Characterization of the Genes Involved in Malic Acid Metabolism from

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Chemistry and Biology of Aroma and Taste

Characterization of the Genes Involved in Malic Acid Metabolism from Pear Fruit and Their Expression Profile after Postharvest 1-MCP/Ethrel Treatment Libin Wang, Min Ma, Yanru Zhang, Zhangfei Wu, Lin Guo, Weiqi Luo, Li Wang, Zhen Zhang, and Shaoling Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02598 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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Journal of Agricultural and Food Chemistry

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Characterization of the Genes Involved in Malic Acid Metabolism from Pear

2

Fruit and Their Expression Profile after Postharvest 1-MCP/Ethrel Treatment

3 4

LIBIN WANG,a,† MIN MA,a,† YANRU ZHANG,a ZHANGFEI WU,a LIN GUO,a WEIQI LUO,b

5

LI WANG,a ZHEN ZHANG, a SHAOLING ZHANG a,*

6 7

a

8

Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing,

9

Jiangsu 210095, China

Centre of Pear Engineering Technology Research, State Key Laboratory of Crop

10

b

11

FL 34945

USDA, ARS, U.S. Horticultural Research Laboratory, 2001 S. Rock Road, Ft. Pierce,

12 13 14 15 16 17 18 19 20 21 22 23

† These authors contributed equally to this paper.

24

* Corresponding author.

25

E-mail address: [email protected] (S.L. Zhang); Tel/Fax: +86 25 84396485

ACS Paragon Plus Environment


26

ABSTRACT: In this study, five genes involved in malic acid (MA) metabolism,

27

including a cytosolic NAD-dependent malate dehydrogenase gene (cyNAD-MDH), a

28

cytosolic NADP-dependent malic enzyme gene (cyNADP-ME), two vacuolar

29

H+-ATPases gene (vVAtp1 and vVAtp2) and one vacuolar inorganic pyrophosphatase

30

gene (vVPp), were characterized from pear fruit based on bioinformatic &

31

experimental analysis. Their expression profile in ‘Housui’ pear was tissue-specific,

32

and their expression patterns during fruit development were diverse. During ‘Housui’

33

pear storage, MA content decreased, which was associated with the downregulated

34

transcripts of MA metabolism-related genes and cyNAD-MDH activity and higher

35

cyNADP-ME activity. The response of MA metabolism to postharvest 1.5 µL L-1

36

1-MCP fumigation and 0.5 mL L-1 ethrel dipping was distinct: 1-MCP fumigation

37

upregulated the gene expression and cyNAD-MDH activity, suppressed cyNADP-ME

38

activity, and thus maintained higher MA abundance when compared with that in

39

control; on the other hand, an opposite behavior was observed in ethrel-treated fruit.

40 41

KEYWORDS: Pear, 1-MCP, ethrel, malic acid, enzyme activity, gene expression

42 43 44 45 46 47 48 49 50


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51

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Organic acids, the composition of which in ripe fruit varies among plant species, are

53

crucial contributors to fruit nutrition and flavor quality 1. In most pear fruit, malic acid

54

(MA) is the predominant organic acid, followed by citric acid 2. Besides its role in

55

flavor quality, MA functions in plant physiology such as an essential storage carbon

56

molecule and a pH regulator 3.

INTRODUCTION

57

The abundance of MA in tree fruit is attributed to the net balance of its synthesis,

58

degradation as well as compartmentation 1. The cytosolic NAD-dependent malate

59

dehydrogenase (cyNAD-MDH) is supposed to be the last and key enzyme involved

60

in MA biosynthesis, while the cytosolic NADP-dependent malic enzyme

61

(cyNADP-ME) plays an important role in its degradation during fruit ripening

62

Over 85% of MA in tree fruit is localized in vacuole 7. Two primary proton pumps,

63

vacuolar H+-ATPase (V-ATPase) and vacuolar inorganic pyrophosphatase (vVPp),

64

proposedly participate in MA transportation across the tonoplast 1. Some genes,

65

including vVAtps (NCBI accession no. AB189963.1 and AB189964.1) and vVPp

66

(NCBI accession no. AB097115.1), involved in this process have been cloned from

67

pear

68

been identified. Previously, a mitochondrial NAD-MDHs (mNAD-MDHs; NCBI

69

accession no. GU256770) and a cyNADP-ME (NCBI accession no. GU256768) were

70

cloned from pear

71

result to verify that cyNADP-ME (NCBI accession no. GU256768) in localized in

72

cytosol; and it is supposed to be localized in chloroplast based on chloroplast transit

73

peptide analysis using Chlorop 1.1 Server 11.

