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VcBBX, VcMYB21, VcR2R3 MYB transcription factors are involved in UVB induced anthocyanin biosynthesis in the peel of harvested blueberry fruit Chau T.T. Nguyen, Sooyeon Lim, Jeong Gu Lee, and Eun Jin Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05253 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 7, 2017

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Journal of Agricultural and Food Chemistry

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VcBBX, VcMYB21, VcR2R3 MYB transcription factors are involved in UV-B

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induced anthocyanin biosynthesis in the peel of harvested blueberry fruit

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Chau T.T. Nguyen, Sooyeon Lim, Jeong Gu Lee, and Eun Jin Lee*

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Department of Plant Science, Research Institute of Agriculture and Life Sciences, Seoul

National University, Seoul 151-921, Korea

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*Corresponding Author: E.J. Lee

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E-mail address: [email protected]

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Phone: (+82)-2-880-4565

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Fax: (+82)-2-873-2056

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ACS Paragon Plus Environment


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ABSTRACT

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This study was carried out to better understand the mechanism responsible for increasing

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the anthocyanins in blueberries after UV-B radiation at 6.0 kJ m-2 for 20 min. UV-B induced up-

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regulation of genes involved in anthocyanin biosynthesis in blueberry fruit compared to a non-

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treated control. Phenylalanine ammonia lyase, chalcone synthase, and flavanone 3’-hydroxylase,

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which are enzymes that function upstream of anthocyanin biosynthesis, were significantly

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expressed by UV-B. Expression levels of VcBBX, VcMYB21, and VcR2R3 MYB transcription

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factors (TFs) were up-regulated by UV-B in the same manner as the anthocyanin biosynthesis

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genes. The significant increase in the expression of TFs occurred immediately after UV-B

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treatment, was then maximized within 3 hours. In accordance with these changes, individual

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anthocyanins contents in the fruits treated with UV-B significantly increased within 6 hours and

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were 2-3 folds higher than the control. Our results indicated that UV-B radiation stimulates an

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increase in anthocyanin biosynthesis which could be up-regulated by TFs studied.

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KEY WORDS: anthocyanins, pigment synthesis, postharvest, transcription factors, treatment,

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UV radiation, Vaccinium corymbosum

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INTRODUCTION

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Blueberries (Vaccinium corymbosum) contain large amounts of anthocyanins, which are

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blue-colored pigments in the skin of fruits found in a wide assortment of healthy foods.

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Anthocyanins have a positive effect on human health and a recent study showed that taking

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individual anthocyanins such as dietary flavonoids can reduce the risk of myocardial infraction in

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young women 1. Five major anthocyanins identified as cyanidin, delphinidin, malvidin, petunidin,

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and peonidin are commonly observed in blueberry fruits 2. In our previous study, three of the

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major anthocyanins malvidin-3-galactoside, delphinidin-3-galactoside, and petunidin-3-

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galactoside were only found in full-ripe blueberry fruits 3. The anthocyanin composition in

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blueberries may be different based on the cultivar, species, or fruit development stage.

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Anthocyanin biosynthesis has been found to involve genes in the phenylpropanoid pathway 2.

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Genes which encode anthocyanin biosynthesis enzymes include phenylalanine ammonium lyase

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(PAL), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavanone 3’-hydroxylase

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(F3’H), flavanone 3’5’-hydroxylase (F3’5’H), dihydroflavonol 4-reductase (DFR), anthocyanidin

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synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). These

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biosynthesis enzymes are controlled by regulatory genes, namely, transcription factors (TFs) 4, 5.

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Light is one of environmental factors regulating plant development and gene expression6.

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Ultraviolet (UV)-B light has been considered as a major environmental factor which could be

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applied to improve fruit quality and antioxidant content in blueberry fruits 7, to enhance

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antioxidant activity of carrot product 8, and to enhance soluble phenolic contents of some other

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crops 9. UV-B radiation in the range between 280 nm and 320 nm affected the plant defense

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system and led to an increase in the number of secondary metabolites 10. UV-B radiation induced 3



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the accumulation of anthocyanins in hypocotyls of radish sprouts 11 and anthocyanins and

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flavonoids in apple skins 12.

