Comparative Transcriptome Analysis of the Skin-Specific

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Comparative transcriptome analysis of the skin-specific accumulation of anthocyanins in black peanut (Arachis hypogaea L.) Jinyong Huang, Minghui Xing, Yan Li, Fang Cheng, Huihui Gu, Caipeng Yue, and Yanjie Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05915 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Journal of Agricultural and Food Chemistry

Comparative transcriptome analysis of the skin-specific accumulation of anthocyanins in black peanut (Arachis hypogaea L.) Jinyong Huang†, ‡, §, Minghui Xing†, ‡, §, Yan Li†, ‡, §, Fang Cheng†, ‡, Huihui Gu ‡ , Caipeng Yue†, ‡, Yanjie Zhang†, ‡, * † School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, People’s Republic of China; ‡ School of Life Sciences, Zhengzhou University, Zhengzhou 450001, People’s Republic of China; § These authors contributed equally to this work. * Corresponding author. Yanjie Zhang, Tel: 008637167781573; Fax: 0086 37167781573; E-mail:[email protected]

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ABSTRACT: As an oil crop with good taste and profuse nutrition, peanut (Arachis

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hypogaea L.) is grown worldwidely, mainly for edible seeds. Black peanuts attract

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more attention for the appealing color and health-promoting anthocyanins. Here, two

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cyanidin-based anthocyanins and four quercetin-based flavonols were separated and

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identified from skins of two black cultivars (Zi Yu and Zi Guan) by

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HPLC-ESI-Q-TOF-MS. To study the anthocyanin accumulation, libraries constructed

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from the mRNA of skins of Zi Yu and white cultivar (Bai Yu) were sequenced and

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4042 differentially expressed genes were identified. Gene ontology and KEGG

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pathway analysis underlined the importance of high expression of flavonoid

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biosynthetic and regulatory genes in seed skin of Zi Yu. Furthermore, expression

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profiles of these genes were analyzed carefully in four representative peanut cultivars.

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Altogether, these results strongly indicate that the up-regulation of transcriptional

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activators (AhMYB1, AhMYB2 and AhTT8) accounts for the skin-specific

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accumulation of anthocyanins in black peanut.

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KEYDORDS: Anthocyanin, Black peanut, skin, HPLC-ESI-Q-TOF-MS, RNA-Seq,

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transcriptome

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INTRODUCTION

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As a group of colorful pigments, anthocyanins widely distribute in most of tissues

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of higher plants, including leaves, stems, roots, flowers, and fruits. These secondary

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metabolites not only play a vital role in controlling the wide range of color from red to

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purple and blue according to the pH and their chemical modifications, but also

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possess some crucial functions in reducing the damage from drought stress, cold, UV


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irradiation and microbial agents in plant tissues.1-4 Besides, flowers and fruits with the

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well-marked colors attract animals and insects to complete seed dispersal and

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pollination.5 As an important subclass of natural flavonoids, anthocyanins exhibit

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plenty of promising effects on human health. Growing evidences have proven that the

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regular intake of anthocyanins can reduce the risk of inflammation, oxidative stress,

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cardiovascular diseases, cancer, diabetes, atherosclerosis and related diseases.6-10

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Recent research shows that cyanidin, a kind of anthocyanidins, is the potent SIRT6

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activator that significantly increased SIRT6 activity (an essential target in

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inflammatory, metabolic diseases and cancer ).11 Owing to the favorite color and

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health-promoting effects, vegetables and fruits rich in anthocyanins are usually more

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valuable and attractive than other cultivars.12

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The flavonoid biosynthetic pathway, as a branch of the phenylpropanoid pathway,

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is responsible for the production of anthocyanins, proanthocynidins and flavonols.

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Moreover, anthocyanin biosynthesis have been widely studied in blood orange

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(Citrussinensis Osbeck L.), grape (Vitis vinifera L.), petunia (Petunia hybrida),

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snapdragon (Antirrhinum majus), maize (Zea mays) and Arabidopsis (Arabidopsis

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thaliana).13-15 Anthocyanin biosynthesis begins with the cleavage of phenylalanine

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catalyzed by phenylalanine ammonia-lyase (PAL), resulting in the production of

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cinnamic acid. Subsequently, the product of previous reaction is catalyzed

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sequentially by cinnamate 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL),

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chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H),

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flavonoid

3'-hydroxylase

(F3'H),

dihydroflavonol,


4-reductase

(DFR)

and


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anthocyanidin synthase (ANS/LDOX), leading to the synthesis of colorful

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anthocyanidins.

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anthocyanidins, leading to the production of stable pigments, anthocyanins.16, 17 It is

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worth mentioning that the basic skeleton of anthocyanins consists of three aromatic

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rings generated by CHS and CHI. F3H catalyzes the oxidation of central ring of

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naringenin, yielding dihydrokaempferol. Moreover, dihydrokaempferol can be further

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hydroxylated by flavonoid 3′-hydroxylase (F3′H) and/or flavonoid 3′ 5′-hydroxylase

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(F3′5′H), resulting in the production of dihydroquercetin and dihydromyricetin,

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respectively. These dihydroflavonols can be further catalyzed by flavonol synthase

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(FLS) in hydrogenation reaction, leading to the synthesis of flavonols (kaempferol,

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quercetin and myricetin). Besides, anthocyanins can be converted to epicatechins by