74 75

4, 8-10

4-6

.

; however, the genes encoding cyNAD-MDH and cyNADP-ME have not

4, 8

. However, there is no bioinformatic analysis or experimental

The ripening & senescence of climacteric fruit is a genetically programmed and developmentally regulated process, where ethylene plays an import role


12, 13

. As an


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efficient ethylene antagonist, 1-methylcyclopropene (1-MCP) has shown a potential

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to delay fruit ripening & senescence and maintain the quality, thus extending the

78

shelf life

79

In a previous study, Liu, Wang, Qin and Tian

80

1-MCP fumigation on ‘Fuji’ apple fruit could suppress MA loss during 20 ℃ storage,

81

which was associated with the altered responses of enzyme activity and gene

82

expression in MA metabolic pathway. Pear, as a climacteric fruit, is characterized by

83

an increase in the respiration rate in association with the accumulation of ethylene

84

upon initiation of ripening

85

1-MCP and ethrel treatments influencing ripening and senescence of pear

86

impact of their application on MA metabolism has not been clarified until recently.

14, 15

, while a opposite phenomenon was observed in ethrel-treated fruit 16. 1

found that postharvest 1.0 µL L-1

14

. Although information is available on postharvest 15-17

, the

87

With the rapid progress in sequencing techniques, our group have sequenced and

88

assembled the pear (Pyrus bretschneideri cv. Dangshansuli) genome 18, which would

89

facilitate the genome-wide identification and analysis of gene family member. In this

90

study, we firstly characterized the genes (cyNAD-MDH, cyNADP-ME, vVAtp1,

91

vVAtp, vVPp) involved in MA metabolism from pear. Subsequently, the impact of

92

postharvest 1-MCP and ethrel treatments on ‘Housui’ pear quality was analyzed.

93

Finally, enzyme activity and genes expression in MA metabolic pathway were

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assayed to illustrate the molecular mechanism of postharvest 1-MCP and ethrel

95

treatments on MA abundance. Pyrus pyrifolia cv. ‘Housui’ pear, a middle-maturity

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cultivar, is widely planted in China because of its high yield, high pest-resistant

97

capacity and good consumption quality.

98 99 100

MATERIALS AND METHODS

Plant Materials and Treatments


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‘Housui’ pear fruit were harvested from homogeneous trees in the experimental

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orchard in Nanjing. After transportation to laboratory, uniform and defect-free fruit

103

were selected, placed on the shelves at 25 °C for 24 h to emanate field heat, and then

104

randomly divided into three treatments with three biological replicates per treatment:

105

H2O dipping for 5 min (control), 0.5 mL L-1 ethrel dipping for 5 min, and 1.5 µL L-1

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1-MCP fumigation for 24 h. After treatments, all samples were dried/ventilated,

107

packed with plastic bags, and stored at 25 ℃. Samples were taken every 6 d until the

108

decay rate over 20%. For the sampling, the first layer of cortex (below the pericarp)

109

from the three fruit per replicates were quickly removed with a sharp parer.

110 111

Identification of Candidate cyNAD-MDH and cyNADP-ME Genes from Pear

112

BLASTP

113

(http://peargenome.njau.edu.cn) with the identified cyNAD-MDH (AT1G04410 and

114

AT5G43330) and cyNADP-ME (AT2G19900, AT5G11670 and AT5G25880) protein

115

sequences from Arabidopsis with an E-valve < 1E-10. Then, all the candidate

116

sequences were subjected to the InterProScan program, Pfam, and SMART databases

117

to confirm the presence of the conserved domain. Finally, the selected sequences and

118

cyNAD-MDH (or NADP-ME) from other plants was aligned by DNAMAN multiple

119

alignment program to confirm the presence of conserved motif 19.

was

used

to

search

the

Pyrus

bretschneideri

genome

120 121

Phylogenetic Analysis

122

The phylogenetic tree was constructed using the maximum likelihood (ML) method

123

with a bootstrap analysis of 1000 replicates and the Jones-Taylor-Thornton (JTT)

124

model using MEGA7.0 software 20.

