Biochemical composition and some anthocyanin biosynthetic genes of

Subscriber access provided by UNIV OF DURHAM

Biotechnology and Biological Transformations

Biochemical composition and some anthocyanin biosynthetic genes of a yellow peeled and pinkish ariled pomegranate (Punica granatum L.) cultivar are differentially regulated in response to agro-climatic conditions Rekha Attanayake, Rasu Eeswaran, Ranil Rajapaksha, Palitha Weerakkody, and Pradeepa Bandaranayake J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02909 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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 Biochemical composition and expression analysis of anthocyanin biosynthetic genes of a yellow peeled

and pinkish ariled pomegranate (Punica granatum L.) cultivar are differentially regulated in response to agro-climatic conditions Rekha Attanayakea, Rasu Eeswaranb, Ranil Rajapakshac, Palitha Weerakkodyc and Pradeepa C.G. Bandaranayakea* a

Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya, Peradeniya

20400, Sri Lanka b

Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI

48824, USA c

Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya 20400,

Sri Lanka *Corresponding author: [email protected]

ACS Paragon Plus Environment


Page 2 of 38

2

1

ABSTRACT

2

The accumulation of beneficial biochemical compounds in different parts of pomegranate (Punica

3

granatum L.) fruit determines fruit quality and highly depends on environmental conditions. We

4

investigated the effects of agro-climatic conditions on major biochemical compounds and on the

5

expression of major anthocyanin biosynthetic genes in the peels and arils of a yellow-peeled and

6

pink-ariled pomegranate cultivar in three agro-climatologically different locations in Sri Lanka.

7

Drier and warmer climates promoted the accumulation of the measured biochemical compounds i.e

8

total phenolic content (TPC), antioxidant capacity (AOX), α, β and total punicalagin in both peels

9

and arils compared to wetter and cooler climates. Pomegranate DFR, F3H and ANS transcripts in

10

both peel and arils showed higher relative expression in hotter and drier regions, compared to those

11

that were cooler and wetter conditions. Therefore, growing pomegranates in drier and warmer

12

environments maximizes the production of beneficial biochemical compounds and associated gene

13

expression in pomegranate fruit.

14

15

KEY WORDS: Pomegranate, Punica granatum L, Punicalagin, Anthocyanin, agro-climatic, Total

16

Phenolic Content (TPC), Antioxidant activity (AOX)

17

18

19

20

21

22

23 ACS Paragon Plus Environment

Page 3 of 38


3 24

INTRODUCTION

25

Pomegranate (Punica granatum L.) has been well known for its medicinal properties. It has gained

26

even more attention due to recent scientific evidences on health promoting and nutritional benefits.1-

27

6.

28

cultivation and its industrial applications are expanding considerably.7 A continuous supply of

29

uniform quality raw materials is a key requirement for marketing and industrial applications of

30

pomegranates including therapeutics.

31

Flavonoids, hydrolysable tannins and condensed tannins are the major bioactive compounds

32

contribute to the medicinal quality of pomegranate fruits.8-9 Flavonoids include flavanols,

33

anthocyanin and phenolic acids, are mainly found in the peel and juice of the pomegranates.10

34

While hydrolysable tannins such as ellagitannins and gallotannins are mainly present in peels and

35

membranes,11 condensed tannins are mainly found in peel and juice.12 Transcriptome sequencing

36

work13 accelerated identification and characterization of the genes and enzymes responsible for the

37

biosynthetic pathways of above valuable bioactive compounds. For example, several major genes

38

responsible for hydrolysable tannin14 and anthocyanin15 biosynthesis were functionally

39

characterized recently. Further, molecular evidences suggest that differential expression of genes in

40

relevant pathways correlate well with the quantity of related products accumulated 14, 16,17

As a result, global pomegranate consumption is increasing and consequently, pomegranate

41

42

It is now known that the quantity and the composition of bioactive compounds in pomegranate peel

43

and aril depend on many pre-harvest and post-harvest factors.18 Some of the key factors are cultivar

44

or genotype, agro-ecological conditions (climate and soil), maturity stages of fruits and crop

45

management 19.

46

Few previous studies on the effects of agro-ecological and seasonal variations on biochemical

47

properties of pomegranate20 have mainly considered the effects of drastic climates such as

48

Mediterranean climate vs dessert climate21or summer vs winter22. Only few studies have evaluated ACS Paragon Plus Environment


Page 4 of 38

4 49

the effects of more than one season in the same location23-24. Furthermore, previous studies have not

50

focused on gene expression in response to environmental factors. In addition, previous studies were

51

restricted to the cultivars with red peel and red arils, while cultivars with yellow peel and pink arils

52

were neglected. Nevertheless, cultivars with yellow peels and pink arils are popular in some

53

countries. The yellow peel, pink aril cultivar, Nimali consists of higher concentrations of beneficial

54

biochemical compounds than popular variety Wonderful, under Sri Lankan conditions.25 As the

55

color change of the peel does not follow that of the arils in pomegranate,21 recognition of harvesting

56

indices is also critical.