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In research on TFs, low-dosage UV-B radiation accumulated more hydroxycinnamic acid

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derivatives and anthocyanins by up-regulation of MYB TFs 13, 14, B-box protein (BBX) 15 and

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MYB-bHLH-WDR (MBW) complex 16. MYB TFs play important roles in synthesis of

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anthocyanins and flavonols in plants. Several studies have shown that MYB family are involved

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in the regulation of anthocyanin synthesis in apples 17, berries 18, and grapes 19, and that R2R3

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MYB and MYBA are key players that determine apple peel color 13, 17. MrMYB1 and a R2R3

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MYB homolog were identified as activators of anthocyanin biosynthesis in Chinese bayberries 20.

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Additionally, UV-B indlinesuced anthocyanin accumulation related to MdCOL11 in the peels of

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apple fruits was found to be due to light induced signal transduction and photomorphogenesis

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through elongated hypocotyl 5 (HY5). HY5 regulated MdCOL11 which further induces the

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expression of MdMYBA 21.

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However, the expression levels of anthocyanin biosynthesis genes in blueberry fruits

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after UV-B radiation are still unknown; in particular, the role of TFs is still unclear. In continuous

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study, thus, we hypothesized that application of UV-B radiation on harvested blueberry fruits

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could stimulate the accumulation of anthocyanins at a certain time, and this could occur through

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the activation of the major genes and related TFs in the anthocyanin biosynthesis pathway. This

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study aims to determine the mechanism mediating the effects of UV-B on expression levels of

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anthocyanin biosynthesis genes as well as TFs such as VcBBX (accession number KX300037.1,

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see Table S1), VcMYB21, and VcR2R3 MYB that are present in the peel tissues of blueberry fruits

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harvested at fully colored stage. To examine the relationships between these genes, correlation

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analysis was also conducted. Individual anthocyanins were also analyzed to confirm the

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accumulation of anthocyanins after UV-B treatment.

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MATERIAL AND METHODS

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Blueberries and UV-B radiation

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The high-bush blueberry (Vaccinium corymbosum) ‘Duke’ cultivar was hand-picked from

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a farm in Korea, in July at the fully colored stage, as indicated by the dark-purple skin color (Fig.

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S1). Approximately 10 kg of blueberry fruits were harvested and sorted by uniform fruit size and

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color. At harvest time, total soluble solid contents of blueberry were ranged from 10 % to 11 %

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and fruit firmness was from 1.75 N to 1.85 N. Within 1 hour after grading, the blueberry fruits

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were treated with UV-B inside the UV radiation device described 3. Approximately 2 kg of the

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fruits were placed in a mesh bag and radiated for 10 min each on the top and bottom side of the

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container until the fruits received a radiation dose of 6 kJ m-2 at 25~27 oC . Control fruits were

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treated in the same manner as those above described without UV-B light. We selected the 6 kJ m-

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dose based on our preliminary study using dosages of 1.6, 4.0, and 6.0 kJ m-2, where a dose of 6

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kJ m-2 remarkably reduced fruit decay of harvested blueberries without causing surface damage 3.

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After UV-B treatment, blueberry fruits were stored in polyethylene containers (20 cm × 10 cm)

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and kept at room temperature for 24 hours. The peel tissues were removed and collected from the

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blueberries (about 50 fruits per container) immediately after harvest without UV-B treatment, and

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immediately, 20 min, 3 hours, 6 hours, and 24 hours after UV-B treatment. Samples were

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immediately frozen in liquid nitrogen and kept at –80oC until analysis.