125



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Physicochemical Parameter, Subcellular Localization, and 3D Structures of

127

Protein

128

Physicochemical parameters of the full-length proteins were calculated with the

129

ProtParam tool (http://web.expasy.org/protparam/) 21, and the subcellular localization

130

was predicted using the CELLO v2.5 server (http://cello.life.nctu.edu.tw/)

131

structures

132

(https://www.swissmodel.expasy.org/interactive/dKBKSG/models/).

of

the

proteins

were

predicted

using

SWISS-MODEL

22

. 3D

server

133 134

Weight Loss, Color, and Decay Rate

135

Weight loss was calculated according to the method of Mahajan, et al. 23. For the color

136

assay of the cortex, the fruit were cut into halves before analysis by a Minolta CR-400

137

chromameter (Konica Minolta Sensing, Inc., Osaka, Japan) based on the instruction of

138

manufacturer. Decay rate was determined according to Jayanty, et al. 24’s method.

139 140

Total Soluble Acid (TSS), Titratable Acid (TA), and Malic Acid

141

TSS and TA were measured by Pocket Brix-Acidity Meter (PAL-BX/ACID12,

142

ATAGO, Japan). Malic acid was measured using the Shimadzu LC-20A series liquid

143

chromatograph (Shimadzu Co., Kyoto, Japan) equipped with a degasser, quaternary

144

pump, 20 µL volume injection autosampler, chromatographic Agilent C18 column and

145

a UV-visible diode array detector. Four g fresh sample was pulverized with liquid N2,

146

diluted in 20 mL of ultra-pure water, and filtered through a 0.45-µm Millipore filter.

147

The mobile phase was a solvent system of 25 mM phosphate buffer (pH 2.4) at a flow

148

rate of 0.5 mL min-1 at 30 ℃. The linearity ranges of the solutions were derived by

149

sequentially diluting a standard stock solution. Malic acid was detected at 210 nm

150

wavelength.


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Enzyme Extraction and Activity Assay

153

Enzyme extraction was performed according to the method of Liu, Wang, Qin and

154

Tian

155

before adding 10 mL extraction buffer (50 mM Hepes-Tris, 250 mM sorbitol, 125 mM

156

KCl, 5 mM EGTA, 10 mM MgSO4, 2 mM PMSF, 1.5% (w/v) PVP, 0.1% (w/v) BSA

157

and 1 mM DTT). The mixture was filtered through microcloth, centrifuged at 1000 g,

158

and recentrifuged at 50,000 g for 1 h at 4 ℃. The supernatant was then collected for

159

enzyme activity assay.

1

with some modification. Two g frozen cortex tissue was ground in liquid N2

160

cyNAD-MDH activity was measured with NAD-MDH assay kit (NMDH-1-Y,

161

Suzhou Comin Biotechnology Co., Ltd., China) according to the protocol of

162

manufacturer, and the result was expressed as nmol min-1 g-1 protein. cyNADP-ME

163

activity was measured with NADP-ME assay kit (NADPME-1-Y, Suzhou Comin

164

Biotechnology Co., Ltd., China) according to the protocol of manufacturer, and the

165

result was expressed as nmol min-1 g-1 protein. Protein determination was carried out

166

based on the BCA method

167

assay kit; A045-4, Nanjing Jiancheng Bioengineering Institute, China).

25

following the instruction of the manufacturer (protein

168 169

Gene Cloning

170

Total RNA was isolated using TRizol Reagents (Invitrogen, Carlsbad, CA) followed

171

by RNase-free DNase treatment (Qiagen, Valencia, CA). Approximately 2 µg of total

172

RNA was used for first-strand cDNA synthesis using TransScript® One-Step gDNA

173

Removal and cDNA Synthesis SuperMix (TRANSGEN, Beijing China).

