Exogenous Î³-Aminobutyric Acid Treatment That Contributes to

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Chemistry and Biology of Aroma and Taste

Exogenous #-Aminobutyric Acid (GABA) Treatment Contributes to Regulation of Malate Metabolism and Ethylene Synthesis in Apple Fruit during Storage Shoukun Han, Yuyu Nan, Wei Qu, Yiheng He, Qiuyan Ban, Yanrong Lv, and Jingping Rao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04674 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Journal of Agricultural and Food Chemistry

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Exogenous γ-Aminobutyric Acid (GABA) Treatment Contributes to

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Regulation of Malate Metabolism and Ethylene Synthesis in Apple

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Fruit during Storage

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Shoukun Han, Yuyu Nan, Wei Qu, Yiheng He, Qiuyan Ban, Yanrong Lv*,

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Jingping Rao*

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College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China

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* Corresponding author：

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Dr. Yanrong Lv (E-mail: [email protected] )

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Prof. Jingping Rao (E-mail: [email protected])

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ACS Paragon Plus Environment


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ABSTRACT: Organic acid is an important indicator of fruit quality, and malate is the

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predominant organic acid in apple fruit. However, the regulation of malate metabolism

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in postharvest fruit is rarely reported. Here, we found that compared with a control

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treatment, a 10 mM γ-aminobutyric Acid (GABA) treatment remarkably delayed the

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loss of titratable acidity and malate and increased the succinate and oxalate contents in

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‘Cripps Pink’ fruit stored in polyethylene bags at room temperature. The higher malate

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levels in GABA-treated fruit were accompanied by higher activities of cyNAD-MDH

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and PEPC but lower cyNADP-ME and PEPCK activities than those seen in control fruit.

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Notably, ethylene production was significantly reduced by GABA treatment,

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paralleling the downregulation of MdACS, MdACO and MdERF expression. Meanwhile,

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GABA treatment also enhanced the activity of the GABA shunt and promoted the

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accumulation of GABA. This study provides new insights into the regulation of malate

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metabolism and reports for the first time the possible interplay between GABA and

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ethylene signaling pathways in apple fruit during postharvest storage.

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KEYWORDS: Apple, γ-GABA, malate metabolism, ethylene, postharvest storage

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INTRODUCTION

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Organic acids are important indicators of fruit taste and, together with sugars and

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aromatic volatiles, determine flavor quality.1 Generally, the level of organic acids in

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fruits increases gradually during the process of fruit growth and development, but

31

decreases continuously during ripening and postharvest storage.2,

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malate and citrate, which technically are the conjugate bases of malic acid and citrate

33

acid and are used to refer to all physiological forms of each compound, are considered

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the main organic acids.4

3

In most fruits,

35

Malate is the predominant organic acid in apple fruit5 and is involved in various

36

metabolic pathways in fruit cells, including glycolysis, the tricarboxylic acid (TCA)

37

cycle, gluconeogenesis, the glyoxylate cycle and the synthesis of complex secondary

38

metabolites.6 Previous studies showed that malate appeared to have a wide range of

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important functions, such as regulating cytosolic pH, controlling stomatal aperture,

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balancing cellular energy supply and increasing resistance to heavy metal toxicity.7-10

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Notably, malate trends to play an important role in postharvest softening and

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susceptibility to bacterial infection.11 In recent years, studies on the accumulation and

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metabolism of malate in fruit cells confirmed that cytosolic nicotinamide adenine

44

dinucleotide-dependent

45

phosphoenolpyruvate carboxylase (PEPC) dominated malate biosynthesis by catalytic

46

reactions, whereas malate degradation was attributed to cytosolic NAD phosphate-

malate

dehydrogenase

3


(cyNAD-MDH)

and


47

dependent malic enzyme (cyNADP-ME) and phosphoenolpyruvate carboxykinase

48

(PEPCK), which would encourage phosphoenolpyruvate (PEP) formation from

49

oxaloacetate (OAA), thereby allowing malate to synthesize OAA.3, 5, 6, 12, 13

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Many studies have been carried out to identify the factors that influence the

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formation of fruit acidity. Environmental temperature, water supply and mineral

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fertilization have all been shown to regulate the metabolism and accumulation of

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organic acids during the process of fruit growth and development.4, 14-16 However, the

54

regulation of malate metabolism in postharvest fruit is still unclear. Liu et al.17 and

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Bekele et al.18 reported that 1-methylcyclopropene (1-MCP) treatment significantly

56

retains the level of malate in apple fruit during storage. Similarly, exogenous ethylene

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application leads to an increase in malate degradation relative to the untreated apple

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fruit during storage, which is attributed to the increase in respiratory consumption.19

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More extensive studies are urgently needed.

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γ-Aminobutyric acid (GABA), a nonprotein amino acid, is recognized as a plant

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signaling molecule.20 GABA is synthesized via the GABA shunt pathway, which

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extends from the TCA cycle. To date, studies have shown that glutamate decarboxylase

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(GAD), GABA transaminase (GABA-T), and succinate semialdehyde dehydrogenase

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(SSADH) are all involved in GABA metabolism.21, 22 In the cytosol, GABA synthesis

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from glutamate is irreversibly catalyzed by GAD, and GABA is then transported into

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the mitochondria, where it is oxidized by GABA-T and SSADH to produce succinate, 4


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which finally flows back to the TCA cycle.21-23 In recent years, studies have shown that

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GABA levels can rapidly increase in response to a range of abiotic and biotic stresses,

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including pathogen infection, hypoxia, drought, wounding, cold and heat shock.24

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Moreover, the GABA shunt pathway is also involved in the regulation of cytoplasmic

71

pH and carbon and nitrogen metabolism.25, 26 Recent studies have shown that GABA

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participates in citrate metabolism and that exogenous GABA treatment significantly

73

increases citrate levels and improves the storage quality of citrus fruit.27, 28 Shang et al.

74

reported that all of the exogenous GABA treatments (1 mM, 5 mM and 10 mM)

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maintain higher titratable acidity (TA) levels in peaches during storage.29 However,

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little is known about the effects of postharvest GABA treatment on malate metabolism

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in apple fruit.

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MATERIALS AND METHODS

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Plant Materials and Treatments

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Fruit of ‘Cripps Pink’ apples (Malus domestica Borkh.) were harvested from a

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commercial orchard in Fuping, Shannxi Province, PR China, and immediately

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transported to the postharvest laboratory at Northwest A&F University. Fruits of

83

uniform size and maturity and free of any physical injuries and defects were selected

84

and then divided randomly into four groups. The four groups were each immersed in a

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solution of distilled water (as a control) or 0.5 mM, 1 mM or 10 mM GABA for 1 h.