57

Therefore, we investigated the effects of three major agro-climatic conditions present in Sri Lanka

58

on total phenolic content (TPC), alpha punicalagin (AP), beta punicalagin (BP), total punicalagin

59

(TP), antioxidant activity (AOX) and the expression of some anthocyanin biosynthetic pathway

60

genes of Nimali fruits over two years. Here we specifically focused on anthocyanin biosynthetic

61

pathway genes; because of the importance of the pathway, having visual color with relative gene

62

expression and its relationship to TPC and AOX 26,27. To the best of our knowledge, this is the first

63

study on the effect of environmental conditions on biochemical composition and anthocyanin gene

64

expression of a yellow peel, pink aril cultivar. Further, we studied the correlation of rainfall and the

65

mean air temperature with quantity of different bioactive compounds. The findings would also

66

enable growers to expand the cultivation of pomegranate in the location/s where the agro-climatic

67

conditions maximize the production of beneficial bioactive compounds.

68

MATERIALS AND METHODS

69

Chemicals and Reagents

70

Standards of punicalagin α & β and gallic acid were purchased from Sigma Aldrich (St. Louis,

71

MO). All reagents used for liquid chromatography were HPLC grade and purchased from Sigma

72

chemicals (Taufkirchen, Germany). All other reagents used for biochemical analysis were analytical

73

grade and obtained either from Sigma Chemicals, St. Louis, MO, USA or Himedia chemicals, ACS Paragon Plus Environment

Page 5 of 38


5 74

Mumbai, India. Taq polymerase, cDNA synthesis kit, dNTPS, DNA ladder were purchased from

75

Pomega Corporation, Madison, WI. Primers were synthesized from IDA Technologies (Coralville,

76

IA).

77

Study locations and sample collections

78

One of the most popular local pomegranate cultivar Nimali, cultivated in three different locations

79

representing two different agro-ecological regions; i.e. Low country dry zone (DL3) and up country

80

intermediate zone (IU3) of Sri Lanka28 was selected for this study. They were Kalpitiya (8.21o N,

81

79.73 o E; DL3), Mullaitivu (9.28 o N and 80.80 o E; DL3) and Teldeniya (7.32 o N, 80.74 o E; IU3)

82

(Supplementary Figure. 1). Each orchard consisted of

83

maintained according to standard management practices, as recommended by the Department of

84

Agriculture, Sri Lanka. Briefly, uninfected healthy seedlings were planted at the beginning of the

85

rainy season using 60 x 60 x 60 cm pits with 3x3 m distance with a underneath coir layering. The

86

pits were filled with cow dung, compost and recommended dosages of chemical fertilizers.

87

Seedlings were irrigated properly until they established well in the orchard. Main stem of the

88

mature plants were pruned at a height of 60-75 cm by plucking the terminal bud to promote

89

flowering and fruiting.

90

Three fruiting trees were randomly selected from the middle of each orchard, and three fully

91

matured (180 Days after initiation), uninfected and undamaged fruits were harvested from each tree.

92

Accordingly, all together 54 fruits (3 fruits x 3 trees x 3 locations x 2 seasons) were sampled during

93

the peak harvesting season i.e. mid-September of two consecutive years.

94

Collected fruit samples were cut into halves, and peels and arils of each half were separately flash

95

frozen in liquid N2 and stored in – 80 oC for the analysis. Differences in external and internal

96

appearances of collected pomegranate fruit samples of from different locations are shown in

97

Supplementary Figure 1.

98

Meteorological Data

25-50 same aged (5 years old) trees,



Page 6 of 38

6 99

Rainfall (RF), maximum temperature (Tmax), minimum temperature (Tmin), maximum relative

100

humidity (RHmax) and minimum relative humidity (RHmin) were collected from the Department of

101

Meteorology of Sri Lanka for the years 2014 and 2015 for three locations.

102

Soil Analysis

103

A composite soil sample for each location was taken by mixing 3 sub-samples per location, at the

104

depth of 0-15 cm. Soil samples were analyzed for soil pH, electrical conductivity (EC), available

105

nitrogen (N), available phosphorous (P), available potassium (K), exchangeable Ca and organic

106

matter (OM) using standard procedures.29 The soil pH was measured using a standard glass

107

electrode pH meter in distilled water using a soil to water ratio of 1:2.5 and the standard electrical

108

conductivity meter was used to measure the EC of 1:5 soil water suspensions. The available N was

109

analyzed using the Kjeldahl method29. The available P and available K were determined by Olsen

110

method.30 Ammonium acetate extraction at pH 7.0 was used to determine the exchangeable Ca29.

111

The Dichromate oxidation method31 was used to analyze the organic matter.

112

Biochemical analysis

113

Other than the sampling and replications mentioned above, all the biochemical tests were repeated

114

three times per each sample.