174

The cDNA obtained was used as a template to amplify the gene using the primers

175

listed in Table S1. Primers used for PCR can amplify the full-length cDNA of the



176

corresponding genes. Polymerase chain reactions (PCRs) were performed with the aid

177

of Q5 High-Fidelity DNA polymerase (New England Biolabs, Inc., USA) in a final

178

volume of 25 µL, containing 11 µL double-distilled H2O, 5 µL 5× reaction buffer, 0.5

179

µL dNTPs, 1.25 µL forward primer, 1.25 µL reverse primer, 0.75 µL cDNA, 5 µL 5×

180

High GC Enhancer, 0.25 µL Q5 High-Fidelity DNA polymerase. PCR conditions

181

were as follows: 98 °C for 30 s, followed by 38 cycles at 98 °C for 10 s, 58 °C for 30

182

s, and 72 °C for 30 s, with a final extension at 72°C for 2 min. PCR products (cDNA

183

of the correspondent gene) were detected by electrophoresis on a 1% agarose gel,

184

from which target bands were cut and purified using an AxyPrep DNA Gel Extraction

185

Kit (Axygen, USA). The gel purified PCR products were then inserted into the

186

pCAMBIA 1302 vector and transformed into Escherichia coli DH5α before

187

sequenced.

188 189

qRT-PCR Analysis

190

The primers of all genes were designed using Primer Premier 6.0, and then we blasted

191

back to the genome database to make sure the primers were all gene specific (Table

192

S1). Total RNA was isolated using TRizol Reagents (Invitrogen, USA) followed by

193

RNase-free DNase treatment (Qiagen, USA). Approximately 2 µg of total RNA was

194

used for first-strand cDNA synthesis using TransScript® One-Step gDNA Removal

195

and cDNA Synthesis SuperMix (TRANSGEN, China).

196

qRT-PCR was performed using the LightCycler 480 SYBR GREEN I Master

197

(Roche, USA) according to the manufacturer’s protocol. Quantitative real-time PCR

198

(qRT-PCR) was performed using 10.0 µL reaction volume, including 5.0 µL

199

LightCycler 480 SYBR GREEN I Master, 0.5 µL forward primer, 0.5 µL reverse

200

primer, 1 µL cDNA, and 3.0 µL RNase-free water. The qRT-PCR was performed on


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the LightCycler 480 (Roche). The qRT-PCR conditions were as follows:

202

pre-incubation at 95 ℃ for 10 min and then 45 cycles of 94 ℃ for 10 s, 60 ℃ for 30 s,

203

72 ℃ for 20 s, with a final extension at 72 ℃ for 3 min. A melting curve was

204

performed from 60 to 95 ℃ in order to check the specificity to the amplified product.

205

Finally, the average threshold cycle (Ct) was calculated. Pyrus tubulin was used as the

206

internal control 26, and the relative expression levels were calculated with the 2-△△Ct

207

method 27.

208 209

Transient Transformation of ‘Dangshansuli’ Pear Fruit

210

Full-length sequences of PbrcyNAD-MDH and PbrcyNADP-ME genes were amplified

211

using high-fidelity KOD-Plus-Ver.2 DNA Polymerase (Toyobo, Osaka, Japan) before

212

insertion into a pSAK277 vector to construct an overexpressing vector with a CaMV

213

35S

214

(unconstructed pSAK277 vector) were transformed into Agrobacterium tumefaciens

215

strain EHA105; and then incubated at 28°C until OD660 was 1.0. After centrifugation

216

and resuspension of the bacterial strain in infiltration buffer (10 mM MgCl2, 10 mM

217

MES, pH 5.5, and 150 µM acetosyringone), 50 µl of solution was slowly injected into

218

the cortex tissue in the pre-defined position (totally eight positions) of the fruit. The

219

injected fruit were then stored at 25 °C for 5 d before sampling. There were three

220

replicates with ten fruit each for each vector.

promoter.

The constructed

overexpressing

vector and

empty

vector

221 222

Statistical Analysis

223

Data presented were the mean values of three biological replicates. SAS Version 9.3

224

(SAS Institute, Gary, NC) was used to analyze the data, using analysis of variance

225

(PROC ANOVA) with multi-comparison correction. Mean separation was determined



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226

by Duncan’s multiple range test at the 0.05 level. Spearman’s correlation analysis was

227

performed to evaluate the association between paired attributes, which was visualized

228

as a heatmap.