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Isolation of total RNA Total RNA was extracted from peel tissues (about 3-5 g) according to the protocol

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described 22, 23 with some modifications. Fruit peels were ground in a mortar under liquid

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nitrogen and 3 g of the frozen powder samples were added to 15 mL of cationic detergent cetyl-

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trimethylammonium bromide (CTAB) based extraction buffer which was then preheated at 65oC

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and incubated for 10 min at 65oC. Next, 15 mL of chloroform/isoamyl alcohol (v/v, 24:1) was

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added and the mixture was vortexed and centrifuged at 11,000 × g for 10 min a 4oC. The

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supernatant was collected and mixed with 6.43 mL of lithium chloride before precipitation

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overnight at 4oC. After 12 hours, the residues were added to 0.5 mL of pre-warmed (65oC)

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sodium chloride, sodium dedocyl sulfate, tris and EDTA (SSTE) buffer after centrifugation at

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21,000 × g for 20 min at 4oC. The suspended RNA was washed once with 0.5 mL of

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chloroform/isoamyl alcohol (24:1, v/v) and twice with 0.5 mL of phenol/chloroform/isoamyl

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alcohol (PCI, 20:20:1, v/v/v). The samples were then centrifuged at 11,000 × g for 10 min at 4oC,

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and washed one more time with 0.7 mL of cold isopropanol. The pellet was collected after

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centrifugation at 21,000 × g for 15 min at 4oC. After washing with 75% ethanol, the RNA pellet

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was suspended in diethylpyrocarbonate (DEPC) treated water and stored at –20oC until analysis.

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The quantity and quality of RNA were assessed by the Nanodrop 2000 UV-visible

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spectrophotometer (Thermo Scientific, Wilmington, USA) and were checked by agarose gel-

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electrophoresis (Fig. S2).

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Analysis of gene expression Gene expression was analyzed using real-time quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). Six genes involved in the anthocyanin biosynthesis 6


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pathway VcPAL, VcCHS, VcF3H, VcF3’H, VcF3’5’H,VcDFR, VcANS, and VcUFGT were

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selected for amplification from the NCBI database based on references 5, 22, 24, 25. Specific primers

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for VcBBX, VcMYB21, and VcR2R3 MYB were designed using the NCBI primer blast tool and

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were based on the sequences reported 21, 26. VcMYB21 and VcR2R3 MYB primers were designed

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based on the nucleotide sequences for V. corymbosum and V. myrtillus, respectively, in the NCBI

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database. The VcBBX primer was designed from MdCOL11, an apple B-box zinc finger protein

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which is involved in UV-B and temperature induced anthocyanin biosynthesis in apple skin 21.

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Information on all primers used in our study is shown in Table S1.

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PCR products of target genes were amplified by RT-PCR using a Bio-Rad T100TM

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Thermal Cycler (Bio-Rad, Foster, USA) and cleaned up for sequencing. All confirmation results

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are presented in Figs. S3 and S4. The qRT-PCR was performed on the CFX ConnectTM using

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Labopass SYBR Green Q Master Mix (2X) by the CFX Connect real-time system (Bio-Rad,

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Hercules, USA). A total reaction volume of 10 µL was used. Each reaction included 2 µL of

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template, 0.4 µL of forward primer, 0.4 µL of reverse primer, and 5 µL SYBR Green along with

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an additional 2.2 µL of RNA-free water. The qRT-PCR was performed using the following

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amplification program: 95oC for 10 min followed by 40 cycles at 95oC for 30 sec and 60oC for 30

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sec. The ∆∆Ct method was used to normalize and calibrate transcript values relative to

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously 27. VcGAPDH

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was used as an internal control 22 and its expression is shown in Fig. S5.

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Analysis of individual anthocyanins High performance liquid chromatography (HPLC) was used to separate and identify individual anthocyanins in blueberry peel tissues according to protocols described 3. Three grams 7



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of peel tissues were macerated in 50 mL of methanol using a Polytron P-10 tissue homogenizer

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(Brinkmann, NY, USA). The extracts were filtered and concentrated using a rotary evaporator

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series (EYELA, Tokyo, Japan) in a water bath at 38°C and then completely dissolved in 3%

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formic acid in water (v/v). All samples were passed through a C18 Sepak cartridge (Waters,

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Milford, MA, USA). The anthocyanins were then recovered with 2 mL absolute methanol

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consisting of 3% formic acid (v/v). Ten microliters of sample were separated by HPLC after

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passing through a 0.42 µm membrane filter.