115

Total phenolic content by Folin-CioCalteau method

116

Total phenolic content (TPC) in the peel and aril (juice extracts) of pomegranate samples were

117

determined by the Folin-CioCalteau colorimetric method described by Thaipong et al32 with some

118

modifications. Accordingly, 10 mg of sample was squeezed and the extract was centrifuged at 5000

119

rpm for 15 minutes. Five hundred microliters of the supernatant was added to 2 ml of distilled water

120

and mixed properly. One milliliter of diluted Folin-CioCalteau reagent was added to the above,

121

mixed thoroughly and kept at room temperature for 3 min and 500 µl of 6% (w/v) sodium carbonate

122

was added. After 120 min, the absorbance was measured in triplicates at 600 nm and the calibration


Page 7 of 38


7 123

curve was performed with Gallic acid and the results were expressed as milligrams of Gallic acid

124

equivalents per grams of fresh sample (mg GAE g-1 (fw)).

125

Antioxidant activity by DPPH radical scavenging method

126

Antioxidant activity (AOX) was quantified by evaluating free radical scavenging activity. The

127

extracts were allowed to react with a stable free radical, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) as

128

previously described by Marxen et al.33

129

HPLC Analysis

130

Peel and aril tissues of pomegranate were ground into very fine powder using liquid N and the

131

punicalagin α & β contents of the samples were determined by HPLC following the method

132

described by Ono et al.13 with some modifications. Thousand micro liters of 40 %(v/v) methanol

133

was added to 200 mg of each samples, sonicated for 20 minutes and centrifuged for 15 minutes at

134

12 000 rpm. About 500 µl of the supernatant was centrifuged at 12000 rpm for 20 minutes and 200

135

µl of the supernatant was used for HPLC analysis in Agilent 1260 system equipped with a Zobax

136

SB-C18, 5 µm, 4.6 x 150 mm column. Ten microliters of sample was injected and separated in a

137

solvent gradient between 0.1 (v/v) Formic acid and 100 % (v/v) Acetonitrile (ACN). The time-ACN

138

combinations were; 5 % (v/v) ACN for 0-3 minutes, 5-25 % (v/v) ACN for 3-24 minutes, 25-35 %

139

(v/v) ACN for 24-29 minutes and 35-45 % (v/v) ACN for 29-30 minutes, at the flow rate of 1ml

140

per minute. Punicalagin α & β were identified with the retention time of the standards and standard

141

curves were generated with commercial standards for quantification of punicalagin isomers.

142

RNA Extraction

143

The total RNA was extracted from 2 g of peel and 8 g of aril samples using a CTAB based method

144

developed combining two protocols from Moser et al34 and Jaakola et al35 with further

145

modifications. Sample was mixed with 500 µl of pre heated CTAB extraction buffer with 2 (w/v)

146

2-mercaptaethanol and the equal volume of chloroform and centrifuged at 3000 rpm for 35 minutes.

147

Four milliliters from that supernatant and 0.25 of the volume of 10 M LiCl were kept in refrigerator ACS Paragon Plus Environment


Page 8 of 38

8 148

overnight and centrifuged at 14000 rpm for 10 minutes. The pellet was washed with 2 M LiCl and

149

re-suspended in 500 µl of 10 mM Tris HCl (pH 7.5). Then, 2 M potassium acetate (pH 5.5) was

150

added in 1/10 volume and centrifuged at 14000 rpm for 10 minutes. The pellet was washed with

151

70% (v/v) ethanol and re-suspended in 20-50 µl water depending its size.

152

The quality and quantity of the extracted RNA were determined by electroporation on 1% (w/v)

153

agarose gel using the Nanodrop spectrophotometer (Thermo ScientificTM Nanodrop 2000). RNA

154

samples were treated with DNAse (Pomega RQ1 RNAse-Free) as 0.5 µl (1 U/ µl) per 1µg of total

155

RNA and the cDNA synthesis was done with total 5 µg of RNA using OligodT primers following

156

the manufactures protocol (Pomega (Madison, Wisconsin, USA)).

157

Gene expression analysis

158

Differential expression of four major genes in the anthocyanin biosynthetic pathway was studied

159

using semi quantitative Polymerase Chain Reaction (PCR). The same primers for CHI, DFR, F3H

160

and ANS used in previous studies were selected Ono et al.36 and Ben-Simhon et al.16

161

(Supplementary Table 1). Pomegranate actin gene36 was used as the housekeeping control.

162 163

Semi-Quantitative PCR was carried out for the selected genes of pomegranate with following

164

conditions: 95 °C for 5 minutes, 29 cycles of denaturation at 95 °C for 10 s , annealing at 58 °C for

165

20 s and extension at 72 °C for 1 minute and a final extension at 72 °C for 10 minutes . Each

166

reaction consisted of 10X PCR Buffer 10 µl, 10 mM dNTPs 2 µl, 50 mM MgCl2 3 µl , 5 µl of each

167

primer (10 µM), 1 µl cDNA, Taq Polymerase (5 U/ L) 0.2 µl , 0.8 mM Spermidine and volumed up

168

to 20 µl with RNAse free water. The PCR products were separated on 2 %(w/v) agarose gel and

169

stained with ethidium bromide.

