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-MCP treatment modulated physiological, biochemical and gene expression

activities of guava during low-temperature storage

Article in Acta Physiologiae Plantarum · September 2022

DOI: 10.1007/s11738-022-03463-x

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Acta Physiologiae Plantarum (2022) 44:125
https://doi.org/10.1007/s11738-022-03463-x

ORIGINAL ARTICLE

1‑MCP treatment modulated physiological, biochemical and gene

expression activities of guava during low‑temperature storage
A. J. Sachin1 · D. V. Sudhakar Rao1 · Kundapura Ravishankar3 · K. Ranjitha1 · C. Vasugi2 · C. K. Narayana1 ·
S. Vijay Rakesh Reddy1

Received: 1 September 2021 / Revised: 5 January 2022 / Accepted: 22 September 2022

© The Author(s) under exclusive licence to Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2022

Abstract
Guava fruits remain biologically active even after harvest as they continue respiration and other physioligical activites. Being
climacteric in nature, guava fruits senesce fast duringstorage. To extend the storage life of guava fruits, they were treated with
1-MCP 500 ppb and found that 1-MCP could extend the storage life of guava. 1-MCP-treated fruits retained greater fruit
texture (2.17 N) with reduced respiration (54.57 C ­ O2 mg/kg/h), ethylene (5.02 µl/kg/h) and pectin methylestaerase activity
(1.21 units/ml) after 21 days of storage period when compared to untreated fruits. To determine the effects of 1-MCP on
ripening, various physiological, biochemcial and gene expression parameters were studied. The results showed that 1-MCP
delayed respiration and ethylene peak by maintaining the quality parameters like ascorbic acid content (129.24 mg/100 g),
antioxidant activity (199.2 AAE/100 g), phenols (528.93 GAE/100 g) and sugar acid ratio (16.05% acid) in the fruits. RNA
isolation protocol was developed using Tris saturated phenol and sodium dodecyl sulfate. This rapid protocol allowed us to
isolate quality RNA from guava pulp. Further, RT-qPCR gene expression analysis of expansin and ACC synthase genes rev-
eled that the texture and ethylene production were significantly down regulated with 1-MCP treatment. The present reserach
work highlighted the effect of 1-MCP in extending the shelf life of guava fruits by regulating physiological and biochemical
processes during friut ripening, and their validation through gene expression studies provide insights for manipulaiton of
genes involved in climacteric fruit ripening.

Keywords Shelf life · 1-MCP · RNA isolation · Gene expression · Expansin · ACC synthase

Introduction cost and high nutritional attributes like rich in vitamin C

Guava (Psidium guajava L.) is one of the most delicious
and nutritious fruits, liked by consumers for its refreshing
taste and pleasant flavor. It is considered as “apple of trop-
ics” or “the poor man’s fruit” because of its low production
Communicated by P. K. Nagar.
* A. J. Sachin
sachin.iihr@gmail.com
1
Division of Postharvest Technology and Agricultural
Engineering, ICAR-Indian Institute of Horticultural
Research, Hesaraghatta, Bengaluru 560089, India
2
Division of Fruit Crops, ICAR-Indian Institute
of Horticultural Research, Hesaraghatta, Bengaluru 560089,
India
3
Division of Basic Sciences, ICAR-Indian Institute
of Horticultural Research, Hesaraghatta, Bengaluru 560089,
India
(150–250 mg/100 g) (Lopez et al. 2021), pectin (0.5–1.9%)
(Salunkhe and Desai 1984), carbohydrates and dietary fib-
ers (Chandra et al. 2011). Under ambient conditions, it has
a limited shelf life of about 4–5 days as it attains full ripe
stage. Like all other tropical climacteric fruits, guava con-
tinues to be biologically active after harvest and carry out
respiration and other biochemical processes, leading to qual-
ity deterioration and render them unacceptable. The role of
autocatalytic production of ethylene leading to rapid rip-
ening and tissue breakdown in climacteric fruits is a well-
established scientific fact (Pech et al. 2008). So, reducing
ethylene production and action is pivotal for improving
the shelf life of climacteric fruits (Reddy et al. 2016). In
recent times, 1-methylcyclopropene (1-MCP) has emerged
as a ray of hope in extending the storage life of climacteric
fruit under ambient as well as low-temperature conditions.
1-MCP acts by binding to the ethylene receptors in a com-
petitive mode so that ethylene cannot bind and elicit action.

