Comparative Transcriptome Profiling in a Segregating Peach

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Omics Technologies Applied to Agriculture and Food

Comparative transcriptome profiling in a segregating peach population with contrasting juiciness phenotypes Talia del Pozo, Simon Miranda, Mauricio Latorre, Felipe Olivares, Leonardo Pavez, Ricardo Gutiérrez, Jonathan Maldonado, Patricio Hinrichsen, Bruno Giorgio Defilippi, Ariel Orellana, and Mauricio Gonzalez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05177 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 12, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

TITLE: Comparative transcriptome profiling in a segregating peach population with

2

contrasting juiciness phenotypes.

3

Talía del Pozo1,3, Simón Miranda1,2, Mauricio Latorre3,4,5,6, Felipe Olivares3, Leonardo Pavez7,8,

4

Ricardo Gutiérrez9, Jonathan Maldonado3, Patricio Hinrichsen10, Bruno G. Defilippi11, Ariel

5

Orellana4,12, Mauricio González*3,4.

6

1

7

Chile. Camino La Pirámide 5750, Huechuraba, Santiago, Chile.

8

2

9

Macul, Santiago, Chile.

Centro Tecnológico de Recursos Vegetales, Faculty of Sciences, Universidad Mayor, Santiago,

Laboratorio de Genética Molecular Vegetal, INTA, Universidad de Chile. Av. El Líbano 5524,

10

3

11

Alimentos, Universidad de Chile. Av. El Líbano 5524, Santiago, Chile.

12

4

13

5

14

Rancagua, Chile.

15

6

16

851, 7th Floor, Santiago, Chile.

17

7

18

Chile.

19

8

20

Gana 1702, Santiago, Chile.

21

9

22

(CECAD), University of Cologne, Cologne, Germany.

23

10

24

Santa Rosa 11610, Santiago, Chile.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los

FONDAP Center for Genome Regulation. Av. Blanco Encalada 2085, Santiago, Chile. Instituto de Ingeniería, Universidad de O’Higgins. Av. Libertador Bernardo O’Higgins 611,

Mathomics, Center for Mathematical Modeling, Universidad de Chile. Av. Almirante Beauchef

Instituto de Ciencias Naturales, Universidad de Las Américas. Av. Manuel Montt 948, Santiago,

Departamento de Ciencias Químicas y Biológicas, Universidad Bernardo O′Higgins. General

Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases

Laboratorio de Biotecnología, Instituto de Investigaciones Agropecuarias, INIA La Platina.

ACS Paragon Plus Environment

1


25

11

26

11610, Santiago, Chile.

27

12

28

Santiago, Chile.

29

*

30

INTA,

31

(+56)229781440. Avenida Macul 5524, Casilla 138-11, Santiago, Chile.

Page 2 of 38

Unidad de Poscosecha, Instituto de Investigaciones Agropecuarias, INIA La Platina. Santa Rosa

Centro de Biotecnología Vegetal, Facultad de Ciencias de la Vida, Universidad Andrés Bello,

Correspondence and requests for materials should be addressed to: Mauricio González, Ph.D. Universidad

de

Chile.

E-mail:

[email protected].


Telephone

number:

2

Page 3 of 38


32

ABSTRACT

33

Cold storage of fruit is one of the methods most commonly employed to extend the postharvest

34

lifespan of peaches (Prunus persica (L.) Batsch). However, fruit quality in this species is affected

35

negatively by mealiness, a physiological disorder triggered by chilling injury after long periods of

36

exposure to low temperature during storage and manifested mainly as a lack of juiciness, which

37

ultimately modifies the organoleptic properties of peach fruit. The aim of this study was to identify

38

molecular components and metabolic processes underlying mealiness in susceptible and non-

39

susceptible segregants. Transcriptome and qRT-PCR profiling were applied to individuals with

40

contrasting juiciness phenotypes in a segregating F2 population. Our results suggest that mealiness

41

is a multiscale phenomenon, since juicy and mealy fruit display distinctive reprogramming

42

processes affecting translational machinery and lipid, sugar and oxidative metabolism. The

43

candidate genes identified may be useful tools for further crop improvement.

44

Keywords: mealiness, gene expression, Prunus persica, cold storage, transcriptome.


3


Page 4 of 38

45

INTRODUCTION

46

Peach and nectarine production in the Southern Hemisphere occurs in the opposite season to their

47

final destination markets. Therefore, long-term cold storage (CS) is often applied during shipping

48

in order to prevent postharvest deterioration. However, cold may induce chilling injury (CI) in

49

fruit, affecting their quality during this period1. Manifestations of CI in peach fruit include hard-

50

textured fruit with no juice (leatheriness), internal browning, red flesh pigmentation (bleeding),

51

loss of flavor and mealiness or woolliness, which is the consumer perception of a dry and wooly

52

texture due to lack of free juice1. Mealiness as a physiological disorder represents a major problem

53

because its symptoms remain unnoticed until peaches reach customers at a ready-to-eat stage1.

54

Different strategies have been attempted during the postharvest period to alleviate symptoms and

55

reduce the economic impact of mealiness losses, such as storage in a controlled atmosphere with

56

elevated CO2 and reduced O2 levels, as well as preconditioning and intermittent warming during

57

shelf life2, 3 and/or exogenous pre-harvest hormone applications4, 5. Although these treatments may

58

modify the moment of appearance or the severity of chilling injury symptoms1, mealiness still

59

represents a major challenge, because its incidence and progression involve multiple factors such

60

as cultivar type, seasonal variations, agricultural practices within each orchard and molecular

61

determinants1.

62

One of the classical hypotheses to explain this physiological disorder states that acclimation during

63

CS would induce changes in cell wall polysaccharide composition, ultimately causing a reduction

64

in tissue fragmentation and juice release, which is perceived as mealy texture6, 7. At a molecular

65

level, mealiness would be associated with incomplete solubilizing of cell wall macromolecules due

66

to an imbalance in the expression of transcripts and enzymatic activity of cell wall-modifying

67

enzymes such as pectin methylesterases (PMEs) and polygalacturonases (PGs)6. This evidence

68

emphasizes the importance of cell wall metabolism in the incidence of mealiness. However, recent


4

Page 5 of 38


69

reports have found the relevance of other processes operating in chilling injury symptoms,

70

including phenolic metabolism and modification of fatty acid desaturation8, 9. Additionally, omic

71

approaches suggest that mealiness is a more intricate disorder related to several metabolic

72

rearrangements, involving hormonal control of ripening and differential accumulation of raffinose,

73

xylose and amino acids10, 11. Previous global analyses performed by our group have been focused

74

on unraveling the mealiness disorder by subjecting peaches to several storage conditions12-14, and

75

comparing cultivar-specific responses to cold15. Contrasting genotypes can be an important

76

approach for the comprehension of physiological and molecular mechanisms involved in the mealy

77

phenotype. To explore the genetic factors involved in mealiness we recently analyzed a ‘Venus’ x

78

‘Venus’ F2 population (VxV herein) to identify QTLs and proteins correlated with this

79

physiological alteration16,

80

reconfiguration operating in the mealiness syndrome, here we expanded our previous studies in

81

VxV peach progeny, combining instrumental, molecular and genetic analysis. A comparison of the

82

transcriptome was performed of individuals representing contrasting phenotypes for mealiness in

83

the framework of an intra-specific population cross. The integrative approach sheds light on the

84

understanding of molecular determinants governing this postharvest quality trait. Identification of

85

candidate genes as molecular markers may also allow selecting cultivars less prone to develop

86

mealiness symptoms.

17.