170

171

Data analysis


Page 9 of 38


9 172

The experiment was arranged in a nested design where trees were considered nested within the

173

locations while fruits were nested within both locations and trees. This specific statistical design

174

was used since the measured variables such as TPC, AOX, AP, BP and TP were affected or nested

175

inside two or more nominal variables. Data on biochemical compounds were analyzed using the

176

SAS 9.4® software package (SAS Institute Inc. Cary, North Carolina, USA) considering the

177

ANOVA of nested design and means were compared by the Duncan’s New Multiple Range Test at

178

5% probability level.37 Correlation analysis38 was done to explore the relationship between agro-

179

climatic conditions and bioactive compounds of the pomegranate fruits using Microsoft Excel

180

2007®. Principal component analysis39 was conducted to identify the dominant components of the

181

variance of measured parameters annual mean temperature, annual rainfall, mean RH, Soil organic matter,

182

Soil pH, Soil EC, Soil N, Soil P & Soil K

183

The relative gene expression was quantified using the constitutively expressed housekeeping gene actin. The

184

intensity of semi-quantitative PCR bands of each fruit sample was calculated using the imageJ software. The

185

ratio of the average of band intensity for each gene over the average for actin was calculated using Microsoft

186

Excel 2007 and the average value for the location data was plotted.

187

188

RESULTS

189

Meteorological conditions during the study period

190

Annual climatological conditions of the selected locations i.e. Kalpitiya (Supplementary Figure 2),

191

Mullaitivu (Supplementary Figure 3) and Teldeniya (Supplementary Figure 4) during 2014 and

192

2015 vary considerably. Nevertheless, the relative humidity was relatively constant among the study

193

locations (Table 1). Kalpitiya, located in low country dry zone (DL3) is the driest location with the

194

lowest rainfall and the highest mean temperatures. Teldeniya, belongs to the upcountry intermediate

195

zone (IU3) received the highest rainfall and experienced the lowest mean temperatures. Though

196

Mullaitivu also belongs to the DL3 zone, that area received relatively higher rainfall and recorded

197

lower average temperature than Kalpitiya (Table 1). In addition, climatology of Kalpitiya is ACS Paragon Plus Environment


Page 10 of 38

10 198

considered unique with proximity to Puttalam lagoon with a very low day and night temperature

199

difference, specific soil properties and relatively high winds.40, 41

200

Soil physical and chemical parameters in the selected locations were not considerably different

201

(Table 2). The lowest EC was recorded in Kalpitiya because of sandy nature. While the highest soil

202

organic matter was found in Teldeniya, the soil pH was in the neutral range in all the locations.

203

Biochemical analysis

204

Interestingly, there was no significant seasonal effect on the total phenolic content (TPC),

205

antioxidant activity (AOX), punicalagin alpha (AP), punicalagin beta (BP) and total punicalagin

206

(TP) in the peel and aril samples collected from different agro-climatic zones (Table 3). Further,

207

there was no significant difference among fruits harvested from the same plant (p0.05). Further, there was no correlation between annual average relative humidity with

298

the biochemical parameters. However, there was a significant correlation between annual average

299

rainfall and average annual temperature with biochemical parameters, TPC, AOX, alpha, beta and ACS Paragon Plus Environment


Page 14 of 38

14 300

total punicalagin in both peel and arils of fruits. Where the climate was cooler and wetter,

301

production of TPC, AOX, alpha, beta and total punicalagin in both peel and arils was significantly

302

reduced. Pomegranate is a sun-loving plant and temperature has a considerable impact on plant

303

growth, fruit development and ripening.41 Our results are comparable with previous work where the

304

Mediterranean climates with hot temperatures (around 30 °C) without much rainfall in the fruiting

305

season was recommended for better quality pomegranates.45 There were higher TPC, hydrolysable

306

tannins, punicalagin contents found in fruit peels of most of the cultivars harvested from desert

307

regions compared to Mediterranean climates.21 The TPC, AOX and punicalagin content their

308

correlations reported here are comparable with previous studies 46-49.

309

Interestingly, there was significant difference among three plants collected from the same location.

310

Floral behavior of Nimali suggests potential of cross-pollination. Nevertheless, currently this variety

311

is mainly propagated through seeds and the vegetative propagation is limited. All the plants used in

312

the current study are seed propagated and shows considerable genetic variation among them, as

313

identified by ISSR finger printing (Data not shown). This genetic diversity was also reflected in

314

biochemical composition.

315

Anthocyanin is one of the major groups of compounds responsible for TPC and AOX of

316

pomegranate27. Further, the products of anthocyanin biosynthetic pathway determines the colour of

317

flower, fruit and arils. Here we observed higher TPC and AOX values than others and relatively

318

brighter color peels and arils in Kalpitiya area. Relatively higher expression of anthocyanin

319

biosynthetic may attribute to such changes.