13
Vol.:(0123456789)
125 Page 2 of 9
1-MCP has higher affinity (10 times) to the receptors when
compared to the ethylene and is also active at very low con-
centrations (at ppb level) compared to ethylene (Sisler and
Serek 1997; Blankenship and Dole, 2003; Porat et al. 2009).
RNA isolation from guava fruit pulp is tricky since the
fruits are rich in polysaccharides, phenols and proteins that
interfere in the process of RNA isolation. Physio-chemical
changes occurring during ripening impart various changes
to fruit in terms of firmness and flavor. These biochemi-
cal events were regulated by several ripening genes and
understanding their underlying mechanisms and inhibition
of ripening genes by novel ethylene inhibitors is of major
interest. Expansins are the major cell wall proteins involved
in breakdown of non-growing tissues through dissocia-
tion of hydrogen bonds between cellulose microfibrils and
crosslinking cell wall glycosides (Ali et al. 2004). The gene
expression studies of expansin genes could suggest the tex-
tural changes occurring during fruit ripening as observed in
few climacteric fruits viz. mango (Reddy et al. 2017), Peach
(Hayama et al. 2003), pear (Hiwasa et al. 2003), and banana
(Harrison et al. 2001). So far, there is very limited work
reported in guava related to expression of ripening genes.
As ethylene triggers the fruit ripening process in climacteric
fruits, studying the expression of genes involved in ethylene
biosynthesis would validate the efficacy of 1-MCP, used as
an ethylene inhibitor. Thus, ACC synthase and expansin are
two good candidate genes for studying the ripening behavior
of guava fruits after exposure to ethylene inhibitor. Usage of
emerging molecular tools for gene expression studies would
provide greater insights and in-depth prospects of fruit rip-
ening and its regulation at genetic level. Keeping in view of
the above gaps, the current research work has been under-
taken to examine and understand the action of 1-MCP in
guava fruit during ripening and storage.
Materials and methods
Plant material and treatments
Guava fruits cv. Arka Mridula were harvested manually
at mature green stage from research plots of ICAR-IIHR,
Bengaluru ­(13o71 N latitude, 7­ 2o291E longitude and 890
MSL altitude) and transported immediately to the laboratory.
Later the fruits were washed, cleaned and sorted to remove
the blemished and diseased fruits from the lot. Eighteen mg
of amorphous 1-MCP was weighed accurately and taken into
a 15 ml test tube. Through the airtight rubber septa fixed to
the test tube, 1 ml of distilled water was injected into it using
a calibrated syringe. 1-MCP powder inside the test tube was
dissolved by shaking gently to liberate 1-MCP gas. From
the liberated 1-MCP in the test tube, calculated amount of
gaseous 1-MCP (0.6 ml for making it to a final concentration
13
Acta Physiologiae Plantarum (2022) 44:125
of 500 ppb) was taken using calibrated syringe and injected
into the 18L capacity airtight desiccators in which 6 kg of
guava fruits were placed. The fruits were removed after 18 h
of exposure and packed in CFB boxes in replications of three
with 3 kg per replication. These boxes were stored in walk-
in cold rooms set at temperatures of 12 ± 1 °C with relative
humidity of 85–90%. Various parameters were studied at
specified intervals of storage.
Observations recorded
Fruit firmness and pectin methylesterase
The firmness of the fruits was measured using Instron-
Universal testing machine (Model 4201, USA). A probe of
8 mm diameter was used, the firmness was calculated and
expressed as Newton (N). PME activity in the guava pulp
was estimated as per the method described by Ashraf and
Asbi (1993) and expressed as units/ml.
Respiration and ethylene production rates
The respiration and ethylene production rate of the treated
and untreated fruits were measured during their storage at
different temperatures using head space technique (Rao and
Rao 2008). Five replications were maintained for each treat-
ment. The ­CO2 evolution was calculated and expressed as
mg ­kg−1 ­h−1. After measuring the respiration, headspace eth-
ylene concentration was measured using ethylene analyser
(Bioconservacion, ppm ethylene). The ethylene evolution
was calculated and expressed as µl ­kg−1 ­h−1.
Ascorbic acid and total antioxidant content
Ascorbic acid content of the guava pulp was estimated by
2,6-dichlorophenol indophenol titration method, with some
modifications (oxalic acid is used in place of meta-phos-
phoric acid) (Ranganna 1986). Total antioxidant capacity
(FRAP and DPPH), free radical scavenging abilities were
estimated using the methods described by (Shivashankara
et al. 2010) and expressed as mg of ascorbic acid equivalent
(AAE) antioxidant capacity for FRAP and percentage (%)
for DPPH.
Total phenolic and total flavonoid content
Total phenols, and flavonoids, were estimated using the
methods as described by (Shivashankara et al. 2010). The
prepared solution for phenols was then kept for 90 min and
absorbance was recorded at 760 nm using a spectrophotom-
eter (T80 + UV/VIS spectrophotometer, PG instruments
Ltd, UK). The total phenolic content was expressed as mg
of gallic acid equivalents. The prepared solution for total
Acta Physiologiae Plantarum (2022) 44:125 Page 3 of 9 125

