Postharvest Biology and Technology 186 (2022) 111846

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

γ-Aminobutyric acid regulates mitochondrial energy metabolism and

organic acids metabolism in apples during postharvest ripening
Jie Zhu a, b, Canying Li a, b, *, Yiting Fan a, b, Linhong Qu a, b, Rui Huang a, b, Jiaxin Liu a, b,
Chenyang Zhang a, b, Yonghong Ge a, b, *
a
College of Food Science and Technology, Bohai University, Jinzhou, 121013, PR China
b
National and Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, Jinzhou,
121013, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: “Golden Delicious” apples were treated with 1.0 mmol L− 1 γ-aminobutyric acid (GABA) to evaluate the changes
Apple fruit of mitochondrial energy metabolism and organic acids metabolism during ripening. Results showed that GABA
γ-Aminobutyric acid dipping enhanced malic, citric, succinic, and tartaric acids contents in apple fruit. GABA treatment enhanced
Energy metabolism
phosphoenolpyruvate carboxylase (PEPC), NADP-dependent isocitrate dehydrogenase (NADP-IDH), cytoplasm
Organic acid
aconitase (Cyt-ACO), citrate synthase (CS), and cytosolic NAD-dependent malate dehydrogenase (NAD-MDH)
activities, but decreased cytosolic NAD phosphate-dependent malic enzyme (NADP-ME) activity. GABA also
enhanced the expressions of MdPEPC, MdNADP-IDH, MdCyt-ACO, MdNAD-MDH and MdCS, and decreased
MdNADP-ME expression in apples. Moreover, higher level of energy charge, adenosine triphosphate (ATP)
content, H+-ATPase, cytochrome C oxidase (CCO), succinic dehydrogenase (SDH), and Ca2+-ATPase activities
were recorded in GABA-treated fruit. Additionally, GABA up-regulated MdSDH, MdCCO, MdH+-ATPase, and
MdCa2+-ATPase expressions of apples. In conclusion, GABA could regulate the key gene expression and enzyme
activity in mitochondrial energy metabolism and organic acids metabolism during ripening to maintain freshness
of apple fruit.

1. Introduction quality of apple fruit during storage.

Apples (Malus domestica Borkh. cv. Golden Delicious), the main
cultivar grows in China, are in large size with golden surface, abundant
juice, and reddish hue when ripening (Ge et al., 2019). Nevertheless,
harvested fruit ripens rapidly to cause quality degradation and fungi
invasion during storage, therefore resulting in huge losses during post­
harvest logistics. It has been suggested that the loss of disease resistance
in horticultural produce after harvest might be due to an energy deficit
(Tang et al., 2010; Zhang et al., 2017). Postharvest benzothiadiazole or
methyl jasmonate treatment enhanced resistance in apples and Nanguo
pears through regulating mitochondrial energy metabolism (Cao et al.,
2014; Li et al., 2021). Study has also shown the supply of cellular energy
and respiratory substrate is crucial for physiology and metabolism of
pineapple fruit after harvest (Li et al., 2018). Postharvest 1-methylcyclo­
propene (Liu et al., 2016a), trisodium phosphate (Ge et al., 2019),
chlorogenic acid (Shu et al., 2020), calcium chloride (Han et al., 2021),
and hydrogen sulfide (Chen et al., 2021) treatments could maintain
γ-Aminobutyric acid (GABA) is known to accumulate in plant tissues
as a compensation for various stressful conditions and involves in
signaling or regulatory mechanisms, nitrogen and carbon metabolism,
and amino acid biosynthesis (Bown and Shelp, 2016; Zhu et al., 2022).
Organic acids in plants can serve as an osmoticum to maintain pH and
link with nitrogen metabolism (Samanta et al., 2020). Malic, citric, and
tartaric acids are the main organic acids in apple fruit, which correlate
with enzyme activities and transcription level of NADP-dependent iso­
citrate dehydrogenase (NADP-IDH), NAD phosphate-dependent malic
enzyme (NADP-ME), and phosphoenolpyruvate carboxylase (PEPC) (Liu
et al., 2016a). It has proven that exogenous GABA treatment affects
GABA shunt to regulate endogenous GABA production, citric acid
degradation, tricarboxylic acid (TCA) cycle, and alleviate cellular
damage from diverse stress in fruit (Famiani et al., 2009; Xiang et al.,
2016; Sheng et al., 2017a, b). A study in Malus hupehensis leaves showed
that exogenous GABA increased organic acid content under alkaline
stress by enhancing citrate synthase (CS), cytosolic NAD-dependent