320

A recent study showed that the presence of functional LDOX (leucoanthocyanidindioxygenase

321

(ANS-anthocyanidin synthase), is critical for the red color phenotype in flowers, peels and arils of

322

pomegranate. Further, it describes that the insertional mutation (PgLDOX gene) in POM-LDOX

323

gene results white color flowers, fruits and arils.15, 16 Our results also support the previous findings

324

and show clear ANS expressions in pink colour arils irrespective of the environmental factors and

325

very low expression in yellowish peel. Among the genes considered CHI did not show a clear ACS Paragon Plus Environment

Page 15 of 38


15 326

differential expression either in peel or arils in response to environmental variation. But there was a

327

considerable up regulation of DFR and F3H genes in peel as well as in arils in the drier and warmer

328

locations, i.e: Kalpitiya and Mullaitivu compared to Teldeniya. Based on gene expression analysis,

329

it could be predicted that the total anthocyanin content in Teldeniya samples would be lower than

330

samples collected from other locations. However, previous work on 11 pomegranate accessions

331

grown under Mediterranean and desert climates in Israel have concluded higher anthocyanin

332

content in fruit arils in the Mediterranean climate, compared to those grown in desert climate.21

333

Similarly, Borochov-Neori et al.22 found that anthocyanin accumulation changed inversely to the

334

environmental temperature during the growing season. However, anthocyanin biosynthetic pathway

335

is also controlled by light intensity, CO2 concentration50 and water availability.51. Lower

336

temperature, high rainfall, greater water holding of soil associated with high soil organic matter and

337

other micro environmental factors associated with high elevation during the fruit development in

338

Teldeniya might have suppressed the expression of anthocyanin biosynthetic pathway genes.

339

Pomegranates perform better in fertile, alluvial soils with good drainage.42 However, Kalpitiya with

340

sandy soils is identified as one of the best regions for growing pomegranate in Sri Lanka because of

341

good drainage. Dry weather and high temperature coupled with other micro environmental

342

conditions facilitates the optimum growth of pomegranates and production of beneficial

343

biochemical compounds. Further, the unique climatic conditions associated with the lagoon and

344

minimum day and night temperature difference and monsoonal winds40,41 would further facilitate

345

higher quality fruits.

346

ABREVIATIONS

347

Total phenolic content (TPC)

348

Antioxidant Activity (AOX)

349

Alpha Punicalagin (AP)

350

Beta Punicalagin (BP) ACS Paragon Plus Environment


Page 16 of 38

16 351

Total Punicalagin (TP)

352

Chalcone Isomerase (CHI)

353

Dihydroflavonol 4-reductase (DFR)

354

Flavonoid 3′-hydroxylase (F3H)

355

Anthocyanidin synthase (ANS)

356

Actine (ACT)

357

Low country Dry zone (DL)

358

Up country Intermediate zone (IU)

359

Rainfall (RF)

360

Maximum temperature (Tmax)

361

Minimum temperature (Tmin)

362

Maximum relative humidity (RHmax)

363

Minimum relative humidity (RHmin)

364

Electical Conductivity (EC)

365

Available nitrogen (N)

366

Aavailable phosphorous (P)

367

Available potassium (K)

368

Organic matter (OM)

369

2, 2-diphenyl-1-picrylhydrazyl (DPPH)

370

High Performance Liquid Chromatography (HPLC)

371

Acetonitrile (ACN). ACS Paragon Plus Environment

Page 17 of 38


17 372

Leucoanthocyanidindioxygenase (LDOX)

373

Galic Acid Equvalent (GAE)

374

Fresh Weight (fw)

375

376

ACKNOWLEDGEMENT

377

Authors wish to thank National Research Council in Sri Lanka for the financial support (Grant

378

number: NRC 12/113) and the staff of the Agricultural Biotechnology Center and the Department of

379

Agriculture, Kalpitiya Research Station for the technical assistance.

380

381

SUPPORTING INFORMATION Supplementary Figure 1. Locations selected for the study

382

Supplementary Figure 2. Annual climatology of Kalpitiya in 2014 (a) and in 2015 (b).

383

Supplementary Figure 3. Annual climatology of Mullaitivu in 2014 (a) and in 2015 (b).

384

Supplementary Figure 4. Annual climatology of Teldeniya in 2014 (a) and in 2015 (b).

385

Supplementary Figure 5. HPLC Elution profiles of punicalagin α and β of peel (column A) and aril (column

386

B) of the pomegranate fruit of 180 DAI recorded at 378 nm in Kalpitiya (1), Mullaitivu (2) and Teldeniya

387

(3). C: Reference spectra of punicalagin α and β; D: Commercial standardes of α and β punicalagin.Two

388

hundred miligrams of tissues were extracted in 40 % (v/v) methanol and sonicated and injected to revese

389

phase HPLC gradient between formic acid 0.1 % (v/v) and accetonitrile 100 %

390

Supplementary Figure 6. Scree plot of principal component analysis. Components included:

391

Annual mean temperature, annual rainfall, mean RH, Soil organic matter, pH, EC, N, P & K Supplementary Table 1. List of primer sequences and annealing temperatures of the primers used



Page 18 of 38

18 392

Supplementary Table 2. Ratio of the average of band intensities of three trees for each gene over the actin

393

gene

394 395

REFERENCES

396

1. Jurenka, J. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern.

397 398 399

Med. Rev. 2008,13,128–144. 2. Ismail, T.; Sestili, P.; Akhtar, S. Pomegranate peel and fruit extracts: A review of potential antiinflammatory and anti-infective effects. J. Ethnopharmacol . 2012,143 2,397–405.