To get deeper insight into the molecular determinants and genic


5


87

Page 6 of 38

MATERIALS AND METHODS

88 89

Plant material and genotyping

90

To achieve a more homogeneous genetic background to study mealiness, this research was

91

conducted on a F2 segregating population composed of 200 siblings resulting from self-pollination

92

of a P. persica cv. ‘Venus’ (freestone, melting and yellow-flesh nectarine) individual. This

93

population is referred to as VxV throughout this paper. The seedlings obtained were planted in a

94

nursery located in Paine, close to Santiago, Chile (-33°48’11,6”S, 70°40’04,9”W; 407 m

95

elevation). To confirm that each field-labeled plant effectively derived from the selfing of ‘Venus’,

96

a set of 19 microsatellite (SSR) markers was used, with emphasis on those heterozygous in the

97

parent. The complete population was genotyped with at least 12 of these markers used in

98

fingerprinting of peaches and nectarines. The heterozygous informative markers used to confirm

99

the VxV selfing condition were the following (ordered according to their assigned linkage groups):

100

LG1: BPPCT-006, BPPCT-016; LG2: UDP98-025, UDP97-402; LG3: BPPCT-007, UDP97-403;

101

LG4: UDP96-003, UDP98-024, CPPCT-028 and UDP97-402; LG 6: LG8: BPPCT-006, UDP98-

102

409. We also used UDP98-420, a marker not yet localized. Note that two of these markers are

103

multi-locus (BPPCT-006 and UDP97-402). The complete population was tested with this

104

informative set of markers, discarding those genotypes that did not fit the corresponding

105

segregation pattern (heterozygotes exhibiting the same allele combination as ‘Venus’ or

106

homozygotes for either of the parent alleles) for at least one of the markers. Using this criterion,

107

the rate of plants not belonging to the VxV population was below 5%. From the F2 population, 68

108

individuals were selected and phenotyped for mealiness by instrumental analysis during two

109

consecutive seasons (2008 and 2009).

110


6

Page 7 of 38


111

Fruit sampling

112

To select the most homogeneous samples at optimum maturity, fruits were harvested at the

113

commercially mature stage (H). 5 - 10 fruit per tree were collected from the middle part of the

114

canopy according to skin background color and pulp firmness. Color, total soluble solids (TSS)

115

and firmness were measured at harvest and after CS (Table S1). A color chart was used to measure

116

the ground color with a scale (from DN1 = green to DN7 = orange) currently used by the peach

117

industry, and all fruit were harvested at DN = 2 and 3 values. TSS was measured by a temperature-

118

compensated refractometer and expressed as the percent of soluble solids in juice. Firmness was

119

assessed in ten fruits per plant, using a penetrometer with an 8 mm plunger in two opposite sides of

120

each previously peeled fruit, avoiding its suture. Fruits of each segregant were subjected to CS in a

121

chamber at 4 °C (RH 95%) for 21 days, plus three days at 20 °C until reaching a ready-to-eat stage

122

(shelf life). In this stage, mealiness was assessed in five to ten fruits per segregant in each season.

123

At least three individuals of the population were selected among the trees whose fruit classified

124

consistently as mealy or juicy in both seasons (the 5% extreme phenotypes). Mesocarp tissue of

125

five fruits per selected individual was pooled and immediately frozen in liquid nitrogen for

126

transcriptome and real time PCR analyses. The conditions for growing, harvesting, sample

127

preparation and analysis were similar in the two seasons considered in this study.

128 129

Instrumental assessment of mealiness

130

Quantitative determination of mealiness was performed as previously described18. Briefly, 40 g of

131

fruit tissue was placed into four layers of cheesecloth for juice extraction with a self-made press.

132

Juice obtained by pressing was centrifuged at 6000 g for 10 min and the supernatant was recovered

133

and weighed to determine the percentage of juice relative to the original fresh weight of the

134

sample. Mealiness was evaluated after CS (21 days at 4 ºC plus three days at 20 ºC), when fruits


7


Page 8 of 38

135

reached a degree of softening equivalent to the ripening and eating stage (i.e. softening to about 1

136

kg force = 9.8 N), which reflects the maximum expression of the disorder. Thus differences in

137

juiciness are to be attributed to the incidence of mealiness, without significant distortion owing to

138

lack of adequate ripeness. According to previous reports17, 18, fruits were classified as follows:

139

mealy, which showed no more than 10% free juice w/w; partially mealy (10-20% w/w) and non-

140

mealy or juicy (>20% w/w).

141 142

RNA extraction, cDNA synthesis and qRT-PCR

143

Total RNA was isolated according to Meisel et al19 from pools of fruits of each selected segregant

144

in two consecutive seasons. RNA quality and quantity were assessed prior to and after DNase

145

digestion by denaturing gel electrophoresis and photometric analysis (A260/280 ratio),

146

respectively. Initially, 7 µg of total RNA was treated with Turbo DNase (Ambion) according to the

147

manufacturer’s instructions. Total RNA (2 µg) was used as a template for reverse transcription

148

reactions to synthesize single-stranded cDNA using M-MLV reverse transcriptase (Promega) and

149

an oligo(dT)15 primer according to standard procedures. Gene-specific primer sets (Table S2) were

150

designed by Primer3Plus to amplify DNA products between 70 and 200 bp. Quantitative RT-PCR

151

(qRT-PCR) reactions were performed in a LightCycler (Roche) using SYBR Green to monitor

152

cDNA amplification. A 1:20 dilution of cDNA was used in each reaction along with 1 µL of

153

FastStart DNA Master SYBR Green I (Roche), 0.8 µL MgCl2 (25 mM) and 2.5 pmol of forward

154

and reverse primers in a total volume of 10 µL. The following standard thermal profile was used:

155

10 min at 95 °C, 40 cycles of 10 s at 95 °C and 15 s at 60 °C, with a final 10 s stage at 72 °C. Data

156

were analyzed using LightCycler Software v.3 (Roche). Efficiency was determined for each

157

sample and gene by LinRegPCR v.7.5 using data obtained from the exponential phase of each

158

amplification plot. The quality of PCR reactions was determined by analysis of the dissociation


8

Page 9 of 38


159

and amplification curves. The products were resolved by 3% agarose gel electrophoresis to

160

confirm the amplification of DNA fragments of expected sizes. The constitutively expressed genes

161

RPII (RNA polymerase subunit), TEF2 (Translation elongation factor 2) and UBQ10 (Ubiquitin

162

10)20 were evaluated for stability, to normalize qRT-PCR reactions. Transcript levels of genes of

163

interest were normalized to the expression values of RPII, which was the most stably expressed

164

gene under the tested conditions according to GeNorm software. qRT-PCR analysis was performed

165

in three biological replicates, corresponding to fruits from three representative segregants (mealy

166

and juicy phenotypes), and differences in gene expression levels between mealy and non-mealy

167

fruits were analyzed by non-parametric Mann-Whitney U-tests, considering p < 0.05 as statistically

168

significant.

169 170

Microarray experiments

171

For microarray experiments, the μPEACH 1.0 platform consisting of 4806 oligos (described in

172

ESTree Consortium, 2005) was used to analyze transcriptome variation. Fruits from each

173

previously selected segregant were subjected to RNA extraction. 1 µg of RNA was amplified and

174

aminoallyl labeled using a MessageAmp II aRNA kit (Ambion) and 5-(3-aminoallyl)-2’-dUTP and

175

then labeled with Cy3 or Cy5 for mealy and juicy genotypes, respectively (Reactive Dye Pack;

176

Amersham). Two slides were used per sample for each comparison. Pre-hybridizations were

177

carried out by soaking whole glass slides in a solution containing 5x SSC, 0.1% SDS and BSA

178

0.1% at 55 ºC for at least 90 min. Then the slides were washed five times with distilled water and

179

dried by centrifuging for 2 min at 800 g. Hybridizations were carried out in 50 µL of hybridization

180

solution (5x SSC, 0.1% SDS, 40% formamide) containing 90 to 100 pmol of Cy3- and Cy5-

181

labelled target cDNA. The glass slides were placed in a hybridization chamber and incubated at 49

182

ºC in a thermal bath for 16 h. Then the slides were washed with 2x SSC 0.1% SDS four times for


9


Page 10 of 38

183

10 min. Six additional washes (three with 0.1x SSC 0.1% SDS and three with 0.1x SSC, for 10

184

min each) at room temperature were performed before drying the glass slides by a brief

185

centrifugation.

