Red walnut: Characterization of the phenolic profiles, and activity and

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Red walnut: Characterization of the phenolic profiles, and activity and gene expression of selected enzymes related to the phenylpropanoid pathway in pellicle during walnut development Martina Persic, Maja Mikulic-Petkovsek, Heidi Halbwirth, Anita Solar, Robert Veberic, and Ana Slatnar J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05603 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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Journal of Agricultural and Food Chemistry

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Red walnut: Characterization of the phenolic profiles, and activity and gene

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expression of selected enzymes related to the phenylpropanoid pathway in

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pellicle during walnut development

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Martina Persic†*, Maja Mikulic-Petkovsek†, Heidi Halbwirth§, Anita Solar†, Robert Veberic†,

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Ana Slatnar†

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† University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Chair for Fruit,

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Vine and Vegetable Growing, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

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§

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Institute for Chemical, Environmental and Biological Engineering, Technical University of

Vienna, Getreidemarkt 9, A-1060 Vienna, Austria

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*

Corresponding author, Tel.: +386 13203110; Fax: 01 256 57 82; E-mail address: [email protected]

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ABSTRACT: A rare variant of walnut with a red seed coat (pellicle) was examined for

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alterations in the phenolic profiles during development. The red walnut (RW) pellicle was

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compared to two commonly-colored walnut varieties; ‘Lara’ (brown) and ‘Fernor’ (light

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brown). Furthermore, the activity of selected enzymes of the phenylpropanoid and flavonoid

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related pathways and the relative expression of phenylalanine ammonia lyase (PAL) and

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anthocyanidin synthase (ANS) structural genes were examined in the pellicles of the three

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varieties. In the pellicle of RW, phenylalanine ammonia lyase (PAL) activity and the related

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PAL expression was most pronounced in August, about one month before commercial

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maturity, suggesting a high synthesis rate of phenolic compounds at this development stage.

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The most pronounced differences between the red and the light/dark brown varieties were the

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increased activity/gene expression of PAL and the increased anthocyanidin synthase (ANS)

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expression of RW in August. The vibrant color of the RW pellicle is based on the presence of

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four derivatives of cyanidin and delphinidin-hexosides.

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KEYWORDS: enzymatic activity, walnut, anthocyanins, tannins, gene expression

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INTRODUCTION

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Walnut (Juglans regia L.) is an important constituent of the Mediterranean diet and a

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significant source of phenolic compounds. It is thought to have multiple centers of origin, but

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Turkey and the Southern Caucasus are considered to be the centers of domestication.1 Walnut

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is today the most cultivated nut in the world,2 with modern cultivation spread across Europe,

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Asia, South Africa, America and Australia. It is also a widespread species in Slovenia,

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traditionally growing in the countryside as well as in commercial plantations.3

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The rich and diverse phenolic profile of walnut kernel is mainly characterized by

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tannins.4 A group of complex polyphenols, hydrolysable tannins (HTs), is present in walnut

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kernels, encompassing both gallo- and ellagitannins.5, 6 The biosynthesis of tannins in walnut

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is still not completely understood. The synthesis of HTs is part of the shikimic acid pathway7

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and it is known that initial steps are mediated by glycosyltransferases, which catalyze the

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synthesis of β-glucogallin.8-10

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A second major group in the phenolic profile of walnut kernels is condensed tannins,

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or proanthocyanidins (PAs). The synthesis of PAs starts with the phenylpropanoid pathway

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from phenylalanine, which is converted to chalcone by a series of reactions catalyzed by

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phenyl alanine ammonium lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA

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ligase (4CL) and chalcone synthase (CHS).11 Chalcone is the first intermediate in the

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flavonoid pathway leading to PAs and anthocyanins, which share the same upstream

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pathway.12 Anthocyanins (consisting of anthocyanidin and glycosides, such as cyanidin-3- O-

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glucoside, peonidin-3- O-glucoside, pelargonidin-3- O-glucoside, delphinidin-3- O-glucoside,

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petunidin-3- O-glucoside and malvidin-3- O-glucoside) are synthesized by the consecutive

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activity of flavonoid 3′-hydroxylase (F3'H), flavanone 3-hydroxylase (F3H), dihydroflavonol

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reductase (DFR), anthocyanidin synthase (ANS, synonym: LDOX), UDP-glucose flavonoid

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3-O-glucosyl transferase (UFGT) and methyltransferases (MT).12 3 ACS Paragon Plus Environment

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PAs can be synthesized either from catechin or epicatechin, which are produced by the

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reduction of leucocyanidin by leucoanthocyanidin reductase (LAR) or of cyanidin by

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anthocyanidin reductase (ANR). The process of polymerization of PAs is still not elucidated.