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Mixed or individual anthocyanin standards were analyzed to confirm the anthocyanins

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extracted from blueberries. Delphinidin-3-galactoside, malvidin-3-arabinoside, and malvidine-3-

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galactoside were purchased from Chromadex (Irvine, CA, USA). They were dissolved in 10 mL

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of 3% formic acid in methanol (v/v) and used as an external standard stock solution for

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generating calibration curves.

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Anthocyanins were analyzed using a YL 9100 HPLC system (YoungLin, Anyang, Korea)

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consisting of a YL9111 binary pump, a YL9160 diode array detector, and a YL9150 autosampler.

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A Phenomenox Bondclon C18 analytical column (4.6 × 250 mm, 10 µm) was used. Mobile

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solvents were 5% formic acid in water (v/v) and acetonitrile (ACN). The solvent system was

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programmed from 5% ACN to 20% ACN for 50 min and flushed with 100% ACN for 5 min. The

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flow rate was 1 mL min-1. Ten microliters of sample were injected. Spectral data between 200

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and 700 nm were collected and anthocyanins were detected at 520 nm.

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Statistical analysis The experiment was carried out using a completely randomized design. Measurements and analysis were repeated in triplicate. The means were expressed with ±standard deviations. The 8


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means and standard deviations were calculated using t-test at P < 0.05 (*), P < 0.01 (**), and at P

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< 0.001 (***) using SPSS software. Figures were generated using Sigma plot 10.0 software.

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Correlation analysis between two sets of variables was conducted using SPSS software and was

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displayed by a heat-map (MeV viewer).

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RESULTS

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Expression of anthocyanin biosynthesis genes The qRT-PCR results for the anthocyanin biosynthesis genes VcPAL, VcCHS, VcF3H,

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VcF3’H, VcF3’5’H, VcDFR, VcANS, and VcUFGT are shown in Fig. 1. We analyzed gene

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expression in peel tissues of blueberry fruits at harvest time, and immediately, 20 min, 3 hours, 6

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hours, and 24 hours after UV-B treatment. The results showed that gene expression was affected

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by UV-B treatment. Blueberries treated with UV-B showed higher gene expression levels

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compared to those without UV-B. The induction of VcPAL, VcCHS, VcF3H, and VcUFGT

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expression in harvested blueberries gradually increased according to the experiment time

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regardless of UV-B treatment, although this phenomenon was more clearly observed in

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blueberries treated with UV-B (Figs. 1A, 1B, and 1H).

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In the UV-treated fruits, a statistically significant induction of gene expression was

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observed for all genes within 3 hours of the treatment, except for VcF3H, VcF3’5’H, and VcANS

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which had a slight increase in transcript level with treatment that was not statistically significant.

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The significant increase in gene expression for VcPAL (P < 0.001), VcCHS (P < 0.01), VcF3’H

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(P < 0.05), VcDFR (P < 0.05), and VcUFGT (P < 0.05) appeared at 3 hours after treatment in the

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UV-treated fruits (Fig. 1). The expression level of VcF3’H immediately showed a strong increase 9



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with a 2-fold change at 20 min after treatment (Fig. 1D). The induction of gene expression in

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blueberry fruits was not significantly different between the control and treated samples after 6

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hours. The UV-B treatment appeared to be effective in stimulating the expression of anthocyanin

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biosynthesis genes. VcPAL, VcCHS, and VcF3’H which are key enzymes involved upstream of

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anthocyanin biosynthesis significantly responded to UV-B within a short time of 3 hours. Higher

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levels of gene expression for VcPAL, VcCHS, and VcF3’H in UV-treated fruits compared to the

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control were confirmed (Fig. 1).

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Expression of TFs Fig. 2 shows the expression levels of three TFs: VcBBX, VcMYB21, and VcR2R3 MYB.

