modulation of mango ripening by chemicals: physiological and biochemical aspects

ORIGINAL PAPER

Modulation of mango ripening by chemicals: physiologicaland biochemical aspects

Rupinder Singh Æ Poorinima Singh Æ Neelam Pathak ÆV. K. Singh Æ Upendra N. Dwivedi

Received: 3 April 2006 / Accepted: 18 July 2007 / Published online: 9 August 2007

� Springer Science+Business Media B.V. 2007

Abstract During ripening, fleshy fruits undergo

textural changes that lead to loss of tissue firmness

and consequent softening due to cell wall dismantling

carried out by different and specifically expressed

enzymes. The effect of various chemical treatments

on the ripening of mango fruit (Mangifera indica)

was investigated at physiological and biochemical

level. Based on changes in respiration, firmness, pH,

total soluble sugar and a cell wall degrading enzyme

pectate lyase (PEL) activity, treatment with 1-meth-

ylcyclopropene (1-MCP), silver nitrate (AgNO3),

gibberlic acid (GA3), sodium metabisulphite (SMS)

and ascorbic acid led to delaying of ripening process

while those of ethrel and calcium chloride (CaCl2)

enhanced the process. PEL of mango was found to be

inhibited by certain metabolites present in dialysed

ammonium sulphate enzyme extract as well as

EDTA. Mango PEL activity exhibited an absolute

requirement for Ca2+ and an optimum pH of 8.5.

Keywords Fruit-ripening � Pectate lyase �Softening � 1-MCP � Mangifera indica

Introduction

Fruit ripening is a genetically programmed event that

is characterized by a number of biochemical and

physiological processes that alter fruit colour, aroma,

flavour, texture and its nutritional value. The onset of

ripening in climacteric fruits is marked by a burst of

CO2 production concomitant with endogenous ethyl-

ene production (Abeles et al. 1992). Studies on

artificial ripening and its regulation are of vital

importance to post-harvest horticulturists and fruit

biologists. In post-harvest management practices,

chemicals are used to both hasten as well as delay

fruit ripening primarily by stimulation or inhibition of

ethylene production (Saltveit 1999). Thus, mature

fruits can be ripened with low doses of ethrel (2-

chloroethyl phosphoric acid) for uniform colour

development. 1-Methylcyclopropene (1-MCP) is an

extensively studied inhibitor of ethylene-action that

delays ripening and improves post-harvest quality of

a wide variety of fruits and vegetables, including

pome fruits (Porat et al. 1999) and tropical fruits

(Feng et al. 2000). It is a cyclic olefin that blocks

ethylene receptors and thus the ethylene-mediated

ripening process (Dong et al. 2002). Silver appears

unique among heavy metals to have anti-ethylene

action and thereby, delay fruit ripening (Mordy et al.

1987). A post-processing dip of 2% (w/v) ascorbic

acid, 1% (w/v) calcium lactate and 0.5% (w/v)

cysteine adjusted to pH 7.0 did significantly extend

shelf-life of ‘Bartlett’ pear slices (Gorny et al. 2002).

R. Singh � P. Singh � N. Pathak � U. N. Dwivedi (&)

Department of Biochemistry, Lucknow University,

Lucknow 226007, India

e-mail: [email protected]

V. K. Singh

Central Institute of Subtropical Horticulture, Lucknow,

India

123

Plant Growth Regul (2007) 53:137–145

DOI 10.1007/s10725-007-9211-1

Plant hormones such as auxins, gibberlins and

cytokinins have been shown to delay fruit ripening

in bananas and tomatoes (Vendrell 1969; Liberman

et al. 1977). Yang (1980) reported that calcium

chloride (CaCl2) induced the ethylene production and

synergistic stimulation of ethylene production with

other metal ions and kinetin.

Ripening is associated with a change in cell wall

hydrolysis enzymes (Giavanonni 2001). Thus pec-

tate lyases (PEL, EC 4.2.2.2), otherwise known as

pectate transeliminases, are known to play a critical

role in the depolymerization process during soften-

ing and catalyse random cleavage of internal a-1,4-

linkages of polygalacturonate or methyl-esterified

pectins through a trans-eliminative mechanism,

thereby generating 4,5-unsaturated oligogalacturo-

nates. While pectin degrading enzymes such as

polygalacturonase have been the focus of significant

research, PEL has been less well studied (Castillejo

et al. 2004).