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The main source of phenolics in walnut kernels is the seed coat (pellicle), which is the

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main physical and chemical defense line of the vulnerable plant embryo. The chemical

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defense properties of the pellicle mainly arise from the high content of various

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phytochemicals. While seeds of some species, such as Prunus spp., accumulate significant

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amounts of cyanogenic glycosides,13 others are protected by high contents of glucosinolates,

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alkaloids or phenolic compounds.14 High levels of proanthocyanidins provide protection,15

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and also (together with anthocyanins) give color to the seed coat. Color mutants have always

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been valuable study objects for obtaining insights into the biosynthetic pathways of

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proanthocyanidins (PCs) and anthocyanins. Zorenc et al.16 thus described differences in the

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polyphenol metabolism in differently colored varieties of red currant (Ribes rubrum L.) and

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Wang et al.17 described the relationship between synthesis and accumulation of anthocyanins

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and flavanols in white- and red-fleshed fruit of malus crabapples.

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The aim of our research was to characterize the phenolic profiles and selected enzymes

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and genes related to the phenylpropanoid pathway in red and brown walnut pellicles and to

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study the dynamics of enzyme activity and gene expression related to the phenylpropanoid

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and flavonoid pathway during the development of walnuts. To the best of our knowledge, this

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is the first time that red walnut has been studied in detail in this way.

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MATERIALS AND METHODS

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Plant material. The samples were collected from three walnut varieties, all of which

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were growing near Maribor (lat. 46° 32' N, long. 15° 39' E, elevation 275 m). The walnut with

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a red pellicle (RW) is probably a natural seedling of unknown origin belonging to Slovenia’s 4 ACS Paragon Plus Environment

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natural walnut population. The medium-sized nuts, with an average in-shell weight of 11.6 g,

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have bright and fairly smooth shells, containing kernels with reddish-brown pellicles and a

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light cream-colored core.

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sampled on 22nd of June (I), 19th of August (II) and 15th of September (III). Immediately on

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harvest, approximately two kilograms of walnuts were collected. Kernels were peeled, and the

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pellicle was subsequently frozen in liquid nitrogen and stored at -80 °C until further analysis.

Green walnuts of the varieties 'Fernor', 'Lara' and RW were

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Extraction, identification and quantification of individual phenolic compounds.

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Phenolic compounds were extracted using the protocol described by Mikulic-Petkovsek et

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al.18 Briefly, 0.15 g of pellicle (ground in liquid nitrogen) was extracted with 2 mL of 3%

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formic acid in methanol (Sigma-Aldrich, Steinheim, Germany). Following 15s stirring in a

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vortex mixer, the samples were further extracted in an ice filled ultrasonic bath for one hour.

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After extraction, the samples were centrifuged at 11500 g for 20 minutes (Eppendorf

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centrifuge 5810 R, Hamburg, Germany), filtered through polyamide Chromafil AO-20/25

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filters (Macherey-Nagel, Düren, Germany) into vials and further analyzed using a HPLC

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system (Thermo Scientific, San Jose, USA). The HPLC conditions were as previously

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described by Mikulic-Petkovsek et al.18 Detection was performed with a diode array detector

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at 280, 350 and 530 nm. All phenolic compounds were identified using mass spectra (Thermo

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Scientific, LCQ Deca XP MAX) with electrospray ionization (ESI) operating in negative and

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positive (for anthocyanins) ion modes. The concentration of individual phenolic compounds

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was calculated from the peak area of the corresponding standard and expressed in mg/kg fresh

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weight (FW) of pellicle.

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Measurements of enzyme activity. The extraction and measurement of enzyme

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activity was based on protocols described in Thill et al.19 Enzyme assays were optimized for

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walnut pellicle (Suppl. Table S1). HEPES 0.1 M; PEG 1500, 1.5%; sucrose, 10%;

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dithioerythritol, 1 mM; ascorbic acid, 0.1 mM; calcium chloride, 0.025 mM, pH 7.3 was used. 5 ACS Paragon Plus Environment

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A total of 0.2 g of pellicle ground in liquid nitrogen, 0.2 g of quartz sand (Sigma-

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Aldrich, Steinheim, Germany), 0.2 g of Polyclar AT (Serva Electrophoresis, Heidelberg,

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Germany) and 3 mL of 0.1 M extraction buffer was homogenized in a mortar. The

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homogenate was centrifuged at 10000 g for 10 minutes at 4 oC. The supernatant was used for

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separation of low molecular compounds on a gel chromatography column (Sephadex G25

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medium; Sigma-Aldrich, Steinheim, Germany). Crude extract was obtained with this

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procedure.