flavonoids was kept for 40 min and absorbance was recorded

at 510 nm using a spectrophotometer. The total flavonoids
content was expressed as mg of catechin equivalents.

Sugar to acid ratio

The method of AOAC (1990) was adopted for estimating

the sugar to acid ratio and calculated using degree brix and
percentage acid and results expressed as percentage acid.

Gene expression study

At regular intervals (7, 14, 21 days), samples were drawn

and processed for RNA isolation immediately using modi-
fied method of Reddy et al. (2015). In the modified protocol,
1–2 g of guava pulp with seeds was taken into DEPC-treated
prechilled mortar and pestle and crushed thoroughly into a
fine powder using liquid nitrogen (− 196 °C). Three ml of
1:1 pre-heated extraction buffer (Table 1) and phenol tris
equilibrated pH 8.0 (SRL pvt. ltd) was added to the fine
powder along with a pinch of PVP (Polyvinyl pyrrolidone),
which froze automatically and allowed to thaw completely
with intermittent grinding, then 500 µl of DEPC-treated
water was added immediately and mixed thoroughly. The
mixture was transferred into 2 ml micro-centrifuge tubes
which were incubated at room temperature (25–32 °C) for
about 10 min and then 200 µl of 100% (v/v) chloroform
was added equally to all the tubes. The tubes were vortexed
thoroughly for 1 min and incubated for 10 min at room tem-
perature prior to centrifugation at 12,500 rpm for 12 min
using table top micro-centrifuge (Spinwin- MC03, Tarsons
products pvt ltd.). The aqueous phase was carefully trans-
ferred into 1.5 ml DEPC-treated Eppendorf tube without
disturbing the phases. Pre-chilled isopropanol of around
600 µl was added to the aqueous phase, followed by thor-
ough vertexing to form a milky white gel form on the top of
the tube and were incubated at room temperature for another
15 min. Immediately after incubation, the tubes were cen-
trifuged using table top micro-centrifuge at 12,500 rpm for
8 min to sediment the RNA in the bottom (Pellet formation
stage). The upper aqueous phase was discarded carefully
without disturbing the pellet, followed by ethanol wash
using 1 ml of prechilled ethanol 75% (MB- grade). Discard
the ethanol carefully and RNA pellet was air dried at room
temperature in a clean area. Based on the pellet size, sterile
Table 1  Extraction buffer components
Components Amount/10 ml
3 M NaOAc 1000 µl
0.5 M EDTA 200 µl
SDS 0.1 g
DEPC water 8.70 ml
Fig. 1  Quality evaluation of RNA isolated from ‘Arka Mridula’ guava
fruits exposed to 1-methylcyclopropene (1-MCP) and stored at 12 °C
[Lane 1: seven days control, Lane 2: seven days 1-MCP treated, Lane
3: 14 days control, Lane 4: 14 days 1- MCP treated, Lane 5: 21 days
control, Lane 6: 21 days 1-MCP treated]
DEPC-treated water was added (15–40 µl) to the tubes con-
taining RNA and incubated at 60 °C for 10–15 min, and
cooled immediately over ice flakes for about 10 min and the
RNA was finally stored at − 80 °C for future use.
RNA quality analysis and its quantification
The quality of isolated RNA was estimated using 10 X TBE
gel electrophoresis (Fig. 1) and quantity was estimated
spectrophotometrically using a nano-spectrophotometer
(Nabi Microdigital UV/VIS NB1-A-190201, South Korea)
(Table 2).
cDNA synthesis and gene expression
The RNA extracted and tested by above methods was
treated with Turbo DNA f­ ree™ kit (Thermo Fisher Scien-
tific, Lithuania) to remove the DNA present in the RNA.
Dnase treated RNA was used for synthesis of cDNA using
Hi-cDNA synthesis kit (HiMedia Laboratories Pvt. ltd,
India). Later gene specific primers were designed using
multiple sequence alignment (CLUSTALW 2.1) to check
Table 2  Quality and yield of RNA isolated from pulp of guava using
modified Reddy et al. (2015) protocol
Sample A260/A280 Concen-
tration
(ng/μl)
Guava pulp 1.98 2653.3
Guava peel 2.02 1856.9