186

Microarray images were acquired at 10 µm pixel resolution and quantified using a Scan Array

187

Express system (PerkinElmer). Array data were analyzed using the R statistical environment,

188

specifically with the microarray analysis tools available from Bioconductor Project. Data were

189

background-subtracted and normalized using the LIMMA Bioconductor package. Data obtained

190

from biological replicates were averaged, then linear models were applied. Differentially expressed

191

genes (DEGs) were determined by a false discovery rate (FDR) ≤5% using empirical Bayesian

192

methods, and p < 0.05 was considered statistically significant. Genes described here have been

193

deposited in NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are

194

accessible through GEO number GSE33027. Log2 ratios of average microarray expression in

195

mealy and juicy tissues were calculated from DEGs and the resulting file was loaded onto the

196

MapMan platform to generate the overview (Table S3). An enrichment analysis of bin categories

197

was performed for the DEGs using Fisher’s exact test with Benjamini–Hochberg multiple testing

198

correction. Categories with corrected p < 0.05 were considered enriched in relation to the total

199

represented categories present in the μPEACH 1.0 platform.

200 201

RESULTS

202 203

Cold storage induces contrasting phenotypes of juiciness in the segregating VxV population.

204

Our aim was to compare the transcription profiles within a cohort of genotypes in a segregating

205

population, whose differences were due only to a contrasting phenotype of juiciness after CS. For

206

this purpose, we selected fruits derived from a F2 population segregating for mealiness: they were


10

Page 11 of 38


207

analyzed for juice content after three weeks at 4 °C plus three days at 20 ºC. Figure 1A shows the

208

juice content distribution of individuals. According to previous reports17, fruit showing no more

209

than 10% w/w juice after the quantitative measurement were classified as mealy, whereas those

210

with more than 20% w/w were considered juicy. Using this criterion, a noticeable seasonal

211

influence on mealiness incidence was observed for most of the genotypes analyzed. During the

212

first harvesting season, 44 genotypes (64%) exhibited juicy behavior versus 13 genotypes (20%)

213

which became mealy after CS. However, this ratio was reversed during the second year (44%

214

mealy genotypes versus 7% juicy). Therefore, to minimize the environmental factor in the analysis,

215

fruits from genotypes consistently exhibiting a mealy or juicy phenotype after CS in both

216

harvesting seasons were sampled. These fruits represented the extreme phenotypes for the trait of

217

the segregating population (5% mealiest and 5% juiciest fruits). The samples were as

218

homogeneous as possible in their maturity parameters (Table S1), since fruit maturity also has

219

been reported to affect chilling injury susceptibility1. Consistent differences in these parameters

220

were not detected either at harvest or after CS (Table S1).

221

Quantitation of juice (Figure 1B) showed that samples with contrasting phenotypes for juiciness

222

exhibited a remarkable difference in juice content, 26.6 ± 2.3% w/w to 32.0 ± 2.3% w/w for juicy

223

fruit, compared to 2.1 ± 1.1% w/w to 2.5 ± 1.5% w/w for mealy fruit. In order to strengthen the

224

characterization of the contrasting phenotypes, we analyzed by qRT-PCR the levels of expression

225

of endopolygalacturonase (PG). Several studies have reported that lower activity, protein

226

accumulation and transcript level of PG correlates with mealiness in peach fruit21, 22. As expected,

227

fruit defined as mealy consistently showed lower levels of PG transcript (Figure 1C): nearly 50%

228

of its expression compared to juicy fruit (0.45 ± 0.09 and 0.46 ± 0.05) in both seasons, confirming

229

our initial fruit classification but also stressing the relevance of this gene as a molecular marker for

230

the mealy phenotype.


11


Page 12 of 38

231

In summary, combining instrumental, genetic and molecular analyses in the VxV progeny we

232

obtained a set of fruit representative of contrasting phenotypes for juiciness among the F2

233

segregating population, which were selected for transcriptome analysis and qRT-PCR profiling.

234 235

Expression profiles in mealy fruit reveal multiple alterations in functional categories of genes

236

compared to juicy fruit

237

The gene expression effect of CS on the occurrence of mealiness symptoms was assessed by

238

comparing the transcriptome profiles, characterizing juicy and mealy fruit by microarray analysis.

239

To focus on genes responsive to CS, only those with a false discovery rate (FDR) ≤5% and ≥1.5-

240

fold change were considered in subsequent analyses. In total, we found 438 differentially

241

expressed genes (DEGs) in the contrasting phenotypes evaluated (Table S3). Of these, 238 genes

242

(54.3%) were up-regulated and 200 genes (45.7%) were down-regulated in juicy fruit compared to

243

fruit exhibiting mealiness.

244

To obtain a better understanding of transcriptome data, genes were classified functionally using

245

MapMan categories. More than 70% of expressed transcripts could be assigned to functional

246

categories and illustrated on pathway overview diagrams (Figure 2). An enrichment analysis of

247

DEGs found in juicy and mealy fruit was also conducted to achieve better comprehension of

248

functional categories correlated with each phenotype (Figure 3; Supplemental Table S3). The

249

overview revealed several up- and down-regulated metabolic pathways operating on contrasting

250

phenotypes after CS. Fruit of the juicy phenotype showed modifications in processes related to cell

251

wall metabolism (Figure 2), such as cellulose and pectin metabolism (Supplemental Table S3).

252

Genes involved in lipid metabolism and fatty acid desaturation were predominantly increased in

253

juicy fruits, along with an important decrease in lipid breakdown, showing transcriptome

254

modulation of cell structural components in response to CS. Interestingly, induction of genes


12

Page 13 of 38


255

encoding for enzymes involved in starch-sucrose interconversion was observed in juicy fruit, along

256

with up-regulation of biosynthetic genes of sugars (raffinose, sorbitol and trehalose).

257

Transcriptional induction of genes involved in terpene and phenylpropanoid biosynthesis was also

258

observed in fruit of the juicy phenotype (Supplemental Table S3). By contrast, mealy fruit showed

259

a general induction of pathways associated with redox impairment, such as the ascorbate-

260

glutathione cycle and mitochondrial electron transport (Figure 2), as well as a general shutdown of

261

minor CHO (Supplemental Table S3). Genes coding for UDP-glycosyltransferases, peroxidases

262

and oxidases also accounted for substantial differences between juicy and mealy fruit.

263

Our analysis revealed genome-wide changes in the transcriptome of fruit in response to CS,

264

leading to differential transcription profiles in contrasting phenotypes. Juicy fruit showed changes

265

in cell wall and lipid components, along with the activation of secondary and sugar metabolism,

266

while mealy fruit exhibited transcription alterations related to redox and energetic processes.