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Enzyme assays for PAL (EC 4.3.1.24), CHS (EC 2.3.1.74)/CHI (EC 5.5.1.6), DFR

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(EC 1.1.1.219), FHT (EC 1.14.11.9) and FLS (EC 1.14.11.23) were performed according to

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Halbwirth et al.20 Buffers, substrates, cofactors and chemicals for stopping the reaction are

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listed in Suppl. Table S1.

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The enzymatic reaction was started with the appropriate cofactor (Suppl. Table S1)

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and incubated for 30 min at 30 °C. After incubation, the reaction was stopped with the

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appropriate chemicals (Suppl. Table S1). After mixing and centrifugation for 3 min at 10000

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g at room temperature, the upper organic phase was used for further analysis.

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For analysis of enzymatic activity of FHT, DFR and FLS, organic phases were

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transferred to pre-coated cellulose plates (Merck, Darmstadt, Germany) for thin-layer

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chromatography (TLC). Chloroform/acetic acid/H2O (10:9:1, v/v/v) was used as a mobile

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phase for FHT and DFR and 30% acetic acid was used as a mobile phase for FLS. The

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conversion rates were then determined with a TLC linear analyser (Berthold, Bad Wildbad,

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Germany). All products were identified as described in Fischer et al.21, using standards

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naringenin (FHT), dihydrokaempferol (FLS) and dihydroquercetin (DFR).

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For analysis of enzyme activity of PAL and CHS/CHI, the upper organic phase mixed

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with 4 mL scintillation cocktail (LSC- UNIVERSAL COCKTAIL ROTISZINT®, Carl Roth,

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Karlsruhe, Germany) was used for product quantification, with a scintillation counter. The

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protein content was quantified by a modified Lowry procedure with crystalline BSA as

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standard22. Enzyme activity was calculated in nkat/kg of protein.

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Gene expression of PAL and ANS. Total RNA was prepared according to Chang et

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al.23 and used for the isolation of mRNA via an µMACS mRNA isolation kit (Miltenyi

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Biotech, Auburn, CA). cDNA was prepared using RevertAid H Minus MuLV reverse

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transcriptase (Fermentas Life Science, St. Leon-Rot, Germany) with an oligo(-dT) anchor,

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Primer GACCACGCGTATCGATGTCGAC(T)16V. Gene expression was quantified using

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the StepOnePlusTM Real-Time PCR System with SYBR_ Green PCR Master Mix (Applied

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Biosystems, Foster City, CA, USA). qPCR primers for PAL and 18S were used according to

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Xu et al.24 The qPCR primer for the ANS primer was designed using a sequence available in

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the database (XM_018991659). The primers used are listed in Table 1. The efficiency of the

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PCR-reaction was determined on the basis of standard curves, which were obtained by

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applying different DNA concentrations. Results were calculated in relation to 18S.

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Statistical Analysis. Statistical analysis was carried out using Statgraphics Plus 4.0

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(Manugistics, Rockville, MD, USA.). For differences in phenolic content, the activity of

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selected enzymes and the expression of structural genes among individual sampling dates,

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one-way analysis of variance (ANOVA) was calculated and the Duncan test was used to

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distinguish among results. p-values lower or equal to 0.05 were considered statistically

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significant. The results are presented as an average of five repetitions ± standard error. For

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graphic interpretation of the results, a heat map was plotted in R-Commander using the gplot

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package (R Formation for Statistical Computina, Anckland, New Zeland) based on

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standardised data (µ=0, σ =1).

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RESULTS

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Phenolic profiles of walnut pellicle. During the last two months of development, a

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similar trend was observed in the accumulation of gallic acid derivatives (Figure 1a),

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flavanols (Figure 1b) and flavonols (Figure 1c) for the three analyzed varieties. Generally, the

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contents of gallic acid derivatives, flavanols, flavonols and total analyzed phenolic

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compounds increased during pellicle development (Figure 1, Figure 3). Of the analyzed

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varieties, RW had the highest content of total analyzed phenolic compounds, followed by

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‘Fernor’ and ‘Lara’ (Figure 1d).

