Diversity among Pomegranate Varieties in Chilling Tolerance and

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Diversity among Pomegranate Varieties in Chilling Tolerance and Transcriptome Responses to Cold Storage Yael kashash, Adi Doron-Faigenboim, Irit Bar-Ya’akov, Kamel Hatib, Rotem Beja, Tali Trainin, Doron Holland, and Ron Porat J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06321 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Journal of Agricultural and Food Chemistry

Diversity among Pomegranate Varieties in Chilling Tolerance and Transcriptome Responses to Cold Storage Yael Kashash,†,§ Adi Doron-Faigenboim,‡ Irit Bar-Ya'akov,ⱡ Kamel Hatib,ⱡ Rotem Beja,ⱡ Taly Trainin,ⱡ Doron Holland,ⱡ and Ron Porat*,† †

Department of Postharvest Science of Fresh Produce, ARO, the Volcani Center,

P.O. Box 15159, Rishon LeZion 7505101, Israel. ‡ Department

of Genomics and Bioinformatics, ARO, the Volcani Center, P.O. Box

15159, Rishon LeZion 7505101, Israel. ⱡ Department

of Fruit Tree Sciences, ARO, Newe Ya'ar Research Center, P.O. Box

1021, Ramat Yishay 30095, Israel § The

Robert H Smith Faculty of Agricultural, Food and Environmental Quality

Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

Corresponding author:

Dr. Ron Porat Department of Postharvest Science of Fresh Produce ARO, the Volcani Center P.O. Box 15159 Rishon LeZion 7505101, Israel

Tel:

972-3-9683617

Fax:

972-3-9683622

E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT: We found great variability in chilling tolerance among 84

2

pomegranate varieties from the Newe Ya'ar collection, among them 'Ganesh' was

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chilling-sensitive whereas 'Wonderful' was relatively chilling-tolerant. In order to

4

evaluate the different molecular responses of these varieties to cold storage, we

5

analyzed the transcriptomic changes in the inner membrane tissues of 'Ganesh' and

6

'Wonderful' fruit after 2 weeks of cold storage at 1°C. By functional categorization of

7

the differentially expressed transcripts using MapMan, we found that many transcripts

8

related to various pathways, such as jasmonic acid biosynthesis and signaling,

9

galactinol, raffinose, phenol and phenylpropanoid biosynthesis, calcium and MAPK

10

signalling, lipid metabolism and various transcription factors and heat-shock proteins,

11

have been massively up-regulated in 'Wonderful', but not in 'Ganesh' fruit. Thus, it is

12

suggested that these pathways most likely participate in imparting chilling tolerance

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in pomegranate fruits.

14 15

KEYWORDS: Chilling; pomegranate; postharvest; transcriptome; varieties.

16 17


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INTRODUCTION

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International trading of pomegranates (Punica granatum L.) requires implementation

20

of cold-quarantine treatments against specific pests. For example, the US Department

21

of Agriculture (USDA) requires the application of a cold-quarantine treatment against

22

the Mediterranean fruit fly (Ceratitus capitate) by exposure of the fruit to an internal

23

temperature below 1.1 °C for at least 14 d.1, 2 Nevertheless, pomegranate fruit are

24

chilling-sensitive and may develop chilling injuries (CI) when exposed to post-harvest

25

temperatures below the optimal recommendation of 7.5 °C. CI in pomegranates

26

appears mainly as browning of the inner white membranes and the white tissue

27

surrounding the arils and as pitting of the outer peel .3, 4

28

We previously described two main factors that dramatically influenced the

29

chilling injury (CI) susceptibility of 'Wonderful' pomegranates after cold storage: the

30

first was harvest time, the second was application of a low-temperature conditioning

31

(LTC) treatment before storage.5 Regarding harvest time, we demonstrated that early-

32

harvested fruit were extremely chilling sensitive, mid-season fruit showed moderate

33

CI symptoms, and late-harvested fruit were quite chilling tolerant and did not develop

34

CI symptoms even after 1 month at 1 °C plus 1 additional week at 20 °C.5, 6

35

Regarding application of the LTC treatment, we found that exposure of the fruit to 15

36

°C for 10 days prior to cold storage considerably reduced CI symptoms after 1 month

37

at 1 °C plus 1 additional week at 20 °C.5, 7 In addition, transcriptomic analysis by

38

RNA Seq of inner membrane tissues revealed that both treatments, i.e., late harvest

39

and LTC application, had remarkable effects on the response of the fruit's

40

transcriptome to cold storage6, 7

41 42

In the present paper we describe another key factor that also had a crucial influence on the susceptibility of pomegranate fruit to chilling stress — the genetic



43

variability in chilling tolerance among pomegranate varieties; some varieties are

44

extremely chilling-sensitive whereas others are relatively chilling-tolerant. The

45

variability in chilling tolerance among distinct pomegranate varieties was examined

46

during two consecutive seasons by evaluating CI indices among 84 distinct

47

pomegranate varieties grown in the Israel Pomegranate Breeding Collection at the

48

Newe Ya'ar Research Center of the Agricultural Research Organization (ARO). The

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genetic variability of the Israel pomegranate collection was determined by single