86

Each treatment contained three replicates (90 fruits per replicate). After being rinsed 5



87

with sterile deionized water, all fruits were air dried, packed in polyethylene film bags

88

and randomly arranged in a storage room set 25 ± 1 ºC. A total of 30 fruit were sampled

89

in three times for immediate analysis to monitor fruit characteristics before treatments

90

(day 0) and the values were used as initial values for all treatments and repetitive

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sampling at 10-day intervals during the 3-month storage period. At each sampling time,

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ten fruits were selected from each replicate and used for determination of soluble solids

93

content (SSC) and TA. All of the samples were cut into small pieces, and immediately

94

frozen in liquid nitrogen and stored at -80 ºC.

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Determination of Fruit Quality Traits

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SSC was measured with a digital handheld refractometer (Atago, Tokyo, Japan) and

98

was expressed as a percentage on the Brix scale. TA was determined by titration with

99

0.01 N NaOH, and the results were expressed as the percentage of malic acid, according

100

to the methods of Liu et al.17

101 102

Measurement of Ethylene Production and Respiration Rate

103

Ethylene production and respiration rate were measured according to the method of

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Han et al. with some modifications.30 Five fruits were enclosed in a 3.6-L airtight vessel

105

for 1 h at 25 ºC, and a 1-mL gas sample was collected with a syringe. Then, the gas 6


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sample was injected into a GC-14A flame ionization detector gas chromatograph

107

(Shimadzu, Kyoto, Japan) to determine ethylene concentration. The ethylene

108

production rate was expressed as μL kg−1 h−1. The respiration rate was measured using

109

a CO2 infrared gas analyzer (TEL7001; GE Telaire) and expressed as mg CO2 kg-1 h-1.

110

Three biological replicates were used.

111 112

Extraction and Determination of Organic Acids

113

Organic acids were extracted according to the method described in previous reports

114

with some modifications.17 Frozen flesh (3 g) was ground into a homogenate in 20 mL

115

of 10% (v/v) ice-cold ethanol with a mortar and pestle and then transferred to 50-mL

116

tubes. After being incubated at 80 ºC for 15 min, the mixture was ultrasonically

117

extracted at 80 W for 40 min (SB-5200DT, Scientz, China) at room temperature and

118

then centrifuged at 8000 × g for 10 min. The supernatant was collected, while the pellet

119

was extracted two more times with 5 mL of 10% (v/v) cold ethanol. All supernatants

120

were collected to a final volume of 30 mL with 10% ethanol. A total of 1 mL of extract

121

solution was evaporated under a vacuum (Eppendorf Concentrate Plus, Germany) at 45

122

ºC, and the residue was resuspended in 0.5 mL of distilled water and filtered through a

123

0.45-μm water syringe filter (Shanghai Xingya Purification Material Factory, China).

124

The filtered solution was used for analysis of organic acids.

7



125

Organic acids were analyzed by high-performance liquid chromatography (HPLC)

126

as described previously with some modifications.31 A 10-μL sample was injected into

127

an ODS C18 column (5 μm, 250 mm×4.6 mm, GL Science, Tokyo, Japan). The flow

128

rate was 0.5 mL min-1, and (NH4)2HPO4 (50 mM, pH of 2.7) was used as the mobile

129

phase. An LC-2010 HPLC system (Shimadzu, Japan) with a Waters 2996 diode array

130

detector (Waters Corporation, USA) was used to separate and quantify organic acids at

131

a wavelength of 210 nm. Individual organic acids were quantified by constructing a

132

standard curve after injection of external organic acid standards and expressed as mg

133

per g of fresh weight (mg g-1 FW). The standard cures were linear in the range of

134

0.001~1.000 mg mL-1 and the correlation coefficients of standard curves were all above

135

0.9998. All the organic acid standards were obtained from Sigma-Aldrich (St. Louis,

136

MO, USA). The data were analyzed with a Waters Empower system.

137 138

Extraction and Determination of GABA

139

GABA was extracted as previously reported32 with some modifications. Frozen flesh

140

(500 mg) was ground in 1.0 mL of 20 mM HCl. The mixture was ultrasonically

141

extracted at 80 W for 15 min and then centrifuged at 13,000 × g for 10 min. The

142

supernatant was passed through a 0.2-μm syringe filter into an Eppendorf tube. The

143

detection was performed by high-performance liquid chromatography-tandem mass

144

spectrometry (HPLC-MS/MS) according to the method of Zhao et al.33 The MS/MS 8


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detection was carried out using an API 5500 Q-TRAP tandem MS instrument (AB

146

SCIEX, Framingham, MA, USA). The results obtained are represented as μg/g FW.

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Enzyme Extraction and Activity Assay

149

Enzyme extraction and purification were performed as described by Yao et al.12 and

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Liu et al.17 with some modifications. Ten grams of frozen tissue was homogenized with

151

a mortar and pestle in 20 mL of ice-cold extract buffer containing 50 mM Hepes-Tris

152

(pH of 7.6), 250 mM sorbitol, 125 mM KCl, 5 mM EGTA, 10 mM MgSO4, 2 mM

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PMSF, 1.5% (w/v) PVP, 0.1% (w/v) BSA and 1 mM DTT. Thereafter, the homogenate

154

was filtered through four layers of cheesecloth into a 50-mL tube and centrifuged at

155

1000 × g for 15 min at 4 ºC. The supernatant was then recentrifuged at 50,000 × g for

156

1 h, and the final supernatant was further desalted by passing it through Sephadex G-

157

25 medium (PD-10, Pharmacia, Uppsala, Sweden). The extracts were then stored at -

158

80 ºC for approximately 16 h and then used for enzyme activity assays of cyNAD-MDH,

159

cyNADP-ME, PEPC and PEPCK.

160

The activities of cyNAD-MDH, cyNADP-ME, PEPC and PEPCK were measured

161

in accordance with the methods of Merio et al.34 and Liu et al.17 with slight

162

modifications. cyNAD-MDH activity was assayed in the direction of malate formation

163

following the oxidation of NAD + hydrogen (NADH). The reaction mixture (1.0 mL) 9



164

contained 50 mM Tris–HCl (pH of 7.8), 0.5 mM EDTA, 2 mM MgCl2, 0.2 mM NADH

165

and 60 μL of crude enzyme extracts. The reaction was started by adding OAA to a final

166

concentration of 2 mM. cyNADP-ME activity was measured in a reaction mixture (1.0

167

mL) containing 80 mM Tris–HCl (pH of 7.5), 1 mM DTT, 0.1 mM EDTA, 0.4 mM

168

MnSO4, 0.2 mM NADP and 300 μL of enzyme extracts. The reaction was initiated by

169

adding 10 mM L-malate. PEPC activity was determined in a reaction mixture (1.0 mL)

170

containing 50 mM Tris-HCl (pH of 8.0), 2 mM DTT, 5 mM MgCl2, 0.2 mM NADH,

171

10 mM NaHCO3, 5 units of NAD-MDH (Shanghai Yuanye Biotechnology Co., Ltd,

172

China), and 60 μL of enzyme extracts. The reaction was started by adding PEP to a

173

final concentration of 2.5 mM. PEPCK activity was assayed in a 1.0-mL reaction

174

mixture containing 100 mM Hepes-KOH (pH of 6.8), 100 mM KCl, 0.14 mM NADH,

175

6 mM MnCl2, 25 mM DTT, 6 mM PEP, 1 mM ADP, 90 mM KHCO3 and 6 U NAD-

176

MDH. All the enzyme activities were expressed in U g-1 FW.