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In all three varieties, derivatives of gallic acid accounted for more than 60 % of total analyzed

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phenolic compounds (Figure 1a, 1d) on all sampling dates. We identified 15 phenolic

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compounds in the group of derivatives of gallic acid: ellagic acid pentoside, ten derivatives of

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hexahydroxydiphenoyl (HHDP) and four isomers of castalagin (Suppl. Table 2). Among

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these, the highest content was measured for galloyl-bis-HHDP hexoside V.

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The flavanol content accounted for approx. 30 % of total analyzed phenolic

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compounds in the pellicle of all three varieties (Figure 1b). In this group, we identified one

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monomeric compound, catechin, and three dimeric and two trimeric procyanidins (Suppl.

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Table 2). In all three varieties, catechin had the lowest concentration in comparison to other

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flavanols. The most abundant flavanols were procyanidin trimers I and II. Their content

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accounted for more than 40 % of total analyzed flavanols in the first sampling, rising to more

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than 50 % in the second and third samplings.

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Flavonols accounted only for 0.1-0.2 % of total analyzed phenolic compounds in the

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walnut pellicle of the three varieties. The increase in flavonol contents was not as distinctive

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as observed in the accumulation of other phenolic groups (Figure 1c). In the flavonol group,

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we identified two quercetin pentoside isomers, present in minor concentrations (Suppl. Table

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2). The lowest content of total analyzed flavonols was measured in the first sampling in the

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pellicle of ‘Lara’ (0.08 ± 0.01 mg/kg), while the highest content was measured in the pellicle

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of RW in the third sampling (0.21 ± 0.01 mg/kg) (Figure 1c).

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In total, 28 phenolic compounds were detected in the pellicles of the analyzed walnuts

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(Suppl. Tables 2-3). Twenty-three phenolic compounds were present in all three varieties

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(Suppl. Table 2), while five anthocyanins were exclusively found in the pellicle of RW

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(Suppl. Table S3). The anthocyanin profile of RW consists of four derivatives of cyanidin

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bound to different sugar moieties (arabinose, galactose, glucose and xylose) and delphinidin

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bound to an unknown hexose (Figure 2). The total analyzed anthocyanin content in the red

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pellicle amounted to 363.20 ± 39.00 µg/kg in the first sampling, which corresponds to 0.4 %

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of total analyzed phenolic compounds. In the second sampling, the anthocyanin (delphinidin-

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hexoside, cyanidin glucoside, cyanidin galactoside, cyanidin arabinoside, cyanidin xyloside)

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content increased by 4.5 times, reaching 1.2 %. On the last sampling date, the content of

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anthocyanins doubled and accumulated at 3167.45 ± 22.92 µg/kg (1.8 % of total analyzed

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phenolic compounds) in the fully ripe red walnut pellicle (Figure 2). For all cyanidin

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derivatives, the content was lowest at the first and highest at the last sampling date. There was

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no difference in the content of delphinidin hexoside between the first and second sampling

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dates. The change in the visible color intensity in the pellicle of the three varieties strongly

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correlated with the change in contents of phenolic compounds, particularly of anthocyanins in

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RW (Suppl. Figure 2).

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Activity of selected phenylpropanoid pathway related enzymes. The activities of

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selected enzymes of the flavonoid pathway were studied in the 3 varieties at the three

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sampling dates. Figure 4 shows a comparison of the 3 varieties at the different stages, while

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the same data are presented in Suppl. Figure 1, with a focus on the changes observed during

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the season. The activity of PAL and DFR enzymes in the walnut pellicles of varieties

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‘Fernor’, ‘Lara’ and RW varied markedly among varieties and sampling dates. For ‘Fernor’,

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the activity of PAL gradually decreased in the last two months of development, being 44%

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lower at the last than at the first sampling. The activity of PAL in the pellicle of cv. ‘Lara’

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more steeply increased over the same time, being 125% higher at the last compared to the first

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sampling (Suppl. Figure 1). In contrast, the activity of PAL in the RW pellicle was highest at

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the second sampling. The activity of PAL at the second sampling was four times higher than

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at the first and two times higher than at the last sampling. A similar increasing trend in the

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activity of CHS/CHI through ripening was observed in the pellicles of ‘Fernor’ and ‘Lara’. At

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the same time, the activity of CHS/CHI in the RW pellicle was highest at the second sampling

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(Suppl. Figure 1). For ‘Fernor’, the activity of FLS was higher at the first than at the second

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sampling. In the pellicle of ‘Lara’, there was no change in the enzymatic activity of FLS

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during the last two months of ripening. For RW, the activity of FLS was at a lower point only

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at the first sampling.

