Preharvest Ultraviolet C Irradiation Increased the Level of Polyphenol

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Preharvest UV-C irradiation increased polyphenols accumulation and flavonoid pathway genes expression in strawberry fruit Yanqun Xu, Marie Thérèse Charles, Zisheng Luo, Benjamin Mimee, Pierre-Yves Véronneau, Daniel Rolland, and Dominique Roussel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04252 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 6, 2017

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Journal of Agricultural and Food Chemistry

Preharvest UV-C irradiation increased polyphenols accumulation and flavonoid pathway genes expression in strawberry fruit Yanqun Xu

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, Marie Thérèse Charles 2*, Zisheng Luo 1*, Benjamin Mimee2, Pierre-Yves

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Veronneau2, Daniel Rolland2, Dominique Roussel2

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1. Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory

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of Agro-Products Postharvest Handling Ministry of Agriculture, 310058, People’s Republic of

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China

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2. Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food

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Canada, Saint-Jean-sur-Richelieu, QC, J3B 3E6, Canada

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*Corresponding author:

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Marie Thérèse Charles

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Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada,

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Saint-Jean-sur-Richelieu, QC, J3B 3E6, Canada E-mail: [email protected] Phone:

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+1-579-2243072

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Zisheng Luo

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College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou

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310058, People’s Republic of China E-mail: [email protected] Phone: +86-571-88982175

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Highlights

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1. Preharvest UV-C in a specific range significantly increase flavonoids and ellagic acid in strawberry

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2. Flavonoid structural genes were up-regulated in the low- and middle-dose groups

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3. The ripening related gene, FaASR, was significantly enhanced in the low dose group

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4. Flavonoid early stage genes inhibition may explain the lack of anthocyanins stimulation

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by the high dose 5. Enzyme activities and genes expression were consistent with UV-C induced flavonoids increment

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Abstract: Preharvest UV-C irradiation is an innovative approach for increasing bioactive

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phytochemicals content in strawberry to increase disease resistance and nutrition value. This

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study investigated the changes in individual flavonoids in strawberry developed with three

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different cumulative doses of preharvest UV-C treatment (low: 9.6 kJ m-2; middle: 15 kJ m-2 and

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high: 29.4 kJ m-2). Significant accumulation (p < 0.05) of phenolics (25 % to 75% increase),

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namely cyanidin 3-glucoside, pelargonidin 3-glucoside/rutinoside, glucoside and glucuronide of

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quercetin and kaempferol, and ellagic acid, was found in the fruit with low and middle

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supplemental doses of UV-C irradiation. The expression of the flavonoid pathway structural

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genes, i.e., FaCHS1, FaCHI, FaFHT, FaDFR, FaFLS and FaFGT, were up-regulated in the low-

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and middle-dose groups, while the early stage genes were not affected by the high dose.

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FaMYB1 was also relatively enhanced in the low- and middle-dose groups, while FaASR was

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only up-regulated in the low dose group. Hormetic preharvest UV-C dose ranges to enhance the

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polyphenols content of strawberries were established for the first time.

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Keywords: Ellagic acid; Flavonoids; Fragaria×ananassa; Hormetic; MYB proteins; Preharvest

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UV-C; RT-qPCR.

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1. Introduction

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Flavonoids, which are ubiquitous in the plant kingdom, are polyphenolic pigments that give

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fruit their characteristic red, blue, and purple colours. Flavonoids perform many important

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biological functions which include protecting against cold, UV irradiation, and pathogen attack

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and increasing fruit postharvest quality and shelf life

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have documented that the consumption of flavonoids is associated with a lower incidence of

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chronic and metabolic diseases 3 through their indirect action on gut microbiota 4, 5.