267 268

qRT-PCR profiling of contrasting phenotypes for juiciness

269

Among the differentially expressed genes obtained by transcriptome analysis, we chose those

270

representative of different functional categories for subsequent validation by qRT-PCR. Figure 4

271

summarizes the genes that showed statistically significant differences in expression levels between

272

mealy and juicy fruit in both harvest seasons. Cell wall-related genes coding for putative ß-D-

273

xylosidase (BXL1, Figure 4A), cinnamyl alcohol dehydrogenase (CAD, Figure 4B) and pectin

274

acetylesterase (PAE, Figure 4C) were differentially altered after CS. In mealy fruit, we observed an

275

increase of 3.3 to 3.8- and 2.4 to 3.0-fold for BXL1 and PAE, respectively. CAD was up-regulated

276

by 1.8 to 2.1-fold in juicy peaches. Similarly, genes related to stress response exhibited noticeable

277

changes between juicy and mealy fruit. Following cold exposure, heat shock protein 70 (Hsp70,

278

Figure 4F) and dehydration-induced protein 22 (RD22, Figure 4G) transcripts were significantly


13


Page 14 of 38

279

down-regulated nearly 2-fold in fruits with mealiness in both seasons. In contrast, stress-responsive

280

genes coding for metallothionein (MT, Figure 4D) and pathogen-related protein 4B (PR-4B, Figure

281

4E) increased between 1.5 to 2.7-fold in mealy fruit compared to healthy fruit. Transcriptome

282

profiling showed that contrasting phenotypes of juiciness resulted in the alteration of lipid

283

metabolism (Figure 3). Genes coding for C-4 sterol methyl oxidase (SMO), ceramide

284

glucosyltransferase (CGT), 3-oxacyl-[acyl-carrier-protein] synthase (FAB1) and lipid transfer

285

protein (LTP) showed higher expression levels in juicy fruit compared to mealy fruit (Figure 4H to

286

K). Global analysis also showed that juicy and mealy fruit had differential transcription patterns

287

for sugar and osmoprotectant metabolism (Figure 3). Among these alterations, proline

288

dehydrogenase (ProDH, Figure 4L), sorbitol dehydrogenase (SDH, Figure 4H) and trehalose 6-

289

phosphate phosphatase (TPP, Figure 4I) were induced in non-mealy fruit. The expression profile

290

obtained by qRT-PCR agrees with transcriptome analysis, validating the transcriptional changes

291

observed after storage at low temperature.

292 293

DISCUSSION

294

Cold storage is a common method for delaying metabolic processes involved in the senescence of

295

peaches, thereby extending the postharvest lifespan of fruit. However, peaches frequently develop

296

chilling injury symptoms during this period. Among these, the mealiness syndrome represents a

297

major problem, because its symptoms are only evident when the fruit is consumed. Mealiness

298

characterization has been often addressed by comparing susceptible and tolerant varieties instead

299

of focusing on the molecular response of individuals with closely related genetic background. We

300

applied a global transcription profiling to individuals representing the contrasting phenotypes of

301

juiciness in a segregating F2 population to identify components and features of molecular

302

reprogramming processes occurring in susceptible and non-mealy fruit. After analyzing this


14

Page 15 of 38


303

population during two seasons, we observed differential transcriptional profiles in juicy and mealy

304

fruit previously exposed to CS. Genes and distinctive metabolic processes were identified in juicy

305

and mealy fruit, which may be involved in adaptation or cold-responsive mechanisms, as discussed

306

below.

307 308

Juicy and mealy fruit display distinctive global profiles of cell wall remodeling

309

Mealiness is characterized by a disruption of cell wall architecture associated with an imbalance of

310

cell wall disassembly1, 6. The alteration of expression, enzymatic activity and abundance of cell

311

wall-degrading and modifying enzymes has been previously described in mealiness susceptibility

312

and progression21. Transcriptome analysis showed that juicy fruit exhibited higher expression of

313

genes encoding for enzymes involved in the synthesis and degradation of cell wall components

314

such as cellulose synthase, cellulase, pectate lyase and ß-1,4-glucanases (Table S3). Among the

315

genes evaluated by qRT-PCR, BXL1 and PAE were induced in mealy fruit. A recent report showed

316

that xylose content increased only in susceptible peach varieties10, which is consistent with the

317

hydrolysis of xylose from hemicellulose substrates due to BXL1 activity. Previous studies have

318

suggested a possible role of BXL1 in chilling injury10,

319

modification of cell wall properties and BXL1 in mealy phenotype deserves further analysis. Cold

320

also induces changes in the activity of pectin-modifying enzymes, which may impact the rigidity

321

of cell wall by changes in the Ca+2-pectin interactions24. Although the role of pectin methylesterase

322

(PME) in the mealy phenotype has been previously addressed1, the role of PAE in the mealiness

323

syndrome has not been yet investigated. PAE affects the physicochemical properties of cell wall

324

polysaccharides, decreasing digestibility of pectins and altering cell extensibility25. Thus PAE

325

could be associated with abnormal pectin depolymerization in the mealy phenotype, acting

326

similarly to PME. However, we found a significant increase of CAD expression in juicy fruit,

23.

However, the link between the


15


Page 16 of 38

327

which is involved in phenylpropanoid and lignin biosynthesis in fruit26. Juicy fruit may up-regulate

328

transcript levels in these routes as a protective mechanism to cold. Similar responses have been

329

previously observed in other fruit models27, 28.

330

Considering that mealy and juicy fruit exhibited differential transcription profiles of processes

331

involved in synthesis and degradation of cell wall components, specific reconfigurations of cell

332

wall structure would occur in fruit susceptible to developing mealiness. Cell wall mechanical

333

properties involve pectate modification mediated by ROS signaling29. It is possible that ROS

334

imbalance observed in mealy fruit, as discussed in the following sections, might affect ROS

335

signaling, leading to an alteration in the metabolism of pectins. However, the impact of this

336

mechanism on the actual cell wall remodeling affecting susceptible fruit remains to be elucidated.

337 338

Cold storage activates differential stress response in contrasting phenotypes for juiciness

339

Fruit exposed to cold temperatures for long periods usually perceive stress signals, activating

340

complex signaling cascades that ultimately induce defense mechanisms. Following exposure to

341

abiotic stress, levels of phytohormones and reactive oxygen species (ROS) are altered, triggering a

342

multiscale response characterized by changes in transcripts, metabolites and proteins that lead to

343

the re-establishment of cell homeostasis30. Phytohormones such as jasmonic acid (JA), abscisic

344

acid (ABA) and ethylene play pivotal roles among the defense mechanisms in regulating stress-

345

response processes that may confer cold tolerance30, 31. Our transcriptome comparison revealed

346

significant up-regulation of JA-responsive elements in juicy fruit (Table S3). Additionally, a

347

remarkable induction of genes involved in terpene and phenylpropanoid synthesis was found in

348

fruit with the ability to retain juice after CS (Figure 2, Table S3). In another report, grapefruit

349

exposed to chilling conditions showed transcriptional alteration of biosynthetic pathways of

350

secondary metabolites, presumably as part of a chilling tolerance response32. Both hormonal and


16

Page 17 of 38


351

secondary metabolism changes observed in this study are likely linked to cold stress response,

352

since components of both categories have been previously associated with abiotic stress in other

353

species33. However, it is also feasible that some of these changes could be involved in ripening

354

processes occurring in fruit susceptible or non-susceptible to mealiness, which may have

355

differential abilities to resume normal ripening, as previously discussed34.

356

Among the molecular responses activated by cold exposure in the contrasting phenotypes, we

357

evaluated the expression profile of stress-related genes. We observed increased abundance of heat

358

shock protein 70 (Hsp70) transcript in juicy fruit (Figure 4D). The induction of heat shock

359

proteins, most notably those of the hsp70 and hsp20 families, has been previously reported as a

360

possible factor contributing to decrease chilling injury in different crops35. Since heat shock

361

proteins may mitigate plasma membrane oxidative damages by enhancing antioxidant enzyme

362

activity at chilling temperatures36, juicy fruits may be capable of inducing larger amounts of Hsp70

363

as an adaptive mechanism, achieving higher cellular stability to withstand cold-induced oxidative

364

stress. A previous study found that small heat shock proteins were up-regulated during CS14,

365

suggesting that early induction of heat shock proteins may contribute to prevent chilling injury in

366

juicy fruit. Global analysis applied to juicy and mealy fruit identified other stress-responsive genes,

367

including pathogen-related protein 4B (PR-4B, Figure 4E) and dehydration-related protein 22

368

(RD22, Figure 4G). Dehydration-induced stress and abscisic acid increase the expression of RD22

369

transcript37. Thus it is possible that cold-triggered induction of Hsp70 and RD22 could be part of

370

an adaptive mechanism after long-term cold exposure. Juicy fruits display protective responses to

371

cope with stress, probably allowing them to resume normal ripening processes after CS16,

372

Interestingly, RD22 co-localizes with EPCCU8503, the nearest molecular marker to QTLs

373

associated with flesh bleeding and endocarp staining identified in linkage group 4 of the

374

‘Venus’x‘BigTop’ genetic map38. EPPCU8503 flanks the Melting Flesh locus (M), which includes


34.