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In terms of differences in the activity of enzymes among varieties at a single sampling

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date (Figure 4), differences were observed in the case of PAL and DFR enzymes at the first

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(I) and second (II) samplings. The activity of PAL at the first sampling was highest in the

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pellicle of ‘Fernor’, followed by activity in RW, which was higher than in ’Lara’. At the

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second sampling, high activity of PAL in the RW pellicle was strongly expressed in

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comparison to the other two analyzed varieties. Similarly, the activity of DFR at the first

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sampling was also highest in the pellicle of RW, while at the second sampling, the activity of

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DFR in RW and ‘Fernor’ was comparable.

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Relative gene expression of selected phenylpropanoid pathway related genes. The

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expression of the structural genes PAL and ANS in the three varieties was examined in

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comparison to 18S as a reference gene (Figure 5). In ‘Fernor’ and RW, ANS had the highest

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relative expression at the second sampling. For ‘Lara’, relative expression had an increasing

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trend through the sampling dates. At the first sampling, the RW pellicle had approximately 6-

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8 times higher relative expression than with the other two varieties. It is interesting that the

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relative expression of ANS in the pellicle of RW at the second sampling was two times higher

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than in ‘Fernor’ and 7.5 times higher than in ‘Lara’, but at the third sampling, five times lower

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than in ‘Fernor’ and 14.7 times lower than in ‘Lara’ (Figure 5).

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The relative expression of the PAL gene was highest at the second sampling for all

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analyzed varieties. Unlike the relative expression of ANS, the expression of the PAL gene at

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the second sampling was more pronounced in the pellicle of ‘Fernor’, followed by the relative

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expression in RW and ‘Lara’, respectively.

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DISCUSSION

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Polyphenols have multiple functions in plants, ranging from roles in repelling or

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attracting other organisms and protection, to regulatory functions in the inner growth

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mechanisms of the plant.25 In addition, polyphenols are to a large extent responsible for taste

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and color, and they also contribute to the aroma of phenol-rich foods. Even though the pellicle

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accounts for only 5% of a walnut kernel’s weight, it significantly contributes to the total

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phenolic content of walnut kernels.26 By comparing six yellow and six red pellicle walnut

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varieties, Trandafir et al.27 showed that yellow pellicle walnut varieties have a significantly

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higher content of total phenolic compounds, antioxidant activity and total flavonoid content

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than varieties with red pellicles. In contrast, the red pellicle of RW in our research had a

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significantly higher content of phenolic compounds in comparison to the pellicles of the two

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brown varieties (Figure 1). Between the brown varieties, ‘Fernor’ and ‘Lara’, the lighter

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colored pellicle of ‘Fernor’ had a significantly higher content of all analyzed phenolics than

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the slightly darker pellicle of ‘Lara’. These finding are in agreement with a recent report by

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Fuentealba et al.,28 who found a higher content of total phenolic compounds and antioxidant

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capacity in extra light walnuts compared to amber walnuts. We can therefore support the

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presented theses that lower TPC may be a consequence of polyphenol oxidation in darker 11 ACS Paragon Plus Environment

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colored pellicles of walnut. Fuentealba et al.28 also reported the first identification of arbutin

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in walnut kernels, irrespective of pellicle color. Our research was focused on the pellicle, in

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which arbutin was not present. We therefore assume that the high reported content of arbutin

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probably derived from the kernel core.

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Specific groups of phenolic compounds have more or less pronounced specific roles.

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Plants accumulate certain groups of phenolic compounds in order to protect vital organs from,

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for instance, UV damage (anthocyanidins) and herbivores (tannins).25-28 We suggest that the

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role of high content of phenolic compounds in the walnut seed coat is indirect protection of

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the seed embryo embedded in the oily endosperm. The protection is not focused on defense

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from predators, since a walnut kernel is encased in a hard shell, which is covered with a

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highly phenolic husk.29,30 The antioxidative potential of phenolic compounds in this case is

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responsible for protection against the oxidation of valuable fatty acids, and thus the internal

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quality of the walnut kernel.

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As a first step in the biosynthesis of phenylpropanoids, the PAL enzyme catalyzes the

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conversion of phenylalanine to trans-cinnamic acid.35 Phenylpropanoids include, among

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other, anthocyanins, flavanols and flavonols.36 Xu et al.24 showed that JrPAL gene expression

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is induced by a variety of abiotic and biotic stresses, and suggested that it is a stress-

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responsive gene. This was later confirmed by Christopoulos and Tsantili10 in a study of the

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participation of PAL in increased content of phenolic compounds in fresh cold stressed

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walnut. The pronounced relative expression of the PAL gene, together with high activity of

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the PAL enzyme, plays an essential role in the elevated content of phenylalanine-pathway

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derived compounds. Our results demonstrate a clear correlation between expression of the

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PAL gene, activity of the PAL enzyme and the content of phenolic compounds (Figure 3-5).