* Corresponding authors at: College of Food Science and Technology, Bohai University, Jinzhou, 121013, PR China.
E-mail addresses: cora_51@163.com (C. Li), geyh1979@163.com (Y. Ge).

https://doi.org/10.1016/j.postharvbio.2022.111846
Received 25 November 2021; Received in revised form 31 December 2021; Accepted 10 January 2022
Available online 13 January 2022
0925-5214/© 2022 Elsevier B.V. All rights reserved.
J. Zhu et al.
malate dehydrogenase (NAD-MDH) and cytoplasm aconitase (Cyt-ACO)
activities (Li et al., 2020a, b). Recent studies have revealed that exog­
enous GABA dipping increased citric acid level and enhanced quality of
citrus fruit during storage (Sun et al., 2013; Sheng et al., 2017a, b). Han
et al. (2018) demonstrated that GABA dipping suppressed the decrease
of malic acid content by repressing malate degradation and promoting
malate biosynthesis, accompanying increased oxalate and succinate
contents in ‘Cripps Pink’ fruit during ripening. Additionally, organic
acids content is also regarded as one of the indicators of mitochondrial
activities, which supplies intermediate material for primary and sec­
ondary metabolism in plant (Fu et al., 2019).
Endogenous GABA is mainly formed from glutamate by cytosolic
enzyme glutamate decarboxylase (GAD), and under the action of GABA
transaminase (GABA-T) and succinic semialdehyde dehydrogenase
(SSADH), GABA eventually generates succinic acid and returns to TCA
cycle, and generate nicotinamide adenine dinucleotide hydrogen
(NADH) which serves as mitochondrial respiratory substrates to produce
ATP (Michaeli et al., 2011; Ma et al., 2015; Wu et al., 2020). A study has
shown that GABA-treated zucchinis have a higher level of endogenous
GABA, ATP and NADH, implying that improving the oxidative phos­
phorylation ability of cells can maintain higher post-harvest quality
(Palma et al., 2019). Studies have proven that Ca2+-ATPase, H+-ATPase,
succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO)
regulated ATP generation through TCA cycle and electron transport
chain in cells (Li et al., 2016; Lyu et al., 2019; Wang et al., 2021; Xie
et al., 2021). Recent study demonstrated that trisodium phosphate
treatment could increase the activities of SDH, Ca2+-ATPase, H+-ATPase
and CCO, and delayed the decline of ATP content and energy charge,
suggesting that postharvest trisodium phosphate maintain the quality of
apples by modulating mitochondrial energy metabolism (Ge et al.,
2019). All these findings imply that there are cross-talk among GABA
shunt, TCA cycle, energy and organic acids metabolism in fruit. How­
ever, there are few studies on the indirect regulatory mechanisms of
organic acids metabolism and energy metabolism by GABA shunt after
exogenous GABA treatment. A previous study in our laboratory
demonstrated that GABA dipping could regulate GABA shunt and reac­
tive oxygen metabolism in apples (Zhu et al., 2022). Therefore, we
proposed a hypothesis that exogenous GABA treatment stimulates GABA
shunt and change organic acids metabolism, which affects the energy
status to maintain freshness of apple fruit.
The current study was carried out to evaluate the influence of GABA
dipping on activities and gene expressions of PEPC, NADP-IDH, Cyt-
ACO, NAD-MDH, CS, NADP-ME, H+-ATPase, SDH, CCO and Ca2+-
ATPase, and energy state and organic acid accumulation in apples
during ripening; to elucidate the relationship among GABA shunt,
organic acids and energy metabolism.
2. Materials and methods
2.1. Fruit and treatment
Apple fruit (Malus domestica Borkh. cv. Golden Delicious) at com­
mercial maturity with 12.52 newton (N) of flesh firmness and 10.71 % of
soluble solid content were collected in Jinzhou (121 ◦ 43′ E, 41 ◦ 32′ N),
China, and transported by car to postharvest laboratory in Bohai Uni­
versity. Harvested fruit (180) without visual defects was randomly
divided into two groups (90 fruit per group). The first group was soaked
in 1.0 mmol L− 1 GABA which was screened by a previous study (Zhu
et al., 2022) for 10 min. The second group was soaked in tapping water
(control) for 10 min. All fruit was desiccated at room temperature (24 ±
1 ◦ C with 75 ± 5 % relative humidity) after air-drying for 2 h.
2.2. Sampling
Flesh tissues were excised from 1.0 cm beneath the epidermis of 15
fruit per treatment at two-day intervals from 0 to 15 d following the
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Postharvest Biology and Technology 186 (2022) 111846
