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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
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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
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1
ABSTRACT
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The accumulation of beneficial biochemical compounds in different parts of pomegranate (Punica
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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
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pink-ariled pomegranate cultivar in three agro-climatologically different locations in Sri Lanka.
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Drier and warmer climates promoted the accumulation of the measured biochemical compounds i.e
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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
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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
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Phenolic Content (TPC), Antioxidant activity (AOX)
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INTRODUCTION
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Pomegranate (Punica granatum L.) has been well known for its medicinal properties. It has gained
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even more attention due to recent scientific evidences on health promoting and nutritional benefits.1-
27
6.
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cultivation and its industrial applications are expanding considerably.7 A continuous supply of
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uniform quality raw materials is a key requirement for marketing and industrial applications of
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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
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While hydrolysable tannins such as ellagitannins and gallotannins are mainly present in peels and
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membranes,11 condensed tannins are mainly found in peel and juice.12 Transcriptome sequencing
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work13 accelerated identification and characterization of the genes and enzymes responsible for the
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biosynthetic pathways of above valuable bioactive compounds. For example, several major genes
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responsible for hydrolysable tannin14 and anthocyanin15 biosynthesis were functionally
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characterized recently. Further, molecular evidences suggest that differential expression of genes in
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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
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It is now known that the quantity and the composition of bioactive compounds in pomegranate peel
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and aril depend on many pre-harvest and post-harvest factors.18 Some of the key factors are cultivar
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or genotype, agro-ecological conditions (climate and soil), maturity stages of fruits and crop
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management 19.
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Few previous studies on the effects of agro-ecological and seasonal variations on biochemical
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properties of pomegranate20 have mainly considered the effects of drastic climates such as
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Mediterranean climate vs dessert climate21or summer vs winter22. Only few studies have evaluated ACS Paragon Plus Environment
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the effects of more than one season in the same location23-24. Furthermore, previous studies have not
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focused on gene expression in response to environmental factors. In addition, previous studies were
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restricted to the cultivars with red peel and red arils, while cultivars with yellow peel and pink arils
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were neglected. Nevertheless, cultivars with yellow peels and pink arils are popular in some
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countries. The yellow peel, pink aril cultivar, Nimali consists of higher concentrations of beneficial
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biochemical compounds than popular variety Wonderful, under Sri Lankan conditions.25 As the
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color change of the peel does not follow that of the arils in pomegranate,21 recognition of harvesting
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indices is also critical.
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Therefore, we investigated the effects of three major agro-climatic conditions present in Sri Lanka
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on total phenolic content (TPC), alpha punicalagin (AP), beta punicalagin (BP), total punicalagin
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(TP), antioxidant activity (AOX) and the expression of some anthocyanin biosynthetic pathway
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genes of Nimali fruits over two years. Here we specifically focused on anthocyanin biosynthetic
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pathway genes; because of the importance of the pathway, having visual color with relative gene
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expression and its relationship to TPC and AOX 26,27. To the best of our knowledge, this is the first
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study on the effect of environmental conditions on biochemical composition and anthocyanin gene
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expression of a yellow peel, pink aril cultivar. Further, we studied the correlation of rainfall and the
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mean air temperature with quantity of different bioactive compounds. The findings would also
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enable growers to expand the cultivation of pomegranate in the location/s where the agro-climatic
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conditions maximize the production of beneficial bioactive compounds.
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MATERIALS AND METHODS
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Chemicals and Reagents
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Standards of punicalagin α & β and gallic acid were purchased from Sigma Aldrich (St. Louis,
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MO). All reagents used for liquid chromatography were HPLC grade and purchased from Sigma
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chemicals (Taufkirchen, Germany). All other reagents used for biochemical analysis were analytical
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grade and obtained either from Sigma Chemicals, St. Louis, MO, USA or Himedia chemicals, ACS Paragon Plus Environment
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Mumbai, India. Taq polymerase, cDNA synthesis kit, dNTPS, DNA ladder were purchased from
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Pomega Corporation, Madison, WI. Primers were synthesized from IDA Technologies (Coralville,
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IA).