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Derived from the phenylpropanoid pathway, 4-coumaroyl-CoA, together with malonyl

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CoA, is a precursor and substrate for the biosynthesis of flavonoids.37 The first steps of this 12 ACS Paragon Plus Environment

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pathway are catalyzed by chalcone synthase CHS and CHI enzymes. Initially, CHS converts

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4-coumaroyl-CoA and three molecules of malonyl CoA into naringenin chalcone, which is

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isomerized by chalcone isomerase (CHI) to the flavanone, naringenin. Flavanones are

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hydroxylated in position 3 by flavanone 3-hydroxylase (FHT), to provide dihydroflavonols.

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Dihydroflavonols serve as substrates for the synthesis of either flavonols (quercetin and/or

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kaempferol and their derivatives) or leucocyanindins.38 Since only low amounts of flavonol

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derivatives were identified (Figure 1), we assume that the dihydroflavonols in a walnut

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pellicle are mainly converted to leucoanthocyanidins by dihydroflavonol 4-reductase (DFR)

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activity. Since ANS activity cannot so far be tested in tissues, we also included ANS gene

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expression studies. ANS is responsible for the synthesis of precursors for anthocyanins and

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proanthocyanidins.37 The red walnut pellicle showed the highest ANS expression in

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comparison to the brown pigmented pellicle (Figure 5). This, together with a general increase

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in precursor production by PAL at sampling date II, was the most striking difference between

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the red and brown pigmented pellicles and correlates well with the intense red pellicle

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coloration of the RW variant.

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The question remains, however, of what role anthocyanins have in the pellicle of

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walnut kernels. Given that the pellicle is not exposed to light, it is obviously not related to UV

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protection, and also not directly related to attraction of pollinators and seed carriers.

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Biosynthesis of anthocyanins is frequently triggered by environmental conditions and stress.36

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This does not seem to be the case for the RW variety, since the red pellicle has been proven to

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be a stable trait, which is supported by McGranahan and Leslie,39 who reported that the seed

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coat (pellicle) of the walnut is a maternal tissue. According to the owner of the tree from

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which the nut samples were taken, the typical red-colored pellicle was observed from the

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beginning of fruiting up to the adult age of the tree. Since there is no information on the origin

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of the red pellicle color of the RW variety, we presume that it might be a result of a random 13 ACS Paragon Plus Environment

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clonal mutation. It is possible that the anthocyanins may contribute to antioxidative potential,

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improving the nutritional and health beneficial roles of RW kernels, which contain added

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nutrients not found in brown walnuts. Anthocyanins may contribute to the protection of the

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kernel’s phenolic complex. Last but not least, for many fruit crops, skin and flesh color are

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increasingly important determinants of consumer preference and marketability, not only for

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optical effects but for the health beneficial value that is particularly associated with red

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pigments. The anthocyanins in the pellicle make RW nuts attractive to consumers and offer

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growers a better price over traditional, light-colored walnuts, as is the case with the red

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‘Robert Livermore’ variety grown and sold in the USA.40 The RW walnut could be used as

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special genetic material in future breeding, with the aim of creating red walnuts that contain

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additional nutrients.

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ABBREVATIONS USED

314

PAL, phenyl alanine ammonium lyase; CHS/CHI, chalcone synthase/chalcone isomerase;

315

FLS, flavonol synthase; FHT, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase;

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HHDP, hexahydroxydiphenoyl; RW, walnut with a red pellicle; HTs, hydrolysable tannins;

317

PAs proanthocyanidins; HPLC, High-Performance Liquid Chromatography

318 319 320 321

ACKNOWLEDGMENTS The research is part of Horticulture Program No. P4-0013-0481, funded by the Slovenian Research Agency (ARRS).

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FIGURE CAPTIONS

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Figure 1: Content of total analyzed derivatives of gallic acid (a), flavanols (b), flavonols (c) and total analyzed phenolics (d) in the pellicles of three analyzed varieties: ‘Fernor’, ‘Lara’ and RW. Comparison of varieties was made separately for each sampling date I (22nd June), II (19th August), III (15th September) and different letters denote statistical differences according to the Duncan test (p