13
125 Page 4 of 9 Acta Physiologiae Plantarum (2022) 44:125

the homology, most similar sequence was taken and prim-
ers were designed using integrated DNA technologies
(IDT) for both the genes (Expansin and ACC synthase) and
synthesized at Bioserve biotechnologies India Pvt. Ltd.
25 s rRNA (AY651067) gene was used as housekeeping
(Reference) gene in RT-qPCR study. The designed primers
were shown below.
Statistical analysis
The observations recorded under each parameter were sta-
tistically analyzed using factorial completely randomized
design. The treatment means were compared using Duncan's
new multiple range test, the analysis was carried out using
R-software (version-3.6.2) with agricolae package.

Primer sequence and their respective annealing temperature

Sl.no Gene specific Primer sequence Annealing Results and discussion
primer tempera-
ture Fruit firmness and PME activity
1 Expansin F- 5’CCT​GCT​ 58 °C
(Pgexp1) ACG​AGA​TGA​ Fruit firmness is one of the important factors determin-
AGT​3’ ing the shelf life of a fruit (Abreu et al. 2012.). Firmness
R- 5’TCC​AGC​
of a fruit is mainly due to the physical properties of the
ACT​TCG​ATA​
TGG​3’ individual cell walls and middle lamella containing pectin-
2 ACC synthase F5’TCA​TCT​ 60 °C substances that act as cementing material. Fruit firmness
(Pgaccsyn1) TCT​AAT​ATC​ at harvest was 3.87 N, which decreased gradually with the
GAA​TCG​AAA​ prolongation of storage period. Guava fruits exposed to
TCG​GTT​CTG​
1-MCP were firm and exhibited higher firmness (2.17 N)
GTG​GA3’
R- 5’ACC​TCT​ during 21 days of storage period at 12 °C while the untreated
TTT​GAT​ fruits had poor firmness (0.23 N) (Fig. 2). The rapid loss
TCTC3’ of firmness in untreated guava fruits could be attributed
3 25 s rRNA F-5’ACA​TTG​ to the pectin solubilization and cell wall degradation with
(AY651067) TCA​GGT​GGG​
increased activity of pectin methylesterase (PME), during
GAG​TT3’
R- 5’CCT​TTT​ storage period. However, rate of increase was higher in
GTT​CCA​CAC​ untreated fruits compared to 1-MCP-treated fruits. Textural
GAG​ATT3’ degradation can be directly correlated to texture degrading
enzyme during storage (Fig. 2). The reduced PME activ-
The PCR product was run on 1.5% agarose gel with ity in 1-MCP-treated fruits was due to retarding effects of
50 bp DNA (Sigma Aldrich- S7025) ladder to check the 1-MCP on fruit softening by delaying the ethylene and res-
amplification and to standardize the annealing tempera- piration peak. Untreated fruits showed twofold higher PME
ture. Further, the amplified primers and annealing temper- activity when compared to treated fruits. The inhibition of
atures were used to conduct qPCR (real time polymerase PME activity by 1-MCP treatment has also been reported
chain reaction) (QuantStudio 7, Thermo Fisher scientific) in persimmon (Khademi et al. 2014), banana (Lohani et al.
using Kapa Biosystems (KAPA SYBR FAST- KK4601)
Kit (Table 3). ...... Fruit firmness ____ PME activity
4 3
3.5 a A
2.5
PME activity (units/ml)

3 b
Fruit firmness (N)

B 2
2.5 c
2 1.5
Table 3  Real-time qPCR Kapa SYBR components CD D C
1.5
1
Sl. No. Components Volume 1 E

used (µl) d
e 0.5
0.5 f
0 0
1 cDNA 2.0 At Harvest 7 14 21
1 Primer forward 0.4 Storage period (days)