method of Ge et al. (2019). The sampled tissues were kept at -80 ◦ C.
2.3. Extraction and determination of organic acids
Malic, citric, succinic, and tartaric acids contents were analyzed
following the method of Liu et al. (2016a) with some modifications.
Frozen tissues (2.0 g) were ground with 4.0 mL of deionized water and
extracted at 50 ◦ C for 30 min. The cooled extract was centrifuged at 4 ◦ C,
8 000×g for 30 min to collect 1.0 mL of the liquid supernatant, and then
added deionized water to 10 mL. After filtering through a 0.45-μm micro
porous membrane, organic acids content was analyzed by
high-performance liquid chromatography (HPLC, 1260 Infinity II)
equipped with a reverse-phase column (XBridge C18, 250 mm × 4.6 mm)
using detection wavelength of 210 nm. Malic, citric, succinic, and tar­
taric acids were eluted by methanol: 10 mmol L− 1 potassium dihydrogen
phosphate (pH 2.5) (5 : 95, v/v) with a flow rate of 0.6 mL min− 1.
Organic acids content was calculated by comparing to the values ob­
tained from a standard curve generated with standard malic, citric,
succinic, and tartaric acids solution, and indicated as g kg− 1 on a fresh
weight.
2.4. Measurement of PEPC, NADP-IDH, Cyt-ACO, NAD-MDH, CS and
NADP-ME activities
Extraction of crude enzyme from apple tissues was performed as
described by Li et al. (2020a, b). Frozen samples (2.0 g) were ground
with 4.0 mL of extraction buffer which was consisted of 0.2 mol L− 1
Tris-HCl (pH 8.2), 10 mmol L-1 isoascorbic acid, and 0.6 mol L-1 sucrose.
The homogenates were centrifuged for 30 min at 8 000×g to collect the
liquid supernatant. Then the supernatant was diluted to 10 mL with
another extraction buffer contained 0.2 mol L-1 Tris-HCl (pH 8.2), 1.0
mol L− 1 Triton X-100, and 10 mmol L-1 erythorbic acid. Four milliliter of
the final solution (10 mL) were centrifuged at 8000×g for 40 min, then
the supernatant was diluted for Cyt-ACO analysis, and the precipitation
was dissolved in extraction buffer for NADP-IDH analysis. Six milliliters
of the final solution was mixed with 6.0 mL of the extraction buffer.
Subsequently, 4.0 mL of the solution were used for NADP-ME and
NAD-MDH analysis, and 8.0 mL of the solution were dialyzed overnight
at 4 ◦ C for PEPC and CS analysis.
PEPC, NADP-IDH, Cyt-ACO, NAD-MDH, CS, and NADP-ME activities
were assayed following Liu et al.’s (2016b) method. The reaction sys­
tems for determination of these enzymes activity are as follows: 200 μL
of 0.4 mol L− 1 Tris-HCl (pH 8.5), 1.5 mL of 0.4 mol L− 1 phosphoenol­
pyruvate, 100 μL of 1.5 mol L− 1 MgCl2, 200 μL of 0.5 mol L− 1 gluta­
thione, 100 μL of 0.2 mol L− 1 KHCO3, 200 μL of deionized water, 300 μL
of 0.5 mol L− 1 NADH, and 500 μL of crude enzyme for PEPC; 1.5 mL of
enzyme solution, 200 μL of 0.6 mol L− 1 NADH, 300 μL of 0.3 mol L− 1
2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid buffer (pH
8.0), 100 μL of deionized water, 100 μL of 0.5 mol L− 1 MnSO4, and 1.0
mL of 5.0 mmol L− 1 sodium isocitrate for NADP-IDH; 1.0 mL of deion­
ized water, 1.0 mL of crude enzyme, 200 μL of 60 mol L− 1 Tris-HCl (pH
8.0), 200 μL of 0.3 mol L− 1 NaCl, and 600 μL of 1.0 mol L− 1 cis-aconitate
for Cyt-ACO; for NAD-MDH, 4.0 mol L− 1 oxaloacetate was instead of
phosphoenolpyruvate, and other reactants were similar to PEPC; 200 μL
of 0.8 mol L− 1 Tris-HCl (pH 9.0), 400 μL of 4.0 mmol L− 1 2-nitrobenzoic
acid, 400 μL of 3.0 mol L− 1 acetyl-CoA, 1.0 mL of crude enzyme, and 1.0
mL of 4.0 mol L− 1 oxaloacetate for CS; 300 μL of 0.4 mol L− 1 Tris-HCI
(pH 7.5), 200 μL of 0.4 mol L− 1 MnSO4, 100 μL of 0.4 mol L− 1 NADP,
800 μL of crude enzyme, 1.4 mL of 5.0 mmol L− 1 malate, and 200 μL of
deionized water for NADP-ME. All enzyme activities were described as
unit (U), where one unit was defined as the oxidation amount of 1.0
mmol L-1 NADH (or NADPH) per minute at 340 nm.
2.5. Assay of ATP, ADP, AMP contents, and energy charge
The contents of ATP, ADP and AMP were measured according to the
J. Zhu et al.
method of Li et al. (2019) by an HPLC equipped with an XBridge C18
reverse-phase column (250 mm × 4.6 mm). AMP, ADP and ATP contents
were expressed as g kg− 1 on a fresh weight which calculated by an
external standard curve. Energy charge was figured up as (ATP + 1/2
ADP)/(ATP + ADP + AMP).
2.6. Assay of Ca2+- ATPase, H+-ATPase, SDH and CCO activities
Crude mitochondria extraction and Ca2+- ATPase and H+-ATPase
activities assay were conducted by inorganic phosphorus kits (Nanjing