400

3. Miguel, M.G.; Neves, M.A.; Antunes, M.D. Pomegranate (Punica granatum L .): A medicinal

401

plant with myriad biological properties - A short review. J. Med. Plants. Res.2010, 4, 25,2836–

402

2847.

403 404

4. Bhandari, P.R. Pomegranate (Punica granatum L). Ancient seeds for modern cure: Review of potential therapeutic applications. Int. J. Nutr. Pharmacol. Neurol. Dis. 2012, 2,3,171–184.

405

5. Viuda-Martos, M.; Ruiz-Navajas, Y.;Martin-Sánchez, A.;Sánchez-Zapata, E.;Fernández-López,

406

.; Sendra, E. Chemical, physico-chemical and functional properties of pomegranate (Punica

407

granatum L.) bagasses powder co-product. J. Food Eng. 2012,110, 220–224.

408 409

6. Landete, J.M. Ellagitannins, ellagic acid and their derived metabolites: A review about source, metabolism, functions and health. Food Res. Int. 2011,44,1150–1160.

410

7. Tzulker, R.; Glazer, I.; Bar-Ilan, I.; Holland, D.; Aviram, M.; Amir, R. Antioxidant activity,

411

polyphenol content, and related compounds in different fruit juices and homogenates prepared

412

from 29 different pomegranate accessions. J .Agric. Food Chem. 2007, 55,23,9559–9570.

413

8. Van Elswijk, D.A.; Schobel, U.P.; Lansky, E.P.; Irth, H.; Van Der Greef, J. Rapid dereplication

414

of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical

415

detection coupled to mass spectrometry. Phytochemistry. 2004,65, 2 ,233–241.

416

9. Seeram, N.P.; Zhang, Y.; McKeever, R.; Henning, S.M.; Lee, R.;Suchard, M.A. Pomegranate

417

Juice and Extracts Provide Similar Levels of Plasma and Urinary Ellagitannin Metabolites in

418

Human Subjects. J. Med. Food. 2008, 11,2,390–394. ACS Paragon Plus Environment

Page 19 of 38


19 419

10. Li, Y.; Guo, C.; Yang, J.; Wei, J.; Xu, J.; Cheng, S. Evaluation of antioxidant properties of

420

pomegranate peel extract in comparison with pomegranate pulp extract. Food Chem.2006,

421

96,2,254–260.

422

11. Fischer, U.A.; Carle, R.; Kammerer, D.R. Identification and quantification of phenolic

423

compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently

424

produced juices by HPLC-DAD-ESI/MSn. Food Chem. 2011, 127, 2,807–821.

425

12. Singh, R.P.; Chidambara, M.K.N.; Jayaprakasha, G.K. Studies on the antioxidant activity of

426

pomegranate (Punica granatum) peel and seed extracts using in vitro models. J. Agric. Food

427

Chem. 2002, 50,1,81–86

428

13. Ono, N.N.; Britton, M.T.; Fass, J.N.; Nicolet, C.M.; Lin, D.; Tian, L. Exploring the

429

Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and

430

SSR Marker Discovery. J. Integr. Plant Biol. 2011,53,10,800–813.

431

14. Ono, N.N.; Qin, X.; Wilson, A.E.; Li, G.;Tian, L. Two UGT84 family glycosyltransferases

432

catalyze a critical reaction of hydrolyzable tannin biosynthesis in pomegranate (Punica

433

granatum). PLoS One. 2016, 5 , 8-20

434 435

15. Zhao, X.;Yuan, Z.; Feng, L.; Fang, Y. Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate. J. Plant Res. 2015, 128,4,687–696

436

16. Ben-Simhon, Z.;Judeinstein, S.;Trainin, T.;Harel-Beja, R.;Bar-Yaakov, I.;Borochov-Neori, H.

437

A “white” anthocyanin-less pomegranate (Punica granatum L.) caused by an insertion in the

438

coding region of the leucoanthocyanidin dioxygenase (LDOX; ANS) gene. PLoS One.

439

2015,10,11,6-13

440

17. Rouholamin, S.; Zahedi, B.; Nazarian-Firouzabadi, F.; Saei, A. Expression analysis of

441

anthocyanin biosynthesis key regulatory genes involved in pomegranate (Punica granatum

442

L.). Scientia Horticulturae. 2015, 186,84-88.

443

18. Mphahlele, R.R.; Fawole, O.A. Stander MA, Opara UL, Preharvest and postharvest factors

444

influencing bioactive compounds in pomegranate (Punica granatum L.)-A review. Sci. Hortic.