2 Reverse primer 0.4 1-MCP 500 ppb Control 1-MCP 500 ppb Control
3 Fast ROX 0.4
4 SYBR 10 Fig. 2  Changes in fruit firmness (N) and pectin methyl esterase
5 Filter sterilized ­H2O 6.8 (units/ml) activity in ‘Arka Mridula’ guava fruit treated with ethylene
Total volume 20.0 inhibitor 1-MCP and stored at 12 ℃. *Means denoted by a different
letter indicate significant differences between treatments

13
Acta Physiologiae Plantarum (2022) 44:125 Page 5 of 9 125

2.5 ...... Ethylene rate ____ Respiration rate

180 45
Relative mRNA expression

2 a a
160 A 40

Respiration rate (CO2 mg/kg/h)

1.5 bc b
140 cd 35
b de

Ethylene rate (µl/kg/h)

b b
1 120 30

0.5 c
d
0 gh
7C 7T 14C 14T
Days after treatment
Fig. 3  RT-PCR expression analysis of expansin (Pgexp1) in ‘Arka
Mridula’ guava after 7, 14, 21 days of 1-MCP treatment stored at
12 ℃ [7C- seven days control, 7 T-seven days 1-MCP treated, 14C-
14 days control, 14 T- 14 days 1-MCP treated, 21C- 21 days control,
21 T-21 days 1-MCP treated]. *Means denoted by a different letter
indicate significant differences between treatments
2004), kiwifruit (Jhalegar et al. 2012) and papaya (Ahmad
et al. 2013). Rawan et al. (2017) and Sachin et al. (2021)
also reported the reduction in firmness during storage of
guava fruits. Therefore, a possible reason for maintaining
the fruit firmness in cv. Arka Mridula was directly related
to inhibition of PME activities. Present study also showed
(Fig. 3) suppressed expansin protein activity due to 1-MCP
action. 1-MCP had significantly repressed the expression of
Pgexp1gene compared to its induction in untreated fruits.
Expression of Pgexp1 transcripts were found to be induced
after 14 days of storage in treated fruits which might be due
to action of 1-MCP in regulating the expansin protein up to
14 days of storage, where 1-MCP-treated fruits had higher
texture with reduced PME activity during storage. A surge
in expression of Pgexp1 gene after 21 days of storage could
be correlated with the excess production of ethylene (Fig. 1).
This ethylene upsurge had initiated fruit ripening process
with augmented textural breakdown, whereas untreated
fruits had clearly exhibited reduced fruit texture with
increased PME activity from the beginning of storage. The
results clearly showed up-regulation of Pgexp1 in untreated
fruits whereas it was downregulated in 1-MCP-treated fruits
(Fig. 3). Similar results of 1-MCP induced down-regulation
of expansin proteins was noticed in climacteric fruits such
as peaches (Obenland et al. 2003), Banana (Trivedi and Nath
2004), melons (Nishiyama et al. 2007), mango (Reddy et al.
2017) and papaya (Zhu et al. 2019).
Respiration and ethylene evolution rate
The rate of respiration was found to accelerate gradually
during ripening of guava fruits. The control guava fruits
exhibited respiratory climacteric (162.1 ­CO2mg/kg/h) on
21st day of storage at low temperature (12 °C), while it was
suppressed (54.57 C ­ O2mg/kg/h) in 1-MCP-treated fruits
(Fig. 4). The delay and suppression of respiratory climacteric
100 f 25
80 20
g
60 ij hi 15
21C 21T jk
40
l 10
D C B
20 5
J J J E
0 I FGH FG F 0
3 7 10 14 18 21
Storage period (days)
1-MCP 500 ppb Control 1-MCP 500 ppb Control
Fig. 4  Changes in rate of respiration ­(CO2 mg/kg/h) and ethylene pro-
duction (µl/kg/h) in ‘Arka Mridula’ guava fruit treated with ethylene
inhibitor 1-MCP and stored at 12 ℃. *Means denoted by a different
letter indicate significant differences between treatments
in 1-MCP-treated fruits could be well correlated with the
reduced rate of ethylene production in treated guava fruits.
However, untreated fruits of guava recorded maximum rate
of ethylene production (40.96 µl/kg/h) on 21st day of storage
at 12 ℃. The reduction in respiration rates of 1-MCP-treated
fruits could be due to suppression of ethylene production
which in turn decelerated the rate of ripening and respira-
tion in fruits. This hypothesis was backed by greater reten-
tion of texture and reduced pectin methylesterase activity in
1-MCP-treated fruits (Fig. 2). The greater delay in ethylene
production rates of 1-MCP-treated fruits was mainly due
to the interference of 1-MCP with autocatalytic production
of ethylene by binding to the available ethylene receptors
(Reddy et al. 2017). Similar reports of ethylene inhibition
by 1-MCP were reported in apple (Watkins 2006), papaya
(Manenoi 2007) and guava (Sachin et al. 2021). Expression
analysis of ACC synthase (Pgaccsyn1) gene showed that the
variation in expression of Pgaccsyn1 at different intervals of
storage was mainly dependent on ethylene production in the
fruit pulp. The ACC synthase gene (Pgaccsyn1) displayed
down-regulation on 7th day of storage owing to the action
of 1-MCP on autocatalytic ethylene production and a sud-
den upsurge/up-regulation was observed from 14th day of
storage (Fig. 5). The untreated control guava fruits exhib-
ited uniform expression of the ACC synthase gene indicat-
ing that the Pgaccsyn1 gene expression was mediated by
ethylene and its up-regulation coincided with the shift in
ripening stage from mature green stage to early ripening
stage (Fig. 4). These results are indicative for the effects
of 1-MCP, which might depend on fruit ripening stage,
cultivar and ethylene receptors (Li et al. 2001; Watkins
2006). The recovery of ethylene sensitivity in treated guava
fruits was mainly due to the appearance of fresh ethylene