Jiancheng Bioengineering Institute, China) following the method of Ge
et al. (2017). The activities of Ca2+- ATPase and H+-ATPase were
expressed as unit (U), where one unit was defined as the release of 1.0
mmol phosphorus per second at 660 nm.
SDH and CCO activities were measured according to the method of
Ge et al. (2017). The reaction solution for SDH activity was composed of
3.0 mL of 50 mmol L− 1 potassium phosphate buffer (pH 7.4), 200 μL of
10 mmol L− 1 2,6-dichlorophenol indophenol, 200 μL of 0.3 mmol L− 1
sodium succinate, and 200 μL of crude mitochondria. For CCO activity
assay, 500 μL of 16 mmol L-1 dimethyl phenylenediamine were added to
the reaction mixture which contained 200 μL of crude mitochondria and
500 μL of 40 mmol L− 1 cytochrome C solution. SDH and CCO activities
were expressed as U, where one unit was defined as the changes of
absorbance for 0.01 at 600 nm and 510 nm, respectively.
2.7. Quantitative real-time PCR (qRT-PCR) analysis
Total RNA of apple tissues was extracted with RNAprep Pure Plant
Kit (Tiangen, Beijing, China), and qRT-PCR analysis was carried out
following Ge et al.’s (2018) method. The gene-specific primers were
designed and synthesized by Sangon Biotech (Shanghai, China) (Sup­
plementary Table 1). qRT-PCR was performed in 96 well plates using
Step One Plus™ instrument (Bio-Rad, USA). Relative expression of
selected gene was calculated using “Comparative 2− ΔΔCT” method with
Mdactin as the reference gene.
2.8. Statistical analysis
All targets were carried out in three independent duplicate and the
triplicate data were presented and plotted with Microsoft Excel 2010.
Statistically significant difference between control and GABA treatment
Fig. 1. Malic (A), citric (B), succinic (C), and tartaric acids (D) contents in apples after GABA treatment during storage at 24 ± 1 ◦ C with a relative humidity of 75 ± 5
%. Vertical bars represent standard errors of the means. Asterisks (*) represent significant difference between control and GABA treatment at level of 0.05 by LSD.
3
Postharvest Biology and Technology 186 (2022) 111846
was performed by least significant difference (LSD) (p < 0.05) using one-
way analysis of variance (ANOVA) of SPSS16.0.
3. Results
3.1. Malic, citric, succinic, and tartaric acids contents
Fig. 1A shows that malic acid content in two groups increased on
days 0–3 and 12–15 of storage, but dropped from 3 to 12 d. Compared
with control, GABA increased malic acid content from 3 to 15 d. On the
third day of storage, the highest amount of citric acid in GABA-treated
fruit was 1.14-times higher than that in control. Additionally, GABA
also significantly increased citric acid content on days 3, 6, and 15
(Fig. 1B). During the storage period, GABA facilitated the production of
succinic acid as compared with control (Fig. 1C). Succinic acid content
in GABA-treated group reached the maximum on day 12, which was
1.47-times higher than that in control. Compared with control, tartaric
acid content in fruit treated with GABA was higher from 3 to 15
d (Fig. 1D). Additionally, tartaric acid content peaked on day 9, which
was 1.34-times higher in fruit treated with GABA than that in control.
3.2. Activities of PEPC, NADP-ME, NADP-IDH, Cyt-ACO, NAD-MDH,
and CS
PEPC activity peaked on day 6 in GABA-treated fruit, whereas two
peaks were recorded on days 3 and 9 in control (Fig. 2A). Moreover,
PEPC activity was lower in untreated fruit than in GABA-treated fruit on
days 3–15 which were 14.81 %, 61.25 %, 20.75 %, 16.23 %, and 41.18
% of control. NADP-ME activity in control maintained at a high level at
first 9 d of storage, and then decreased over the remaining days, which
were consistent with the tendency in fruit treated with GABA (Fig. 2B).
GABA reduced NADP-ME activity in fruit after 6 d which were 26.63 %,
34.66 %, 38.15 %, and 40.51 % of control. Compared with control,
NADP-IDH activity was enhanced by GABA from 0 to 15 d (Fig. 2C). The
activity of Cyt-ACO increased on days 3–15 by 1.21-, 1.16-, 1.07-, 1.14-,
and 1.23-times in fruit treated with GABA as high than control (Fig. 2D).
Fig. 2E shows that NAD-MDH activity in control increased at initial 9
d of stage, but declined afterwards. Compared with control, NAD-MDH
activity in fruit treated with GABA retained at higher level during
postharvest storage, with differences from 3 to 15 d. CS activity indi­
cated similar tendency in fruit treated with GABA and control, which
J. Zhu et al. Postharvest Biology and Technology 186 (2022) 111846