Mango (Mangifera indica) var. Dashehari is a fruit

of prime economic importance to India. The objective

of this work was to evaluate the effects of different

chemicals like 1-MCP, ethrel, sodium metabisulphite

(SMS), silver nitrate (AgNO3), ascorbic acid, calcium

chloride and gibberlic acid (GA3) on ripening asso-

ciated physiological and biochemical parameters in

mango var. Dashehari. We also report the modulation

of the activity of a cell wall degrading enzyme

namely PEL. This information should help to clarify

the possible inter-relationships between chemicals

and plant hormone in regulating the ripening process.

To the best of our knowledge this is first report of a

study of this type in mango.

Experimental

Plant material

Mango (M. Indica) var. Dashehari fruits were

collected from Central Institute of Subtropical

Horticulture (CISH) orchard, Lucknow, India.

Mature unripe fruits free from disease were selected,

washed with distilled water, air dried and pulp and

peels of the samples were taken for analysis. Ten

fruits of similar sizes were used for each treatment

and analysed and three replicates were used per

treatment.

Determination of fruit firmness

Firmness of fruits was measured by puncture analysis.

Firmness was measured at three points per fruit (without

peel) using a ‘McCormick fruit tester FT 327’ pene-

trometer with head diameter of 11 mm. Based on whole

fruit compression analysis the changes in fruit softening

were determined by measuring firmness during ripening

of fruit in both treated and untreated samples. Ten fruits

of similar sizes were used for each treatment and

analysed and three replicates were used per treatment.

Fruit firmness was expressed in Newtons.

Determination of total soluble solids (TSS)

Total soluble solids (TSS) was measured by using

Erma refractometer (0–32%). Results were expressed

in percentage (%) TSS. Ten fruits of similar sizes

were used for each treatment and analysed and three

replicates were used per treatment.

Determination of acidity

Fruit samples were homogenized and 20 g of the

tissue was suspended in H2O up to a volume of 100 ml

(AOAC 1980). Changes in the acidity of fruits during

ripening were determined by measuring the pH of the

homogenate of mango pulp using pH meter. Ten fruits

of similar sizes were used for each treatment and

analysed and three replicates were used per treatment.

Determination of respiration rate

Single fruit samples were kept in CO2 detector chamber

(ECG Gas analyser). The change in CO2 concentration

was recorded at every 15 min for 1 h at 20�C. The

results were expressed as ml h�1 kg�1 FW. Ten fruits of

similar sizes were used for each treatment and analysed

and three replicates were used per treatment.

Chemical treatments

1-MCP treatment

Fruits (10) were placed in 20-l containers and

exposed to 1-MCP (2 mg kg�1) for 12 h at 20�C

138 Plant Growth Regul (2007) 53:137–145

123

and 85% relative humidity (RH). Immediately fol-

lowing 1-MCP treatment, fruit were removed from

the chambers, placed in cardboard boxes with holes.

Control fruit were maintained in identical containers

without 1-MCP at room temperature.

Ethrel treatment (Etephon)

Fruits were dipped uniformly in 750 mg kg�1 con-

sisting 1.8 ml l�1 ethrel in hot water at 52 ± 2�C for

5 min. Fruits were air dried and placed in cardboard

boxes with holes.

Treatment of AgNO3, GA3, ascorbic acid, SMS

and CaCl2

All fruits were treated with labolene (2%) and then

with 2% AgNO3, SMS and CaCl2 while GA3 was

added at the rate of 0.5% w/v.