445

Elsevier. 2014, 178,1,114–123. ACS Paragon Plus Environment


Page 20 of 38

20 446

19. Cristofori, V.;Caruso, D.; Latini, G.; Dell’Agli, M.; Cammilli, C.; Rugini, E.;Bignami, C.;

447

Muleo, R. Fruit quality of Italian pomegranate (Punica granatum L.) autochthonous

448

varieties. European Food Res. and Techno. 2011,232,3, 397-403

449

20. Caliskan, O.;

Bayazit, S.Morpho-pomological and chemical diversity of pomegranate

450

accessions grown in Eastern Mediterranean region of Turkey. J. Agric. Sci. Technol. 2013,15

451

(SUPPL),1449–1460

452

21. Shwartz, E.; Glazer, I.;Bar-Ya’akov, I.; Matityahu, I.;Bar-Ilan, I.; Holland, D. Changes in

453

chemical constituents during the maturation and ripening of two commercially important

454

pomegranate accessions. Food Chem. 2009, 115,3,965–973.

455

22. Borochov-Neori, H.; Judeinstein, S.; Harari, M.; Bar-Ya’akov, I.;Patil, B.S.; Lurie, S. Climate

456

Effects on Anthocyanin Accumulation and Composition in the Pomegranate (Punica granatum

457

L.) Fruit Arils. J. Agric. Food Chem. 2011, 59,10,5325–5334

458

23. Fawole, O.A.; Opara, U.L. Seasonal variation in chemical composition, aroma volatiles and

459

antioxidant capacity of pomegranate during fruit development. African J. Biotechnol.

460

2013,12,25,4006–4019

461

24. Li, X.; Wasila, H.; Liu, L.; Yuan, T.;Gao, Z.; Zhao, B. Physicochemical characteristics,

462

polyphenol compositions and antioxidant potential of pomegranate juices from 10 Chinese

463

cultivars and the environmental factors analysis. Food Chem. 2015,175,575–584

464

25. Obeyesekera, U.G.;Silva, B.F.S.; Bandaranayake, P.C.G. Development of wholesome lassi with

465

incorporation of pomegranate (Punica granatum) juice by using most cultivated local variety

466

(Nimali) and imported variety (Wonderful). In: FAuRS-2014. Faculty of Agricultulture

467

Undergraduate Research Symposium Peradeniya, Sri Lanka, 2014; pp. 145-152.

468

26. Eddebbagh, M.; Massoudi, M.; Abourriche, A. Correlation of the Cytotoxic and Antioxidant

469

Activities of Moroccan Pomegranate (Punica Granatum) with Phenolic and Flavonoid

470

Contents. J Pharm Pharmacol. 2016, 4, 9, 511-519.

471

27. Vergara-Salinas, J.R.; Cuevas-Valenzuela, J.; Pérez-Correa, J.R. Pressurized hot water

472

extraction of polyphenols from plant material. In: Biotechnology of Bioactive Compounds: ACS Paragon Plus Environment

Page 21 of 38


21 473 474

Sources and Applications, Hoboken; Wiley; 2015; pp. 63–101 28. Punyawardena,

B.V.R.;Bandara,

T.M.J.;Munasinghe,

M.A.K.;

Jayaratne

Banda,

M.;

475

Pushpakumara, S.M.V. Agro-Ecological Regions of Sri Lanka (Map). Natural Resources

476

Management Center, Department of Agriculture, Peradeniya, Sri Lanka, 2003; pp. 23-25

477

29. Ranst, E.V.;Verloo, M.;Demeyer, A.; Pauwels, J.M. Manual for the soil chemistry and fertility

478

laboratory: Analytical methods for soils and plants equipment, and management of

479

consumables. Faculty of Agricultural and Applied Biological Sciences, University of Ghent,

480

Belgium, 1999; pp. 25-28

481 482

30. Kuo, S. Chemical Methods. In: Methods of Soil Analysis. Soil Science Society of America, Madison, WI, USA, 1996; pp. 869–919

483

31. Wlakey, A. A critical examination of a rapid method for determination of organic carbon in

484

soils - effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci.

485

1947, 63,25,251–257.

486

32. Thaipong, K.;Boonprakob, U.;Crosby, K.;Cisneros-Zevallos, L.; Hawkins Byrne, D.

487

Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from

488

guava fruit extracts. J. Food Compos. Anal. 2006,19,669–675.

489

33. Marxen, K.;Vanselow, K.H.; Lippemeier, S.;Hintze, R.; Ruser, A.;Hansen, U.P. Determination

490

of DPPH radical oxidation caused by methanolic extracts of some microalgal species by linear

491

regression analysis of spectrophotometric measurements. Sensors. 2007, 7,10,2080–2095.

492

34. Moser, C.; Gatto, P.; Moser, M.;Pindo, M.;Velasco, R. Isolation of functional RNA from small

493

amounts of different grape and apple tissues. Appl Biochem Biotechnol - Part B Mol.

