2003). Inthe current study, fruit softening was more pronounced in ABA-treated fruit compared to the control (Fig. 3A). This response couldhave been either a direct consequence of ABA application and/oran indirect effect of ABA application, brought about by an ABA-induced increase in ethylene production (Fig. 1). The ABA treatmentpromoted endo-PG activity and reduced PE activity in pulp tissueduring ripening (Fig. 3B and C). In untreated fruit, softening wassubstantially increased on the second day of the ripening period,which may be due to the accumulation of endogenous ABA (Zaharahet al., 2012) and this was further enhanced when ABA was applied(Fig. 3A). Similarly, Parikh et al. (1990) reported that softening inABA-treated mango fruit is associated with loss of cellular integritydue to cell wall hydrolysis and increased starch degradation dur-ing ripening of ‘Alphonso’ mango. Fruit softening was delayed inNDGA-treated fruit (Fig. 3A), possibly due to a direct inhibition ofABA accumulation and/or an indirect effect mediated by the sup-pression in ethylene production (Fig. 1).

PG is responsible for degrading the (1–4)-linked galacturonicacid residues, and has been reported in mango fruit during ripening(Abu-Sarra and Abu-Goukh, 1992). Our results show that ABA-treated fruit exhibited higher activity of endo-PG on the second

day of ripening, while NDGA-treated fruit only exhibited a smallpeak on day 4 of ripening (Fig. 3B). The increased activities of endo-PG in ABA-treated fruit, may also be due to either a direct effect ofthe increase in the endogenous level of ABA (Zaharah et al., 2012)

ology

abrb

grTmoktactiisoiiapBfttsd

4a

slarAlmatingsieagrtfids

dmsaAar(

S.S. Zaharah et al. / Postharvest Bi

nd/or an indirect effect of the ABA-induced increase in ethyleneiosynthesis (Figs. 1 and 2A–C). The exo-PG enzyme has also beeneported to play a key role in mango fruit softening (Ali et al., 2004)ut ABA treatment did not alter the activity of exo-PG.

PE activity in the pulp of ‘Kensington Pride’ mango fruit pro-ressively declined in ABA-treated fruit with advancement of fruitipening and the activity was higher in NDGA-treated fruit (Fig. 3C).he activities of PE have also been reported to decrease in variousango cultivars such as ‘Kitcher’ and ‘Dr Knight’ with progression

f fruit ripening (Abu-Sarra and Abu-Goukh, 1992). It is also wellnown that PE catalyses the de-esterification of pectin into pec-ate and methanol. The effect of ABA and NDGA treatment on PEctivity possibly results in an interaction between pectate and freealcium to form either loser or stronger cell walls, respectively, inhe pulp of mango fruit, leading to changes in fruit softening dur-ng ripening. Similarly, the activity of PE was found to be highern apple fruit on harvest day and it declined during ripening andoftening (Goulao et al., 2007). The application of ABA (5 mg L−1

r 100 �M) showed inconsistent effects on the change of PE activ-ty during fruit ripening in ‘Zihua’ mango (Zhou et al., 1996), whilst,n ‘Robusta Harichhal’ banana, ABA application (100 �M) enhancedctivity of PE through enhanced ethylene production, consequentlyromoting fruit softening (Lohani et al., 2004). Similarly, Roe andruemmer (1981) reported increased activity of PE and decreased

ruit firmness during ripening of ‘Keitt’ mangoes. Our results onhe role of ABA in mango fruit softening suggest that ABA or NDGAreatment regulates the activity of endo-PG and PE and the resultshowed no significant effects on exo-PG and EGase activities in pulpuring the ripening period in ‘Kensington Pride’ mango fruit.

.3. The concentration of total and individual sugars and organiccids

ABA application to fruit advanced the accumulation of totalugars and sucrose, whilst, NDGA treatment delayed their accumu-ation during fruit ripening as compared with the control (Fig. 4And B). The faster and slower rates of sugar accumulation duringipening in ABA and NDGA-treated fruit, respectively, suggest thatBA is involved in regulating their production, possibly by control-

ing carrier-mediated transport across the tonoplast and plasmaembrane, thus increasing the degradation of starch and organic

cids as reported earlier by Kobashi et al. (1999). Sucrose seemso be more highly influenced by the application of ABA and itsnhibitor than other sugars (such as glucose and fructose) (dataot shown). This observation may be consistent with the sug-estion that in tomato, ABA treatment might increase activity ofucrose synthase (SUS) and the expression of SUS gene(s), lead-ng to increased accumulation of sucrose during ripening (Bastiast al., 2011). Palejwala et al. (1988) have also reported that ABApplication (10−6 M) increased the concentration of sugars in ‘Lan-ra’ mango compared with the control. In the current study, theeduction in the concentration of total sugars and sucrose in ABA-reated fruit on days 8 and 10 of ripening relative to the controlruit may be due to the more advanced sugar accumulation earliern the ripening period (Fig. 4A and B), and also the use of starchuring respiratory processes instead of forming sugar at the ripetage (data not shown).