229 230

231

Characterization of MA Metabolism-related Genes from Pear

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RESULTS

Characterization of cyNAD-MDH Gene from Pear Fruit. A total of seven

233

candidate

234

Pfam:Ldh_1_C) and a highly conserved motif “IWGNH” which has been reported in

235

plant MDHs and is responsible for the catalytic activity of the dehydrogenase 19, have

236

been identified in Pyrus bretschneideri genome (Figure S1). Of these, only

237

Pbr002553.1 and Pbr030554.1 possessed the NAD-binding motif ‘‘TGAAGQI’’,

238

which was conserved in plant cyNAD-MDHs 19 (Figure S1). In combination with the

239

transcriptome analysis (the expression of Pbr002553.1 was undetectable in mature

240

‘Yali’ fruit, while the FPKM of Pbr030554.1 is over 300; those are unpublished data)

241

as well as hydropathy analysis (a strong hydrophobic domain containing about 24

242

amino acids downstream from the N-terminal nine amino acids (Figure 1a), which

243

was reported in plant cyNAD-MDHs

244

PbrcyNAD-MDH and plays an important role in fruit MA biosynthesis. To further

245

confirm the role of Pbr030554.1 in pear MA biosynthesis, fruit transiently

246

over-expressing PbrcyNAD-MDH were constructed. As shown in Figure 1b, a higher

247

MA content was detected in transgenic fruit than that in control (empty vector) after

248

storage at 25°C for 5 d, which was associated with higher PbrcyNAD-MDH mRNA

249

(data not shown). Subsequently, we cloned the cyNAD-MDH from Pyrus pyrifolia cv.

250

‘Housui’ and constructed a phylogenetic tree with cyNAD-MDH proteins from other

members,

possessing

the

conserved

19

domain

(Pfam:Ldh_1_N;

), Pbr030554.1 proposedly encodes a


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251

plants (Table S2). As shown in Figure 1c, PbrcyNAD-MDH and PpcyNAD-MDH

252

were

253

PbrcyNAD-MDH was analyzed by SWISS-MODEL server. As shown in Figure 1d,

254

PbrcyNAD-MDH harbored several secondary structures, such as α-helix, β-turn and

255

random coil.

grouped

with

MdcyNAD-MDH.

Furthermore,

the

3D

structure

of

256 257

Characterization of cyNADP-ME Gene from Pear Fruit. A total of three

258

candidate members, possessing the conserved domain (Pfam: malic; Pfam: Malic_M)

259

and five highly conserved motifs which has been reported in plant NADP-ME 28, have

260

been identified in Pyrus bretschneideri genome (Figure S2). Based on chloroplast

261

transit peptide analysis (Figure 2a), Pbr008772.1 is selected as the candidate

262

cyNADP-ME in pear, while the other two are proposed to be chloroplast NADP-MEs

263

(clNADP-MEs). Furthermore, a phylogenetic tree of plant NADP-MEs was

264

constructed (Table S3). As shown in Figure 2b, clNADP-MEs and cyNADP-MEs

265

from plants are categorized into two groups, and Pbr008772.1 is categorized with

266

cyNADP-MEs. To further confirm its role in MA biosynthesis, fruit transiently

267

over-expressing PbrcyNADP-ME were constructed. As shown in Figure 2c, MA

268

content was considerably lower in transgenic fruit than that in control (empty vector)

269

after 5-d storage at 25 ℃, which was associated with higher PbrcyNADP-ME mRNA

270

(data not shown). Subsequently, we cloned the cyNADP-ME from Pyrus pyrifolia cv.

271

‘Housui’ and found that the amino acid sequence of PpcyNAD-MDH was very similar

272

to that of PbrcyNAD-MDH (Table S3). Hydropathy analysis and 3D structure of

273

PbrcyNADP-ME were analyzed and illustrated in Figure 2d and Figure 2e as well. As

274

shown in Figure 2d, there is a strong hydrophilic domain in the N-terminal of the

275

protein, and PbrcyNADP-ME harbored several secondary structures, such as α-helix,



276

β-turn and random coil (Figure 2e).