50

nucleotide polymorphism (SNP) analysis, which revealed that the collection could be

51

divided into two main distinguished genetic branches: the central Asian branch which

52

includes ornamental, inedible pomegranate accessions, as well as accessions from

53

India, China and Iran (the G1 group); and the second branch which includes

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accessions from the Mediterranean region, Central Asia and California (the G2

55

group).8

56

In order to unravel the different molecular responses to cold storage of

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chilling-sensitive and relatively chilling-tolerant pomegranate varieties, we chose two

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distinct varieties with notable differences in sensitivities to low temperatures —

59

'Ganesh', which is chilling-sensitive and belongs to the G1 group of the Israeli

60

collection, and 'Wonderful', which is relatively chilling-tolerant and belongs to the G2

61

group of the Israeli collection — and conducted RNA-Seq analysis of their inner

62

membrane tissues on the day of harvest (T0) and after a 2-week cold-quarantine

63

treatment at 1 °C. Comparisons between the various transcriptional responses to cold

64

storage of the chilling-sensitive 'Ganesh' and those of the relatively chilling-tolerant

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'Wonderful' yielded numerous differentially expressed transcripts involved in

66

regulation, metabolism, and stress-adaptation mechanisms that were specifically

67

down-regulated or unaffected in the chilling-sensitive 'Ganesh' but up-regulated in the


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relatively chilling-tolerant 'Wonderful'. The present findings shed light on some key

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mechanisms putatively involved in imparting chilling stress tolerance of pomegranate

70

fruits.

71 72

MATERIALS AND METHODS

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Plant Material. We evaluated the chilling susceptibilities of 84 distinct

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pomegranate varieties held in the Israel Pomegranate Breeding Collection at the Newe

75

Ya'ar Research Center — all the varieties are registered in the Israeli Gene Bank for

76

Agricultural Crops (IGB; Web site: http://igb.agri.gov.il). The varieties were

77

harvested at full maturity during the 2011 and 2012 growth seasons. The commercial

78

name of P.G. 160-61 is 'Ganesh' and that of P.G. 101-2 is 'Wonderful'. Further

79

analyses of chilling susceptibility of these two varieties, and RNA Seq analyses were

80

conducted on fruits harvested in the 2016 growth season.

81

Storage Conditions. The chilling susceptibilities of 84 distinct pomegranate

82

varieties was evaluated after 6 weeks of storage at 5 °C plus 1 additional week of

83

shelf-life at 20 °C. Chilling susceptibilities of 'Ganesh' and 'Wonderful' were further

84

evaluated after 4 weeks of storage at 5 °C plus 1 additional week of shelf-life at 20

85

°C. Samples for RNA Seq analysis were collected after 2 weeks of cold storage at 1

86

°C.

87

Chilling Injury Assessment. CI index was assessed as the extent and severity

88

of internal browning of the white tissue and inner membranes after cold storage, as

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described by Kashash et al., and ranged from 0 (none) to 3 (severe).5 The CI indices

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of the 84 varieties represent means ± S.E. of data obtained during two consecutive

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growth seasons (2011 and 2012); 15-20 fruits of each variety were assessed. The CI



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indices of 'Ganesh' and 'Wonderful' from the 2016 growth season were calculated as

93

means ± S.E. of four replications, each including 9–11 fruits.

94

Fruit Ripening Indices. Total soluble solids (TSS) content in the juice was

95

measured by a PAL-1 digital refractometer (Atago, Tokyo, Japan), and acid

96

percentage was measured by titration to pH 8.3 with 0.1 M NaOH using an automatic

97

titrator Model CH-9101 (Metrohm, Herisau, Switzerland). Peel color was quantified

98

by a Chromometer, Model CR-400 (Minolta, Tokyo, Japan).

99

RNA Isolation, cDNA Library Construction and RNA-Seq. The inner

100

membranes of 'Ganesh' and 'Wonderful' fruits were collected on the day of harvest

101

and after 2 weeks at 1 °C and were immediately frozen in liquid nitrogen and stored at

102

-80 ºC until RNA extraction. Each treatment included 3 samples collected from 3

103

different fruits. Extraction of total RNA was carried-out according to the CTAB

104

protocol9 and RNA concentrations were determined with a NanoDrop ND-1000

105

spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA purity was

106

further examined with an RNA BioAnalyzer, Model 2100 (Agilent Technologies,

107

Santa Clara, CA, USA). Library preparation and sequencing were performed at the

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Crown Institute for Genomics, in the Weizmann Institute of Science, Rehovot, Israel,

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as described previously7. 12 single-end RNA-Seq libraries with a length of 125

110

nucleotides were sequenced with a Hiseq 2000 instrument (Illumina Inc., San Diego,

111

CA, USA) and separated on two different lanes.