ABA application to fruit accelerated, whilst, NDGA-treatmentelayed degradation of total organic acids and citric acid, duringango ripening, relative to the control (Fig. 5A and B). This is con-

istent with an earlier report that showed a decrease in total organiccids in ABA-treated ‘Langra’ mango fruit (Palejwala et al., 1988).

BA and NDGA may have influenced the rate of reduction of organiccid concentrations during ripening by either directly or indi-ectly affecting the expression of citrate synthase in mitochondrialmCS) and glioxisomal (gCS) compartments as well as isocitrate

and Technology 75 (2013) 37–44 43

dehydrogenase in mitochondria (mICDH), as has been reported ear-lier in tomato (Bastias et al., 2011).

In this study we have shown that ABA application promotesthe activities of the ethylene biosynthesis enzymes (ACS, ACO),increases ACC content and therefore accelerates ethylene produc-tion during ripening in mango fruit. ABA application was alsoshown to promote fruit softening by regulating the activity ofsoftening enzymes including endo-PG and PE, increase the concen-tration of total sugars and sucrose, and decrease the concentrationof total organic acids and citric acid. We show that ABA promotesthe activity of ACS and ACO and increases ACC content and hencepromotes ethylene production. This demonstrates that the effect ofABA on mango fruit ripening is, at least in part, mediated by changesin ethylene production. It is not possible to nominate which of thesetwo hormones is the “primary” signal, as we have shown that bothare critically important to the ripening process, with ABA workingat least in part by increasing the level of ethylene synthesis enzymesand the immediate precursor of ethylene. The effects of ABA on 1-MCP treated fruit may provide evidence of the direct effects of ABAon fruit ripening, and separate those from the ripening processesthat are mediated by ABA induced increases in ethylene levels. Thisis not likely to yield any new information because the ‘Kensing-ton Pride’ mango fruit does not respond well to 1-MCP treatment.Recently, Sivakumar et al. (2012) also reported that the responseof mango fruit to 1-MCP treatment is cultivar specific. We there-fore propose a model whereby endogenous ABA plays a key role inthe regulation of mango fruit ripening and its effects are, at least inpart, mediated by changes in ethylene production.

Acknowledgements

Zaharah, S.S. gratefully acknowledges the Ministry of HigherEducation Malaysia and Universiti Putra Malaysia for financial sup-port and study leave during her PhD studies respectively. She alsogratefully thanks Curtin University, Western Australia, for award-ing a Completion Scholarship during her final year of PhD study. Weacknowledge Ms. S. Petersen and Mr. I. Iberahim for their technicalsupport.

References

Abu-Sarra, A.F., Abu-Goukh, A.A., 1992. Changes in pectinesterase, polygalactur-onase and cellulase activity during mango fruit ripening. Journal of HorticulturalScience 67, 561–568.

Adato, I., Gazit, S., Blumenfeld, A., 1976. Relationship between changes in abscisicacid and ethylene production during ripening of avocado fruits. Australian Jour-nal of Plant Physiology 3, 555–558.

Ali, Z., Chin, L.H., Lazan, H., 2004. A comparative study on wall degrading enzymes,pectin modifications and softening during ripening of selected tropical fruits.Plant Science 167, 317–327.

Bastias, A., Lopez-Climent, M., Valcarcel, M., Rosello, S., Gomez-Cadenas, A.,Casaretto, J.A., 2011. Modulation of organic acids and sugar content in tomatofruits by an abscisic acid-regulated transcription factor. Physiologia Plantarum141, 215–226.

Bradford, M.M., 1976. A rapid and sensitive method for the quantification of micro-gram quantities of protein utilizing the principles of protein–dye binding.Analytical Biochemistry 72, 248–254.

Buesa, C., Dominguez, M., Vendrell, M., 1994. Abscisic acid effects on ethyleneproduction and respiration rate in detached apple fruits at different stagesof development. Revista Espanola de Cienciay Tecnologia de Alimentos 34,495–506.