13
 125 Page 6 of 9 Acta Physiologiae Plantarum (2022) 44:125

Relative mRNA expression

9
a
declined thereafter in untreated fruits, while in 1-MCP-
8 treated fruits the raise of AAC was delayed due to reduced
7
6
ripening rate associated with suppressed ethylene action.
5 However, even after 21 days of storage 1-MCP-treated fruits
4 recorded an increasing trend in AAC without showing any
3
signs of declining indicating that, 1-MCP-treated fruits were
2 b
1
b
c
b still at mature green stage during 21 days of storage under
d
0 low temperature (Fig. 6). Similar trend of increasing AAC
7C 7T 14C 14T 21C 21T content during fruit ripening and further declining during
Days after treatment
storage period was noticed in jujube (Zhong and Xia 2007),
and mango (Reddy et al. 2017). Total antioxidant content
Fig. 5  RT-PCR expression analysis of ACC synthase (Pgaccsyn1) in
‘Arka Mridula’ guava after 7, 14, 21 days of 1-MCP treatment stored
(TAC-FRAP) was in direct correlation with the AAC of the
at 12 ℃ [7C- seven days control, 7 T-seven days 1-MCP treated, 14C- guava fruits. TAC was maintained throughout the storage
14 days control, 14 T- 14 days 1-MCP treated, 21C- 21 days control, period in 1-MCP-treated fruits and maximum (251.77 mg
21 T-21 days 1-MCP treated]. *Means denoted by a different letter AAE/100 mg) was recorded at seven days of storage. The
indicate significant differences between treatments
retention of TAC in 1-MCP-treated fruits might be due to
slower ripening and retention of phenolic compounds and
receptors in due course of storage. However, the expression ascorbic acid. The reduction in TAC content in untreated
of Pgaccsyn1 was regulated significantly by 1-MCP mol- fruits could be correlated with reduced AAC content dur-
ecule through inhibition of autocatalytic ethylene production ing the storage period. Similar, retention in TAC in 1-MCP-
resulting in slow down of guava fruit ripening (Fig. 5). The treated loquat fruits was reported by Cao et al. (2011).
results were in conformity with the findings of Reddy et al.
(2017) and Li et al. (2020) in mango who reported similar DPPH and FRAP
regulation of ethylene by 1-MCP using ACC synthase gene
expression studies. Antioxidants are molecules which reduce the oxidation pro-
cess which in turn reduces the production of free radicals.
Ascorbic acid content (AAC) and total antioxidant In the present study, free radical scavenging activity (RSA)
content (TAC) in fruits tend to decline from the day of harvest (Fig. 7).
Maximum RSA was recorded at the day of harvest (98.36%
Ascorbic acid content (AAC) in guava fruits exhibited a DPPD), but the RSA was retained and rate of reduction was
sharp raise initially followed by a gradual decline during seen reduced in 1-MCP-treated fruits. At the end of 21 days
ripening and storage in general (Fig. 6). However, the actual of storage 1-MCP-treated fruits had 82.49% DPPH of RSA,
AAC in guava fruits depends on the stage of maturity/ripen- whereas control fruits showed 55.64% DPPH. Reduced TAC
ing and also on its integral varietal character. AAC reached in fruits might be due to deduction in phenols and ascorbic
maximum (171.34 mg/100 g) on 14th day of storage and acid during the storage period, the results were in conformity

...... TAC ____ AAC FRAP ............