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Both VcBBX and VcMYB21 were significantly up-regulated at 20 min by UV-B and their

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expression levels reached a 2.66-fold change for VcBBX and 3.04-fold change for VcMYB21

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(Figs. 2A and 2B). The significant increase of VcR2R3 MYB occurred 3 hours after UV-B

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treatment and reached a 3.87-fold change (Fig. 2C). After 6 and 24 hours, the expression levels

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of all TFs decreased and stably maintained at around a 1-fold change without a statistically

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significant difference. These results are displayed by a heat-map (Fig. 3). The highest level of

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expression of VcBBX was recorded at 20 min after UV-B treatment, whereas the expression of

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VcMYB21 and VcRER3MYB increased not only after 20 min but also after 3 hours and was

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maintained after 6 hours. These findings suggest that VcBBX is a major TF for inducing an

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immediate response to UV-B for the accumulation of anthocyanins in blueberry fruit skins. The

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expression of VcBBX after UV-B treatment was clearly distinguished from the control compared

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to the other TFs analyzed.

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Correlation analysis The correlation analysis between all studied genes is shown in Table 1. VcBBX had a high

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significant correlation with VcMYB21 and VcF3’H (r = 0.62 and r = 0.66 at P < 0.001).

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VcMYB21 had a significant correlation with VcR2R3 MYB and VcF3’H (r = 0.46 and r = 0.50 at

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P < 0.05 and P < 0.01, respectively). VcR2R3 MYB had not only a high correlation with VcF3’H

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(r = 0.48 at P < 0.05) but also had a significant correlation with VcUFGT (r = 0.46 at P < 0.05).

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The genes involved in the anthocyanin biosynthesis pathway had positive correlations with each

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other.

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Analysis of individual anthocyanin compounds Changes in the three individual anthocyanins in blueberry fruit skins are shown in Fig. 4:

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delphinidin-3-galactoside (Del-3-Gal) (Fig. 4A), malvidin-3-galactoside (Mal-3-Gal) (4B), and

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malvidin-3-arabinoside (Mal-3-Ara) (4C). All of the individual anthocyanins increased

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significantly within 3 hours after UV-B treatment. At 3 hours, the concentrations of Del-3-Gal

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and Mal-3-Gal reached 197.38 µg g-1 and 228.05 µg g-1 fresh weight in the UV-treated fruits,

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respectively. After 6 hours, the concentrations of the three anthocyanins Del-3-Gal, Mal-3-Gal,

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and Mal-3-Ara reached maximum levels of 248.56 µg g-1, 409.75 µg g-1, and 376.63 µg g-1 fresh

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weight, respectively, in the UV-B treated fruits. The total amount of anthocyanins after UV-B

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treatment was ~103 mg 100 g-1 higher than the control (data not shown).

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DISCUSSION

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Among postharvest technologies, UV radiation is a technique that may be more effective

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in reducing microbial growth than other immersing methods, especially since it does not leave a

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residue after treatment. UV radiation at low doses from 0.25 to 8 kJ m-2 affects the DNA of

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microorganisms by blocking microorganism development 28. UV radiation also induces stress

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responses and secondary plant metabolites which lead to enhanced antioxidant compounds and

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improved anthocyanin accumulation 29. In our previous work 3, we applied three types of UV-

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light (A, B, and C) at 6 kJ m-2 to harvested blueberry fruits in order to investigate the effect of

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UV on fruit quality and anthocyanins during 4 weeks of cold storage at 0oC. We found a reduced

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decay in UV-treated fruits and a remarkable increase in individual anthocyanins after 3 hours of

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UV-B and -C treatment. In this work, we confirmed that UV-B light at 6 kJ m-2 was effective in

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increasing the levels of anthocyanin compounds (Fig. 3) by significantly increasing the

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expression levels of the anthocyanin biosynthetic genes VcPAL, VcCHS, VcF3’H, VcDFR, and

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VcUFGT (Fig. 1) and all of the studied TFs VcBBX, VcMYB21, and VcR2R3 MYB (Fig. 2).