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Transcriptome Analysis. Analysis of the transcriptome was performed as

113

previously described.7 In brief, raw reads were filtered and cleaned and adaptors were

114

removed, and read-end nucleotides with quality scores < 30 were trimmed. The

115

observed clean reads were aligned to the Punica granatum (pomegranate) genome

116

obtained from the US National Center for Biotechnology Information (NCBI).


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Differential Expression Analysis. Differential expression was analysed using the

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DESeq2 R package.10 Differentially expressed transcripts from cold-stored tissues

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were considered as those that differed from T0 by a factor greater than 4, with an

120

adjusted p-value of no more than 0.001.11 Heat map visualization was performed with

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the R Bioconductor software using heatmap.3 function.12 Venn diagrams were created

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with the Bioinformatics & Evolutionary Genomics tool

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(bioinformatics.psb.ugent.be/webtools/Venn/). Functional categorizations were

124

analysed using MapMan software.13

125 126

RESULTS

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Diversity among Pomegranate Varieties in Chilling Tolerance. We

128

evaluated the chilling susceptibility of 84 distinct pomegranate varieties. As shown in

129

Figure 1, we observed wide variation in chilling sensitivity among the varieties, as

130

indicated by the appearance of internal browning symptoms after 6 weeks of cold

131

storage at 5 °C plus 1 additional week at 20 °C. Some varieties were extremely

132

chilling-sensitive and had very high CI indices, up to 2.50 on a scale of 0–3, whereas

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other varieties were much more chilling-tolerant, some even had a CI index of 0.

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To reveal the molecular mechanisms that may play a role in the responses of

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different pomegranate varieties to cold storage, we chose two specific varieties with

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distinctively different sensitivities to chilling: 'Ganesh' (P.G. 160-61), which is

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relatively chilling-sensitive (CI index = 1.52), and 'Wonderful' (P.G. 101-2), which is

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relatively chilling-tolerant (CI index = 0.25) (Figure 1). The ripening indices of

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'Ganesh' were: TSS = 14.8%, acidity = 0.51%, and L*, a*, b* color scores of 67, 18

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and 40, respectively; and the ripening indices of 'Wonderful' were: TSS = 16.5%,

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acidity = 1.64%, and L*, a*, b* color scores of 44, 42 and 22, respectively. We chose



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these particular varieties for two main reasons: first, they exhibited differing chilling

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sensitivities; and second, we already have a segregating F2 population generated from

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a cross between 'Ganesh' and 'Wonderful', therefore the present transcriptomic data

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will support our genetic mapping studies and the recognition of quantitative trait loci

146

(QTLs) for chilling-tolerance traits.14

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Another evaluation of the chilling susceptibility of 'Ganesh' and 'Wonderful'

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pomegranates was conducted in the 2016 season under a different cold storage regime

149

of 4 weeks at 1 °C plus 1 additional week at 20 °C. It confirmed the differences in

150

chilling susceptibility between these varieties: 'Ganesh' was chilling-sensitive, with a

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CI index of 2.5, whereas 'Wonderful' was relatively chilling-tolerant, with a much

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lower CI index of 0.44 (Figure 2).

153 154 155

Effects of Cold Storage on the Transcriptomes of 'Ganesh' and 'Wonderful' Pomegranate Fruits.

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Differentially Expressed Transcripts. To identify molecular mechanisms

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triggered in response to chilling stress in the two varieties 'Ganesh' and 'Wonderful', a

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high-throughput RNA sequencing analysis was conducted, using RNA obtained from

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inner membrane tissues collected on the day of harvest (designated 'T0') and after 2

160

weeks of cold storage at 1 °C (designated 'chilling'). Each of the cDNA libraries

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yielded between 17.1 million and 21.3 million clean single-end reads of 125 bp in

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length. About 95% of the cleaned reads were mapped to the Punica granatum

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(pomegranate) genome. Pair-wise comparisons revealed transcripts of 'Ganesh' and

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'Wonderful' varieties that showed significant changes in their expression patterns in

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response to cold storage, and transcripts that were induced or repressed by a factor of

166

at least 4 (2 ≥ log2 ≤ -2) compared with their initial levels at T0, and that differed


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significantly by padj  0.001 (DESeq2) were selected. This comparison yielded

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3,132 transcripts from 'Ganesh' fruits, whose expression were modified by cold

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storage conditions — 1,354 up-regulated and 1,778 down-regulated — and 5,605

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transcripts from 'Wonderful' fruits, whose expression were modified by cold storage

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conditions — 3,379 up-regulated and 2,226 down-regulated — (Table 1). It should be

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noted that in the chilling-sensitive 'Ganesh' most (56.8%) differentially expressed

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transcripts were down-regulated by low-temperature storage, whereas in the relatively

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chilling-tolerant 'Wonderful' most (60.3%) differentially expressed transcripts were

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up-regulated.