17


Page 18 of 38

375

endoPG, responsible for a major QTL in linkage group 4 controlling both mealiness and

376

bleeding22, 39. Pathogen-related proteins are frequently associated with pathogen defense, but PR-4

377

proteins are inducible by wounding, phytohormones such as abscisic acid (ABA) or ethylene, and

378

sugar starvation40, 41. Members of the PR family are part of response mechanisms to cold stress,

379

since they are induced by chilling injury42. The exacerbated mitochondrial response in the mealy

380

phenotype (Figure 2) may suggest greater oxidative damage mediated by ROS, which would

381

correlate with the induction of PR gene expression.

382 383

Juicy fruits display lipid rearrangements conferring membrane stability that decreases

384

chilling injury

385

Lipid metabolism plays an important role in regulating the fluidity of the plasma membrane; its

386

participation in cold stress tolerance has been well documented43. Plants often respond to low

387

temperature exposure by increasing unsaturation or decreasing chain length of membrane lipid

388

components43. Cold stress-triggered membrane remodeling has been described as a complex

389

process that includes modifying lipid composition and protein abundance in the plasma membrane,

390

which maintains structural stability but also regulates signaling processes43,

391

transcriptome analysis showed that several genes involved in lipid metabolism were differentially

392

expressed in juicy fruit. These genes are involved in fatty acid and sphingolipid synthesis and lipid

393

degradation (Figure 2, Table S3). Sterols and sphingolipids such as sitosterol, stigmasterol and

394

ceramide glucoside participate in the regulation of the fluidity of the plasma membrane, since they

395

can form lipid microdomains with membrane-stabilizing properties in plant cold acclimation45.

396

Interestingly, DEGs involved in the synthesis of these molecules were found to be up-regulated in

397

juicy fruit (Table S3), including SMO and CGT (Figure 4H, I), two genes encoding for enzymes

398

involved in the last steps of phytosterol and ceramide glucoside biosynthesis, respectively. In


44.

In this study,

18

Page 19 of 38


399

addition, a member of the lipid transfer protein (LTP) family was also enhanced in juicy fruits

400

(Figure 4M). These proteins form a hydrophobic cavity that binds to acyl chains of fatty acids,

401

leading to lipid stabilizing effects46. Another LTP gene was found to be induced during and after

402

CS14, indicating that LTP plays a protective role in peach fruit both in cold acclimation and after

403

storage. Thus increased gene expression level in lipid metabolism observed in juicy fruits agrees

404

with its protective role in preserving membrane structure during temperature transition from CS to

405

normal ripening. Alternatively, it is also possible that modulation of lipid components of plasma

406

membrane could have a signaling role during cold stress perception17, 43.

407 408

Cold triggers differential gene expression profiles of sugar and oxidative metabolism

409

Cold produces complex modifications in redox state, ultimately leading to oxidative stress

410

responses. Cold stress increases reactive oxygen species (ROS) generated as an intermediate

411

cellular stress response47, which is one of the main factors leading to chilling injury. ROS can

412

affect membrane lipids, proteins and DNA and promote transcriptional changes in diverse target

413

genes, but also induce mechanisms of cold acclimation47. Transcription profiling showed induction

414

of oxidative stress-related processes such as the ascorbate-glutathione cycle and mitochondrial

415

electron transport in mealy fruit (Figure 3, Table S3), suggesting that cold exposure generates an

416

important oxidative imbalance in susceptible fruit. Several transcripts including thioredoxins

417

(TRXs) and glutaredoxins (GRXs) were up-regulated in juicy fruit (Table S3). GRXs are small

418

ubiquitous oxidoreductases of the TRX family, usually defined as redox regulators playing a role

419

in maintaining cellular redox homeostasis48. Interestingly, ectopic expression of AtGRXS17 in

420

tomato plants generates an acquired tolerance to chilling stress49. Transgenic plants exhibited

421

lower ion leakage, increased photochemical efficiency and accumulated higher levels of soluble

422

sugar compared to wild-type plants after chilling stress. Moreover, chilling tolerance correlated


19


Page 20 of 38

423

with increased antioxidant enzyme activities and lower levels of H2O2. Thus the accumulation of

424

these proteins may be an important factor in fine-tuning ROS levels under stressful conditions49.

425

Similarly, higher ascorbate peroxidase and MT transcript levels in mealy fruit (Figure 4D, Table

426

S3) may play a role in coping with high levels of oxidative stress in fruit exhibiting an impaired

427

redox state. Previous reports showed that ascorbate peroxidase and other ROS scavengers are up-

428

regulated after CS, suggesting that mealy fruit respond to the excess of ROS generated by ripening

429

and/or chilling injury12,

430

Arabidopsis thaliana, acting as H2O2 scavengers under cold stress50. An Arabidopsis mt2a

431

insertion mutant exhibited lower tolerance to cold stress, while a catalase cat2 mutant had higher

432

expression of the metallothionein gene after exposure to low temperature51. We also showed in the

433

Mapman overview (Figure 2) and Table S3 that several genes involved in saccharide-related

434

pathways were up-regulated in juicy fruit. Transcript levels involved in trehalose biosynthesis

435

showed increased levels in juicy fruit. Trehalose has been traditionally considered as an

436

osmoprotectant, since high levels of trehalose correlate with membrane and protein integrity52. A

437

previous transcriptome study proposed that trehalose can act as a putatively compatible solute that

438

may function together with other solutes while tolerance is induced53. Interestingly, several genes

439

related to the intermediate trehalose-6-phosphate (T6P) were up-regulated (Table S3). T6P has

440

been recently described as a sensor for available sucrose, shaping the kind of response to changing

441

environmental conditions54.

442

The transcriptional up-regulation of the raffinose oligosaccharide pathway found in juicy fruit may

443

result in an increase of monosaccharides and disaccharides53. A metabolomic analysis showed that

444

resistance to mealiness specifically correlated with higher levels of raffinose10. A number of

445

reports have suggested that disaccharides, galactinol, fructans and sugar alcohols may function

14.

MT proteins have also been shown to mediate ROS balance in


20

Page 21 of 38


446

directly as antioxidants rather than as osmoprotectants55,

56.

447

control damage due to ROS overproduction.

448

Our results also showed an important up-regulation of genes encoding for enzymes involved in the

449

biosynthesis of osmoprotectant metabolites. The transcript level of ProDH was decreased in mealy

450

fruit, which should lead to a higher level of proline as a possible mechanism. Other authors

451

reported that the oxidative-alleviating molecule GABA exogenously applied to peach fruits

452

reduces chilling injury by increasing proline levels due to decreased ProDH enzymatic activity

453

together with increased activity of proline biosynthetic enzymes57.

Thus trehalose and raffinose could

454 455

Cold induces global changes in protein metabolism

456

Intriguingly, a large number of transcripts involved in protein synthesis, degradation and

457

modification were enhanced in mealy fruit (Figure 3, Table S3). Our results agree with previous

458

studies that have also shown alterations in protein metabolism differentially affecting susceptible

459

and non-susceptible fruit12,

460

characterized by preferential synthesis of specific proteins functionally connected to cold stress

461

would occur in susceptible fruit. Considering that translation is a highly energy-demanding

462

process, it is possible that under severe stress conditions such as CS the translation machinery is

463

reduced, which correlates with the down-regulation of mitochondrial electron transport and ATP

464

synthesis components observed in juicy fruit (Figures 2, 3 and Table S3). Interestingly, the

465

extremophile Thellungiella halophila, a plant model for stress-tolerance58, shows expansion of the

466

ubiquitin-dependent protein modification gene family compared to its salt-sensitive relative A.