1, 2

. Furthermore, epidemiologic studies

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The strawberry (Fragaria×ananassa, Duch), which has a considerable variety and

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abundance of polyphenolic constituents, is an economically important fruit crop worldwide and

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enjoys considerable popularity among consumers. Flavonoids in strawberry, represented by

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anthocyanins, flavonols and flavanols, are synthesized from phenylalanine via the

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phenylpropanoid and flavonoid pathways (Figure 1), which have been extensively studied at the

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genetic, biochemical and molecular levels

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acid by the action of phenylalanine ammonia lyase is the initial step in the phenylpropanoid

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pathway; trans-cinnamic acid is subsequently transformed into 4-coumaroyl-CoA. The resulting

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phenylpropanoids are then directed into the flavonoid pathway by the action of chalcone

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synthase (CHS). Further sequential reactions involving chalcone isomerase (CHI), flavanone 3ß-

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hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) lead

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to the synthesis of anthocyanindins. An important branch of this pathway is that of the enzyme

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flavonol synthase (FLS), which catalyzes the production of flavonols from dihydroflavonols; two

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other branches yield proanthocyanins. Lastly, various flavonoid glycosyltransferases (FGTs)

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modify anthocyanins and flavonols through the addition of sugar molecules, which modulates

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their physiological activities by increasing their polarity, solubility, reactivity and interaction

6, 7

. Conversion of L-phenylalanine to trans-cinnamic

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with cellular targets 8. In addition, genes of the flavonoid pathway are known to be coordinately

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induced by transcription factors 1. MYB proteins are known to serve as essential regulators in the

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biosynthesis of pigments in strawberry through interaction with MYC-like basic helix-loop-helix

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(bHLH) and WD40-repeat proteins 9. Another essential factor, the abscisic acid (ABA)-, stress-

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and ripening-induced gene (FaASR), contributes to the acceleration of strawberry fruit ripening

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and the accumulation of anthocyanin under stress 10, 11.

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Although the flavonoid content of strawberry is largely determined by genetic factors,

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external and environmental cues can also affect the quantitative and qualitative composition of

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flavonoids in ripening fruit. The expression of structural flavonoid biosynthesis genes in

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developing strawberry fruit is highly regulated by light conditions, temperature, chilling stress

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and nutritional status

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hormones

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flavonoid accumulation in stored strawberries.

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13

12

. Postharvest treatments such as the application of exogenous plant

, water-deficit stress stimulation

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and modified storage conditions

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influence

Application of ultraviolet C (UV-C) light is identified as an environmentally friendly 15, 16

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approach for enhancing health promoting phytochemicals in fruit

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irradiation was found to increase total anthocyanin and phenolic compounds with concurrent

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stimulation of flavonoid pathway transcripts in stored strawberry

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there is a lack of detail knowledge on fruit responses to preharvest application of UV-C radiation.

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A few and fairly recent studies have examined the potential such an approach offers in relation to

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pathogen resistance 19-22 and fruit quality improvement 23-25 . Both Xie et al.23 and de Oliviera et

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al. 25 demonstrated that applying a unique cumulative dose of UV-C to strawberries imparted

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changes in polyphenols content. Xie et al. 23 showed that the response was cultivar dependent. In

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other plant systems, the effect of preharvest UV-C treatment on biochemical stimulation also

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. Postharvest UV-C

and tomato

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. However,

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showed strong dose dependency. Significant enhancement of trans-resveratrol accumulation was

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found in preharvest UV-C treated grapes, with this effect being dependent not only on the dose

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but also on how the dose was applied in terms of power output and exposure time 26. In a recent

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study, Xu et al.,

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strawberry, was differently affected by a range of preharvest UV-C treatments. Therefore, in the

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present study three different cumulative UV-C radiation doses were applied to strawberry plants

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in an attempt to establish the effective range for increase health-related polyphenols in

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strawberry fruit. The molecular analysis focused on the structural genes and transcription factors

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of the flavonoid pathway with the aim of identifying the major steps regulating of the impact of

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the different UV-C doses.

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2. Materials and methods

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2.1 Plant material

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have shown that abscisic acid, a key player in anthocyanins accumulation in

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Strawberry plants (Fragaria × ananassa Duch, cv. Albion) were planted in 15-cm-diameter

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pots filled with mixed growth medium (PRO-MIX, Rivière-du-Loup, Canada) and grown in a

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growth chamber (Conviron, PGV40, Manitoba, Canada). The cultivation conditions were as

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follows: 20 °C (day) and 15 °C (night), 50 % relative humidity and 15-h photoperiod with light

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intensity of 500 µmol m-2 s-1. The plants were fertilized with a nutrient solution containing 200

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ppm of nitrogen, 200 ppm of phosphorus and 71 ppm of potassium from Monday to Friday and

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received only water on Saturday and Sunday. The nutrient solution was prepared using Plant-

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Prod 6-11-31 Hydroponic and calcium nitrate (PlantProducts, Leamington, ON, Canada). No

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fungicides were applied during the experiment. At the onset of flowering, 108 plants with similar

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growth status were selected and randomly separated into four groups of 27 plants, providing

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three replicates of nine plants. Three groups were assigned to receive the different doses of

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experimental UV-C irradiation and one group was set as the control.

