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

311

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

312

additional nutrients.

313

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;

316

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).

322

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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

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

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