177 178

RNA Extraction and Real-Time Quantitative PCR (qRT-PCR) Analysis

179

The total RNAs of all samples were extracted according to the protocol described by

180

Gasic et al.35 A total of 1 μg of RNA was used for first-stand cDNA synthesis using a

181

PrimeScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa, Dalian,

182

China). The synthesized cDNA was diluted 10-fold for the following qRT-PCR

183

analysis. 10


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qRT-PCR was performed to analyze the transcript levels of malate metabolizing

185

genes (MdMDH (GenBank Acc. No. DQ221207), MdPEPC (GenBank Acc. No.

186

EU315246), MdME (GenBank Acc. No. JX971885), MdPEPCK (GenBank Acc. No.

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KT454965)), GABA metabolizing genes (MdGAD1 (GenBank Acc. No. KC812242),

188

MdGAD2 (GenBank Acc. No. KC812243), MdGAD3 (GenBank Acc. No. KC812244),

189

MdGABA-T1/T2 (GenBank Acc. No. JX276380/ JX276381), MdSSADH (GenBank

190

Acc. No. XM_008357890)) and ethylene-related genes (MdACS (GenBank Acc. No.

191

DQ137849), MdACO (GenBank Acc. No. X98627), MdERF (GenBank Acc. No.

192

AB288347)) in a 20-μL reaction volume using SYBR Premix Ex Taq (TaKaRa, Kyoto,

193

Japan) with an iCycler iQ5 real-time PCR system (Bio-Rad, United States) according

194

to the manufacturer’s protocol. All the specific primers were designed with Oligo 7.0

195

and listed with identification information of genes in Table S1. The comparative 2-ΔΔCT

196

method36 was used to calculate the relative expression level of each target gene and β-

197

Actin (GenBank Acc. No. AB638619)17 was used as a reference gene. qRT-PCR was

198

conducted with three biological replicates.

199 200

Statistical Analysis

201

All the experiments were performed in triplicate, and the data were analyzed using the

202

Data Processing Software DPS-9.5 to eliminate outlier and then checked for normality

203

using Kolmogorov-Smirnov (K-S) test and for assumptions of homogeneity of 11



204

variances across treatments by using Levene test and log-transformed if required. When

205

no homogeneity was observed, nonparametric test was preformed to detect differences

206

(Kruskal-Wallis test). All the statistical analysis was performed using SPSS Statistics

207

version 22.0 (SPSS-IBM Inc., Chicago, IL, USA) and one-way analysis of variance

208

(ANOVA) was applied between GABA-treated and control fruit for each storage period.

209

Differences were determined by using Tukey’s test and were considered statistically

210

significant if p < 0.05. Similarly, significant differences in some related indicators as a

211

function of time were analyzed in the above methods and the results were provided as

212

supplementary figures (S1-S14). All results are presented as the mean ± standard errors.

213 214

RESULTS

215

Effects of GABA Treatment on Fruit Quality

216

Changes in the SSC and TA of apples during storage are shown in Table S2. The SSC

217

increased rapidly during the early stage of storage and then decreased constantly until

218

the end of storage in all treatments. The highest values of SSC occurred at 20 days in

219

control fruit and 0.5 mM and 1 mM GABA-treated fruit but at 30 days in 10 mM

220

GABA-treated fruit. After 20 days of storage, the SSC in all the GABA-treated fruit

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was significantly higher than that in untreated fruit. The changes in TA declined

222

throughout storage, and the 10 mM GABA treatment significantly suppressed the 12


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223

decrease compared with the control. Taken together, these results showed that the

224

GABA treatment is beneficial for maintaining fruit acidity and quality during storage.

225 226

Ethylene Production and Respiration Rate

227

Ethylene production exhibited typical ethylene climacteric patterns, and the peaks

228

occurred at 30 days of storage in both control and GABA-treated fruit (Figure 1A, S3).

229

During most storage periods, GABA treatment greatly inhibited ethylene production.

230

Maximum ethylene production in the 0.5 mM and 1 mM GABA-treated fruit was

231

reduced to approximately 37% and 42% of that in the control fruit, respectively, while

232

the value increased to approximately 65% in 10 mM GABA-treated fruit. These results

233

suggested that GABA is involved in ethylene synthesis and that a 10 mM concentration

234

treatment can effectively reduce ethylene production.

235

Similarly, the respiration rate showed a characteristic climacteric pattern

236

throughout storage (Figure 1B, S4). Unlike ethylene production, respiration rate had

237

two climacteric peaks, which occurred at 10 days and 30 days, in all the treatments

238

except 10 mM GABA treatment. All the GABA treatments led to a significantly lower

239

respiration rate, especially during the climacteric peaks, and the 10 mM GABA

240

treatment was the most effective in decreasing respiration rate in parallel with the

241

effects on ethylene production. 13



242 243

Effects of GABA Treatment on Organic Acids

244

The concentrations of three main organic acids (malate, oxalate, succinate) and GABA

245

in ‘Cripps Pink’ fruit were detected. Among these acids, malate had the highest

246

concentration in postharvest apple fruit, followed by succinate and oxalate (Figure 2A,

247

B, C). During storage, changes in malate concentration were similar to those in TA.

248

Malate level decreased gradually with the prolongation of storage time, and GABA

249

treatment remarkably restrained the decline, especially during the earlier stage of

250

storage. Despite showing patterns of change that differed from those in malate content,

251

the succinate and oxalate contents in GABA-treated fruit were higher than those in the

252

control fruit at most of the time points. Furthermore, the GABA concentration declined

253

during storage, but it accumulated significantly in all the control and GABA-treated

254

fruits at the end of storage (Figure 2D). The GABA concentration in 10 mM GABA-

255

treated fruit was significantly higher than that in the control and other treatments.

256

Notably, 10 mM GABA treatment showed better effects in maintaining malate,

257

succinate and oxalate contents than 0.5 mM and 1 mM GABA treatments. Hence, we

258

focused on the 10 mM treatment in the following analysis.

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Effects of Exogenous GABA on the Activities of Enzymes and Expression Patterns 14


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of Crucial Genes Related to Malate Metabolism

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To determine whether malate metabolism was affected by exogenous GABA treatment,

263

the activities of enzymes and expression profiles of crucial genes related to malate

264

metabolism were examined. As shown in Figure 3A, cyNAD-MDH activity gradually

265

increased in control fruit, reaching a peak at 30 days (21.78 U g-1 FW), and then

266

decreased. Similarly, the cyNAD-MDH activity peak was observed in GABA-treated

267

fruit at the same point (24.89 U g-1 FW) but was approximately 14% higher than that in

268

the control fruit. In addition, PEPC activity showed the same pattern of change as

269

cyNAD-MDH activity in both GABA-treated fruit and control fruit, but peak PEPC

270

activity occurred at 20 days (Figure 3B). In contrast, cyNADP-ME activity increased

271

sharply from 0 to 30 days and then decreased until the end of storage in control fruit,

272

while in GABA-treated fruit, cyNADP-ME activity increased moderately and was

273

significantly lower than that in the control throughout storage (Figure 3C). PEPCK

274

activity was constantly enhanced during storage, and GABA treatment apparently

275

mitigated this trend (Figure 3D).