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There have been several studies investigating the effect of UV radiation on the

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expression of anthocyanin biosynthesis-related genes in Arabidopsis, apple, grape, and bilberry

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fruits 16, 21, 29-34. Many studies have focused on the expression of main genes in the

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phenylpropanoid biosynthetic pathway and well demonstrated the effect of UV on gene

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expression. However, the effects of UV-B on the genes involved in the anthocyanin biosynthesis

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pathway in harvested blueberry fruit skins have not yet been characterized, so we examined this

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through the current study. The increases in expression levels of the early-stage biosynthetic genes

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CHS and F3H induced the expression of the late-stage biosynthetic genes DFR, ANS, and UFGT,

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which is similar to reports 35, 36. In our study, up-expression of VcF3’H occurred after 20 min

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while increased expression of VcDFR and VcUFGT was only detected 3 hours after UV-B 12


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treatment (Fig. 1). Similar results were found in the enhanced expression of VcPAL, VcCHS, and

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VcDFR in skin tissues of peach fruits 33 and apple fruits 12, 16, 37, as well as the over expression of

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UFGT in Pyrus pyrifolia (‘Nakai’) 34, 38 after UV-B treatment.

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In addition, the expression of VcPAL was significantly positively correlated with VcCHS,

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VcF3H, VcDFR, VcANS, and VcUFGT (Table 1). This result indicated that although VcPAL was

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involved in the early-biosynthetic gene pathway, it may be correlated with the expressions of

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VcCHS, VcF3H, VcDFR, VcANS, and VcUFGT. It has been shown that stress responses can

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affect the plant defense system and lead to an increase in secondary metabolites 10, 39. CHS plays

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an important role in providing a common chalcone precursor for the production of all

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intermediate and final products in the flavonoid biosynthesis pathway 19. In this work, the

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expression level of VcCHS was highly correlative with VcF3H, VcF3’5’H, VcDFR, VcANS, and

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VcUFGT (Table 1) which clearly confirmed that the expression of the early-biosynthetic genes of

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CHS and F3’H affected the late-biosynthetic genes of DFR, ANS, and UFGT. The MYB and BBX families have been identified as light responsive regulatory factors

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and are involved in controlling the transcription of anthocyanin genes in apple 13, 17. The BBX

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family is a class of zinc finger TFs that contain one or two B-box domains with specialized

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tertiary structures that are stabilized though the binding of Zn ions 40. BBX has also been known

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as a CO-like (COL) family protein. After UV-B treatment, the over expression of MdCOL11 in

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Arabidopsis was observed 21. Similar to the increase in MdCOL11 in apple skin under UV-B light

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21

, we found an enhanced-expression of VcBBX in the peels of blueberry fruits 20 min after

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treatment with UV-B radiation (Fig. 2). A hypothetical working mechanism proposed that

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MdHY5 directly binds to the MdMYBA promoter to induce its expression. MdHY5 also regulates

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the expression of MdCOL11 which induces the expression of MdMYBA 21. MYBA and R2R3 13



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MYB are known TFs that are related to fruit flavonoid synthesis. MYBA has been shown to

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control anthocyanin biosynthesis in grape and bilberry fruits 26, 41. The expression of MYBA

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responded to light, tissue type, and maturity of fruit 20, 42.

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The role of the MYB TFs and BBX families in harvested blueberry fruits after UV-B

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light treatment has not been well characterized. In our study, analysis of VcMYB21 was designed

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based on nucleotide sequences of MYBA in studies on V. myrtillus 26 and VcR2R3 MYB analysis

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was based on nucleotide sequences of V. corymbosum 5 (Table S1). VcMYB21 and VcR2R3 MYB

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had a high significant correlation with each other (Table 1) which suggested that they have a

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close relationship. The R2R3 MYB protein has a long C-terminal sequence 43 and it specifically

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controls the anthocyanin biosynthetic pathway genes as well as anthocyanin conjugation and

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transport into the vacuole 44. R2R3 MYB is also a key player that determines the effect of

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anthocyanins on the color of apple fruit 13, 17. During the ripening stage in bilberry fruits,

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VmR2R3 MYB was involved in the process of anthocyanin accumulation 26. In our study, the