176

Because many differentially expressed transcripts represented similar genes,

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they were also compared with the Arabidopsis thaliana database

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(http://www.arabidopsis.org) by BLASTx searches (E < 10−5) and their corresponding

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Arabidopsis homologs were identified.15 Overall, 2,253 and 3,591 transcripts from

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cold-stored 'Ganesh' and 'Wonderful' fruits, respectively, that corresponded to

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Arabidopsis genes have been identified (Table 1). (Figure 3). This analysis revealed:

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754 transcripts — 292 up-regulated and 462 down-regulated — that were affected by

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cold storage exposure only in 'Ganesh'; 1,556 transcripts — 690 up-regulated and 866

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down-regulated — that were affected by cold storage exposure in both 'Ganesh' and

185

'Wonderful'; and 2,182 transcripts — 1,336 up-regulated and 846 down-regulated —

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that were affected by cold storage exposure only in 'Wonderful' (Figure 3). In light of

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these findings we classified the observed differentially expressed transcripts into three

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main regulons: 'Ganesh-specific'; 'common'; and 'Wonderful-specific' (Figure 3).

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Functional Categorization. Transcripts of the 'Ganesh-specific', 'common'

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and 'Wonderful-specific' regulons were categorized using MapMan (Table 2). The

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functional bins in which we noticed the most notable effect of cold storage were lipid 9 ACS Paragon Plus Environment


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metabolism, secondary metabolism, hormone metabolism, stress, RNA, and signaling.

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In these bins, in response to cold storage, the differentially expressed transcripts were

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largely up-regulated in relatively chilling-tolerant 'Wonderful' fruit but down-

195

regulated in 'Ganesh' fruit (Table 2). The sub-bins most affected by exposure to cold

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storage were 'raffinose family', 'lipid degradation', 'jasmonates', 'phenylpropanoids',

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'flavonoids', 'simple phenols', 'abiotic stress', 'RNA regulation of transcription',

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'receptor kinases', 'calcium signalling', and 'MAP kinases signaling' (Table 2).

199

Jasmonate Biosynthesis and Signaling. A significant up-regulation of

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transcripts involved in jasmonate biosynthesis and signalling was observed

201

specifically in the relatively chilling-tolerant 'Wonderful' variety, in response to cold

202

storage (Figure 4). These transcripts included two lipoxygenase (LOX) genes —

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LOX1 and LOX3 —, allene oxide synthase (AOS), three 12-oxo-phytodienoic acid

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reductase (OPR) genes — OPR1, OPR2 and OPR3 — and the jasmonate-responsive

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transcription factor MYC2 (Figure 4).

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Carbohydrate Metabolism- A significant up-regulation of transcripts

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encoding galactinol synthase and raffinose synthase was observed in response to cold

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storage only in the relatively chilling-tolerant 'Wonderful' variety, but not in the

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relatively chilling-sensitive 'Ganesh' (Figure 5). More specifically, this up-regulation

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included two galactinol synthase transcripts and two raffinose synthase transcripts.

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Moreover, it is worth notice that transcript levels of galactinol synthase 2 were

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significantly down-regulated in 'Ganesh' (Figure 5).

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Phenylpropanoids, Phenols and Flavonoids Pathways. Transcripts

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involved in the phenylpropanoid biosynthesis pathway, including phenylalanine

215

ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase

216

(4CL), were significantly up-regulated by cold storage, specifically in 'Wonderful'


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fruits (Figure 6). Furthermore, in the relatively chilling-tolerant variety, 'Wonderful'

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we observed significant up-regulation of transcripts encoding enzymes that belong to

219

the phenol metabolism pathway, including caffeoyl-CoA O-methyltransferase

220

(COMT), 4CL, cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase

221

(CAD), peroxidase (POD) and laccase (LAC) (Figure 6). In contrast, in 'Wonderful'

222

we observed decreases in transcript levels of most flavonoid biosynthesis genes,

223

including chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and

224

dihydroflavonol 4-reductase (DFR) (Figure 6). Overall, these findings indicate that in

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cold-stored 'Wonderful' fruits there is induction of the phenylpropanoids pathway and

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diversion towards the phenol metabolism pathway rather than towards the flavonoids

227

biosynthesis pathway (Figure 6). Ca2+ and MAPK Signaling Pathways. Enormous up-regulation of

228 229

transcripts involved in Ca2+- and mitogen-activated protein kinase (MAPK) signaling

230

pathways after cold storage was observed specifically in the relatively chilling-

231

tolerant variety, 'Wonderful' but not in the ‫לא השתמשנו עד עכשיו במילה הזאת עבור‬

232

‫ רק עבור וונדרפול‬,‫גנאש‬chilling-sensitive 'Ganesh' (Figure 7). This activation of Ca2+

233

signaling included up-regulation of calmodulin (CaM) and calmodulin-like proteins

234

(CMLs), calcium-dependent protein kinases (CDPKs), calcineurin B-like proteins

235

(CBLs), CBL-interacting protein kinases (CIPKs) and calcium/calmodulin-regulated

236

receptor-like kinases (CRLKs) (Figure 7). Furthermore, downstream to CRLK1 and

237

CDPKs, we also observed significant up-regulation of five MAPKKK transcripts, a

238

MAPKK1 transcript and three MAPK transcripts; this occurred only in 'Wonderful',

239

not in 'Ganesh', which indicated a marked enhancement of the MAPK signaling

240

cascade in 'Wonderful' (Figure 7).