276

As shown in Figure 4, the expression of MdcyNAD-MDH and MdPEPC was

277

upregulated by GABA treatment at all time points during storage, whereas the

278

transcription of MdPEPCK was suppressed by GABA treatment. However, except for

279

at 40 days, no differences in the expression levels of MdcyNADP-ME were observed

280

between the control and GABA-treated fruits. 15



281 282

Effects of Exogenous GABA Treatment on the Expression Patterns of Genes

283

Related to Ethylene Biosynthesis

284

1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-

285

carboxylate oxidase (ACO) are two crucial enzymes involved in ethylene biosynthesis,

286

and ethylene-responsive factor (ERF) is an important transcription factor in the ethylene

287

signal transduction process. Previous studies have demonstrated that, for apple fruit,

288

MdACS and MdACO are primarily expressed in the ripening stage and more susceptible

289

to ethylene induction compared with other homologous genes.37, 38 Similarly, MdERF

290

has been proved to express predominately in fruit tissues and respond to ethylene signal

291

in most apple varieties.39 Therefore, the expression profiles of these genes were

292

analyzed in the present study. We found that the 10 mM GABA treatment significantly

293

inhibited the expression levels of MdACS and MdACO until 30 days of storage (Figure

294

5A, B), that is, the ethylene climacteric period. The transcript levels of MdERF were

295

also suppressed by GABA treatment during the early stage of storage but increased

296

visibly at 40 days and 70 days of storage compared with those in the control (Figure

297

5C). These results suggested that exogenous GABA participated in the inhibition of

298

ethylene biosynthesis before the ethylene climacteric period and played multiple roles

299

in apple fruit at different stages of storage.

300 16


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Effects of Exogenous GABA on Expression Patterns of Genes Involved in the

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GABA Pathway

303

Three cytosolic MdGADs (MdGAD1, MdGAD2 and MdGAD3) and two MdGABA-Ts

304

(MdGABA-T1 and MdGABA-T2) were identified and functionally characterized in

305

previous studies.40, 41 Since MdSSADH is highly homologous (78%) to the Arabidopsis

306

SSADH amino acid sequence (At1G79440.1), we hypothesized that the gene also has

307

similar functions in GABA pathway. Hence, the expression levels of these genes were

308

analyzed in the present study. MdGADs showed different expression patterns in apple

309

fruit during storage (Figure S11, S12, S13). The expression level of MdGAD1 increased

310

gradually with the prolongation of storage time, whereas the expression level of

311

MdGAD2 increased until 30 days and then decreased in both control fruit and GABA-

312

treated fruit. Furthermore, the transcript abundance of MdGAD1 and MdGAD2 were

313

significantly enhanced in GABA-treated fruit when compared with the control fruit at

314

all stages of storage (Figure 6A, B). Overall, the transcript level of MdGAD3 declined

315

especially at the end of storage and 10 mM GABA treatment significantly suppressed

316

the expression abundance compared with that of the control at 30 days and 40 days of

317

storage (Figure 6C). Moreover, the transcript levels of MdGABA-T1/T2 increased

318

gradually from 0 to 30 days, reaching peaks at the same time as the ethylene climacteric

319

peak (30 days), and then decreased until the end of storage in the control fruit (Figure

320

S14). At the same time, exogenous GABA treatment significantly promoted the 17



321

expression of MdGABA-T1/T2 compared with that of the control at some time points

322

(Figure 6D). Furthermore, the transcript level of MdSSADH was significantly enhanced

323

in 10 mM GABA-treated fruit compared with that in the control fruit at most of the time

324

points (Figure 6E). These results indicated that exogenous GABA treatment could still

325

stimulate the activity of the GABA pathway, which tended to promote the accumulation

326

of GABA, although the activity of the GABA pathway appeared to be enhanced along

327

with the increased ethylene production.

328

DISCUSSION

329

As a regulator of cytosolic pH, GABA is likely to be closely related to organic

330

acid metabolism. The application of exogenous GABA contributed to an increase in the

331

accumulation of organic acids (malic acid, succinic acid and oxalic acid) and of serval

332

amino acids (glutamic acid, aspartic acid, alanine, threonine, serine, and valine) in

333

creeping bentgrass.42 Recent studies have also demonstrated that the GABA pathway

334

plays an important role in citrate metabolism and that exogenous GABA treatment of

335

postharvest citrus fruit effectively increases citrate content and improves storage

336

quality.28 In this study, similar results were obtained in ‘Cripps Pink’ fruit during

337

postharvest storage. Specifically, we found that exogenous GABA treatments of

338

different concentrations (0.5 mM, 1.0 mM and 10 mM) significantly inhibited the

339

decline in TA and malate contents (Table S2 and Figure 2A). Among these

340

concentrations of GABA, 10 mM was considered to be the most effective. In addition, 18


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exogenous GABA treatment also effectively inhibited the decrease in SSC (Table S2),

342

indicating the role of GABA in maintaining fruit postharvest quality. Moreover,

343

compared with the control, GABA treatment significantly improved the succinate and

344

oxalate contents during storage (Figure 2B, C), whereas citrate could not be detected,

345

which may have been due to the very small amount of citrate in ‘Cripps Pink’ fruit.

346

Hence, we can speculate that GABA is also involved in malate metabolism to some

347

extent. Furthermore, we found that GABA was continuously consumed with the

348

prolongation of storage time and that exogenous GABA treatment effectively

349

maintained the GABA concentration of fruit pulp at high levels (Figure 2D). The

350

dramatic accumulation of GABA content may be a response to the aging of apple fruit

351

at the end of storage.