467

thaliana, suggesting that abiotic stresses trigger translational adjustment as a stress-regulatory

468

response59. Considering that over-represented processes in juicy fruit included other functional

469

categories, these metabolic rearrangements would probably confer increased tolerance to stress,

34.

As previously proposed34, a feasible ribosome response


21


Page 22 of 38

470

whereas mealy fruit may display other strategies to counteract this condition, such as increasing

471

preferential translational machinery.

472

Examining the genetic resources and discerning the molecular mechanisms of mealiness are

473

needed to implement an efficient strategy for the breeding of cultivars less prone to develop

474

mealiness symptoms. In this study we performed a transcriptome analysis after CS, comparing the

475

contrasting juiciness phenotypes in a P. persica segregating population with identical genetic

476

background. Our results reveal that complex gene networks are tightly connected to juiciness,

477

deepening our understanding of this quality trait. The set of genes identified could be validated in

478

other peach cultivars with different sensitivities to CS to prospect for breeding tools in order to

479

improve less tolerant cultivars. Integrating our findings with other information layers involving

480

broader omics data will improve the selection of molecular markers for breeding purposes.

481 482

Abbreviations

483

CS, Cold Storage; CI, Chilling Injury; RH, Relative Humidity; LG, Linkage Group; TSS, Total

484

Soluble Solids; SSC, Saline-Sodium Citrate buffer; SDS, Sodium Dodecyl Sulfate; BSA, Bovine

485

Serum Albumin; qRT-PCR, quantitative reverse transcription PCR; DEG, Differentially Expressed

486

Gene; FDR, False Discovery Rate; ABA, Abscisic Acid; SA, Salicylic Acid; JA, Jasmonic Acid;

487

GABA, Gamma-Aminobutyric Acid; T6P, Trehalose-6-Phosphate; M-MLV, Moloney Murine

488

Leukemia Virus.

489 490

Funding Sources

491

This study was supported by Funding Agency Grant CONICYT-Fondecyt 11160899, Redes

492

REDI170422 and PEP I-2018033 to TdP, Fondecyt 11150679 to ML, Basal Program AFB 170004

493

to AO and FONDAP 15090007 to MG and AO.


22

Page 23 of 38


494 495

Conflict of interest

496

The authors declare no competing financial interest.

497 498

Supporting information

499

The Supporting Information is available free of charge on the ACS Publications website at DOI:

500

Table S1. Fruit quality parameters for juicy and mealy fruits in both harvest seasons. Table S2.

501

Primer set used for RT-qPCR analysis. Table S3. Up- and down-regulated DEGs in mealy and

502

juicy fruits.


23


Page 24 of 38

503

References

504

1.

505

2005, 37, 195-208.

506

2.

507

A.; Twyman, R. M.; Nora, F. R.; Nora, L.; Silva, J. A.; Rombaldi, C. V., Effect of ethylene,

508

intermittent warming and controlled atmosphere on postharvest quality and the occurrence of

509

woolliness in peach (Prunus persica cv. Chiripá) during cold storage. Postharvest Biol. Technol.

510

2005, 38, 25-33.

511

3.

512

effects on Chilling injury, mitochondrial dysfunction, and the ascorbate-glutathione (AsA-GSH)

513

cycle in postharvest peach fruit. J. Agric. Food Chem. 2016, 64, 4665-4674.

514

4.

515

harvest gibberellic acid spraying on gene transcript accumulation during peach fruit development.

516

J. Plant Growth Regul. 2011, 65, 231-237.

517

5.

518

treatments for reducing the chilling injury on peach fruit. J. Agric. Food Chem. 2012, 60, 1209-

519

1212.

520

6.

521

metabolism during the development of chilling injury in cold-stored peach fruit: association of

522

mealiness with arrested disassembly of cell wall pectins. J. Exp. Bot. 2004, 55, 2041-2052.

523

7.

524

development in peaches and nectarines: A review. Sci. Hortic. 2014, 180, 1-5.

Lurie, S.; Crisosto, C. H., Chilling injury in peach and nectarine. Postharvest Biol. Technol.

Girardi, C. L.; Corrent, A. R.; Lucchetta, L.; Zanuzo, M. R.; Costa, T. S. d.; Brackmann,

Song, L.; Wang, J.; Shafi, M.; Liu, Y.; Wang, J.; Wu, J.; Wu, A., Hypobaric treatment

Pegoraro, C.; Clasen C., F.; Dal Cero, J.; Girardi, C. L.; Valmor R., C., Effect of pre-

Yang, Z.; Cao, S.; Zheng, Y.; Jiang, Y., Combined salicyclic acid and ultrasound

Brummell, D. A.; Dal Cin, V.; Lurie, S.; Crisosto, C. H.; Labavitch, J. M., Cell wall

Fruk, G.; Cmelik, Z.; Jemric, T.; Hribar, J.; Vidrih, R., Pectin role in woolliness


24

Page 25 of 38


525

8.

Wang, K.; Yin, X. R.; Zhang, B.; Grierson, D.; Xu, C. J.; Chen, K. S., Transcriptomic and

526

metabolic analyses provide new insights into chilling injury in peach fruit. Plant Cell Environ.

527

2017, 40, 1531-1551.

528

9.

529

reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and

530

phenolic metabolism. Food Chem. 2018, 245, 659-666.

531

10.

532

Lara, M. V.; Fernie, A. R.; Drincovich, M. F., Differential metabolic rearrangements after cold

533

storage are correlated with chilling injury resistance of peach fruits. Front Plant Sci. 2016, 7, 1478.

534

11.

535

Madesis, P.; Fernie, A.; Molassiotis, A., Exploring priming responses involved in peach fruit

536

acclimation to cold stress. Sci. Rep. 2017, 7, 11358.

537

12.

538

Cambiazo, V.; Campos-Vargas, R.; González, M.; Orellana, A.; Silva, H., Comparative EST

539

transcript profiling of peach fruits under different post-harvest conditions reveals candidate genes

540

associated with peach fruit quality. BMC Genomics 2009, 10, 423.

541

13.

542

González, M.; Meisel, L. A.; Retamales, J.; Silva, H.; Orellana, A., Proteomic analysis of peach

543

fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE). BMC

544

Genomics 2010, 11, 43.

545

14.

546

treatments on gene expression in Prunus persica fruit: Normal and altered ripening. Postharvest

547

Biol. Technol. 2013, 75, 125-134.

Gao, H.; Lu, Z.; Yang, Y.; Wang, D.; Yang, T.; Cao, M.; Cao, W., Melatonin treatment

Bustamante, C. A.; Monti, L. L.; Gabilondo, J.; Scossa, F.; Valentini, G.; Budde, C. O.;

Tanou, G.; Minas, I. S.; Scossa, F.; Belghazi, M.; Xanthopoulou, A.; Ganopoulos, I.;

Vizoso, P.; Meisel, L. A.; Tittarelli, A.; Latorre, M.; Saba, J.; Caroca, R.; Maldonado, J.;

Nilo, R.; Saffie, C.; Lilley, K.; Baeza-Yates, R.; Cambiazo, V.; Campos-Vargas, R.;

Pavez, L.; Hödar, C.; Olivares, F.; González, M.; Cambiazo, V., Effects of postharvest


25


Page 26 of 38

548

15.

González-Agüero, M.; Pavez, L.; Ibañez, F.; Pacheco, I.; Campos-Vargas, R.; Meisel, L.

549

A.; Orellana, A.; Retamales, J.; Silva, H.; González, M.; Cambiazo, V., Identification of

550

woolliness response genes in peach fruit after post-harvest treatments. J. Exp. Bot. 2008, 59, 1973-

551

1986.

