Comparative Transcriptome Analysis Reveals Effects of Light on

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Biotechnology and Biological Transformations

Comparative Transcriptome Analysis Reveals Effects of Light on Anthocyanin Biosynthesis in Purple Grain of Wheat Fang Wang, Yu Xiu Dong, Xiao Zhen Tang, Tian Li Tu, Bin Zhao, Na Sui, Daolin Fu, and Xian Sheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05435 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Journal of Agricultural and Food Chemistry

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Comparative Transcriptome Analysis Reveals Effects of

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Light on Anthocyanin Biosynthesis in Purple Grain of

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Wheat

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Fang Wang1§, Yu Xiu Dong1§, Xiao Zhen Tang2§, Tian Li Tu1, Bin Zhao1, Na Sui3,

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Dao Lin Fu1, Xian Sheng Zhang1*

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1

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Agricultural University, Tai’an, 271018, Shandong, China

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2

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271018 , Shandong, China

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong

College of Food Science and Engineering，Shandong Agricultural University, Tai’an,

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Shandong Normal University, Jinan, 250014, China

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§

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Corresponding Author

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*E-mail: [email protected]

Shandong Provincial Key Laboratory of Plant Stress, College of Life Science,

These authors contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT

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To study mechanism of anthocyanin biosynthesis regulation, we examined

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light-regulated gene expression involved in anthocyanin biosynthesis in purple grain

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of wheat. Ten kinds of anthocyanins were identified from a purple-grain wheat

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cultivar by HPLC ESI-MS/MS analysis. The libraries constructed from total RNA of

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purple grain under light (L) or dark (D) conditions for 15 and 20 days were sequenced.

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In total, 1,874 differentially expressed genes (DEGs) in L20 vs. L15, 1,432 DEGs in

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D20 vs. D15, 862 DEGs in D15 vs. L15, and 1,786 DEGs in D20 vs. L20 were

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identified. DEG functional enrichments suggested that light signal transduction is

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critical to anthocyanin biosynthesis. 911 DEGs were referred to as LDEGs

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(light-regulated DEGs) involved in a number of genes in anthocyanin biosynthesis,

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transcription regulation, sugar- and calcium-signaling pathways and hormone

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metabolisms. These findings lay the foundation for future studies on regulatory

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mechanism of anthocyanin biosynthesis in purple grain of wheat.

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KEY WORDS: anthocyanin biosynthesis, light, RNA-seq, purple-grain wheat,

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transcriptomic analysis

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INTRODUCTION

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Anthocyanins are a class of secondary metabolites that contribute to the red, blue, and

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purple colors in plants. In flowers, these pigments attract pollinators, and in fruit skin

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they attract animals to aid in seed dispersal. Anthocyanins are also important for

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maintaining human health by preventing cardiovascular disease, carcinogenesis,

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inflammation, and many other pathological states.

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Most of the genes involved in anthocyanin biosynthesis and its regulation have

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been isolated and characterized in a number of plant species. 1 The genes involved in

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the early step of dihydroflavonol biosynthesis include phenylalanine ammonia lyase

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(PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone

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3-hydroxylase (F3H), and those involved in successive reactions in anthocyanin

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production include dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS),

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and

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biosynthesis is regulated by the combined action of R2R3-MYB and bHLH

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transcription factors (TFs), as well as WD40-repeat proteins.3-5

UDP-glucose, flavonoid 3-O-glucosyltran-ferase

(UFGT).2

Anthocyanin

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The purple colors are not natural for seeds of common hexaploid wheat.

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Cyanidin-3-glucoside and peonidine-3-glucoside are the main anthocyanins in the

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grain coat of purple-grain wheat.6 Most of the structural genes for anthocyanin

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biosynthesis in wheat have been identified. In previous studies, two closely linked,

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highly homologous genes encoding phenylalanine ammonia-lyase (PAL1 and PAL2)

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were isolated from the wheat genomic library, 7 and the loci for PAL, CHS, and CHI in

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wheat were determined using Southern blot hybridization analyses. 8 Analyses of four



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early flavonoid biosynthesis genes (CHS, CHI, F3H, and DFR) in wheat showed that

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they were expressed at higher levels in the grain coat tissue of red-grained lines than

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in white-grained ones.9 Three homoeologous full-length CHI copies were isolated and

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precisely mapped to the long arms of chromosomes 5A, 5B, and 5D.10 F3H-A1,