352

A decrease in malate content is usually attributed to respiratory consumption,

353

which mainly involves three metabolic pathways, namely, glycolysis, the TCA cycle

354

and gluconeogenesis.6 The application of 1-MCP, an inhibitor of ethylene perception,

355

effectively maintained fruit acidity in apple during postharvest storage.17 Therefore, the

356

respiratory rate and ethylene production rate were analyzed in control and GABA-

357

treated fruits during storage. Interestingly, exogenous GABA treatments not only

358

reduced the respiratory rate but also obviously inhibited the production of ethylene

359

(Figure 1). At the peak of ethylene release, the 10 mM GABA treatment significantly

360

reduced the peak value compared with the other treatments and the control. Furthermore, 19



361

the expression levels of MdACO, MdACS and MdERF were restrained by the 10 mM

362

GABA treatment to varying degrees, suggesting that GABA participated in the

363

regulation of ethylene biosynthesis and signal transduction at the molecular level

364

(Figure 5). Although the possible relationship between GABA and ethylene synthesis

365

in fruit has not been directly clarified, it is not entirely absent in plants. During

366

controlled atmosphere (CA) storage, high CO2 or low O2 and high CO2 conditions

367

significantly affected the GABA accumulation in various postharvest fruits and 1-

368

MCP- treated fruit presented an increase in GABA content in apple fruit.43-45 In poplar

369

woody plant, exogenous GABA regulated the key genes for ethylene production and

370

abscisic acid (ABA) biosynthesis in response to salt stress, demonstrating the potential

371

role of GABA as a signal transducer.46 However, GABA promoted ethylene production

372

by acting on ACS in the cotyledons and leaves of sunflower.47 Similarly, ethylene

373

production drastically increased (156.7%) after 48 h of treatment with 10 mM GABA

374

in NaCl-treated roots of C. intermedia.48 These studies suggest that interactions

375

between GABA and ethylene biosynthesis occur and may differ among plant species.

376

Thus, further specific studies are needed to clarify the regulatory metabolism of the

377

GABA and ethylene signal pathways. Considering that ethylene accelerates the

378

respiratory metabolism of postharvest fruit, we further speculate that the effects of

379

exogenous GABA on the metabolism of organic acids are partly achieved through the

380

regulation of ethylene biosynthesis and signal transduction in ‘Cripps Pink’ fruit. 20


Page 20 of 49

Page 21 of 49


381

The expression profiles of crucial genes involved in malate metabolism were

382

consistent with the changes in malate content in ‘Cripps Pink’ fruit during storage. In

383

contrast to control fruit, GABA-treated fruit showed higher expression levels of

384

MdcyMDH and MdPEPC along with lower activities of cyNAD-MDH and PEPC but

385

lower transcript levels of MdPEPCK and higher PEPCK and cyNADP-ME activities

386

(Figure 3 and Figure 4). However, no obvious difference was found in the expression

387

pattern of MdcyME between GABA-treated and control fruit (Figure 4C), which might

388

have been due to the posttranslational regulation of cytosolic pH and malate

389

concentration. This result coincided with the effects of 1-MCP treatment on the

390

expression levels of MdcyNADP-ME in postharvest apple fruit.17 Moreover, the

391

multiple forms of genes associated with malate metabolism have not been taken into

392

account here.

393

The effects of GABA treatment on the GABA pathway were also observed in

394

‘Cripps Pink’ fruit during storage. GAD, the key enzyme in the GABA shunt, was partly

395

activated by both acidic pH in cytosol and Ca2+/CaM.21, 40 Sheng et al.28 reported that

396

exogenous GABA treatment significantly inhibited the expression of GAD in citrus fruit

397

due to the feedback inhibition of the GABA synthesis precursor glutamate, which

398

rapidly accumulated after treatment during storage. However, GABA-treated fruit

399

presented higher GAD activity than control fruit in peach after long-term cold storage.29

400

In other words, the effects of exogenous GABA treatment on GABA metabolism are 21



401

likely to vary across fruits of different species. MdGAD1 and MdGAD2 were regulated

402

by Ca2+/CaM at physiological pH, but MdGAD3 was proved to be insensitive to pH.40

403

These indicate that multiple isoforms may have different responses to exogenous

404

GABA treatment. In the present study, we found that exogenous GABA treatment

405

induced higher expression of MdGAD1 and MdGAD2 in apple fruit during storage,

406

whereas the expression level of MdGAD3 was suppressed in GABA-treated fruit at the

407

ethylene climacteric period (Figure 6A, B, C). Furthermore, compared with the levels

408

in the control, the transcript levels of MdGABA-T1/T2 and MdSSADH in the exogenous

409

GABA treatment were enhanced (Figure 6D, E), indicating that the activity of the

410

GABA shunt was higher in GABA-treated fruit than in control fruit. Moreover, the

411

expression levels of MdGAD1, MdGAD2, MdGABA-T1/T2 and MdSSADH all have

412

strong responses to storage in postharvest ‘Cripps Pink’ apple fruit, which were similar

413

to those in ‘Empire’ apple fruit stored under low temperature or CA storage.49 Since

414

succinate is the final product of the GABA shunt and flows to the TCA cycle, the higher

415

succinate content in GABA-treated fruit than in control fruit throughout storage most

416

likely results from the increased activity of the GABA shunt and the inhibited

417

respiratory metabolism. This result was consistent with those of previous studies

418

suggesting that succinate concentration could be affected by the accumulation of

419

GABA.45 However, due to the absence of direct evidence, we cannot conclude that an

420

increase in GABA shunt activity leads to the accumulation of malate. 22


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421

In summary, our study suggested that exogenous GABA treatment with a suitable

422

concentration (10 mM) was effective in suppressing the consumption of malate and

423

maintaining fruit quality in ‘Cripps Pink’ fruit during storage. The application of

424

exogenous GABA apparently reduced the decrease in malate content by accelerating

425

malate biosynthesis and inhibiting its degradation. In addition, the activity of the GABA

426

shunt was promoted in GABA-treated fruit through the upregulation of MdGAD,

427

MdGABA-T and MdSSADH, which may result in the accumulation of succinate and

428

GABA. Notably, GABA treatment also reduced ethylene biosynthesis, especially

429

before the ethylene climacteric peak, and this effect appeared to be one of the most

430

important factors for the maintenance of malate content and fruit quality during storage,

431

although the interaction still requires further investigation.

432 433

ABBREVIATIONS USED

434

GABA, γ-Aminobutyric Acid; cyNAD-MDH, cytosolic nicotinamide adenine

435

dinucleotide-dependent

436

phosphoenolpyruvate carboxylase (EC4.1.1.31); cyNADP-ME, cytosolic NAD

437

phosphate-dependent malic enzyme (EC 1.1.1.40); PEPCK, phosphoenolpyruvate

438

carboxykinase (EC4.1.1.32); 1-MCP, 1-methylcyclopropene; GAD, glutamate

439

decarboxylase

440

(EC2.6.1.96); SSADH, succinate semialdehyde dehydrogenase (EC 1.2.1.24); SSC,

malate

(EC4.1.1.15);

dehydrogenase

GABA-T,

(EC1.1.1.38);

gamma-aminobutyrate

23


PEPC,

transaminase


441

Page 24 of 49

soluble solids content; TA, titratable acidity

442 443

AUTHOR INFORMATION

444

Corresponding Author

445

*Dr. Yanrong Lv

446

*Prof.

447

[email protected]

448

ORCID

449

Jingping Rao: 0000-0002-8726-947X

450

Notes

451

All authors declare that they have no conflict of interest.

Jingping

Telephone: +86-029-87082336 Rao

Telephone:

E-mail: [email protected]

+86-029-87182613

E-mail:

452 453

ACKNOWLEDGMENTS

454

This research is supported by the National Key Research and Development Program

455

(2016YFD0400100). We thank Dr. Ye Han (Gansu Agricultural University) and Dr. Yali

456

Hou (Shenyang Agricultural University) for providing some support with the statistical

457

analysis. 24


Page 25 of 49


458 459

SUPPORTING INFORMATION DESCRIPTION

460

The supplementary materials including two supplementary tables and fourteen

461

supplementary figures for this paper can be found online at http://pubs.acs.org.