Enzyme preparation

Mango fruits were peeled and the flesh was sliced

into small pieces. 5g of mango pulp was homoge-

nized, using liquid nitrogen, in 20 ml of extraction

buffer [0.02 M Na-PO4 buffer, pH 7.0, 0.02 M freshly

prepared cysteine-HCl and 1% (v/v) Triton X-100] in

a chilled mortar and pestle to obtain 25% homoge-

nate. The homogenate was centrifuged at 15,000g for

25 min in Sorvall RC5C at 4�C. Supernatant was

collected and total protein was precipitated using

ammonium sulphate and the precipitate suspended in

0.02 M sodium phosphate buffer, pH 7.0 and dialysed

overnight at 4�C. The preparation constituted a crude

enzyme extract.

Enzyme assay and protein estimation

The PEL activity was determined by monitoring the

increase in absorbance at 232 nm as described by

Collmer et al. (1988). The assay system consisted of

0.45 ml polygalacturonic acid in 0.02 M Tris–HCl

buffer, pH 8.5, 0.45 mM of CaCl2, crude enzyme

extract (20 ll) and water in a total volume of 3.0 ml.

The increase of absorbance was noted. One unit of

enzyme was defined as 1 lmol of digalacturonide

formed in 1 min under conditions of assay. Total

soluble protein in the enzyme preparation was

determined by the Bradford method (Bradford 1976).

Statistical analysis

Statistical analysis of data was performed by ANO-

VA test by using PRISM software.

Results

Effect of chemical treatment on respiration rate of

fruit during ripening

The rate of respiration during mango fruit ripening is

shown in Fig. 1. The peak in respiration for control

fruit occurred on day 3 (1,490 ml h�1 kg�1 FW).

However, fruits treated with ethrel and CaCl2 pro-

duced CO2 peak after 24 h. In contrast, fruits treated

with GA3 showed delayed the peak of CO2 production

by around 2 days compared to untreated controls. 1-

MCP delayed the peak of CO2 production by 4 days

compared to untreated control. Only the 1-MCP

treatment showed a respiration peak at around 7 days.

Days0 8 10 12

CO

2 Pro

duct

ion(

ml h

-1kg

-1 F

W)

200

400

600

800

1000

1200

1400

1600

1800

2000

2200Ethrel

Calcium Chloride

GA3

Ascorbic Acid

1-MCP

SMS

Silver Nitrate

Control

2 4 6

Fig. 1 Effect of chemical treatments on respiration at room

temperature during ripening of mango fruit in absence (d) and

presence of Ethrel (u), CaCl2 (e), 1-MCP (�), AgNO3 (.),

GA3 (5), SMS (h) and AA (j). Each value represents a

mean ± SD of three independent experiments of each in

triplicate. The data were analysed by Newman–Keuls multiple

method and was found to be significant (P < 0.05)

Plant Growth Regul (2007) 53:137–145 139

123

Effect of chemical treatment on changes in

firmness of fruit during ripening

A decrease in fruit firmness was directly related with

ripening. Data are presented in Table 1. Firmness of

untreated mango fruit (control) showed decrease from

181.3 to 112.7 N during the ripening period in this

study. Treatment with ethrel and CaCl2 increased the

rate of softening as compared to their respective

controls. In contrast, 1-MCP, AgNO3, GA3, did not

reduce softening compared to their respective con-

trols. 1-MCP was the most effective in delaying

softening.

Effect of chemical treatment on changes in pH of

the fruit during ripening

Ripening of mango fruit was associated with an

increase in pH as shown in Table 2. Thus in untreated

fruits, pH increased from 2.4 to 4.7 over the study

period. Treatment of fruits with ethrel and CaCl2 led

to an increase in pH (from 2.5 to 5.6 and 2.4 to 5.0,

respectively), as compared to untreated controls.

Effect of chemical treatment on changes in fresh

weight of fruit during ripening

The fresh weight of fruit decreased gradually during

ripening in both treated and untreated controls

(Table 3). The fresh weight of untreated mango fruits

decreased by 11.3% during the study period. Treat-

ment with ethrel caused an 18.2% decrease in fresh

weight while CaCl2 reduced fresh weight by 15.6%

compared to the controls. The decrease in fresh

weight for 1-MCP treated fruit was 6.8% which was

significantly less compared to various treatments. The

decrease in tissue browning was also observed in fruit

treated with ascorbic acid but AgNO3 led to greater

browning of the peel.