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Study locations and sample collections
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One of the most popular local pomegranate cultivar Nimali, cultivated in three different locations
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representing two different agro-ecological regions; i.e. Low country dry zone (DL3) and up country
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intermediate zone (IU3) of Sri Lanka28 was selected for this study. They were Kalpitiya (8.21o N,
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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)
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(Supplementary Figure. 1). Each orchard consisted of
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maintained according to standard management practices, as recommended by the Department of
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Agriculture, Sri Lanka. Briefly, uninfected healthy seedlings were planted at the beginning of the
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rainy season using 60 x 60 x 60 cm pits with 3x3 m distance with a underneath coir layering. The
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pits were filled with cow dung, compost and recommended dosages of chemical fertilizers.
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Seedlings were irrigated properly until they established well in the orchard. Main stem of the
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mature plants were pruned at a height of 60-75 cm by plucking the terminal bud to promote
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flowering and fruiting.
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Three fruiting trees were randomly selected from the middle of each orchard, and three fully
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matured (180 Days after initiation), uninfected and undamaged fruits were harvested from each tree.
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Accordingly, all together 54 fruits (3 fruits x 3 trees x 3 locations x 2 seasons) were sampled during
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the peak harvesting season i.e. mid-September of two consecutive years.
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Collected fruit samples were cut into halves, and peels and arils of each half were separately flash
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frozen in liquid N2 and stored in – 80 oC for the analysis. Differences in external and internal
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appearances of collected pomegranate fruit samples of from different locations are shown in
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Supplementary Figure 1.
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Meteorological Data
25-50 same aged (5 years old) trees,
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Rainfall (RF), maximum temperature (Tmax), minimum temperature (Tmin), maximum relative
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humidity (RHmax) and minimum relative humidity (RHmin) were collected from the Department of
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Meteorology of Sri Lanka for the years 2014 and 2015 for three locations.
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Soil Analysis
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A composite soil sample for each location was taken by mixing 3 sub-samples per location, at the
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depth of 0-15 cm. Soil samples were analyzed for soil pH, electrical conductivity (EC), available
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nitrogen (N), available phosphorous (P), available potassium (K), exchangeable Ca and organic
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matter (OM) using standard procedures.29 The soil pH was measured using a standard glass
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electrode pH meter in distilled water using a soil to water ratio of 1:2.5 and the standard electrical
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conductivity meter was used to measure the EC of 1:5 soil water suspensions. The available N was
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analyzed using the Kjeldahl method29. The available P and available K were determined by Olsen
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method.30 Ammonium acetate extraction at pH 7.0 was used to determine the exchangeable Ca29.
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The Dichromate oxidation method31 was used to analyze the organic matter.
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Biochemical analysis
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Other than the sampling and replications mentioned above, all the biochemical tests were repeated
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three times per each sample.
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Total phenolic content by Folin-CioCalteau method
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Total phenolic content (TPC) in the peel and aril (juice extracts) of pomegranate samples were
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determined by the Folin-CioCalteau colorimetric method described by Thaipong et al32 with some
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modifications. Accordingly, 10 mg of sample was squeezed and the extract was centrifuged at 5000
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rpm for 15 minutes. Five hundred microliters of the supernatant was added to 2 ml of distilled water
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and mixed properly. One milliliter of diluted Folin-CioCalteau reagent was added to the above,
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mixed thoroughly and kept at room temperature for 3 min and 500 µl of 6% (w/v) sodium carbonate
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was added. After 120 min, the absorbance was measured in triplicates at 600 nm and the calibration
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curve was performed with Gallic acid and the results were expressed as milligrams of Gallic acid
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equivalents per grams of fresh sample (mg GAE g-1 (fw)).
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Antioxidant activity by DPPH radical scavenging method
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Antioxidant activity (AOX) was quantified by evaluating free radical scavenging activity. The
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extracts were allowed to react with a stable free radical, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) as
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previously described by Marxen et al.33
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HPLC Analysis
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Peel and aril tissues of pomegranate were ground into very fine powder using liquid N and the
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punicalagin α & β contents of the samples were determined by HPLC following the method
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described by Ono et al.13 with some modifications. Thousand micro liters of 40 %(v/v) methanol
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was added to 200 mg of each samples, sonicated for 20 minutes and centrifuged for 15 minutes at
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12 000 rpm. About 500 µl of the supernatant was centrifuged at 12000 rpm for 20 minutes and 200
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µl of the supernatant was used for HPLC analysis in Agilent 1260 system equipped with a Zobax
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SB-C18, 5 µm, 4.6 x 150 mm column. Ten microliters of sample was injected and separated in a
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solvent gradient between 0.1 (v/v) Formic acid and 100 % (v/v) Acetonitrile (ACN). The time-ACN
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combinations were; 5 % (v/v) ACN for 0-3 minutes, 5-25 % (v/v) ACN for 3-24 minutes, 25-35 %
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(v/v) ACN for 24-29 minutes and 35-45 % (v/v) ACN for 29-30 minutes, at the flow rate of 1ml
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per minute. Punicalagin α & β were identified with the retention time of the standards and standard
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curves were generated with commercial standards for quantification of punicalagin isomers.