DPPH
300 200 120 300
TAC- (mg ascorbic acid eqivalence/100g)

mg ascorbic acid eqivalence/100g

a 180 275
A 1
A
250 100 B2 250
160
B aa c 225
C
AAC (mg/100g)

140
200 c 80 bb 200
b 120 d C3
DPPH (%)

175
150 d 100 ee ff
60 150
E 4
80
100 e D f E 125
60 40 D F 100
F 75
40
50
20 20 50
0 0
25
At Harvest 7 14 21 0 0
At Harvest 1 2 3
Storage period (days)
Storage period (Weeks)
1-MCP 500ppb Control 1-MCP 500ppb Control
1-MCP CONTROL

Fig. 6  Changes in total antioxidant content (mg ascorbic acid equiva-

lence/100 g) and ascorbic acid content (mg/100 g) of ‘Arka Mridula’ Fig. 7  Changes in total antioxidant content (DPPH and FRAP) of
guava fruit treated with ethylene inhibitor 1-MCP and stored at 12 ℃. ‘Arka Mridula’ guava fruit treated with ethylene inhibitor 1-MCP and
*Means denoted by a different letter indicate significant differences stored at 12 ℃. *Means denoted by a different letter indicate signifi-
between treatments cant differences between treatments

13
Acta Physiologiae Plantarum
with the findings of (Lata et al. 2018) in papaya fruits. Kim
et al. (2007) also reported the reduction in antioxidant activ-
ity in 1-MCP-treated mangoes during the storage period.
Kolniak et al. (2014) reported a reduction in the loss of
DPPH activity in apples by 1-MCP during storage. However,
reduction in DPPH and FRAP during storage was related
to the oxidation of polyphenolic compounds, and other
phenolic compounds or transformation of active phenolic
compounds into inactive compounds (Hossain et al. 2021).
Total phenol content and total flavonoids content
Phenolic compounds show a good antioxidant activity and
are integral part of human diet, which reduces the risk of
dangerous human diseases. Irrespective of the treatments
total phenol content (TPC) was decreased gradually during
the storage, at harvest total phenol content was 714.58 mg
GAE/100 g FW from which the deviation was noticed
(Fig. 8). Control fruits recorded and the maximum decrease
in TPC when compared to 1-MCP-treated fruits. Control
fruits recorded lowest TPC (120.75 mg GAE/100 g FW) on
21st day of storage. The highest TPC (651.16 mg GAE/100 g
FW) was recorded in the 1-MCP-treated fruits on 7th day of
storage. Interestingly, 1-MCP-treated fruits had higher TPC
(528.93 mg GAE/100 g FW) even after 21st day of storage at
12 °C. This might be due to the action of 1-MCP in reducing
the respiration rates and slower ripening of the fruits which
had helped in retention of TPC in guava fruits. Lee et al.
(2017) recorded maximum TPC content in 1-MCP-treated
apple fruits when compared to control. The higher oxidation
rate of total phenols by PPO could have contributed to the
reduction of total phenols in control fruits. Similar results
where reduction in TPC content during storage was noticed
in litchi fruits by (Hossain et al. 2021).
TPC
800
700
mg galic acid equivalence/100g
600
500
400 d E
300
200
100
0 0
At Harvest 1 2
1-MCP
Fig. 8  Changes in total phenolic content (mg GAE/100 g) and total
flavonoids content (mg CE/100 g) of ‘Arka Mridula’ guava fruit
treated with ethylene inhibitor 1-MCP and stored at 12 ℃. *Means
denoted by a different letter indicate significant differences between
treatments
(2022) 44:125 Page 7 of 9 125
Total flavonoids are the major compounds contributing
for the total antioxidants, total flavonoids content (TFC)
was seen increasing during the storage period. At harvest
TFC was 123.25 mg catechin equivalence/100 g FW which
was gradually increased as the storage period prolonged.
In the current study, maximum TFC was recorded at full
ripe stage compared to mature green stage, control fruits
recorded maximum TFC (334.03 mg CE/100 g FW) after
21 days of storage while 1-MCP-treated fruits exhibited
211.50 mg CE/100 g FW after 21 days of storage indicating
the slower rates of ripening in 1-MCP-treated fruits when
compared to control fruits. The increased TFC in control
fruits might be due to the increased respiration and ripen-