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F3H-B1, and F3H-D1 were mapped to chromosomes 2A, 2B, and 2D of wheat, and

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were shown to be highly expressed in red grains and coleoptiles.11 Three copies of

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DFR were mapped to homoeologous group 3 chromosomes,12 and five copies of ANS

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were mapped to chromosomes 6A, 6B, and 6D.13 In addition, a partial UFGT

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sequence has been isolated from the wheat genome.14

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Light signaling plays a pivotal role in controlling plant morphogenesis, metabolism,

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and development. Light is sensed by plants through several classes of photoreceptors

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such as the red- and far-red light-sensing phytochromes, the blue/ultraviolet

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(UV)-A-perceiving cryptochromes and phototropins, as well as the UV-B-sensing

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photoreceptor UVR8.15 Upon light activation, photoreceptors induce developmental

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responses such as seedling deetiolation, phototropism, and flowering induction, as

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well as affecting metabolism. In Arabidopsis, anthocyanins accumulate in light-grown

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plants but not in dark-grown ones, implying that light is required for anthocyanin

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biosynthesis. However, little is known about the regulatory genes that control the

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biosynthesis of anthocyanins in response to light signals.16 Wheat (Triticum aestivum

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L.) cultivars possessing purple grain are thought to be more nutritious to human

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because of their high anthocyanin contents in their grain coats. In this study, the

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components of anthocyanin in the purple-grain cultivar (Luozhen No.1) were


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examined by HPLC ESI-MS/MS analysis. Because light is critical to regulate the

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production of anthocyanins in grain coats, we made the transcriptome analysis on the

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coat tissues of purple-grain wheat from plants grown under light conditions for 15 and

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20 days and under dark conditions for 15 and 20 days, respectively. The results of this

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study might provide important information for understanding mechanism of

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light-mediated anthocyanin biosynthesis in purple-grain wheat.

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MATERIALS AND METHODS

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Plant Materials and Growth Conditions. The purple-grain wheat cv. Luozhen No.1

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was used for RNA-seq analyses. The wheat plants were cultivated in a field at the

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Experimental Station of Shandong Agricultural University. Whole ears were covered

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with two layers of dark paper bags for dark treatment. The grains were harvested at 0,

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10, 15, 20, 25, and 30 DAP. The grain coats were isolated and collected, frozen in

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liquid nitrogen, and stored at −80℃ for RNA extraction.

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Total anthocyanins analysis. Total anthocyanins were measured using a

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spectrophotometric differential pH method. 1.0 g grains were crushed into powder

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and extracted separately with 2 mL of pH 1.0 buffer containing 50 mM KCl and 150

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mM HCl, as well as 2 mL of pH 4.5 buffer containing 400 mM sodium acetate and

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240 mM HCl. The mixtures were centrifuged at 10,000g for 3 min. Supernatants were

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collected and diluted for direct measurement of absorbance at 510 nm. Total

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anthocyanin content was calculated using the following equation:

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Amount (mg/g) = (A510 at pH 1.0−A510 at pH 4.5)×445.2/29,600×dilution factor.

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The number 445.2 is the molecular mass of cyanidin-3-glucoside and 29,600 is its



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molar absorptivity (ε) at 510 nm. Each sample was analyzed in triplicate and the

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results were expressed as the average of the three measurements.

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Anthocyanin Extraction and HPLC-ESI-MS/MS Analysis. Coats of purple-grain

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wheat were used for the anthocyanin components analysis. About 1.0 g grain coats

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were weighed, and then, 5 mL of acidified ethanol (ethanol (95%) and HCl (1.0 N),

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85:15, v/v) was added. The solution was mixed and adjusted to pH 1 with 4 N HCl,

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and shaken for 20 min. The solution was centrifuged at 4,800 rpm for 5 min. The

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extraction was repeated four times. The supernatant was poured into a 50 mL

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volumetric flask and vaporized under 40°C by a rotary evaporator. The pigments were

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dissolved in 5 ml 0.1% methanoic acid and purified using SPE C18 column (Waters).

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The samples were then analyzed by ACQUITY Ultra Performance Liquid

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Chromatography (Waters, USA), equipped with mass spectrum system (Micromass

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Quattro Ultima IMPT, Waters, USA). The chromatographic separation was performed

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on Sun FireTM C18 column (4. 6 mm × 150 mm, 5 μm, Waters, USA). The injection

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volume was 10.0 μL. Elution was performed using mobile phase A (aqueous 0.1%

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formic acid solution) and mobile phase B (methanol). The column oven temperature

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was set at 40 °C. The flow rate was 0.8 mL/min. The gradient program is described as

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follows: 0−13 min, 10-52% B; 13−15 min, 52-90% B; 15−17.5 min, 90−10% B.