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enhanced-expression of VcR2R3 MYB was induced at 3 hours after the enhanced-expression of

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VcBBX and VcMYB21 that occurred immediately after UV-B treatment (Fig. 2). Moreover, the

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close correlation between TFs and VcF3’H, especially between VcR2R3 MYB and VcF3’H or

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VcUFGT (Table 1), confirmed that the enhanced-expression of these TFs activated the

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expression of VcUFGT, a key enzyme in the last consecutive step in the anthocyanin biosynthesis

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pathway. On the other hand, UV-B radiation induced stress in the peels of blueberry fruits

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immediately, which activated an increase in the late-biosynthetic genes leading to anthocyanin

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accumulation after 3 hours.

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Anthocyanins are one of the most common pigments in plants. UV-B induces anthocyanin accumulation in plant tissues via the activation of anthocyanin biosynthetic genes 45. 14


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In this work, levels of Del-3-Gal, Mal-3-Gal, and Mal-3-Ara were quickly increased by UV-B

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treatment in peels of harvested blueberry fruits which confirmed our previous results that UV-B

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and -C at 6 kJ m-2 induced an increase in nine individual anthocyanins of blueberry fruits after 3

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hours 3. This anthocyanin accumulation might be induced by significant increases in the

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expression levels of the phenylpropanoid pathway related genes (Figs. 1 and 2), and by UV-B

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acting on stresses that induce the plant defense system and lead to an increased number of

319

secondary metabolites 10, 39. A recent report on the red Chinese sand pear found that anthocyanins

320

did not accumulate in the dark 38. Under light, individual anthocyanins accumulated in bilberry

321

fruits 46. Anthocyanins accumulated in both the pulp and peel of bilberry fruits while the

322

accumulation occurred in the skin of blueberry fruits 47.

323

In conclusion, anthocyanins accumulated in the peels of harvested ‘Duke’ blueberry fruits

324

due to treatment with UV-B at 6 kJ m-2. UV-B induced increased expression of the TFs VcBBX,

325

VcMYB21, and VcR2R3 MYB, and the anthocyanin biosynthesis genes VcPAL, VcCHS, VcF3’H,

326

VcDFR, and VcUFGT, thereby promoting the accumulation of individual anthocyanins that

327

appeared quickly after UV-radiation. Nucleotide sequences of VcBBX were determined using an

328

online alignment search (BLAST) program to obtain the accession number KX300037, and we

329

confirmed that this TF was regulated by UV-B light. Similar to anthocyanin accumulation in the

330

skins of apple fruits by UV-light 21, anthocyanin accumulation in the skins of blueberry fruits

331

might also be due to the activation of MYB21 and R2R3 MYB. Our findings could aid in

332

revealing the molecular mechanism of anthocyanin biosynthesis by UV-light and contribute to the

333

development of postharvest technologies allowing healthier fruit consumption.

334 335

ACKNOWLEDGMENTS 15



336 337

This work was supported by the Basic Science Research Program (NRF-

338

2013R1A1A1057658) through the National Research Foundation of Korea funded by the

339

Ministry of Education, Science, and Technology and by the Aspiring Researcher Program

340

through Seoul National University (SNU) in 2014.

341 342

ABBREVIATIONS

343 344

ACN, acetonitrile; ANS, anthocyanidin synthase; CTAB, cetyl-trimethylammonium

345

bromide; CHS, chalcone synthase; Del-3-Gal; delphinidine-3-galactoside; DEPC,

346

diethylpyrocarbonate; DFR, dihydroflavonol 4-reductase; F3’H, flavanone 3’-hydroxylase; F3H,

347

flavanone 3-hydroxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high

348

performance liquid chromatography; Mal-3-Ara, malvidine-3-arabinoside; Mal-3-Gal, malvidine-

349

3-galactoside; PCI, phenol/chloroform/isoamyl alcohol; PAL, phenylalanine ammonium lyase;

350

qRT-PCR, quantitative real-time polymerase chain reaction; TFs, transcription factors; UFGT,

351

UDP-glucose:flavonoid 3-O-glucosyltransferase; UV, ultraviolet

352 353

SUPPORTING INFORMATION AVAILABLE

354 355

Supporting information material is available free of charge via the internet at

356

http://www.pubs.acs.org

357

Table S1. Primer pairs used for qRT-PCR.