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anthocyanin reductase (ANR), while leucoanthocyanidins can be directly converted to

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catechins under the activity of leucoananthcyanidin reductase (LAR). Both catechins

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and epicatechins can be polymerized to form proanthocyanidins.18

Then,

glucosyltransferases

catalyze

the

glycosylation

of

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Transcriptional regulation of anthocyanin biosynthesis has been widely studied in

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many plant species.19 The expression of anthocyanin structural genes are directly

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transcriptional activated by a ternary protein complex (MBW complex) consisting of

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R2R3 MYB transcription factors (TFs), basic helix-loop-helix (bHLH) TFs and

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WD40 proteins. On the contrary, MYB4, as a R2R3-MYB class repressor, negatively

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regulate the transcription of the C4H, in responding to UV-B irradiation.20 Besides,

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recent studies proved that both CPC (MYB transcription factor) and MYBL2 can

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repress the anthocyanin biosynthesis by inhibiting the assemblage of ternary MBW


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complex.21,

22

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indirectly by other TFs such as CIP7, WRKY, NAC(ATAF, NAM, CUC), PIF3 and

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HY5. 23-27

Nevertheless, the structural genes can also be regulated directly or

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Peanut (Arachis hypogaea L), as a major source of edible oil and protein, is ranked

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as the second most important cultivated grain legume and the fourth largest edible

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oilseed crop in the world.28-30 The leaves, stems, hulls and skins of peanut have been

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identified as sources of phenolic compounds.31-35 As viewed from outer appearance,

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the color of testa varies widely (such as red, white, purple, tan and wine). Compared

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with anthocyaninless cultivars, black peanuts attract more attention because of the

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appealing color and health-promoting ingredients in the purple/black skins. Although

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several kinds of flavonoids including anthocyanins and flavonols have been

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indentified in various peanut cultivars in previous articles, studies on the genetic

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regulation of anthocyanins and derivatives have not been reported.36-38 In this study,

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two anthocyanins and four flavonols were separated and identified from skins of two

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representative balck peanut cultivars (Zi Yu and Zi Guan) using the high performance

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liquid chromatography coupled with electrospray ionization quadrupole time-of-flight

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mass spectrometry (HPLC-ESI-Q-TOF-MS). Moreover, RNA-Seq analysis was

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caught out to investigate differentially expressed genes in the skins of two peanut

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cultivars (Zi Yu and Bai Yu) differing greatly in color. Accordingly, the expression

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levels of biosynthetic and regulatory genes were analyzed by quantitative real-time

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polymerase chain reaction (qRT-PCR) in four representative cultivars with different

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colors in testa (Zi Yu, Zi Guan, Hong Yu and Bai Yu). This work expands our



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understanding on skin-specific accumulation of anthocyanins in black peanuts at both

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metabolic and molecular levels.

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MATERIALS AND METHODS

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Plant Materials. Four representative peanut cultivars (Arachis hypogaea L.) were

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selected for this study. In detail, black cultivars (Zi Yu and Zi Guan) display dark

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purple pigmentation in the skins of peanut kernels, while the anthocyaninless cultivars

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exhibit red skin (Hong Yu) or white skin (Bai Yu) (Figure 1). All plants of four

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peanut cultivars were grown in greenhouse under the same growth conditions. The

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seed coats, kernel embryos, and mature leaves of four peanut cultivars were carefully

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separated and harvested 30 days after anthesis. All these tissues were immediately

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flash-frozen in liquid nitrogen and stored in -80°C freezer for RNA and metabolite

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extraction.

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Chemicals and reagents

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Water of ultra pure grade was prepared by a Milli-Q purification system. Methanol,

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acetonitrile, acetic acid, formic acid, quercetin and cyainidin-3-O-glucoside of HPLC

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grade were bought from Sigma–Aldrich.

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RNA Extraction and Quantitative Real-time PCR Analysis. Total RNA were

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extracted from leaves, embryos and skins of different peanut cultivars using the Plant

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RNA Kit (OMEGA, USA) according to the manufacturer's instructions. The purity

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and integrity of RNA sample were tested with RNase free agarose gel electrophoresis

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and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), respectively.

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The qRT-PCR analysis was carried out as the protocols mentioned before and Actin


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was selected as reference gene.39 The sequences of the primer pairs used for the

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qRT-PCR are listed in Table S1.

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Library Preparation and High-throughput Sequencing.

The total RNA

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extracted from skins of Zi Yu and Bai Yu was used to construct RNA-Seq libraries

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and every sample was analyzed in three replicates. The prepared libraries were

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sequenced and analyzed by Berry Genomics Corporation (Beijing, China) on an

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Illumina HiSeq 2000 platform.

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Bioinformatics Analysis. Filtered clean reads were mapped to peanut reference

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genome (https://peanutbase.org/peanut_genome) with Bowtie software and mapped to

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peanut reference genes with BWA software.40 The original digital gene-expression

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levels

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kilobase-of-transcript-per-million-fragments-mapped (FPKM). The differentially

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expressed genes (DEGs) were identified based on the ratio of the FPKM values. The

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expressed FPKM value of each gene in each sample was calculated by using RSEM

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(V1.2.15) software. The method of false discovery rate (FDR) control was used to

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calculate the threshold of the P-value. Only transcripts with an absolute value of log2

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ratio ≥2 and an FDR significance score

Comparative Transcriptome Analysis of the Skin-Specific

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