241

Regulation of Transcription. The expression levels of many transcription

242

factors belonging to the AP2/ERFs, heat-shock factors (HSFs) and WRKYs families

243

significantly increased in response to cold storage, specifically in the relatively

244

chilling-tolerant variety, 'Wonderful' (Figure 8). Within the AP2/ERFs family, we

245

noticed specific up-regulation of the stress-related C-repeat binding factor

246

(CBF)/dehydration-responsive element binding (DREB) and RAP2 TFs. Among the

247

CBF/DREB1 family, transcripts of DREB1E and DREB1F were significantly up-

248

regulated in 'Wonderful', whereas two transcripts of DREB1D/CBF4 were

249

significantly down-regulated in 'Ganesh' but did not change in 'Wonderful' (Figure

250

8A). Among the RAP2 family, transcript levels of RAP2-1 and RAP2-3 increased

251

significantly in response to chilling specifically in 'Wonderful' (Figure 8A). We also

252

observed remarkable induction of ethylene response factors (ERFs) in the relatively

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chilling-tolerant 'Wonderful', in which levels of 19 ERF transcripts were significantly

254

up-regulated in response to cold storage, as compared with just two transcripts up-

255

regulated in 'Ganesh' (data not shown). Among the HSF family, in response to cold

256

storage, transcript levels of HSFA4C, HASFB2B, HSFC1 and of two HSF4A

257

significantly increased in 'Wonderful' but did not alter significantly in 'Ganesh'

258

(Figure 8B); furthermore, transcript levels of HSFB4 decreased significantly only in

259

the chilling-sensitive 'Ganesh'. In contrast, transcript levels of HSFA2 decreased

260

significantly only in 'Wonderful' fruits (Figure 8B). Among the WRKY family, we

261

found that almost all of the transcripts (18 out of 19) were significantly up-regulated

262

only in 'Wonderful', except for WRKY4, whose expression level was down-regulated

263

(Figure 8C).

264

Heat-Shock Proteins. Transcripts of 21 heat-shock proteins (HSPs)

265

significantly increased in response to cold storage in the relatively chilling-tolerant


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'Wonderful', whereas those of only five HSPs were up-regulated in chilling-sensitive

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'Ganesh' fruit (Figure 9). Also, five other HSP transcripts — HSP26.5, ClpB, HSP-

268

TPR, HSP17.4, and Clp — were significantly down-regulated only in chilling-

269

sensitive 'Ganesh' (Figure 9).

270

Lipid Metabolism. We identified 21 transcripts involved in lipid metabolism

271

that were up-regulated and eight that were down-regulated in response to cold storage,

272

particularly in the relatively chilling-tolerant variety, 'Wonderful' (Figure 10). The

273

up-regulated transcripts encode enzymes that are involved in remodelling of fatty

274

acids, formation of signaling molecules, and biosynthesis of sphingolipids,

275

triacylglycerol and sterols (Figure 10). For example, carboxylesterase (CXE) is

276

involved in the remodelling of fatty acids by hydrolysing esters into short fatty

277

acids;16 diacylglycerol kinase (DGK) and phospholipase D (PLD) are involved in the

278

generation of the important signaling molecule phosphatidic acid and in membrane

279

trafficking;17, 18 long-chain base biosynthesis protein (LCB) is the first enzyme in the

280

sphingolipids biosynthesis pathway;19 phospholipid diacylglycerol acyltransferase

281

(PDAT) catalyses the synthesis of triacylglycerols;20 and squalene synthase (SS) is a

282

key enzyme in the biosynthesis of sterols.21 In contrast, the down-regulated

283

transcripts mainly encode enzymes that participate in degradation of fatty acids via

284

the β- and ω-oxidation pathways (Figure 10). For example, enoyl-CoA hydratase

285

catalyses the second stage of β- oxidation22 and long-chain fatty alcohol oxidase 3

286

(FAOD3) is involved in the degradation of lipids into aldehydes via the ω-oxidation

287

pathway.23

288 289

DISCUSSION



290

We hereby demonstrated the existence of great variability in chilling

291

sensitivity among pomegranate varieties (Figures 1, 2). To reveal the molecular

292

mechanisms that could be involved in imparting chilling tolerance in pomegranate

293

fruits we used RNA Seq analysis to investigate the transcriptomes of two varieties —

294

'Ganesh' and 'Wonderful' — that greatly differ in their chilling sensitivities after a 2-

295

week cold storage period at 1° C. In the chilling-sensitive 'Ganesh' we noticed a

296

substantial decrease in transcript levels in response to cold storage, whereas in the

297

relatively chilling-tolerant 'Wonderful' we noticed a substantial increase in transcript

298

levels, indicating activation of molecular defense mechanisms against cold stress. We

299

obtained similar findings of down- and up-regulation, respectively, of transcript levels