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2.2 UV-C radiation exposure

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The UV-C treatment was carried out in a modified growth chamber with three supplemental

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UV-C lamps (254 nm, 160W; Clean Light Inc., Vineland Station, ON, Canada) placed

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horizontally on the ceiling. The UV-C light intensity was monitored using a portable radiometer

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(ML1400A; Miltec UV, Stevensville, MD, USA) equipped with a SEL240 #6090 sensor

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(ML1400A; Miltec UV). The potted plants in each group were transferred to the chamber when

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it was time for them to be treated. UV-C lamps were at a distance of 70 cm from the top of the

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plants. Irradiation at 0.6 kJ m-2 was applied to the plants as described by Xie et al.

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rate of 8.57 J m-2 s-1. There were four irradiation conditions, namely a low dose group in which

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the plants were exposed every three days and received a total cumulative dose of 9.6 kJ m-2 by

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the end of treatment; a middle dose group receiving 15 kJ m-2 by irradiating every two days; a

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high dose group receiving 29.4 kJ m-2 by irradiating daily; and a control group that received no

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irradiation. UV-C radiation was always applied at the onset of the dark cycle of the photoperiod

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and the treated plants were kept in darkness until the next day, when the light cycle of the

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photoperiod resumed. The irradiation treatments started when the first flowers were wide open.

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UV-C treatments lasted 7 weeks, corresponding to the entire period of fruit production, and were

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stopped when the plants no longer produced well-developed flowers and fruit. All flowers were

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hand-pollinated using a feather, and under the experimental conditions the ripening time was

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about 26 days. At the end of the experiment none of the treated plants displayed visible signs of

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damage. To promote fruit development, the light and temperature conditions provided during the

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treatment period were as follows: for the first 3 weeks of treatment, 20 °C (day), 15 °C (night)

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at a dose

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with 10 h photoperiod, to maintain flower production; and for the last 4 weeks, 25 °C (day),

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15 °C (night) with 13 h photoperiod, to facilitate fruit ripening. The fruit were harvested once

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they were fully red, at the commercial maturity level.

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The fruit were cut into small pieces, frozen in liquid nitrogen and further broken to smaller

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pieces with the aid of mortar and pestle and stored at -80 °C. All the fruit with marketable quality

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produce during the entire experimental period were collected. Fruit from same replicate,

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harvested at different days were mixed together at the end of the experiment to have composite

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samples (around 40 fruits totally with 300–400 g for each replicate) from which two sub-

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replicates were drawn for the biochemical analysis.

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2.3 Identification and quantification of individual flavonoids by UPLC-PDA

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Three grams of frozen tissues were homogenized with 15 mL of acetone. The homogenate

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was then centrifuged at 10,000 g for 20 min at 4 °C. A volume of 7 ml of the acetone fraction

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was taken and evaporated under N2 at 37 °C. The residue was re-dissolved with 5 mL of 3 %

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formic acid. The mixture was loaded onto a C18 Sep-Pak cartridge (Waters Ltd, Mississauga,

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ON, Canada) that was preconditioned with 5 ml of methanol, followed by 6 mL of H2O-MS and

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7 ml of 3 % formic acid. The phenolic compounds were eluted with acidified MeOH (3 mL, 3 %

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formic acid), and 3 ml of H2O was added to the elution vial. The extracts were passed through a

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0.20-µm filter and then transferred to injection bottles.