Effect of chemical treatment on total soluble

solids (TSS) of fruit during ripening

The TSS increased gradually during fruit ripening in

both treated and control samples (Table 4). In

untreated controls there was 1.88-fold increase in Ta

ble

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123

TSS during the study period. Treatment with 1-MCP,

AgNO3, GA3, SMS and ascorbic acid reduced the

increase in TSS compared to untreated controls.

These changes were statistically significant (Table 4).

The difference in TSS between days 0 and 11 was

11% in control and 14% in ethrel treated fruit.

Optimization of pectate lyase extraction

The PEL was found to be readily inactivated during

isolation. Therefore conditions were optimized for its

isolation. No PEL activity was detected in the crude

extract when mango pulp tissue was homogenized in

sodium phosphate buffer. All activities were subse-

quently measured after ammonium sulphate

precipitation of the homogenate (90%).

Characterization of the PEL enzyme

The effect of pH on the activity of the enzyme was

investigated. Optimum pH for PEL activity was

found to be 8.5. Mango PEL activity exhibited an

absolute requirement for Ca2+ ions. Maximum activ-

ity was found at 0.45 mM CaCl2, whilst activity

decreased at higher concentrations and activity was

undetectable in its absence (data not presented). The

maximum rate of product formation was observed

using polygalacturonate as the substrate and optimum

activity was obtained by the addition of 0.45 ml of

0.36% PGA in an assay system. It was concluded that

the depolymerizing activity of polygalacturonate was

characteristic of a PEL as indicated by the formation

of unsaturated products (digalacturonide). The mango

PEL activity was completely inhibited by 5 mM

EDTA.

Effect of various chemicals on PEL activity

during ripening

The activity of PEL during ripening of mango was

investigated in untreated and treated fruits. Results

are shown in Fig. 2. The activity of PEL in control

fruit increased gradually for the first 3 days after

treatment and thereafter declined. Compared to the

control fruit, 1-MCP, AgNO3 and GA3 treated

samples exhibited a delay in the peak of fruit PELTa

ble

4E

ffec

to

fch

emic

altr

eatm

ents

on

tota

lso

lub

leso

lid

(TS

S)

con

ten

to

fm

ang

ofr

uit

du

rin

gri

pen

ing

(sto

red

atro

om

tem

per

atu

rean

d8

5%

rela

tiv

eh

um

idit

y)

Day

sT

SS

(%)