494

Biotechnol. 2004, 26,2,95–99

495 496

35. Jaakola, L.; Pirttilä, A.M.;Halonen, M.;Hohtola, A. Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Mol. biotechnol. 2001, 19,2, 201-203

497

36. Ono, N.N.; Bandaranayake, P.C.G.; Tian, L. Establishment of pomegranate (Punica granatum)

498

hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway.

499

Planta. 2012,236,931–941. ACS Paragon Plus Environment


Page 22 of 38

22 500 501 502 503 504 505

37. Steel, R.G.D.; Torrie, J,H. Principles and Procedures of Statistics, 2nd ed. NY, USA, McGraw Hill Book Co. Inc.,New York, 1980; pp.153-156. 38. Ergle, W.D. Introductory Statistics with a Minitab Guide. Duxbury Press, North Scituate, Massachusetts, 1995; pp. 200-215 39. Jolliffe I. Principal component analysis. In International encyclopedia of statistical science 2011 (pp. 1094-1096). Springer, Berlin, Heidelberg

506

40. Wijeratne, E.M.S.; Rydberg, L. Modelling and observations of tidal wave propagation,

507

circulation and residence times in Puttalam Lagoon, Sri Lanka. Estuar. Coast. Shelf Sci.

508

2007,74,4,611–622

509

41. Jayasingha, P.; Pitawala, A.; Dharmagunawardhane, H.A.Vulnerability of coastal aquifers due

510

to nutrient pollution from agriculture: Kalpitiya, Sri Lanka. Water Air Soil Pollut. 2011, 219,1–

511

4,563–577

512

42. Teixeira da Silva, J.A.;Rana, T.S.; Narzary, D.;Verma, N.;Meshram, D.T.; Ranade, S.A.

513

Pomegranate Biology And Biotechnology: A Review. Sci. Hortic. (Amsterdam). 2013,160, 85–

514

107

515

43. Moretti, C.L.; Mattos,L.M.;Calbo, A.G.; Sargent, S.A. Climate changes and potential impacts

516

on postharvest quality of fruit and vegetable crops: A review. Food Res. Int. 2010, 43,1824–

517

1832 .

518 519 520 521

44. Venkata, C.;Prakash, S.;Prakash, I. Bioactive Chemical Constituents from Pomegranate (Punica granatum) Juice, Seed and Peel-A Review. Int. J. Res. Chem. Environ. 2011,1,1,1–181. 45. Levin, G.M. Pomegranate roads. A Soviet b. B.L. Baer ed.; Floreat Press.; Forestville, CA, 2006; pp. 15-183

522

46. Gil, M.I.; Tomas-Barberan, F.A.; Hess-Pierce, B.; Holcroft, D.M.;Kader, A.A. Antioxidant

523

activity of pomegranate juice and its relationship with phenolic composition and processing. J.

524

Agric. Food Chem. 2000, 48,10,4581–4589

525

47. Kalaycıoğlu, Z.;Erim, F.B. Total phenolic contents, antioxidant activities, and bioactive

526

ingredients of juices from pomegranate cultivars worldwide. Food Chem. 2017, 221,496–507 ACS Paragon Plus Environment

Page 23 of 38


23 527

48. Mousavinejad, G.;Emam-Djomeh, Z.; Rezaei, K.;Khodaparast, M.H.H. Identification and

528

quantification of phenolic compounds and their effects on antioxidant activity in pomegranate

529

juices of eight Iranian cultivars. Food Chem. 2009, 115,4,1274–1278.

530

49. Tezcan, F.;Gültekin-Özgüven, M.; Diken, T.;Özçelik, B.;Erim, F.B. Antioxidant activity and

531

total phenolic, organic acid and sugar content in commercial pomegranate juices. Food Chem.

532

2009, 115,3,873–877

533

50. Holcroft, D.M.;Gil, M.I.; Kader, A.A. Effect of Carbon-Dioxide on Anthocyanins,

534

Phenylalanine Ammonia-Lyase and Glucosyltransferase in the Arils of Stored Pomegranates. J

535

.Am. Soc. Hortic. Sci. 1998, 123,1,136–140

536

51. Laribi, A.I.; Palou, L.; Intrigliolo, D.S.;Nortes, P.A.; Rojas-Argudo, C.; Taberner ,V.Effect of

537

sustained and regulated deficit irrigation on fruit quality of pomegranate cv. “Mollar de Elche”

538

at harvest and during cold storage. Agric. Water. Manag. 2013, 125:61–70.

539 540

FIGURE CAPTIONS

541

Figure 1. Esoteric appearance (a-c) and cross-sectional view (d-f) of fruits of Nimali accessions

542

collected from different locations

543 544

Figure 2. Effect of agro-climatic conditions on total phenolic content (TPC) of peel (a) and aril

545

(b) of the pomegranate fruit harvested at an average 60 cm canopy level in season 1 and season

546

2. Results are presented as mean ± standard deviation values of three samples measured from

547

each technical replicate and three technical replicates (fruits) from each plant and 3 biological

548

replicates (plants) for each location. Values denoted by same letters in a given season are not

549

significantly different at P

Biochemical composition and some anthocyanin biosynthetic genes of

Recommend Documents