and pinkish ariled pomegranate (Punica granatum L.) cultivar are

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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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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

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

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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

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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.

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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

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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

145

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

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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

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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

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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

193

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),

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

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(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

298

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

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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

306

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

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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.

310

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

314

biochemical composition.

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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

323

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

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

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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

335

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

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

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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.

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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

345

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)

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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)

375

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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

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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).

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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

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

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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

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

549

significantly different at P

and pinkish ariled pomegranate (Punica granatum L.) cultivar are

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