Fig. 2. PEPC (A), NADP-ME (B), NADP-IDH (C), Cyt-ACO (D), NAD-MDH (E) and CS (F) activities in apples after GABA treatment during storage at 24 ± 1 ◦ C with a
relative humidity of 75 ± 5 %. Vertical bars represent standard errors of the means. Asterisks (*) represent significant difference between control and GABA treatment
at level of 0.05 by LSD.

increased at initial 6 d of storage and decreased subsequently (Fig. 2F).
Higher CS activity in fruit treated with GABA was recorded after 6 d,
which were 1.44-, 1.53-, 1.63-, and 1.96-times higher than those in
control, respectively.
3.3. MdPEPC, MdNADP-ME, MdNADP-IDH, MdCyt-ACO, MdNAD-
MDH, and MdCS expressions
MdPEPC expression increased from 0 to 6 d and 12–15 d, but
declined from 6 to 12 d in GABA-treated fruit, and the un-treated group
showed an irregular trend (Fig. 3A). GABA promoted MdPEPC expres­
sion on days 6–15, and the peak value was recorded on day 6, which was
14.7-times higher than that in control. MdNADP-ME expression peaked
on day 6 in control, and decreased subsequently except day 9, which
showed 1.42-times higher than GABA-treated fruit (Fig. 3B). MdNADP-
IDH expression in fruit treated with GABA showed an increase from 0 to
9 and 12 to 15 d with decrease from 9 to 12 d. Moreover, MdNADP-IDH
expression was higher in GABA-treated fruit than that in control from 3
to 15 d (Fig. 3C). The transcription of MdCyt-ACO was also increased by
GABA from 3 to 15 d, which were 10.65-, 3.81-, 1.39-, 8.04-, and 1.11-
times higher than that in control (Fig. 3D). The expression of MdNAD-
MDH in control increased slightly within the initial 3 d of storage, then
declined rapidly (Fig. 3E). Whereas, MdNAD-MDH expression in fruit
treated with GABA reached the maximum on day 6, which was 2.66-
times higher than that in control. GABA treatment enhanced the tran­
scription level of MdCS as compared with control, which was 1.08-,
1.04-, 1.81-, 4.22-, and 7.89-times higher than those in control on days
3–15 (Fig. 3F).
3.4. AMP, ADP, ATP contents, and energy charge
ATP content in fruit treated with GABA increased from 0 to 6 d and 9
to12 d, but declined from 6 to 9 d and 12 to 15 d, whereas ATP content
did not change so much from 0 to 15 d in control (Fig. 4A). GABA
significantly increased ATP content from 3 to 12 d, which were 1.04-,
1.16-, 1.06-, and 1.07-times higher than those in control. The peak of
ADP content was observed on day 12 in control, which was 1.97-times
higher than that in GABA-treated fruit (Fig. 4B). However, GABA
significantly reduced the content of ADP in apple fruit from 3 to 6 d and
12 to 15 d of storage. AMP content with or without GABA treatment
increased continuously with the progression of the storage period
(Fig. 4C). Moreover, GABA suppressed AMP content on days 3–15,
which were 15.07 %, 17.31 %, 11.03 %, 19.72 %, and 25.64 % lower
than those in control. Fig. 4D shows that GABA enhanced energy charge