Buta, J.G., Spaulding, D.W., 1994. Changes in indole-3-acetic acid and abscisic acidlevels during tomato (Lycopersicon esculentum Mill.) fruit developing and ripen-ing. Journal of Plant Growth Regulation 13, 163–166.

Chen, J.Y., Chen, M., Gan, L., 2005. Effects of ethephon and ABA treatments on phys-iology of ‘Jinkui’ kiwifruit during its ripening and softening. Acta Agriculturae27, 6–11.

Chen, K.S., Li, F., Zhang, S.L., Ross, G.S., 1999. Role of abscisic acid and indole-3-aceticacid in kiwifruit ripening. Acta Horticulturae Sinica 26, 81–86.

Chourasia, A., Sane, V.A., Singh, R.K., Nath, P., 2008. Isolation and characterizationof the MiCel1 gene from mango: ripening related expression and enhancedendoglucanase activity during softening. Plant Growth Regulation 56, 117–127.

4 ology

C

D

G

G

G

G

I

J

K

K

K

K

K

L

L

M

P

P

4 S.S. Zaharah et al. / Postharvest Bi

hourasia, A., Sane, V.A., Nath, P., 2006. Differential expression of pectate lyaseduring ethylene-induced postharvest softening of mango (Mangifera indica var.Dashehari). Physiologia Plantarum 128, 546–555.

eytieux, C., Geny, L., Doneche, B., 2005. Relation between hormonal balance andpolygalacturonase activity in grape berry. Acta Horticulturae 682, 163–170.

iovannoni, J., 2001. Molecular biology of fruit maturation and ripening. AnnualReview of Plant Physiology 52, 725–749.

iovannoni, J., 2004. Genetic regulation of fruit development and ripening. PlantCell 16, 170–180.

orny, J.R., Kader, A.A., 1996. Controlled-atmosphere suppression of ACC synthaseand ACC oxidase in ‘Golden Delicious’ apples during long-term cold storage.Journal of the American Society for Horticultural Science 12, 751–755.

oulao, L.F., Santos, J., de Sousa, I., Oliveira, C.M., 2007. Patterns of enzymatic activityof cell wall-modifying enzymes during growth and ripening of apples. Posthar-vest Biology and Technology 43, 307–318.

naba, A., Ishida, M., Sobajima, Y., 1976. Changes in endogenous hormone concentra-tions during berry development in relation to the ripening of Delaware grapes.Journal of the Japanese Society for Horticultural Science 45, 245–252.

iang, Y.M., Joyce, D.C., 2003. ABA effects on ethylene production, PAL activity, antho-cyanin and phenolic contents of strawberry fruit. Plant Growth Regulation 39,171–174.

han, A.S., Singh, Z., 2007. 1-MCP regulates ethylene biosynthesis and fruit softeningduring ripening of ‘Tegan Blue’ plum. Postharvest Biology and Technology 43,298–306.

obashi, K., Gemma, H., Iwahori, S., 1999. Sugar accumulation in peach fruit asaffected by abscisic acid (ABA) treatment in relation to some sugar metabolizingenzymes. Journal of the Japanese Society for Horticultural Science 68, 465–470.

ojima, K., 1996. Distribution and change of endogenous IAA and ABAin asparagus spear and orange fruit. Chemical Regulation of Plants 31,68–71.

ondo, S., Inoue, K., 1997. Abscisic acid (ABA) and 1-aminocyclopropane-1-cyclopropane acid (ACC) content during growth of ‘Satohnishiki’ cherry fruit,and the effect of ABA and ethephon application on fruit quality. Journal of Hor-ticultural Science 72, 221–227.

ondo, S., Sungcome, K., Setha, S., Hirai, N., 2004. ABA catabolism during develop-ment and storage in mangoes: influence of jasmonates. Journal of HorticulturalScience & Biotechnology 76, 891–896.

alel, H.J.D., Singh, Z., Tan, S.C., 2003. The role of ethylene in mango fruit aromavolatiles biosynthesis. Journal of Horticultural Science & Biotechnology 278,485–496.

ohani, S., Trivedi, P.K., Nath, P., 2004. Changes in activities of cell wall hydroly-ses during ethylene-induced ripening in banana: effect of 1-MCP, ABA and IAA.Postharvest Biology and Technology 31, 119–126.