Co

ntr

ol

1-M

eth

yl

cycl

op

rop

ene

Sil

ver

nit

rate

Gib

ber

lic

acid

Asc

orb

ic

acid

So

diu

mm

eta

bis

ulp

hit

e

Eth

rel

Cal

ciu

m

chlo

rid

e

01

2.5

±0

.08

12

.5±

0.2

01

2.0

±0

.26

13

.0±

0.3

21

3.5

±0

.32

12

.5±

0.3

21

2.0

±0

.33

13

.0±

0.3

3

11

3.0

±0

.10

12

.5±

0.9

41

2.5

±0

.94

13

.0±

0.3

31

4.0

±0

.32

13

.0±

0.3

31

3.0

±0

.38

13

.5±

0.2

4

31

4.5

±0

.15

13

.0±

0.3

11

4.0

±0

.23

14

.5±

0.2

61

4.5

±0

.41

14

.5±

0.2

91

7.5

±0

.32

15

.0±

0.3

3

51

7.5

±0

.20

13

.5±

0.2

01

4.5

±0

.16

15

.0±

0.3

21

5.5

±0

.29

15

.5±

0.2

91

9.0

±0

.33

17

.5±

0.1

2

71

9.0

±0

.23

14

.5±

0.2

31

6.0

±0

.32

17

.0±

0.3

21

7.5

±0

.36

17

.5±

0.2

82

2.5

±0

.32

19

.5±

0.2

3

92

1.5

±0

.21

17

.5±

0.2

01

8.5

±0

.32

19

.5±

0.3

21

9.5

±0

.32

20

.0±

0.4

02

4.5

±0

.32

21

.5±

0.3

2

11

23

.5±

0.2

31

9.5

±0

.20

20

.5±

0.3

22

1.5

±0

.32

21

.5±

0.3

22

2.0

±0

.20

26

.0±

0.3

32

5.5

±0

.43

To

tal

dif

fere

nce

inT

SS

in1

1d

ays

11

.07

.08

.58

.58

.09

.51

4.0

12

.5

Eac

hv

alu

ere

pre

sen

tsa

mea

n±

SD

of

thre

ein

dep

end

ent

exp

erim

ents

of

each

intr

ipli

cate

.T

he

dat

aw

ere

anal

yse

db

yN

ewm

an–

Keu

lsm

ult

iple

met

ho

dan

dw

asfo

un

dto

be

sig

nifi

can

tfe

wtr

eatm

ents

(P<

0.0

5)


123

activity. The peak of PEL activity was detected after

7 days for 1-MCP and after 5 days for AgNO3 and

GA3, treatments, respectively. Ascorbic acid and

SMS also delayed the activity but they were not that

effective as compared to 1-MCP. The peak of PEL

activity for mango treated with ethrel and CaCl2 was

advanced to day 1 suggesting enhancement of

ripening.

Discussion

Results presented in this paper show that certain

chemicals were effective in enhancing ripening while

others were effective in delaying mango ripening.

Ethrel and CaCl2, enhanced ripening while 1-MCP,

AgNO3, GA3, SMS and ascorbic acid were effective

in delaying ripening, with 1-MCP being the most

effective.

Significant changes in fruit firmness during ripen-

ing were observed here under different chemical

treatments as with other fruits (Baritelle et al. 2001).

The ethrel and calcium chloride mediated increased

in softening is probably due to increased hydrolysis

of cell wall components (Tateishi et al. 2005).

Mango ripening was associated with an increase in

total fruit soluble solids, which appears linked in

increase to cell wall hydrolysing enzyme during

ripening as reported in banana (Pathak and Sanwal

1998). The major compositional changes during

mango ripening were an increase in reducing sugars.

Starch, which constitutes 20–25% of the fresh weight

of climacteric fruits is almost entirely converted into

soluble sugars during ripening with *2–5%, being

lost via respiration (Chang and Hwang 1990). Fan

et al. (2000) found that the onset of ethylene

production and fruit softening were delayed, and the

respiration rate was reduced on ‘Perfection’ apricots

treated with 1 mg kg�1 1-MCP for 4 h at 20�C. Here,

with mango treated with 1-MCP (2 mg kg�1), the

ripening process was slowed but not completely

inhibited. The potent suppressive action of the 1-

MCP treatment was consistent with the role of

ethylene in softening-related metabolism in ripening

fruits (Saltveit 1999). Furthermore, in peach fruit 1-

MCP has been shown to have direct effect on

ethylene perception particularly through its effect

on the expression of ethylene receptor genes (Rasori

et al. 2002). A family of six genes encoding ethylene

receptors have been characterized from tomato, from

which the ethylene receptor LeETR3 varies dramat-

ically during ripening (Klee 2002). To the best of our

knowledge only one ethylene receptor has been

characterized from mango (Martınez et al. 2001).

Tassoni et al. (2006) reported that maximal ethylene

production occurred in control fruit on day 4 after

harvest, gradually decreasing from days 6 to 16. In

contrast, 1-MCP-treated mango fruit showed a small

peak in ethylene production at the same time as the

control on day 4, followed by a decline to similar the

original rate prior to increasing rapidly from around

day 8 with a maximum on day 12, which was similar

in magnitude to the ethylene peak of the controls.

This effect was consistent with other reports for

tomato that show that 1-MCP causes a temporary

delay in ripening and not a complete inhibition

(Hoeberichts et al. 2002; Wills and Ku 2002; Most-

olfi et al. 2003). A delayed increase in ethylene

production can be associated with a delay in gene

transcription of ethylene biosynthetic enzymes, as

noted by Hoeberichts et al. (2002) and Nakatsuka

et al. (1997, 1998). These results demonstrate that the

reacquisition of ripening competence coincides with

the recovery of ethylene receptor gene transcription.