Fig. 3. Relative expressions of MdPEPC (A), MdNADP-ME (B), MdNADP-IDH (C), MdCyt-ACO (D), MdNAD-MDH (E) and MdCS (F) in apples after GABA treatment
during storage at 24 ± 1 ◦ C with a relative humidity of 75 ± 5 %. Vertical bars represent standard errors of the means. Asterisks (*) represent significant difference
between control and GABA treatment at level of 0.05 by LSD.

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J. Zhu et al. Postharvest Biology and Technology 186 (2022) 111846

Fig. 4. The contents of ATP (A), ADP (B), AMP (C), and energy charge (D) in apples after GABA treatment during storage at 24 ± 1 ◦ C with a relative humidity of 75
± 5 %. Vertical bars represent standard errors of the means. Asterisks (*) represent significant difference between control and GABA treatment at level of 0.05 by LSD.

from 0 to15 d as compared with control, which was 1.08-, 1.09-, 1.07-,
1.14-, and 1.13-times higher than those in control, respectively.
3.5. Activities of Ca2+-ATPase, H+-ATPase, SDH, and CCO
Fig. 5A demonstrates that Ca2+-ATPase activity in two groups
showed a similar tendency and GABA treatment promoted the peak in
advance. Ca2+-ATPase activity was significantly higher in GABA-treated
fruit throughout the storage time except day 12. The highest level of
Ca2+-ATPase activity in fruit treated with GABA was 1.36-times higher
than that in control on day 6. The activity of H+-ATPase peaked on day 9
in both GABA-treated and control fruit (Fig. 5B). While GABA treatment
enhanced H+-ATPase activity, which were 26.55 %, 36.52 %, 45.22 %,
42.48 %, and 30.97 % higher than those in control from 3 to 15 d,
respectively. SDH activity in fruit treated with GABA was higher than
that in control from 6 to 15 d. The activity of SDH in GABA-treated fruit
increased 1.63-times as compared to control on day 6 (Fig. 5C).
Additionally, GABA significantly induced CCO activity on days 6, 9, and
12, which were 2.13-, 1.18-, and 2.21-times higher than those in control
(Fig. 5D).
3.6. MdCa2+-ATPase, MdH+-ATPase, MdSDH, and MdCCO expressions
MdCa2+-ATPase expression was induced by GABA treatment
throughout the storage time, which was 7.23-, 2.14-, 2.21-, 5.73-, and
2.53-times higher than those in control, respectively (Fig. 6A). In con­
trol, MdH+-ATPase expression gradually increased at the initial 9 d of
storage, and then decreased sharply (Fig. 6B). While MdH+-ATPase
expression in GABA-treated fruit was enhanced from days 3 to 15, which
were 3.61-, 6.84-, 2.14-, 2.23-, and 8.97-times higher than those in
control, respectively. MdSDH expression in fruit with treated GABA
increased gradually to a peak on day 6, and decreased in the following
period (Fig. 6C). Transcription levels of MdSDH in fruit treated with
GABA were 5.21-, 3.47-, and 2.53-times higher than that in control on

Fig. 5. Ca2+-ATPase (A), H+-ATPase (B), SDH (C) and CCO (D) activities in apples after GABA treatment during storage at 24 ± 1 ◦ C with a relative humidity of 75 ±
5 %. Vertical bars represent standard errors of the means. Asterisks (*) represent significant difference between control and GABA treatment at level of 0.05 by LSD.