urti, G.S.R., Upreti, K.K., 1995. Changes in some endogenous growth substancesduring fruit development in mango. Plant Physiology and Biochemistry 122,44–47.

alejwala, V.V., Amin, B., Parikh, H.R., Modi, V.V., 1988. Role of abscisic acid in theripening of mango. Acta Horticulturae 231, 662–667.

arikh, H.R., Nair, G.M., Modi, V.V., 1990. Some structural changes during ripeningof mangoes (Mangifera indica var. Alphonso) by abscisic acid treatment. Annalsof Botany 65, 121–127.

and Technology 75 (2013) 37–44

Rodrigo, M.J., Marcos, J.F., Alferez, F., Mallent, M., Zacarias, L., 2003. Characterizationof pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration thatresults in yellow pigmentation and decreased ABA content. Journal of Experi-mental Botany 54, 727–738.

Roe, B., Bruemmer, J.H., 1981. Changes in pectic substances and enzymes duringripening and storage of ‘Keitt’ mangoes. Journal of Food Science 46, 186–189.

Rudnicki, I., Machnik, J., Pieniazek, J., 1968. Accumulation of abscisic acid duringripening of pears (Clapp’s Favourite) in various storage conditions. Bulletin de lAcademie Polonaise des Sciences-Serie des Sciences Biologique 16, 509–512.

Sheng, J., Ruan, Y., Liu, K., Shen, L., 2008. Spatiotemporal relationships betweenabscisic acid and ethylene biosynthesis during tomato fruit ripening. Acta Hor-ticulture 774, 59–65.

Singh, Z., Janes, J., 2001. Effects of postharvest application of ethephon on fruit ripen-ing, quality and shelf life of mango under modified atmosphere packaging. ActaHorticulture 553, 599–602.

Singh, Z., Singh, S.P., 2011. Mango. In: Rees, D., Orchard, J. (Eds.), Crop Post-harvest:Science and Technology Volume 3: Perishables. Blackwell Publishing Ltd., UK,pp. 108–142.

Sivakumar, D., Van Deventer, F., Terry, L.A., Polanta, G.A., Korsten, L., 2012. Combi-nation of 1-methylcyclopropene treatment and controlled atmosphere storageretains overall fruit quality and bioactive compounds in mango. Journal of theScience of Food and Agriculture 92, 821–830.

Suzuki, Y., Asoda, T., Matsumoto, Y., Terai, H., Kato, M., 2005. Suppression of theexpression of genes encoding ethylene biosynthesis enzymes in harvested broc-coli with high temperature treatment. Postharvest Biology and Technology 36,265–271.

Wang, H.C., Huang, H.B., Huang, X.M., 2007. Differential effects of abscisic acid andethylene on the fruit maturation of Litchi chinensis Sonn. Journal of Plant GrowthRegulation 52, 1573–5087.

Zaharah, S.S., Singh, Z., 2011a. Mode of action of nitric oxide in inhibiting ethylenebiosynthesis and fruit softening during ripening and cool storage of ‘KensingtonPride’ mango. Postharvest Biology and Technology 62, 258–266.

Zaharah, S.S., Singh, Z., 2011b. Post-harvest fumigation with nitric oxide at the pre-climacteric and climacteric rise stages influences ripening and quality in mangofruit. Journal of Horticultural Science & Biotechnology 86, 645–653.

Zaharah, S.S., Singh, Z., Symons, G.M., Reid, J.B., 2012. Role of brassinosteroids, ethy-lene, abscisic acid, and indole-3-acetic acid in mango fruit ripening. Journal ofPlant Growth Regulation 31, 363–372.

Zhang, M., Leng, P., Zhang, G., Li, X., 2009a. Cloning and functioning analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme duringabscisic acid biosynthesis from peach and grape fruits. Journal of Plant Physiol-ogy 166, 1241–1252.

Zhang, M., Yuan, B., Leng, P., 2009b. The role of ABA in triggering ethylenebiosynthesis and ripening of tomato fruit. Journal of Experimental Botany 60,1579–1588.

Zhou, Y.C., Tang, Y.L., Tan, X.J., Guo, J.Y., 1996. Effects of exogenous ABA, GA3 and cell-

wall-degrading enzyme activity, carotenoid content in ripening mango fruit.Acta Phytophysiology Sinica 22, 21–426.

Zhu, B.Z., Wei, S.C., Luo, Y.B., 2003. Relationship between calcium and ABA inethylene synthesis in tomato fruit. Journal of Agricultural Biotechnology 11,359–364.