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Quantification of the different anthocyanins was based on peak areas and calculated

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as equivalents of the standard compounds (Standard substances, purity > 90%, i.e.

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cyanidin-3-glucoside, peonidin-3-glucoside and delphinidin-3-glucoside). Scanning

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wavelength range of diode array detector was 200-800 nm. Data were acquired by


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Masslinx 4.0 software (Micromass, Beverly, MA) with Quan-Optimize option for the

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fragmentation study. The experimental conditions were as follows: ionization mode is

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atmospheric pressure electrospray particle source (ESI), positive ion mode; capillary

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voltage 1.50kV, cone voltage 50V, Ion source temperature, 120 ℃, dissolvent gas

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temperature, 450 ℃, cone-hole gas flow, 81L / h, desolvent gas flow, 618L / h. The

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voltage of photoelectric multiplier is 650 V. MS/MS, scan from m/z 200 to 1500; ion

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trap, scan from m/z 200 to 1500; source accumulation, 50 ms; ion accumulation time,

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300 ms; flight time to acquisition cell, 1 ms; smart parameter setting (SPS),

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compound stability, 50%; trap drive level, 60%.

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RNA extraction and library construction. Total RNAs were extracted using an

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RNeasy Plus Micro kit (Qiagen, Valencia, CA, USA). At least 500 ng total RNAs

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were extracted from each material used for library construction. First, mRNAs

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extracted from each material were enriched by using oligo(dT) magnetic beads

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(Illumina, San Diego, CA, USA). The purity and quantity of total RNAs were

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checked. Then, mRNAs were further enriched by removing rRNAs from the total

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RNAs. Mixed with the fragmentation buffer, the mRNAs were fragmented into short

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fragments. Then cDNA was synthesized using the mRNA fragments as templates.

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Short fragments were purified and resolved with EB buffer for end reparation and

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single nucleotide A (adenine) addition. After that, the short fragments were ligated

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with adapters. After agarose gel electrophoresis, the suitable fragments were selected

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for the PCR amplification as templates. During the quality control (QC) steps, Agilent

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2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used for



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quantification and qualification of the sample libraries.

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Illumina sequencing and data analysis. Each library was sequenced using the

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Illumina HiSeq™ 2000 system in the Beijing Genomics Institute (Shenzhen, China).

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Raw reads were subjected to QC that determines if a resequencing step was needed.

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After QC, raw reads were filtered into clean reads which will be aligned to the

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reference sequences. QC of alignment was performed to determine if resequencing is

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needed. The alignment data was utilized to calculate distribution of reads on reference

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genes and mapping ratio. If alignment result passed QC, we could proceed with

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downstream analysis. RNA-seq data were obtained in three biological replicates and

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deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress)

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under accession number E-MTAB-5975 and E-MTAB-6398.

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The filtered clean reads were mapped to the wheat reference genome

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(ftp://ftp.ensemblgenomes.org/pub/plants/release-26/fasta/triticum_aestivum/dna/Triti

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cum_aestivum.IWGSC2.26.dna.toplevel.fa.gz) using Bowtie software, and mapped to

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the

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(ftp://ftp.ensemblgenomes.org/pub/plants/release-26/fasta/triticum_aestivum/cds/Triti

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cum_aestivum.IWGSC2.26.cds.all.fa.gz) using BWA software. Mismatches of less

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than two bases were allowed in this process. According to the alignment, clean reads

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were divided into unmapped reads, multi-position matched reads, and unique matched

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reads. For all mapped transcripts with unique matched reads, the original digital gene

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expression levels were calculated using fragments Per Kilobase of transcript per

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Million fragments mapped (FPKM) method. The DEGs were identified based on a

wheat

reference


gene

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-fold change of ≥2 and diverge probability ≥0.8. Gene annotations were obtained

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from the wheat sequence (http://plants.ensembl.org/Triticum_aestivum/Info/Index).

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Genes were classified using MapMan software.

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The gene ontology (GO) analysis was conducted using the singular enrichment

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analysis tool (http://bioinfo.cau.edu.cn/agriGO/analysis.php). GO terms with p-value

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Comparative Transcriptome Analysis Reveals Effects of Light on

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