16


Page 16 of 31

Page 17 of 31

358

Figure S1. Blueberry fruit according to skin coloration. Stage 5 (fully dark-purple colored blueberry) was used for UV-B radiation in this study.

359 360


Figure S2. Gel electrophoresis analysis to determine RNA quality extracted from

361

blueberry fruit peel tissues. HT, at harvest time; BT, after 20 min without UV-B treatment

362

(controls); AT, at 20 min; 3H, at 3 hours; 6H, at 6 hours; 20H, at 20 hours; and 24H, at 24

363

hours after UV-B treatment (treatments).

364

Figure S3. Gel electrophoresis analysis for qRT-PCR products.

365

Figure S4. Specific primers used for confirmation of all examined genes.

366

Figure S5. Gel electrophoresis analysis to determine GADPH quality as an internal standard and M values.

367 368 369

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497

Page 24 of 31

Table 1. Descriptive statistics and correlation matrix between studied variables. VcBBX VcBBX

1

VcMYB21

0.62***

VcMYB21 VcR2R3 MYB VcPAL VcCHS

VcF3H VcF3’H VcF3'5'H VcDFR VcANS

1

VcR2R3 MYB 0.35ns

0.46*

1

VcPAL

0.34ns

0.11ns

0.06ns

1

VcCHS

-0.07ns

-0.27ns

0.12ns

0.56**

1

VcF3H

-0.09ns

-0.34ns

-0.05ns

0.40*

0.60***

1

VcF3’H

0.66***

0.50**

0.48*

0.29ns

0.05ns

-0.25ns 1

VcF3'5'H

-0.10ns

-0.27ns

0.12ns

0.34ns

0.47*

-0.2ns

-0.2ns

1

VcDFR

0.19ns

0.30ns

0.34ns

0.43*

0.43*

0.09ns

0.27ns

-0.07ns

1

VcANS

0.23ns

-0.22ns

-0.04ns

0.59*** 0.64***

0.6***

0.02ns

0.41*

0.29ns

VcUFGT

0.22ns

0.09ns

0.46*

0.66**

0.49**

0.13ns

0.42*

0.64*** 0.58**

0.65**

498 499

***

VcUFGT

Correlation is significant at the 0.001 level, ** at the 0.01 level, * at the 0.05 level, or is NS non-significant.

24


1 1

Page 25 of 31

500


Figure Captions

501 502

Fig. 1. Real-time quantitative PCR analysis of changes in expression of anthocyanin biosynthesis

503

genes (A, VcPAL; B, VcCHS; C, VcF3H; D, VcF3’H; E, VcF3’5’H; F, VcDFR; G, VcANS;

504

H, VcUFGT) in the peels of harvested ‘Duke’ blueberry fruits without (control) or with

505

UV-B treatment at 6 kJ km-1 for 20 min (UV-B). Data were obtained from samples at

506

harvest, at 20 min, and at 3, 6, and 24 hours at room temperature. Data were expressed as

507

the mean ± SD of three replicates. *, significant at P < 0.05; **, significant at P < 0.01; ***,

508

significant at P < 0.001.

509 510

Fig. 2. Real-time quantitative PCR analysis of changes in gene expression of transcription factors

511

(A, VcBBX; B, VcMY21B; C, VcR2R3 MYB) in the peels of harvested ‘Duke’ blueberry

512

fruits without (control) or with UV-B treatment at 6 kJ km-1 for 20 min (UV-B). Data were

513

obtained from samples at harvest, at 20 min, and at 3, 6, and 24 hours at room temperature.

514

Data are the mean ± SD of three replicates. *, significant at P < 0.05; **, significant at P

VcBBX, VcMYB21, and VcR2R3MYB Transcription ... - ACS Publications

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