277 278

Characterization of vVAtps and vVPp Genes from Pear Fruit. vVAtps and vVPp

279

are ancient genes in plant kingdom and have been retained during plant evolution 29, 30.

280

In a previous study, the sequence of vVAtps and vVPp from Pyrus communis and

281

Pyrus pyrifolia have been reported 8. Based on this, we conducted a BLASP against

282

the Pyrus bretschneideri genome (http://peargenome.njau.edu.cn) and designed the

283

primers to clone the correspondent genes in Pyrus bretschneideri. As shown in Figure

284

S3a, all vVAtps from plants have four conserved domains (Pfam: ATP-synt_ab_N;

285

Pfam: ATP-synt_ab_Xtn; Pfam:ATP-synt_ab; Pfam:ATP-synt_ab_C); meanwhile, the

286

vVAtps from pear were clustered with MdvVAtp based on phylogenetic analysis

287

(Figure 3a), suggesting their amino acid sequence were very similar (Table S4 and

288

S5). Similarly, three highly conserved motifs, which has been reported in vVPps, have

289

been identified in vVPps from pear; and a catalytic domain “DVGADLVGKVE” is

290

localized in the first motif (Figure S3b) 29. Besides, PbrvVPp has 13 transmembrane

291

regions (Figure 3b).

292 293

Expression Profile of MA metabolism-related Genes in Different Tissues of

294

‘Housui’ pear as well as during Pear Fruit Development

295

As shown in Figure 4a, MA metabolism-related genes were expressed in leaf, flower,

296

seed, shoot and pericarp of ‘Housui’ pear by qRT-PCR analysis. Most members,

297

including PpcyNAD-MDH, PpcyNADP-ME, PpvVAtp1 and PpvVAtp2, showed the

298

highest transcripts in flower, but presented relatively lower level in pericarp or shoot

299

(Figure 4a). On the other hand, seed possessed the highest mRNAs of PpvVPp, whose

300

transcripts were lowest in pericarp (Figure 4a).


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301

In a previous study, our unit carried out a survey on the expression patterns of

302

MA metabolism-related genes in cortex during pear (‘Housui’, ‘Yali’, ‘Kuerlexiangli’,

303

‘Nanguoli’, ‘Starkrimson’) fruit development, including the fruit-setting stage (period

304

1), physiological fruit dropping stage (period 2), fruit rapid enlargement stage (period

305

3), a month after fruit enlargement stage (period 4), commercially mature stage

306

(period 5). As shown in Figure 4b, the expression patterns of five genes in each

307

cultivar were diverse. Taken ‘Starkrimson’ pear as an example, the mRNAs of

308

cyNAD-MDH, vVAtp1, vVAtp2 and vVPp accumulated to the highest level at

309

commercially mature stage, while cyNADP-ME transcripts decreased in contrast.

310

Besides, the expression patterns of each gene were different across cultivars (Figure

311

4b).

312 313

Dynamic Change in Quality Attributes after Postharvest 1-MCP and Ethrel

314

Treatments

315

As shown in Table 1, both weight loss and decay rate of control accumulated during

316

‘Housui’ fruit storage. Taking control fruit as an example, the weight loss increased

317

from 0.0% to 3.1% after 24-d storage at 25 ℃; the decay occurred on the 12th d (1.3%),

318

and then increased to 39.3% at the end of storage (Table 1). No significant difference

319

in weight loss was observed between treatments. On the other hand, postharvest

320

1-MCP fumigation delayed the occurrence of decay in fruit until 18th d, and

321

significantly suppressed the decay rate (p < 0.05), while a opposite behavior was

322

observed after ethrel dipping (Table 1). In addition to weight loss and decay rate, there

323

was also a color transition from light green to bronze in pericarp (Figure 5a); and the

324

whiteness index of the cortex increased from 61.3 to 65.3 during the first 12-d storage,

325

then decreased to 63.3 at the end of storage (Table 1). 1-MCP fumigation deterred the



326

color alternation, which was accelerated by ethrel dipping (Table 1 and Figure 5a).