Days (Room Temperature)0 4 10 12

PE

L S

peci

fic A

ctiv

ity(U

nits

. mg

prot

ein-

1 )

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

MCP

GA3

AgNO3

CaCl2

Ethrel

SMS Control

Ascorbic Acid

2 6 8

Fig. 2 Developmental profile of PEL activity during ripening

of mango fruit in the absence (d) and presence of chemicals

Ethrel (u), CaCl2 (e), MCP (�), AgNO3 (.), GA3 (5), SMS

(h) and AA (j). Each value represents a mean ± SD of three

independent experiments of each in triplicate. The data were

analysed by Newman–Keuls multiple method were found to be

significant (P < 0.05)


123

1-MCP is assumed to act via receptor binding thereby

blocking ethylene perception. It appears that this

mode of action also suppresses the immediate

transcription of ethylene receptor genes rather than

enhancing this transcription to compensate for the

lack of functional receptors. The continued produc-

tion of new ethylene receptors, to which 1-MCP has

not bound, is therefore an explanation of the renewed

sensitivity to further ethylene produced during rip-

ening (Feng et al. 2004). The delayed increase in

ethylene receptor gene transcription was particularly

associated with only ripening genes, namely LeE-

TR4, 5 and 6 but not all of ethylene receptors (Klee

and Tieman 2002) whereas no relationship was found

in LeETR1 and 2, which are expressed in all tissues

during development (Tieman and Klee 1999). It has

been reported that PEL activity increases with

ripening and in our case the PEL activity of samples

treated with 1-MCP and AgNO3 were significantly

higher than that of control for the reasons unknown,

this needs to be explored further.

Data on silver nitrate suggested a role similar to

that of 1-MCP. Silver inhibits ethylene biosynthesis

via reducing perception of endogenous levels of

ethylene (Edwards et al. 1983). Furthermore, silver

may compete with copper cofactor within the ethyl-

ene receptor protein (Yang 1980). Gibberellins

mediated delay in ripening, as observed in present

study, has been suggested due to antagonistic action

of gibberellins on ethylene perception (Ben-Arie

et al. 1995; Scott and Leopold 1967).

Ethylene released by the breakdown of ethrel was

the cause of softening of fruit and hastened the onset

of ripening of mango. Calcium mediated increased

softening of mango fruits during ripening, is due to

increase in PEL activity. Reports on calcium mediated

activation of PEL from actinomycetes Amycolata

(Bruhlmann 1995) and bacterium E. chrysanthemi

(Yoder et al. 1993) are available. Furthermore, Ca2+

has been suggested to form ionic cross-links between

some of the carbohydrates of the galacturonic acid

residues in homogalacturonan followed by degrada-

tion of pectin (Hofmann 2002; Koornneef et al. 2003).

In vitro characterization of PEL activity revealed

presence of activity only in dialysed preparation but

not in crude (undialysed) preparations suggesting the

presence of dialysable interfering substances in the

crude extract. Similar report of high PEL activity in

dialysed preparation are apparent (Collmer et al.

1988) in the banana fruit pulp extract. Activating

effect of cysteine (20 mM) on PEL activity was

probably due to prevention of oxidation caused by

phenolics. Similar observation has been made in

banana (Payasi and Sanwal 2003). A concentration of

0.45 mM Ca2+ was required for maximum activity of

the enzyme and 5 mM EDTA inhibited the PEL

activity completely.

There is still a lot to learn about the enzymatic

degradation of pectic substances and it is anticipated

that results arising from this study will, in the future,

be coupled to data provided by other functional

genomic tools (proteonomics and metabolomics)

would facilitate a more thorough understanding of

fruit ripening, one of the most economically impor-

tant processes in agriculture.

Acknowledgements The financial assistance from the

Department of Biotechnology (DBT), New Delhi, India (in

the form of DBT-JRF to Rupinder Singh) and DST (YS-Fast

Track to Neelam Pathak) are gratefully acknowledged. We are

also thankful to DST-FIST and CSIR for their support in the

form of infrastructural facilities.

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modulation of mango ripening by chemicals: physiological and biochemical aspects

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