300

in chilling-sensitive and -tolerant fruits in a previous study, in which we compared the

301

transcriptomic responses of early-harvested chilling-sensitive and late-harvested

302

relatively chilling-tolerant 'Wonderful' fruits.6

303

The main transcripts and corresponding metabolic pathways that were up-

304

regulated in relatively chilling-tolerant 'Wonderful' fruits but were unaffected or

305

down-regulated in chilling-sensitive 'Ganesh' fruits in response to cold storage were

306

related to: jasmonic acid biosynthesis and signaling; activation of galactinol and

307

raffinose biosynthesis; activation of the phenylpropanoid pathway and its deflection

308

towards phenol biosynthesis; activation of calcium and MAPK signaling pathways;

309

activation of different stress-related transcription factors, such as AP2/ERFs, HSFs

310

and WRKYs; expression of heat-shock proteins; and induction of transcripts involved

311

in lipid remodeling and synthesis. All of these transcripts are known to be involved in

312

abiotic stress responses, including cold stress, in other plant species.24, 25

313 314

Jasmonic acid is an important regulator of biotic and abiotic stress responses , including cold stress,26, 27 and it has been demonstrated that cold stress led to


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increased levels of jasmonate in some plant species, such as pine and rice.28, 29

316

Moreover, application of exogenous jasmonate resulted in increased chilling

317

resistance of various fruits and vegetables.30 In the present study we noted marked

318

up-regulation of transcripts encoding key enzymes in the jasmonate biosynthesis

319

pathway, including LOX, AOS, and OPR, and also of MYC2, the key transcriptional

320

activation factor involved in transmission of jasmonate signaling,31 specifically in the

321

relatively chilling-tolerant variety, 'Wonderful' (Figure 4). These findings indicate

322

that jasmonate probably plays a major role in imparting chilling tolerance in

323

pomegranate fruits. This hypothesis is further supported by findings of Mirdehghan

324

and Ghotbi, who described that exogenous application of jasmonates reduced chilling

325

injury in pomegranate fruits. 32

326

It is well known that an increase in soluble solutes contributes to cold stress

327

tolerance in plants.33 Accordingly, wenoted significant increases of galactinol

328

synthase and raffinose synthase transcripts, specifically in the relatively chilling-

329

tolerant variety 'Wonderful' (Figure 5). Several researchers reported increases in

330

galactinol and raffinose contents during cold acclimation,34-36 and found that

331

overexpression of galactinol synthase resulted in enhanced cold-stress tolerance in

332

rice.37 Therefore, we suggest that up-regulation of galactinol and raffinose synthase

333

transcripts may take part in imparting chilling tolerance in pomegranate as well.

334

One of the preeminent stress responses in plants is the induction of

335

phenylpropanoid metabolism.38 It is well known that phenylpropanoids and phenols

336

act as antioxidants and are able to protect plant cells from oxidative damage that may

337

be caused by accumulation of reactive oxygen species (ROS) in response to

338

environmental stresses.39 In this study, we observed up-regulation of transcript levels

339

of all major phenylpropanoid and phenol biosynthetic genes, specifically in the



340

relatively chilling-tolerant variety 'Wonderful' (Figure 6). Previous studies found that

341

phenylpropanoid biosynthesis genes were induced by low temperatures and were key

342

regulators of cold-stress responses, 40 and that accumulation of phenols and lignin

343

were involved in plant responses to chilling stress.41,42 These observations indicate

344

that induction of phenylpropanoids and phenols metabolism is among the metabolic

345

adjustments that occur in the relatively chilling-tolerant 'Wonderful' in response to

346

cold storage.

347

Stress signals, including cold stress, lead to enhanced concentrations of

348

cytosolic Ca2+, which acts as a second messenger to various extracellular stimuli.43, 44

349

Calcium sensors in plants can be divided into three main classes: CaM/CMLs, CDPKs

350

and CBLs-CIPKs.45, 46 It is also well documented that the MAPK signaling cascade is

351

a key pathway in cold-stress responses because it regulates expression of CBF and

352

cold-responsive genes.47, 48 The present findings demonstrate marked up-regulation of

353

Ca2+-dependent signaling pathways via CaM/CMLs, CDPKs and CBLs-CIPKs, as

354

well as via the MAPK signaling pathway, in response to cold storage, specifically in

355

the 'Wonderful' fruits (Figure 7). Notably, we detected up-regulation of CRLK1,

356

which was recently shown to interact specifically with MEKK1 in imparting cold

357

tolerance on Arabidopsis thaliana.49, 50

358

Transcription factors (TFs) regulate plant stress responses, and in the present

359

study we observed significant alterations in transcript levels of various stress-related

360

TFs, mainly AP2/ERFs, HSFs and WRKYs, in response to cold storage (Figure 8).