552

16.

553

Vargas, R.; Orellana, A.; Blanco-Herrera, F.; Meneses, C., Identification of candidate genes

554

associated with mealiness and maturity date in peach [Prunus persica (L.) Batsch] using QTL

555

analysis and deep sequencing. Tree Genet. Genomes 2015, 11.

556

17.

557

Infante, R.; Defilippi, B. G.; Campos-Vargas, R.; Orellana, A., Proteomic analysis of a segregant

558

population reveals candidate proteins linked to mealiness in peach. J. Proteomics 2016, 131, 71-

559

81.

560

18.

561

(Prunus persica) flesh mealiness. Postharvest Biol. Technol. 2002, 25, 151-158.

562

19.

563

R.; González, M.; Orellana, A.; Retamales, J.; Silva, H., A rapid and efficient method for purifying

564

high quality total RNA from peaches (Prunus persica) for functional genomics analyses.

565

Biological Research 2005, 38, 83-88.

566

20.

567

gene expression studies in peach using real-time PCR. BMC Mol. Biol. 2009, 10, 71.

568

21.

569

enzymes and cell wall changes in ‘Flavortop’ nectarines: mRNA abundance, enzyme activity, and

570

changes in pectic and neutral polymers during ripening and in wooly fruit. J. Amer. Soc. Hort. Sci.

571

2000, 125, 630-637.

Nuñez-Lillo, G.; Cifuentes-Esquivel, A.; Troggio, M.; Micheletti, D.; Infante, R.; Campos-

Almeida, A. M.; Urra, C.; Moraga, C.; Jego, M.; Flores, A.; Meisel, L. A.; González, M.;

Crisosto, C. H.; Labavitch, J. M., Developing a quantitative method to evaluate peach

Meisel, L. A.; Fonseca, B.; González, S.; Baeza-Yates, R.; Cambiazo, V.; Campos-Vargas,

Tong, Z.; Gao, Z.; Wang, F.; Zhou, J.; Zhang, Z., Selection of reliable reference genes for

Zhou, H.-W.; Sonego, L.; Khatchitski, A.; Ben-Arie, R.; Lers, A.; Lurie, S., Cell wall


26

Page 27 of 38


572

22.

Peace, C. P.; Crisosto, C. H.; Garner, D. T.; Dandekar, A. M.; Gradziel, T.; Bliss, F. A.,

573

Genetic control of internal breakdown in peach. Acta Hortic. 2006, 713, 489-496.

574

23.

575

a PR-4B precursor identified as genes accounting for differences in peach cold storage tolerance.

576

Funct. Integr. Genomics 2011, 11, 357-368.

577

24.

578

metabolism in response to abiotic stress. Plants (Basel) 2015, 4, 112-166.

579

25.

580

mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant

581

reproduction. Plant Cell 2012, 24, 50-65.

582

26.

583

Comprehensive Reviews in Food Science and Food Safety 2010, 9, 398-416.

584

27.

585

during postharvest cold storage in zucchini fruit. Postharvest Biol. Technol. 2015, 108, 68-77.

586

28.

587

transcriptionally regulates chilling-induced lignification of loquat fruit via physical interaction with

588

an atypical CAD-like gene. J. Exp. Bot. 2017, 68, 5129-5136.

589

29.

590

sensing and signaling pathways involved in plant cell wall remodeling in response to abiotic stress.

591

Plants (Basel) 2018, 7.

592

30.

593

stress: molecular mechanisms. Plants (Basel) 2014, 3, 458-475.

594

31.

595

14, 5312-5337.

Falara, V.; Manganaris, A.; Bonghi, C.; Ramina, A.; Kanellis, A., A beta-D-xylosidase and

Le Gall, H.; Philippe, F.; Domon, J. M.; Gillet, F.; Pelloux, J.; Rayon, C., Cell wall

Gou, J. Y.; Miller, L. M.; Hou, G.; Yu, X. H.; Chen, X. Y.; Liu, C. J., Acetylesterase-

Singh, R.; Rastogi, S.; Dwivedi, U., Phenylpropanoid metabolism in ripening fruits.

Carvajal, F.; Palma, F.; Jamilena, M.; Garrido, D., Cell wall metabolism and chilling injury

Ge, H.; Zhang, J.; Zhang, Y. J.; Li, X.; Yin, X. R.; Grierson, D.; Chen, K. S., EjNAC3

Novakovic, L.; Guo, T.; Bacic, A.; Sampathkumar, A.; Johnson, K. L., Hitting the wall-

Rejeb, I. B.; Pastor, V.; Mauch-Mani, B., Plant responses to simultaneous biotic and abiotic

Miura, K.; Furumoto, T., Cold signaling and cold response in plants. Int. J. Mol. Sci. 2013,


27


Page 28 of 38

596

32.

Maul, P.; McCollum, G. T.; Popp, M.; Guy, C. L.; Porat, R., Transcriptome profiling of

597

grapefruit flavedo following exposure to low temperature and conditioning treatments uncovers

598

principal molecular components involved in chilling tolerance and susceptibility. Plant Cell

599

Environ. 2008, 31, 752-768.

600

33.

601

metabolites in plants. Plant Signal. Behav. 2011, 6, 1720-1731.

602

34.

603

genomics-expression approach reveals components of the molecular mechanisms beyond the cell

604

wall that underlie peach fruit woolliness due to cold storage. Plant Mol. Biol. 2016, 92, 483-503.

605

35.

606

temperatures. Plant Physiol. 1998, 117, 651-658.

607

36.

608

biochemical markers for postharvest chilling stress in fruits and vegetables. Sci. Hortic. 2013, 160,

609

54-64.

610

37.

611

Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene

612

expression. The Plant Cell 1997, 9, 1859-1868.

613

38.

614

A.; Gogorcena, Y., Chilling injury susceptibility in an intra-specific peach [Prunus persica (L.)

615

Batsch] progeny. Postharvest Biol. Technol. 2010, 58, 79-87.

616

39.

617

H., Molecular genetic dissection of chilling injury in peach fruit. Acta Hortic. 2007, 738, 633-638.

Ramakrishna, A.; Ravishankar, G. A., Influence of abiotic stress signals on secondary

Pons, C.; Marti, C.; Forment, J.; Crisosto, C. H.; Dandekar, A. M.; Granell, A., A genetic

Sabehat, A.; Lurie, S.; Weiss, D., Expression of small heat-shock proteins at low

Aghdam, M. S.; Sevillano, L.; Flores, F. B.; Bodbodak, S., Heat shock proteins as

Abe, H.; Yamaguchi-Shinozaki, K.; Urao, T.; Iwasaki, T.; Hosokawa, D.; Shinozaki, K.,

Cantín, C. M.; Crisosto, C. H.; Ogundiwin, E. A.; Gradziel, T.; Torrents, J.; Moreno, M.

Ogundiwin, E. A.; Peace, C. P.; Gradziel, T.; Dandekar, A. M.; Bliss, F. A.; Crisosto, C.


28

Page 29 of 38


618

40.

Bravo, J. M.; Campo, S.; Murillo, I.; Coca, M.; San Segundo, B., Fungus- and wound-

619

induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein

620

4 (PR-4) family of maize. Plant Mol. Biol. 2003, 52, 745-759.

621

41.

622

characterization of six cDNAs expressed during glucose starvation in excised maize (Zea mays L.)

623

root tips. Plant Mol. Biol. 1995, 28, 473-485.

624

42.

625

K., Pathogenesis-Related Protein 1b1 (PR1b1) Is a major tomato fruit protein responsive to

626

chilling temperature and upregulated in high polyamine transgenic genotypes. Front Plant. Sci.

627

2016, 7, 901.

628

43.

629

Unravelling the response to cold stress in Arabidopsis and its extremophile relative Eutrema

630

salsugineum. Plant Sci. 2017, 263, 194-200.