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RNA Extraction
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The total RNA was extracted from 2 g of peel and 8 g of aril samples using a CTAB based method
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developed combining two protocols from Moser et al34 and Jaakola et al35 with further
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modifications. Sample was mixed with 500 µl of pre heated CTAB extraction buffer with 2 (w/v)
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2-mercaptaethanol and the equal volume of chloroform and centrifuged at 3000 rpm for 35 minutes.
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Four milliliters from that supernatant and 0.25 of the volume of 10 M LiCl were kept in refrigerator ACS Paragon Plus Environment
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overnight and centrifuged at 14000 rpm for 10 minutes. The pellet was washed with 2 M LiCl and
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re-suspended in 500 µl of 10 mM Tris HCl (pH 7.5). Then, 2 M potassium acetate (pH 5.5) was
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added in 1/10 volume and centrifuged at 14000 rpm for 10 minutes. The pellet was washed with
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70% (v/v) ethanol and re-suspended in 20-50 µl water depending its size.
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The quality and quantity of the extracted RNA were determined by electroporation on 1% (w/v)
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agarose gel using the Nanodrop spectrophotometer (Thermo ScientificTM Nanodrop 2000). RNA
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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
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the manufactures protocol (Pomega (Madison, Wisconsin, USA)).
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Gene expression analysis
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Differential expression of four major genes in the anthocyanin biosynthetic pathway was studied
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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
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(Supplementary Table 1). Pomegranate actin gene36 was used as the housekeeping control.
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Semi-Quantitative PCR was carried out for the selected genes of pomegranate with following
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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
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reaction consisted of 10X PCR Buffer 10 µl, 10 mM dNTPs 2 µl, 50 mM MgCl2 3 µl , 5 µl of each
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primer (10 µM), 1 µl cDNA, Taq Polymerase (5 U/ L) 0.2 µl , 0.8 mM Spermidine and volumed up
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to 20 µl with RNAse free water. The PCR products were separated on 2 %(w/v) agarose gel and
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stained with ethidium bromide.
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Data analysis
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The experiment was arranged in a nested design where trees were considered nested within the
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locations while fruits were nested within both locations and trees. This specific statistical design
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was used since the measured variables such as TPC, AOX, AP, BP and TP were affected or nested
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inside two or more nominal variables. Data on biochemical compounds were analyzed using the
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SAS 9.4® software package (SAS Institute Inc. Cary, North Carolina, USA) considering the
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ANOVA of nested design and means were compared by the Duncan’s New Multiple Range Test at
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5% probability level.37 Correlation analysis38 was done to explore the relationship between agro-
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climatic conditions and bioactive compounds of the pomegranate fruits using Microsoft Excel
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2007®. Principal component analysis39 was conducted to identify the dominant components of the
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variance of measured parameters annual mean temperature, annual rainfall, mean RH, Soil organic matter,
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Soil pH, Soil EC, Soil N, Soil P & Soil K
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The relative gene expression was quantified using the constitutively expressed housekeeping gene actin. The
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intensity of semi-quantitative PCR bands of each fruit sample was calculated using the imageJ software. The
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ratio of the average of band intensity for each gene over the average for actin was calculated using Microsoft
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Excel 2007 and the average value for the location data was plotted.
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RESULTS
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Meteorological conditions during the study period
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Annual climatological conditions of the selected locations i.e. Kalpitiya (Supplementary Figure 2),
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Mullaitivu (Supplementary Figure 3) and Teldeniya (Supplementary Figure 4) during 2014 and
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2015 vary considerably. Nevertheless, the relative humidity was relatively constant among the study
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locations (Table 1). Kalpitiya, located in low country dry zone (DL3) is the driest location with the
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lowest rainfall and the highest mean temperatures. Teldeniya, belongs to the upcountry intermediate
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zone (IU3) received the highest rainfall and experienced the lowest mean temperatures. Though
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Mullaitivu also belongs to the DL3 zone, that area received relatively higher rainfall and recorded
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lower average temperature than Kalpitiya (Table 1). In addition, climatology of Kalpitiya is ACS Paragon Plus Environment
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considered unique with proximity to Puttalam lagoon with a very low day and night temperature
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difference, specific soil properties and relatively high winds.40, 41
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Soil physical and chemical parameters in the selected locations were not considerably different
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(Table 2). The lowest EC was recorded in Kalpitiya because of sandy nature. While the highest soil
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organic matter was found in Teldeniya, the soil pH was in the neutral range in all the locations.