361

Particularly worth noticing is the up-regulation of DREB1E and DREB1F transcripts,

362

specifically in the relatively chilling-tolerant 'Wonderful', because they are key

363

regulators of abiotic stress responses, including exposure to low temperatures.51 For

364

instance, overexpression of DREB1F increased drought, salt, and chilling tolerance in


Page 16 of 43

Page 17 of 43


365

rice and Arabidopsis plants.52 In contrast to DREB1E and DREB1F, transcript levels

366

of DREB1D/CBF4 were down-regulated in 'Ganesh' but were unchanged in

367

'Wonderful' in response to cold storage; however, this gene was reported to be

368

induced by drought but not by cold stress.53 In addition to DREBs, transcripts of

369

RAP2 and HSFs — TFs that are also associated with the regulation of abiotic stress

370

responses — were also up-regulated in 'Wonderful' fruits.54, 55 HSPs have an important role in conferring thermotolerance on plants.56, 57 In

371 372

this study, we observed a marked up-regulation in HSP transcript levels in the

373

relatively chilling-tolerant variety 'Wonderful' in response to cold storage (Figure 9).

374

Increases in HSP levels were reported to be related to chilling tolerance,58, 59 and

375

heterologous expression of a small plant HSP enhanced Escherichia coli viability

376

under high- and low-temperature stresses.60 Thus, our results suggest that induction of

377

HSPs is probably also involved in imparting chilling tolerance in pomegranate fruits

378

as well.

379

Lipids contents and composition play a remarkable role in governing cold

380

tolerance.61, 62 Our present findings demonstrate that the relatively chilling-tolerant

381

variety 'Wonderful' was more amenable to modify its fatty acids and membranes

382

compositions in response to cold storage than the chilling-sensitive 'Ganesh'. These

383

modifications included up-regulation of transcripts involved in remodeling of fatty

384

acids; synthesis of fatty acid-derived signaling molecules; and biosynthesis of

385

sphingolipids, triacylglycerol and sterols; and down-regulation of transcripts involved

386

in degradation of fatty acids via the β- and ω-oxidation pathways (Figure 10). In this

387

context, previous studies revealed the roles of DGK and PLD in accumulation of the

388

signaling molecule phosphatidic acid and regulation of cold stress responses.63, 64



389

Overall, we demonstrated that various regulatory (jasmonates, calcium and

390

MAPK signaling; TFs), metabolic (carbohydrates, phenols and phenylpropanoids) and

391

stress-adaptation (HSPs) transcripts were specifically induced in 'Wonderful' in

392

response to cold storage and therefore might be involved in imparting the chilling

393

tolerance of this variety. Most of these molecular mechanisms were also induced by

394

other factors that increased chilling tolerance in 'Wonderful' pomegranate, e.g., a pre-

395

storage LTC treatment and late harvest.5 More specifically, pre-storage LTC treatment

396

also increased transcript levels related to calcium and MAPK signaling, and

397

transcripts of HSFs and WRKYs TFs and HSPs ;7 whereas late harvest increased

398

transcripts involved in jasmonate synthesis and signaling, AP2/ERFs, HSFs and

399

WRKYs, galactinol and raffinose biosynthesis, and HSPs.6

400


Page 18 of 43

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401

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Table 1. Effects of pomegranate variety and chilling exposure on the transcriptome of pomegranate inner membrane tissue. Data include transcripts differentially expressed at P  0.001 according to DESeq2 R package, and induced or repressed by a factor of at least 4. Numbers in parentheses indicate the number of transcripts with annotations to Arabidopsis. Pair-wise comparison

Transcripts differentially expressed at P  0.001 and induced or repressed by a factor of at least 4 Up-regulated

Down-regulated

Total

Ganesh + chilling/ T0

1,354 (1134)

1,778 (1,494)

3,132 (2,628)

Wonderful + chilling/ T0

3,379 (2,026)

2,226 (1,968)

5,605 (4,598)



Page 28 of 43

Table 2. Functional categorizations of transcripts belonging to the 'Ganesh', 'Common' and 'Wonderful' regulons according to the MapMan software. Bin

Functional categorization

Ganesh Up Down

Common Up Down

Wonderful Up Down

3 3.1

minor CHO metabolism minor CHO metabolism.raffinose family minor CHO metabolism.raffinose family.galactinol synthases lipid metabolism lipid metabolism.lipid degradation secondary metabolism secondary metabolism.phenylpropanoids secondary metabolism.flavonoids secondary metabolism.simple phenols hormone metabolism hormone metabolism.jasmonate stress stress.abiotic stress.abiotic.heat RNA RNA.regulation of transcription RNA.regulation of transcription.MYB domain transcription factor family RNA.regulation of transcription.WRKY domain transcription factor family signaling signaling.receptor kinases signaling.calcium signaling.MAP kinases