5
J. Zhu et al.
Fig. 6. Relative expressions of MdCa2+-ATPase (A), MdH+-ATPase (B), MdSDH (C), and MdCCO (D) in apples after GABA treatment during storage at 24 ± 1 ◦ C with a
relative humidity of 75 ± 5 %. Vertical bars represent standard errors of the means. Asterisks (*) represent significant difference between control and GABA treatment
at level of 0.05 by LSD.
days 3, 6, and 9, respectively. MdCCO expression in control had little
change during storage, whereas the relative expression of MdCCO was
significantly enhanced by GABA treatment through the storage, which
was 1.56-, 2.43-, 5.15-, 6.79-, and 1.79-times higher than those in
control (Fig. 6D).
4. Discussion
Sweetness and acidity are the major factors that affect flavor of
“Golden Delicious” apples (Kolniak-Ostek et al., 2014). Organic acids
accumulated during fruit growth are metabolized as respiratory sub­
strates during fruit ripening, which is the reason for acidity decrease
after harvest (Ma et al., 2015). We found that GABA greatly increased
the contents of malic, citric, succinic, and tartaric acids in apple fruit.
The higher content of organic acids in fruit is conducive to provide
sufficient substrate to keep fruit quality after harvest (Wu et al., 2020).
Study has revealed that malic acid plays a pivotal role in regulation of
carbohydrate metabolism, fruit ripening, and postharvest softening (Lee
et al., 2021). Studies have confirmed that GABA shunt is the major
pathway for citrate degradation, and citrate is one of the most critical
fluxes in mitochondria to maintain intercellular metabolic redox (Sheng
et al., 2017a, b; Zhao et al., 2020). Succinate, one of the respiratory
intermediates, increases the resistance ability of fruit by regulating
alternate respiration under stress conditions (Sun et al., 2013). Higher
succinate content in fruit treated with GABA is most likely due to the
increased activity of GABA shunt and inhibition of respiratory meta­
bolism (Han et al., 2018). These evidences imply that organic acids may
be closely connected with physiological changes during fruit ripening
and senescence process.
TCA cycle is a bridge for the metabolic transformation of carbohy­
drate, organic acid, and amino acid in fruit (Etienne et al., 2013). The
accumulation of various organic acids in fruit is determined by the
balance of acid synthesis, degradation and utilization (Yang et al.,
2019). Malic, citric, succinic, and tartaric acids are the major organic
acids in apple fruit which regulated by PEPC, NADP-ME, NADP-IDH,
Cyt-ACO, NAD-MDH, and CS directly or indirectly (Ma et al., 2015,
2021). PEPC catalyzes phosphoenolpyruvic acid to oxaloacetate, sub­
sequently degrades to malate in the presence of NAD-MDH or to citrate
by CS, and malate can be further degraded to pyruvate by NADP-ME
(Borsani et al., 2009; Chen et al., 2012; Li et al., 2018). Additionally,
citrate can be decomposed to ketoglutaric acid and carbon dioxide by
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Postharvest Biology and Technology 186 (2022) 111846
ACO and NAD-IDH (Lin et al., 2016). We demonstrated that GABA
increased the activities and gene expressions of PEPC, NADP-IDH,
Cyt-ACO, NAD-MDH, and CS, but decreased the activity and gene
expression of NADP-ME in apple fruit. This finding might partially
associate with the accumulation of malic, citric, succinic, and tartaric
acids in GABA-treated apples. Study in apples also showed that the
transcription level of MdVHA-A, MdVHP, MdNAD-MDH, and MdPEPC
was up-regulated by 1-methylcyclopropene, which resulted in the
retention of malate in vacuole (Liu et al., 2016a). Sheng et al. (2017a, b)
reported that the accumulation of citrate inducing by GABA was closely
related to the low activity of the GABA shunt and block of the TCA cycle.
Similarly, GABA treatment leads to higher malic acid content in apples
accompanying by the higher activities of PEPC and cyNAD-MDH, as well
as lower PEPCK and cyNADP-ME activities (Han et al., 2018). These
results implied that GABA inhibited the degradation of organic acids and
maintained sufficient respiratory substrates by regulating the activities