631

44.

632

proteomics for improving cold tolerance. Front. Plant Sci. 2013, 90.

633

45.

634

Moreau, P.; Bessoule, J. J.; Simon-Plas, F.; Mongrand, S., Lipids of plant membrane rafts. Prog.

635

Lipid Res. 2012, 51, 272-299.

636

46.

637

stability and its related proteins in the tolerance of peach fruit to chilling injury. Amino Acids 2010,

638

39, 181-194.

639

47.

640

hormonal and molecular mechanisms regulat1ing chilling injury in horticultural species.

641

Postharvest technologies applied to reduce its impact. J. Sci. Food Agric. 2009, 89, 555-573.

Chevalier, C.; Bourgeois, E.; Pradet, A.; Raymond, P., Molecular cloning and

Goyal, R. K.; Fatima, T.; Topuz, M.; Bernadec, A.; Sicher, R.; Handa, A. K.; Mattoo, A.

Barrero-Sicilia, C.; Silvestre, S.; Haslam, R. P.; Michaelson, L. V., Lipid remodelling:

Takahashi, D.; Li, B.; Nakayama, T.; Kawamura, Y.; Uemura, M., Plant plasma membrane

Cacas, J. L.; Furt, F.; Le Guedard, M.; Schmitter, J. M.; Bure, C.; Gerbeau-Pissot, P.;

Zhang, C.; Ding, Z.; Xu, X.; Wang, Q.; Qin, G.; Tian, S., Crucial roles of membrane

Sevillano, L.; Sanchez-Ballesta, M. T.; Romojaro, F.; Flores, F. B., Physiological,


29


Page 30 of 38

642

48.

Meyer, Y.; Belin, C.; Delorme-Hinoux, V.; Reichheld, J. P.; Riondet, C., Thioredoxin and

643

glutaredoxin systems in plants: molecular mechanisms, crosstalks, and functional significance.

644

Antioxid. Redox Signal. 2012, 17, 1124-1160.

645

49.

646

A.; Cheng, N.; Hirschi, K. D.; White, F. F.; Park, S., Tomato expressing Arabidopsis glutaredoxin

647

gene AtGRXS17 confers tolerance to chilling stress via modulating cold responsive components.

648

Hortic. Res. 2015, 2, 15051.

649

50.

650

Metallothionein, AtMT2a, mediates ROS balance during oxidative stress. J. Plant Biol. 2009, 52,

651

585-592.

652

51.

653

Vanacker, H.; Miginiac-Maslow, M.; Van Breusegem, F.; Noctor, G., Conditional oxidative stress

654

responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key

655

modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in

656

the regulation of H2O2-induced cell death. Plant J. 2007, 52, 640-657.

657

52.

658

Plant Sci. 1995, 112, 1-9.

659

53.

660

Global transcript levels respond to small changes of the carbon status during progressive

661

exhaustion of carbohydrates in Arabidopsis rosettes. Plant Physiol. 2008, 146, 1834-1861.

662

54.

663

development. Front. Plant Sci. 2011, 2, 70.

664

55.

665

function to protect plants from oxidative damage. Plant Physiol. 2008, 147, 1251-1263.

Hu, Y.; Wu, Q.; Sprague, S. A.; Park, J.; Oh, M.; Rajashekar, C. B.; Koiwa, H.; Nakata, P.

Zhu, W.; Zhao, D.-X.; Miao, Q.; Xue, T.-T.; Li, X.-Z.; Zheng, C.-C., Arabidopsis thaliana

Queval, G.; Issakidis-Bourguet, E.; Hoeberichts, F. A.; Vandorpe, M.; Gakiere, B.;

Müller, J.; Boller, T.; Wiemken, A., Trehalose and trehalase in plants: recen developments.

Usadel, B.; Blasing, O. E.; Gibon, Y.; Retzlaff, K.; Hohne, M.; Gunther, M.; Stitt, M.,

Ponnu, J.; Wahl, V.; Schmid, M., Trehalose-6-phosphate: connecting plant metabolism and

Nishizawa, A.; Yabuta, Y.; Shigeoka, S., Galactinol and raffinose constitute a novel


30

Page 31 of 38


666

56.

Peshev, D.; Vergauwen, R.; Moglia, A.; Hideg, E.; Van den Ende, W., Towards

667

understanding vacuolar antioxidant mechanisms: a role for fructans? J. Exp. Bot. 2013, 64, 1025-

668

1038.

669

57.

670

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

671

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

672

58.

673

essential and critical components of abiotic stress tolerance in plants. Mol. Plant 2009, 2, 3-12.

674

59.

675

reprogramming of the Eutrema salsugineum (Thellungiella salsuginea) transcriptome in response

676

to UV and silver nitrate challenge. BMC Plant. Biol. 2015, 15, 137.

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

Amtmann, A., Learning from evolution: Thellungiella generates new knowledge on

Mucha, S.; Walther, D.; Muller, T. M.; Hincha, D. K.; Glawischnig, E., Substantial


31


Page 32 of 38

677

Figure captions

678

Figure 1. Characterization of VxV F2 segregating population. A) The population assayed was a

679

F2 segregating population obtained from a controlled intra-specific cross of P. persica VxV.

680

Frequency of individuals (y-axis) and juice content (x-axis, informed as the percentage of juice)

681

for 68 F2 genotypes in two consecutive harvest seasons (Seasons 1 and 2, respectively) evaluated

682

after CS for 21 days at 4 °C plus three days at 20 ºC are shown. Dashed line indicates the criterion

683

used to establish mealiness incidence (below 10% w/w). B) Percentage of juice in contrasting

684

phenotypes for juiciness. C) Relative expression of polygalacturonase PG normalized to RPII

685

determined in mesocarp tissue of peach fruit showing juicy or mealy phenotypes (white and black

686

bars, respectively). Data represent the mean of three biological replicates by condition (panels B

687

and C), corresponding to the representative trees of the most contrasting phenotypes (5% juiciest

688

and 5% very mealy) of a population segregating for mealiness in two seasons. Asterisks indicate

689

significant differences between juicy and mealy peaches (Mann-Whitney U-test, p < 0.05).

690 691

Figure 2. MapMan overview of cold storage-mediated gene expression changes involved in

692

different metabolic processes. The images were obtained using MapMan, showing different

693

functional metabolic categories in peach fruit after CS for 21 days at 4 °C plus three days at 20 ºC.

694

Log2 ratios of average microarray expression in mealy and juicy fruit tissues were calculated and

695

the resulting file was loaded on the MapMan platform to generate the overview (Table S3).

696

Positive log2 values represented by red color indicate up-regulation and signify that those

697

transcripts are induced in juicy fruit, while negative values shown by blue color denote down-

698

regulation of the transcripts in juicy fruit. In both cases, color saturates at 1.5-fold change.

699


32

Page 33 of 38


700

Figure 3. Differentially expressed genes (up- and down-regulated) grouped by functional

701

categories. Functional categories: 1: PS, 2: major CHO metabolism, 3: minor CHO metabolism, 4:

702

glycolysis, 5: fermentation, 7: OPP, 8: TCA / org transformation, 9: mitochondrial electron

703

transport / ATP synthesis, 10: cell wall, 11: lipid metabolism, 13: amino acid metabolism, 15:

704

metal handling, 16: secondary metabolism, 17: hormone metabolism, 18: Co-factor and vitamin

705

metabolism, 20: stress, 21: redox, 23: nucleotide metabolism, 26: miscellaneous, 27: RNA, 28:

706

DNA, 29: protein, 30: signaling, 31: cell, 33: development, 34: transport, 35: not assigned (total

707

data in Table S3). Asterisks indicate statistically enriched classes (hypergeometric distribution, p

Comparative Transcriptome Profiling in a Segregating Peach

Recommend Documents