5 1

3 1

8 2

6 1

8 3

11 0

0

0

1

0

2

0

6 1 6 2

8 3 12 3

17 8 20 5

8 5 22 6

31 12 40 13

17 6 32 8

0 0 5 2 12 9 4 45 37 2

5 0 24 0 30 20 8 62 54 5

5 1 21 2 37 17 6 59 53 4

7 1 34 4 27 15 3 84 77 3

10 4 48 7 81 37 19 165 148 15

8 1 26 1 35 18 6 104 91 8

2

2

7

2

14

1

15 6 3 2

37 19 8 1

50 21 15 0

52 32 6 0

133 60 33 6

55 24 10 1

3.1.1 11 11.9 16 16.2 16.8 16.10 17 17.7 20 20.2 20.2.1 27 27.3 27.3.25 27.3.32 30 30.2 30.3 30.6


Page 29 of 43


FIGURE LEGENDS

Figure 1. Diversity in chilling sensitivity among 84 pomegranate varieties from the Israel Pomegranate Collection at the Newe Ya'ar Research Center, presented as internal browning index (0–3). The chilling injury index was evaluated after 6 weeks of cold storage at 5 °C + 1 week at 20 °C. The 'Ganesh' (P.G. 160-61) and 'Wonderful' (P.G. 101-2) varieties are indicated by black arrows. Data are means ± S.E. of two replications of two consequent seasons, each comprising 9–11 fruits.

Figure 2. Effects of cold storage on CI development in 'Ganesh' and 'Wonderful' pomegranate varieties. The fruits were stored for 4 weeks at 1 °C + 1 week at 20 °C. A. Chilling injury indices of 'Ganesh' and 'Wonderful' fruits. B. Photographs of 'Ganesh' and 'Wonderful' varieties after cold storage. Data are means ± S.E. of four cartons, each containing 9–11 fruits.

Figure 3. Venn diagram illustrating the overlapping and differences between transcript expression patterns after exposure of 'Ganesh' and 'Wonderful' pomegranates to 2 weeks of cold storage at 1 °C. The table at the bottom indicates the total numbers of differentially expressed transcripts homologous to Arabidopsis genes in the 'Ganesh- specific', 'common', and 'Wonderful-specific' regulons.

Figure 4. Differentially expressed transcripts belonging to jasmonate biosynthesis and signal transduction pathways. A. Diagram of jasmonate biosynthesis and signaling pathway. Transcripts up-regulated by chilling in the 'Wonderful- specific' regulon are marked in red. B. Heat-map analysis of differentially expressed



transcripts related to jasmonate biosynthesis and signaling in 'Ganesh' and 'Wonderful' varieties, at T0 and after cold storage. Red and green colors represent high and low expression levels. The units on the color scale are standard deviations.

Figure 5. Differentially expressed transcripts belonging to galactinol and raffinose biosynthesis pathway. A. Diagram of galactinol and raffinose biosynthesis pathway. Transcripts up-regulated by chilling in the 'Wonderful-specific' regulon are marked in red. B. Heat-map analysis of differentially expressed transcripts related to the galactinol and rafinnose biosynthesis pathway, in 'Ganesh' and 'Wonderful' varieties, at T0 and after cold storage. Red and green colors represent high and low expression levels, respectively. The units on the color scale are standard deviations.

Figure 6. Differentially expressed transcripts belonging to the phenylpropanoids, flavonoids and phenols biosynthesis pathways. A. Diagram of phenylpropanoids, flavonoids and phenols biosynthesis pathways. Transcripts up-regulated by chilling in the 'Wonderful-specific' regulon are marked in red; those down-regulated by chilling in the 'Wonderful-specific' regulon are marked in green. B. Heat-map analysis of differentially expressed transcripts related to the phenylpropanoids, flavonoids and phenols biosynthesis pathways, in 'Ganesh' and 'Wonderful' varieties, at T0 and after cold storage. Red and green colors represent high and low expression levels, respectively. The units on the color scale are standard deviations.

Figure 7. Differentially expressed transcripts belonging to Ca2+ and MAPK signaling pathways. A. Diagram of Ca2+ and MAPK signaling pathways. Transcripts upregulated by chilling in the 'Wonderful-specific' regulon are marked in red. B. Heat-


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map analysis of differentially expressed transcripts related to Ca2+ and MAPK signaling pathways in 'Ganesh' and 'Wonderful' varieties, at T0 and after cold storage. Red and green colors represent high and low expression levels, respectively. The units on the color scale are standard deviations.

Figure 8. Heat-map analysis of differentially expressed transcripts encoding TFs in 'Ganesh' and 'Wonderful' varieties, at T0 and after cold storage. A. AP2/ERFs. B. HSFs C. WRKYs. Red and green colors represent high and low expression levels, respectively. The units on the color scale are standard deviations.

Figure 9. Heat-map analysis of differentially expressed transcripts encoding HSPs in 'Ganesh' and 'Wonderful' varieties, at T0 and after cold storage. Red and green colors represent high and low expression levels, respectively. The units on the color scale are standard deviations.

Figure 10. Heat-map analysis of differentially expressed transcripts encoding lipid metabolism-related transcripts in varieties 'Ganesh' and 'Wonderful', at T0 and after cold storage. Red and green colors represent high and low expression levels, respectively. The units on the color scale are standard deviations.



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TOC Graphics


Diversity among Pomegranate Varieties in Chilling Tolerance and

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