327

TSS accumulated during storage to the highest level on the 12th d (14.6%), then

328

decreased to 12.7% after 24-d storage (Figure 5b). Postharvest 1-MCP and ethrel

329

treatments did not change the expression pattern, but the highest TSS in ethrel-treated

330

fruit was observed after 6-d storage, while 1-MCP delayed the peak to 18th d (Figure

331

5b). On the other hand, the abundances of TA and MA gradually degraded (Figure 5c

332

and 5d). Postharvest 1-MCP fumigation suppressed such reduction, while lower TA

333

and MA were observed in ethrel-treated fruit in comparison with those in control

334

(Figure 5c and 5d). For TSS/TA, it accumulated during storage (Figure 5e).

335 336

Dynamic Change in the Activities of Enzymes Involved in MA Metabolism after

337

Postharvest 1-MCP and Ethrel Treatments

338

During ‘Housui’ pear storage, cyNAD-MDH activity gradually decreased from 779.0

339

nmol min-1 g-1 protein at the beginning of storage to 443.5 nmol min-1 g-1 protein after

340

24-d storage with a 43.0% reduction (Figure 6a). Postharvest 1-MCP fumigation

341

mitigated such trend, which was accelerated by ethrel dipping (Figure 6a). Taken 18th

342

d as an example, cyNAD-MDH activity in control, 1-MCP, and ethrel treated fruit

343

were 481.8, 612.3, and 378.1 nmol min-1 g-1 protein, respectively (p < 0.05) (Figure

344

6a).

345

On the other hand, the cyNADP-ME activity accumulated from 35.7 to 75.5 nmol

346

min-1 g-1 protein during storage (Figure 6b). The response of cyNADP-ME activity to

347

postharvest 1-MCP and ethrel treatments was distinct: a lower activity was observed

348

in 1-MCP-treated fruit, while ethrel dipping upregulated cyNADP-ME activity. In 12th

349

d, cyNADP-ME activity in control, 1-MCP, and ethrel treated fruit were 60.0, 46.6,

350

and 82.2 nmol min-1 g-1 protein, respectively (p < 0.05) (Figure 6b).


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351 352

Dynamic Change in the Expression Level of Genes Involved in MA Metabolism

353

after Postharvest 1-MCP and Ethrel Treatments

354

As shown in Figure 7a-e, the expression of MA metabolism-related genes suffered a

355

reduction during ‘Housui’ pear storage. The transcripts of PpcyNAD-MDH,

356

PpcyNADP-ME, PpvVAtp1, PpvVAtp2, and PpvVPp in control fruit were

357

downregulated by 57.4%, 73.4%, 68.9%, 61.7%, 49.2%, respectively, at the end of

358

the storage (Figure 7a-e). Such phenomenon agreed with our previous study via

359

RNA-seq analysis (unpublished data, Figure 7f).

360

The response of gene expression in MA metabolic pathway to postharvest 1-MCP

361

and ethrel was distinct. As shown in Figure 7a-e, the expression of PpcyNAD-MDH,

362

PpcyNADP-ME, PpvVAtp1, PpvVAtp2, and PpvVPp was upregulated by 1-MCP

363

fumigation, while ethrel dipping suppressed their transcripts, which was consistent

364

with the result of our previous study (unpublished data, Figure 7f).

365 366

Correlation coefficient between MA content, enzyme activity and gene expression

367

As shown in Figure 8a, the reduction of MA during pear fruit ripening was

368

positively correlated with the downregulated cyNAD-MDH activity and expression of

369

five MA-metabolizing genes (p < 0.05). Meanwhile, a positive correlation was

370

observed between cyNAD-MDH activity and cyNAD-MDH transcripts (p < 0.05). A

371

similar result was observed in 1-MCP -treated fruit during ripening (Figure 8b) (p
50 % were displayed. (d) 3D structures of PbrcyNAD-MDH.


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Figure 2. Characterization of cyNADP-ME gene from pear. (a) Chloroplast transit peptide

was

analyzed

by

Chlorop

1.1

Server

(http://www.cbs.dtu.dk/services/ChloroP/?_ga=2.83445334.1169137693.1525100201475886990.1525100201). (b) Phylogenetic relationship of plant NADP-MEs. (c) Overexpression of PbrcyNADP-ME on MA. (d) Hydropathy analysis of the PbrcyNADP-ME by TMpred program. Amino acid residues are numbered from left to right on the horizontal axes, and the hydrophobic and hydrophilic positions are plotted



above and below the ordinate, respectively. (e) 3D structures of PbrcyNAD-MDH.