462

Table S1. Primer sequences used for quantitative real-time PCR

463

Table S2. Changes in SSC and TA of the control and GABA-treated apples during

464

storage at room temperature.

465

Figure S1-S14. Changes in the related quality indicators, ethylene production rate,

466

respiratory rate, metabolites, enzyme activity and gene transcript level as a function of

467

time in the control and GABA-treated apples.

468 469

REFERENCES

470

1.

471

S.; Sa'ar, U.; Davidovitz-Rikanati, R.; Baranes, N.; Bar, E.; Wolf, D.; Petreikov, M.;

472

Shen, S.; Ben-Dor, S.; Rogachev, I.; Aharoni, A.; Ast, T.; Schuldiner, M.; Belausov,

473

E.; Eshed, R.; Ophir, R.; Sherman, A.; Frei, B.; Neuhaus, H. E.; Xu, Y.; Fei, Z.;

474

Giovannoni, J.; Lewinsohn, E.; Tadmor, Y.; Paris, H. S.; Katzir, N.; Burger, Y.;

475

Schaffer, A. A. The PH gene determines fruit acidity and contributes to the evolution

Cohen, S.; Itkin, M.; Yeselson, Y.; Tzuri, G.; Portnoy, V.; Harel-Baja, R.; Lev,

25



476

of sweet melons. Nat Commun. 2014, 5, 4026.

477

2.

478

concentration and activities of acid-metabolising enzymes during the fruit

479

development of two loquat (Eriobotrya japonica Lindl.) cultivars differing in fruit

480

acidity. Food Chemistry. 2009, 114, 657-664.

481

3.

482

C.; Chen, K. Transcriptome and metabolome analyses of sugar and organic acid

483

metabolism in Ponkan (Citrus reticulata) fruit during fruit maturation. Gene. 2015,

484

554, 64-74.

485

4.

486

fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. J Exp

487

Bot. 2013, 64, 1451-1469.

488

5.

489

expression gene network modules regulating fruit acidity in diverse apples. BMC

490

Genomics. 2015, 16, 612.

491

6.

492

of malate metabolism in grape berry and other developing fruits. Phytochemistry

493

2009, 70, 1329-1344.

494

7.

Chen, F.; Liu, X.; Chen, L. Developmental changes in pulp organic acid

Lin, Q.; Wang, C.; Dong, W.; Jiang, Q.; Wang, D.; Li, S.; Chen, M.; Liu, C.; Sun,

Etienne, A.; Genard, M.; Lobit, P.; Mbeguie, A. M. D.; Bugaud, C. What controls

Bai, Y.; Dougherty, L.; Cheng, L.; Zhong, G. Y.; Xu, K. Uncovering co-

Sweetman, C.; Deluc, L. G.; Cramer, G. R.; Ford, C. M.; Soole, K. L. Regulation

Fernie, A. R.; Martinoia, E., Malate. Jack of all trades or master of a few? 26


Page 26 of 49

Page 27 of 49


495

Phytochemistry 2009, 70, 828-832.

496

8.

497

activated vacuolar chloride channel required for stomatal opening in Arabidopsis. Nat

498

Commun. 2013, 4, 1804.

499

9.

500

Plantarum 2004, 120, 21-26.

501

10. Liu, J.; Zhou, M. The ALMT Gene Family Performs Multiple Functions in

502

Plants. Agronomy 2018, 8, 20.

503

11. Centeno, D. C.; Osorio, S.; Nunes-Nesi, A.; Bertolo, A. L.; Carneiro, R. T.;

504

Araujo, W. L.; Steinhauser, M. C.; Michalska, J.; Rohrmann, J.; Geigenberger, P.;

505

Oliver, S. N.; Stitt, M.; Carrari, F.; Rose, J. K.; Fernie, A. R. Malate plays a crucial

506

role in starch metabolism, ripening, and soluble solid content of tomato fruit and

507

affects postharvest softening. Plant Cell 2011, 23, 162-184.

508

12. Yao, Y.-X.; Li, M.; Liu, Z.; You, C.-X.; Wang, D.-M.; Zhai, H.; Hao, Y.-J.

509

Molecular cloning of three malic acid related genes MdPEPC, MdVHA-A, MdcyME

510

and their expression analysis in apple fruits. Sci. Hortic. 2009, 122, 404-408.

511

13. Tang, M.; Bie, Z.-l.; Wu, M.-z.; Yi, H.-p.; Feng, J.-x. Changes in organic acids

512

and acid metabolism enzymes in melon fruit during development. Sci Hortic. 2010,

513

123, 360-365.

De Angeli, A.; Zhang, J.; Meyer, S.; Martinoia, E. AtALMT9 is a malate-

Scheibe, R. Malate valves to balance cellular energy supply. Physiologia

27



514

14. Lin, Q.; Qian, J.; Zhao, C.; Wang, D.; Liu, C.; Wang, Z.; Sun, C.; Chen, K. Low

515

temperature induced changes in citrate metabolism in ponkan (Citrus reticulata

516

Blanco cv. Ponkan) fruit during maturation. PLoS One 2016, 11, e0156703.

517

15. Rienth, M.; Torregrosa, L.; Sarah, G.; Ardisson, M.; Brillouet, J. M.; Romieu, C.

518

Temperature desynchronizes sugar and organic acid metabolism in ripening grapevine

519

fruits and remodels their transcriptome. BMC Plant Biol. 2016, 16, 164.

520

16. Hummel, I.; Pantin, F.; Sulpice, R.; Piques, M.; Rolland, G.; Dauzat, M.;

521

Christophe, A.; Pervent, M.; Bouteille, M.; Stitt, M.; Gibon, Y.; Muller, B.

522

Arabidopsis plants acclimate to water deficit at low cost through changes of carbon

523

usage: an integrated perspective using growth, metabolite, enzyme, and gene

524

expression analysis. Plant Physiol. 2010, 154, 357-372.

525

17. Liu, R.; Wang, Y.; Qin, G.; Tian, S. Molecular basis of 1-methylcyclopropene

526

regulating organic acid metabolism in apple fruit during storage. Postharvest Biology

527

and Technology 2016, 117, 57-63.

528

18. Bekele, E. A.; Beshir, W. F.; Hertog, M. L.; Nicolai, B. M.; Geeraerd, A. H.

529

Metabolic profiling reveals ethylene mediated metabolic changes and a coordinated

530

adaptive mechanism of 'Jonagold' apple to low oxygen stress. Physiol Plant. 2015,

531

155, 232-247.

532

19. Bruno B.G.; Dandekar, A. M.;Kader A.A. Impact of suppression of ethylene 28


Page 28 of 49

Page 29 of 49


533

action or biosynthesis on flavor metabolites in apple (Malus domestica. Borkh) fruits.