ing rates. Bhandari and Lee (2016) also noticed increased
flavonoids content during ripening stage when compared to
unripe stage in tomato. Zhang et al. (2013) also reported
that 1-MCP treatment remarkably inhibited the accumula-
tion of total flavonoids in avocado. Addai et al. (2013) also
reported the increase in TFC as the storage period prolonged
and maximum TFC was observed during fully ripen stage.
Sugars to acid ratio (S:A ratio)
Sugars to acid ratio was significantly higher in untreated
fruits (Fig. 9). Generally, total sugars tend to increase and
TA decreases as guava fruit ripens. A decline in S:A ratio
was noticed in 1-MCP-treated guavas; it was clearly evident
that the effect of 1-MCP in delaying the increase of S:A
ratio by inhibiting the biochemical reactions related to the
ripening. The observed reduction in S:A ratio was mainly
due to the increase in titratable acidity (TA). The higher TA
in 1-MCP-treated fruits could be due to the inhibitory effect
of 1-MCP in reducing the respiration of fruits along with
reduced loss of existing acids during ripening. Sivakumar
et al. (2012) also noticed reduced S:A ratio in 1-MCP-treated
mango fruits. Untreated fruits showed higher S:A ratio at
seven days after storage thereafter a decline in S:A ratio
TFC .............
400
was observed which might be due to the utilization of major
a A 350
mg catechin equivalence/100g
b
c 300
B 30
C 250
Sugar : Acid Ratio (% acid)
a
25
D 200 b c
e 20
150 e de d
F 15
100
f 10
50
5
3 0
At Harvest 1 2 3
Storage period (Weeks) Storage period (weeks)
CONTROL 1-MCP CONTROL
1-MCP CONTROL
Fig. 9  Changes in sugar to acid ratio (% acid) of ‘Arka Mridula’
guava fruit treated with ethylene inhibitor 1-MCP and stored at 12 ℃.
*Means denoted by a different letter indicate significant differences
between treatments
13
125 Page 8 of 9
acids and sugars for increased respiration. Sakhale et al.
(2018) also noticed increase in total sugars during ripen-
ing of mango fruits due to breakdown of complex organic
metabolites into simple molecules. Further decrease in S:A
ratio might be due to utilization of other acids and sugars
as primary substrate for respiration. The results were also
in conformity with the findings of (Faasema et al. 2012) in
1-MCP-treated mango fruits.
Conclusion
1-MCP treatment aided in extending the storage life of guava
fruits cv. Arka Mridula for 21 days at 12 ℃. The treated
fruits retained firmness owing to lower PME enzyme activ-
ity. The fruit quality assessed at different intervals revealed
that, all the biochemical and physiological parameters were
significantly influenced by 1-MCP treatment. Further, the
RT-qPCR gene expression analysis validated the posi-
tive effect of 1-MCP in altering the expression of texture
(expansin- Pgexp1) and ethylene biosynthesis (ACC syn-
thase- Pgaccsyn1) related genes. The current study opens up
the potential for biotechnological manipulations of ripening
genes in guava fruits, and simultaneously provides the vali-
dation opportunity for other climacteric fruits.
Author contribution statement SAJ—Research Fellow,
conducted postharvest physiology experiments, biochemical
analysis for the experiment. Dr. DVSR—conceptualization
of the experiment conducted. Dr. RK—contributed in ana-
lytical work and manuscript preparation. Dr. VC—contrib-
uted in quality analysis, manuscript preparation and original
breeder of the variety. Dr. CKN—contributed in manuscript
preparation and first authors advisory council member. Dr.
KR—contributed in manuscript preparation and first authors
advisory council member. Dr. VRR—contributed in analyti-
cal work and manuscript preparation.
Acknowledgements The authors would like to acknowledge P.G.
School, ICAR-IARI, New Delhi, India for providing the Ph.D. fellow-
ship for the first author. We also acknowledge ICAR-IIHR, Bengaluru
for providing Laboratory facilities.
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