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Biochemical analysis
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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,
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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
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the biochemical parameters. However, there was a significant correlation between annual average
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rainfall and average annual temperature with biochemical parameters, TPC, AOX, alpha, beta and ACS Paragon Plus Environment
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total punicalagin in both peel and arils of fruits. Where the climate was cooler and wetter,
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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
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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
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season was recommended for better quality pomegranates.45 There were higher TPC, hydrolysable
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tannins, punicalagin contents found in fruit peels of most of the cultivars harvested from desert
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regions compared to Mediterranean climates.21 The TPC, AOX and punicalagin content their
308
correlations reported here are comparable with previous studies 46-49.
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Interestingly, there was significant difference among three plants collected from the same location.
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Floral behavior of Nimali suggests potential of cross-pollination. Nevertheless, currently this variety
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is mainly propagated through seeds and the vegetative propagation is limited. All the plants used in
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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
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biochemical composition.
315
Anthocyanin is one of the major groups of compounds responsible for TPC and AOX of
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pomegranate27. Further, the products of anthocyanin biosynthetic pathway determines the colour of
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flower, fruit and arils. Here we observed higher TPC and AOX values than others and relatively
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brighter color peels and arils in Kalpitiya area. Relatively higher expression of anthocyanin
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biosynthetic may attribute to such changes.
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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
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pomegranate. Further, it describes that the insertional mutation (PgLDOX gene) in POM-LDOX
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gene results white color flowers, fruits and arils.15, 16 Our results also support the previous findings
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and show clear ANS expressions in pink colour arils irrespective of the environmental factors and
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very low expression in yellowish peel. Among the genes considered CHI did not show a clear ACS Paragon Plus Environment
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differential expression either in peel or arils in response to environmental variation. But there was a
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considerable up regulation of DFR and F3H genes in peel as well as in arils in the drier and warmer
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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
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samples collected from other locations. However, previous work on 11 pomegranate accessions
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grown under Mediterranean and desert climates in Israel have concluded higher anthocyanin
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content in fruit arils in the Mediterranean climate, compared to those grown in desert climate.21
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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
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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
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Teldeniya might have suppressed the expression of anthocyanin biosynthetic pathway genes.
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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
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higher quality fruits.
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ABREVIATIONS
347
Total phenolic content (TPC)
348
Antioxidant Activity (AOX)
349
Alpha Punicalagin (AP)
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Beta Punicalagin (BP) ACS Paragon Plus Environment
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Total Punicalagin (TP)
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Chalcone Isomerase (CHI)
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Dihydroflavonol 4-reductase (DFR)
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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)
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High Performance Liquid Chromatography (HPLC)
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Acetonitrile (ACN). ACS Paragon Plus Environment
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Leucoanthocyanidindioxygenase (LDOX)
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Galic Acid Equvalent (GAE)
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Fresh Weight (fw)
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ACKNOWLEDGEMENT
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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
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SUPPORTING INFORMATION Supplementary Figure 1. Locations selected for the study
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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).
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Supplementary Figure 4. Annual climatology of Teldeniya in 2014 (a) and in 2015 (b).
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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
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Supplementary Table 2. Ratio of the average of band intensities of three trees for each gene over the actin
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gene
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REFERENCES
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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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
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Figure 1. Esoteric appearance (a-c) and cross-sectional view (d-f) of fruits of Nimali accessions
542
collected from different locations
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Figure 2. Effect of agro-climatic conditions on total phenolic content (TPC) of peel (a) and aril
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(b) of the pomegranate fruit harvested at an average 60 cm canopy level in season 1 and season
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2. Results are presented as mean ± standard deviation values of three samples measured from
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each technical replicate and three technical replicates (fruits) from each plant and 3 biological
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replicates (plants) for each location. Values denoted by same letters in a given season are not
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significantly different at P