534

J Agric Food Chem. 2004, 52, 5694-5701.

535

20. Ramesh, S. A.; Tyerman, S. D.; Gilliham, M.; Xu, B. gamma-Aminobutyric acid

536

(GABA) signalling in plants. Cell Mol Life Sci. 2017, 74, 1577-1603.

537

21. Bouche, N.; Fromm, H., GABA in plants: just a metabolite? Trends Plant Sci.

538

2004, 9, 110-115.

539

22. Zhao, G. C.; Xie, M. X.; Wang, Y. C.; Li, J. Y. Molecular mechanisms

540

underlying gamma-aminobutyric acid (GABA) accumulation in giant embryo rice

541

seeds. J Agric Food Chem. 2017, 65, 4883-4889.

542

23. Yu, G. H.; Zou, J.; Feng, J.; Peng, X. B.; Wu, J. Y.; Wu, Y. L.; Palanivelu, R.;

543

Sun, M. X. Exogenous gamma-aminobutyric acid (GABA) affects pollen tube growth

544

via modulating putative Ca2+-permeable membrane channels and is coupled to

545

negative regulation on glutamate decarboxylase. J Exp Bot. 2014, 65, 3235-3248.

546

24. Shelp, B.J.; Bown, A.W.; Zarei A. 4-Aminobutyrate (GABA) a metabolite and

547

signal with practical significance. Botany 2017, 11,1015-1032.

548

25. Crawford, L.A.; Bown, A.W.; Breitkreuz, K.E.; Guinel, F.C. The synthesis of γ-

549

aminobutyric acid in response to treatments reducing cytosolic pH. Plant Physiology

550

1994, 865-871.

551

26. Michaeli, S.; Fait, A.; Lagor, K.; Nunes-Nesi, A.; Grillich, N.; Yellin, A.; Bar, D.; 29



552

Khan, M.; Fernie, A. R.; Turano, F. J.; Fromm, H. A mitochondrial GABA permease

553

connects the GABA shunt and the TCA cycle, and is essential for normal carbon

554

metabolism. Plant J. 2011, 67, 485-498.

555

27. Sheng, L.; Shen, D.; Yang, W.; Zhang, M.; Zeng, Y.; Xu, J.; Deng, X.; Cheng, Y.

556

GABA pathway rate-limit citrate degradation in postharvest citrus fruit evidence from

557

HB pumelo (Citrus grandis) x Fairchild (Citrus reticulata) hybrid population. J Agric

558

Food Chem. 2017, 65, 1669-1676.

559

28. Sheng, L.; Shen, D.; Luo, Y.; Sun, X.; Wang, J.; Luo, T.; Zeng, Y.; Xu, J.; Deng,

560

X.; Cheng, Y. Exogenous gamma-aminobutyric acid treatment affects citrate and

561

amino acid accumulation to improve fruit quality and storage performance of

562

postharvest citrus fruit. Food Chem. 2017, 216, 138-145.

563

29. Shang, H.; Cao, S.; Yang, Z.; Cai, Y.; Zheng, Y. Effect of exogenous gamma-

564

aminobutyric acid treatment on proline accumulation and chilling injury in peach fruit

565

after long-term cold storage. J Agric Food Chem. 2011, 59, 1264-1268.

566

30. Han, Y.; Zhu, Q.; Zhang, Z.; Meng, K.; Hou, Y.; Ban, Q.; Suo, J.; Rao, J.

567

Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes and diverse

568

roles of isoenzymes during persimmon fruit development and postharvest softening.

569

PLoS One 2015, 10, e0123668.

570

31. Chen, M.; Jiang, Q.; Yin, X.-R.; Lin, Q.; Chen, J.-Y.; Allan, A. C.; Xu, C.-J.; 30


Page 30 of 49

Page 31 of 49


571

Chen, K.-S. Effect of hot air treatment on organic acid- and sugar-metabolism in

572

Ponkan (Citrus reticulata) fruit. Sci. Hortic. 2012, 147, 118-125.

573

32. Zhang, Y.; Li, P.; Cheng, L. Developmental changes of carbohydrates, organic

574

acids, amino acids, and phenolic compounds in ‘Honeycrisp’ apple flesh. Food

575

Chemistry 2010, 123, 1013-1018.

576

33. Zhao, Y.; Tan, D.-X.; Lei, Q.; Chen, H.; Wang, L.; Li, Q.; Gao, Y.-N.; Kong, J.

577

Melatonin and its potential biological functions in the fruits of sweet cherry. J. Pineal

578

Res. 2012, 55, 79-88.

579

34. Merlo, L.; Ferretti, M.; Ghisi, R.; Passera, C. Developmental changes of enzymes

580

of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation

581

in peach leaves. Physiologia Plantarum 1993, 89, 71-76.

582

35. Gasic, K.; Hernandez, A.; Korban, S. S. RNA extraction from different apple

583

tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant

584

Molecular Biology Reporter 2004, 22, 437a-437g.

585

36. Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using

586

real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25,

587

402-408.

588

37. Yang, X.; Song, J.; Campbell-Palmer, L.; Fillmore, S.; Zhang, Z. Effect of

589

ethylene and 1-MCP on expression of genes involved in ethylene biosynthesis and 31



590

perception during ripening of apple fruit. Postharvest Biology and Technology 2013,

591

78, 55-66.

592

38. Zhu, Y.; Barritt, B. H. Md-ACS1 and Md-ACO1 genotyping of apple (Malus x

593

domestica Borkh.) breeding parents and suitability for marker-assisted selection. Tree

594

Genetics & Genomes 2008, 4, 555-562.

595

39. Wang, A.; Tan, D.; Takahashi, A.; Li, T. Z.; Harada, T. MdERFs, two ethylene-

596

response factors involved in apple fruit ripening. J Exp Bot. 2007, 58, 3743-3748.

597

40. Trobacher, C. P.; Zarei, A.; Liu, J.-Y.; Clark, S. M.; Bozzo, G. G.; Shelp, B. J.

598

Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in

599

apple fruit. BMC Plant Biology 2013, 144.

600

41. Trobacher, C. P.; Clark, S. M.; Bozzo, G. G.; Mullen, R. T.; DeEll, J. R.; Shelp,

601

B. J. Catabolism of GABA in apple fruit: Subcellular localization and biochemical

602

characterization of two γ-aminobutyrate transaminases. Postharvest Biology and

603

Technology 2013, 75, 106-113.

604

42. Li, Z.; Yu, J.; Peng, Y.; Huang, B. Metabolic pathways regulated by gamma-

605

aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass

606

(Agrostis stolonifera). Sci Rep. 2016, 6, 30338.

607

43. Lum, G. B.; Brikis, C. J.; Deyman, K. L.; Subedi, S.; DeEll, J. R.; Shelp, B. J.;

608

Bozzo, G. G. Pre-storage conditioning ameliorates the negative impact of 132


Page 32 of 49

Page 33 of 49


609

methylcyclopropene on physiological injury and modifies the response of antioxidants

610

and γ-aminobutyrate in ‘Honeycrisp’ apples exposed to controlled-atmosphere

611

conditions. Postharvest Biology and Technology 2016, 116, 115-128.