and expressions of key enzymes in organic acid metabolism.
Study has proven that organic acid metabolism is a complex physi­
ological process, which not only affects the electron transport chain, but
also indirectly affects fruit energy status (Rienth et al., 2016). Studies
have demonstrated that ATP directly affects the synthesis and degra­
dation of organic acids which closely relate to the normal metabolic
process in fruit and vegetable (Cao et al., 2014; Lyu et al., 2019). Besides
participating in various metabolic pathways in plants, organic acids also
supply NADH and ATP in cells through the TCA cycle (Wang et al.,
2021). Additionally, an increase in certain amino acids, such as
phenylalanine, and GABA, is another common response of fruit to
postharvest stress which related to carbon and nitrogen metabolism
(Zhou et al., 2019). Our previous study in apple fruit has proven that
postharvest application of GABA can induce a large amount of endog­
enous GABA accumulation (Zhu et al., 2022). Therefore, we speculate
that endogenous GABA serves as a supplier of carbon and nitrogen
which provided ATP in physiological metabolism after harvest. Study
has shown that GABA is the central metabolite of a biochemical bypass
of TCA cycle, and affects CCO, SDH, H+-ATPase and Ca2+-ATPase ac­
tivities, also involves in oxidative phosphorylation and ATP synthesis in
fruit (Zhao et al., 2019; Xie et al., 2021). Apart from plenty of trans­
porters and channels in tonoplast, Ca2+-ATPase, H+-ATPase, and P-type
ATPase also help to transport organic acids from cytosol to vacuole to
maintain vacuolar pH (Huang et al., 2021). This study showed the in­
crease of ATP content and energy charge, and the decrease of ADP and
J. Zhu et al.
AMP contents in apples after GABA treatment. GABA also promoted the
activities and gene expressions of CCO, SDH, H+-ATPase and
Ca2+-ATPase in apple fruit. Studies in zucchini and citrus fruit have
shown that GABA treatment improved the antioxidant enzymes activ­
ities and ATP content (Sheng et al., 2017a, b; Palma et al., 2019).
Similarly, study in apples demonstrated that benzothiadiazole treatment
enhanced ATP level through energy metabolism and act as a resistance
irritant to enhance disease tolerance (Li et al., 2020a, b). Likewise,
postharvest trisodium phosphate treatment increased energy charge and
ATP content in apple fruit, and kept higher activities of ATPase, SDH and
CCO which played pivotal roles in mediating respiration and mito­
chondrial energy metabolism (Ge et al., 2019). In Arabidopsis, AttDT is
found to transport citrate into vacuole and maintain vacuolar pH and
citrate homeostasis by manipulating Ca2+ and H+ channels and
ATP-dependent transporters, demonstrating that organic acids meta­
bolism is inseparable from energy metabolism (Huang et al., 2021).
Therefore, GABA treatment could be a very promising strategy for
supplying sufficient energy for postharvest apples through stimulating
organic acids and energy metabolism.
5. Conclusions
Postharvest GABA treatment can activate organic acids and mito­
chondrial energy metabolism in apples, which lead to the deposition of
malic, citric, succinic, and tartaric acids, and higher ATP content and
energy charge. These substances provide intermediates for the TCA cycle
to regulate respiration metabolism, energy status and improve stress
tolerance and quality of apple fruit during ripening.
CRediT authorship contribution statement
Jie Zhu: Methodology, Writing - original draft. Canying Li:
Conceptualization, Writing - review & editing. Yiting Fan: Investiga­
tion, Formal analysis. Linhong Qu: Data curation, Formal analysis. Rui
Huang: Investigation, Data curation. Jiaxin Liu: Validation, Investi­
gation. Chenyang Zhang: Investigation. Yonghong Ge: Supervision,
Funding acquisition, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This study was supported by the National Natural Science Founda­
tion of China (31801595).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.postharvbio.2022.111
846.
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