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Figure 3. Characterization of vVAtps and vVPp genes from pear. (a) Phylogenetic relationship of plant vVAtps. Percentage bootstrap scores of

>

50

%

were

displayed.

(b)

Transmembrane

helices

in

PbrvVPp

(http://www.cbs.dtu.dk/services/TMHMM/).


was

predicted

by

TMHMM

Server

v.

2.0


Figure 4. Expression profile of MA metabolism-related genes in different tissues of ‘Housui’ pear as well as during pear fruit development. (a) Heatmap of expression profile of MA metabolism-related genes in different tissues (leaf, flower, seed, shoot and pericarp) of


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‘Housui’ pear. The expression level of PpcyNAd-MDH in leaf was set as 1. The genes are located on the right and tissues at the bottom. The color scale represents normalized log 2-transformed (mean value of the three biological replicates + 1), where light red indicates a high level, light green indicates a low level and black indicates a medium level. (b) Expression patterns of MA metabolism-related genes during pear fruit development. ‘Housui’, ‘Yali’, ‘Kuerlexiangli’, ‘Nanguoli’, ‘Starkrimson’ fruit were harvest from a commercial field at five developmental stages, including fruit-setting stage (period 1), physiological fruit dropping stage (period 2), fruit rapid enlargement stage (period 3), a month after fruit enlargement stage (period 4), commercially mature stage (period 5). Data were adapted from transcriptome data 35.



Figure 5. Impact of postharvest 1-MCP and ethrel treatments on visual quality of pericarp and cortex (a), TSS (b), TA (c), MA (d) and TSS/TA (e) during ‘Housui’ pear storage. Different capital letters with the same treatment mean significant difference among samples, and different small letters in the same sampling data mean significance among treatments (p < 0.05).


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Figure 6. Impact of postharvest 1-MCP and ethrel treatments on cyNAD-MDH and cyNADP-ME activities during ‘Housui’ pear storage. Different capital letters with the same treatment mean significant difference among samples, and different small letters in the same sampling data mean significance among treatments (p < 0.05).



Figure 7. Impact of postharvest 1-MCP and ethrel treatments on the expression of MA metabolism-related genes during ‘Housui’ pear storage. (a-d) Response of gene expression to postharvest 1-MCP/ethrel treatment by qRT-PCR analysis in this study. The expression level of PpNAD-MDH at the beginning of storage was set as 1. Different capital letters with the same treatment mean significant difference among samples, and different small letters in the same sampling data mean significance among treatments (p < 0.05). (c) Heatmap of the expression profile of MA metabolism-related genes after 1-MCP/ethrel treatment (unpublished data). ‘Housui’ fruit were harvested from in a commercial orchard in Xuzhou on 25th August in 2016, and treated with H2O (control), 1.5 µL L-1 1-MCP and 0.5 mL L-1 ethrel treatment


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before storage at 25 ℃. Cortex was sampled for transcriptome analysis when the decay rate of samples was over 30% (The decay rate of ethrel-treated fruit and control was over 30% on the 24th d and 29th d, respectively). The color scale represents normalized log 2-transformed (RPKM + 1), where light red indicates a high level, light green indicates a low level and black indicates a medium level.



Figure 8. Correlation coefficient between MA content, enzyme activity and gene expression in control (a), 1-MCP-fumigated (b) or ethrel-treated (c) fruit. ** and * represent the significance level at 0.01 and 0.05, respectively


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Cytosol

1-MCP Postharvest Storage

Light green

Bronze

PbrVAtps

Control Malic acid

PbrVPp

Malic acid

PbrNADP-ME

Pyruvic acid

Malic acid PbrNAD-MDH Oxaloacetic acid

Ethrel

2.00

Content (mg g-1 FW)

Vacuolar

Pyrus bretschneideri cv. Dangshansuli


1.50

1.00

0.50

0.00 0

6

12

18

24

Storage time (d)

30

Characterization of the Genes Involved in Malic Acid Metabolism from

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