612

44. Deewatthanawong, R.; Nock, J. F.; Watkins, C. B. γ-Aminobutyric acid (GABA)

613

accumulation in four strawberry cultivars in response to elevated CO2 storage.

614

Postharvest Biology and Technology 2010, 57, 92-96.

615

45. Mae, N.; Makino, Y.; Oshita, S.; Kawagoe, Y.; Tanaka, A.; Aoki, K.;

616

Kurabayashi, A.; Akihiro, T.; Akama, K.; Koike, S.; Takayama, M.; Matsukura, C.;

617

Ezura, H. Accumulation mechanism of gamma-aminobutyric acid in tomatoes

618

(Solanum lycopersicum L.) under low O2 with and without CO2. J Agric Food Chem.

619

2012, 60, 1013-1019.

620

46. Ji, J.; Yue, J.; Xie, T.; Chen, W.; Du, C.; Chang, E.; Chen, L.; Jiang, Z.; Shi, S.

621

Roles of γ‑aminobutyric acid on salinity‑responsive genes at transcriptomic level in

622

poplar involving in abscisic acid and ethylene‑signalling pathways. Planta 2018

623

( https://doi.org/10.1007/s00425-018-2915-9)

624

47. Kathiresan, A.; Tung, P.; P. T., Chinnappa,C. C.; Reid, D. M. γ-aminobutyric

625

acid stimulates ethylene biosynthesis in sunfIower. Plant Physiology 1997, 115, 129-

626

135.

627

48. Shi, S. Q.; Shi, Z.; Jiang, Z. P.; Qi, L. W.; Sun, X. M.; Li, C. X.; Liu, J. F.; Xiao, 33



628

W. F.; Zhang, S. G. Effects of exogenous GABA on gene expression of Caragana

629

intermedia roots under NaCl stress: regulatory roles for H2O2 and ethylene

630

production. Plant Cell Environ. 2010, 33, 149-62.

631

49. Brikis, C. J.; Zarei, A.; Chiu, G. Z.; Deyma, K. L.; Liu, J. Y.; Trobacher, C. P.;

632

Hoover, G. J.; Subedi, S.; DeEll, J. R.; Bozzo, G. G.; Shelp, B. J. Targeted

633

quantitative profiling of metabolites and gene transcripts associated with 4-

634

aminobutyrate (GABA) in apple fruit stored under multiple abiotic stresses.

635

Horticulture Research 2018, 5, 61.

Figure captions

Figure 1. Ethylene production rate (A) and respiration rate (B) of the control and GABA-treated apples (at 0.5 mM, 1.0 mM and 10 mM) during storage at room temperature. The value measured before treatment was used as initial value for all treatments at 0 time. Data are the mean ± standard error from three biological replicate 34


Page 34 of 49

Page 35 of 49


assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

Figure 2. Changes in malate (A), succinate (B), oxalate (C), and GABA contents of the control and GABA-treated apples (at 0.5 mM, 1.0 mM and 10 mM) during storage at room temperature. The value measured before treatment was used as initial value for all treatments at 0 time. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P ＜ 0.05) for each sampling date among treatments.

Figure 3. Effects of 10 mM GABA treatment on the activities of cyNAD-MDH (A), PEPC (B), cyNADP-ME (C) and PEPCK (D) in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments, and ns indicate no significant difference.

Figure 4. Effects of 10 mM GABA treatment on expression levels of MdcyNAD-MDH (A), MdPEPC (B), MdcyNADP-ME (C) and MdPEPCK (D) compared with the control in apples during storage at room temperature. Data are the mean ± standard error from 35



three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

Figure 5. Effects of 10 mM GABA treatment on expression levels of crucial genes related to ethylene biosynthesis and signal transduction compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

Figure 6. Effects of 10 mM GABA treatment on expression levels of genes related to the GABA shunt compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

36


Page 36 of 49

Page 37 of 49


Figure graphics

Figure 1. Ethylene production (A) and respiration rate (B) of control and GABAtreated apples (at 0.5 mM, 1.0 mM and 10 mM) during storage at room temperature. The value measured before treatment was used as initial value for all treatments at 0 time. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments, and ns indicate no significant difference.

37



Figure 2. Changes in malate (A), succinate (B), oxalate (C) and GABA (D) contents of the control and GABA-treated apples (at 0.5 mM, 1.0 mM and 10 mM) during storage at room temperature. The value measured before treatment was used as initial value for all treatments at 0 time. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P ＜ 0.05) for each sampling date among treatments.

38


Page 38 of 49

Page 39 of 49


Figure 3. Effects of 10 mM GABA treatment on activities of cyNAD-MDH (A), PEPC (B), cyNADP-ME (C) and PEPCK (D) in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments, and ns indicate no significant difference.

39



Figure 4. Effects of 10 mM GABA treatment on expression levels of MdcyNAD-MDH (A), MdPEPC (B), MdcyNADP-ME (C) and MdPEPCK (D) compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

40


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Figure 5. Effects of 10 mM GABA treatment on expression levels of crucial genes related to ethylene biosynthesis and signal transduction compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

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Figure 6. Effects of 10 mM GABA treatment on expression levels of genes related to the GABA shunt compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments.

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TOC Figure

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Ethylene production (A) and respiration rate (B) of control and GABA-treated apples (at 0.5 mM, 1.0 mM and 10 mM) during storage at room temperature. The value measured before treatment was used as initial value for all treatments at 0 time. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments, and ns indicate no significant difference. 309x115mm (150 x 150 DPI)


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Changes in malate (A), succinate (B), oxalate (C) and GABA (D) contents of the control and GABA-treated apples (at 0.5 mM, 1.0 mM and 10 mM) during storage at room temperature. The value measured before treatment was used as initial value for all treatments at 0 time. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments. 240x180mm (150 x 150 DPI)



Effects of 10 mM GABA treatment on activities of cyNAD-MDH (A), PEPC (B), cyNADP-ME (C) and PEPCK (D) in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments, and ns indicate no significant difference. 247x188mm (150 x 150 DPI)


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Effects of 10 mM GABA treatment on expression levels of MdcyNAD-MDH (A), MdPEPC (B), MdcyNADP-ME (C) and MdPEPCK (D) compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments. 258x177mm (150 x 150 DPI)



Effects of 10 mM GABA treatment on expression levels of crucial genes related to ethylene biosynthesis and signal transduction compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments. 225x161mm (150 x 150 DPI)


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Effects of 10 mM GABA treatment on expression levels of genes related to the GABA shunt compared with the control in apples during storage at room temperature. Data are the mean ± standard error from three biological replicate assays. Different letters show significant differences (P＜0.05) for each sampling date among treatments. 166x207mm (150 x 150 DPI)


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