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The extracts were analyzed using an ultra-performance liquid chromatography (UPLC)

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system (Acquity, H-Class, Waters Corp., Milford, MA, USA) equipped with a quaternary solvent

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manager, a sample manager, a column compartment, and a PDA eλ detector. The instrument was

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operated using MassLynx 4.1 software. An injection volume of 2 µl was used. The separation of

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all compounds was carried out with a Waters Acquity UPLC Cortecs C18 column (150 mm × 2.1

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mm, 1.6 µm) in series with a Waters Acquity UPLC Cortecs VanGuard C18 (5 mm × 2.1 mm,

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1.6 µm) pre-column. The mobile phase consisted of acidified water (1% formic acid) (A) and

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acidified methanol (1 % formic acid) (B). The gradient system involved a 26-min elution period

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at a flow rate of 0.216 mL min-1: linear gradient from 1 to 5 min with solvent A from 85 % to

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70 %, followed by an isocratic mixture for another 2 min; then solvent A changed from 70 % to

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20 % in 2 min, and this composition was maintained for 3 min; afterward, solution A decreased

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to 0% in 1 min and was maintained for 2 min; the system returned to the initial conditions (85 %

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solution A) in 1 min and was maintained without change for the last 10 min.

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The signals were monitored between 210 and 750 nm and identified on the PDA detector.

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The phenolic compounds of the samples were identified based on their retention time and their

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UV-visible absorption spectra by comparison with external standards. The quantification of

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individual compounds was performed according to their respective maximum absorption

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wavelengths as follows: 280 nm for ellagic acid (EA); 365 nm for kaempferol 3-glucoside (K3G),

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kaempferol 3-glucuronide (K3Gr), quercetin 3-glucoside (Q3G) and quercetin 3-glucuronide

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(Q3Gr); 503 nm for pelargonidin 3-glucoside (P3G) and pelargonidin 3-rutinoside (P3R); 519

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nm for cyanidin 3-glucoside (C3G). The results were expressed as mg kg-1 on a fresh weight

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basis. Pure EA and P3R were purchased from Apin Chemicals (Abingdon, UK) and the other

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standards were from Sigma Aldrich (Oakville, ON, Canada).

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2.4 Activities of PAL and TAL

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Enzyme extraction was performed based on the method of Montero et al.

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with some

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modifications. Two sub-replicates of frozen fruit samples (5 g) were homogenized with cold

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acetone and the insoluble residue was filtered and dried under vacuum. The residue was then

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vortexed with 10 ml of extraction buffer containing 100 mM sodium borate (pH 8.8), 5 mM ß-

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mercaptoethanol, 2 mM EDTA and 1 g polyvinylpyrrolidone (PVP). The mixture was kept at

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4 °C for 1 h and then centrifuged at 4 °C for 15 min. The supernatant was used as a crude

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enzyme extract.

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PAL (EC 4.3.1.5) and TAL (EC 4.3.1) activities were determined from the production of

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trans-cinnamic acid from L-phenylalanine and the formation of p-coumaric acid from L-tyrosine,

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respectively (Montero et al., 1998). Enzyme extract (0.5 mL) was added to 2.7 ml of 30 mM

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sodium borate buffer (pH 8.8) containing 6 mM L-phenylalanine for PAL assays or 3 mM L-

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tyrosine for TAL assays. The mixture was incubated at 37 °C for 2 h and the absorbance changes

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at 290 nm (PAL) and 333 nm (TAL) were recorded. Molar extinction coefficients of 19207 for

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trans-cinnamic acid and 6886 for p-coumaric acid were used. One enzyme unit (U) was defined

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the production of 1 mmol trans-cinnamic acid h-1 for PAL and 1 mmol p-coumaric acid h-1 for

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PAL. The specific activity was expressed as U kg-1 protein, where the total protein content was

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determined according to Lowry et al. 29

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2.5 RNA isolation and first-strand cDNA synthesis

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Frozen fruit samples comprising about 5 g in each replicate were ground to a fine power in

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liquid nitrogen using a mortar and pestle. Total RNA was extracted from approximately 100 mg

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of tissue powder using Spectrum Plant Total RNA kit (Sigma Aldrich, Oakville, ON, Canada).

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Contaminating genomic DNA was removed by RNAse-free DNAase I (NEB, Pickering, ON,

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Canada) with a 10-min incubation at 37 °C. The integrity and quality of the extracted RNA were

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determined using a Bioanalyzer 2100 (Agilent, Cedar Creek, TX, USA) with the RNA 6000

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Nano kit (Agilent, Cedar Creek, TX, USA) according to the manufacturer’s protocol, and an

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RNA integrity number over 8.6 was accepted. First-strand cDNA was synthesized from 1 µg of

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total RNA with 200 ng of oligo-d(T)17 using SuperScript III (Invitrogen, Burlington, ON, Canada)

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according to the manufacturer’s instructions in a total volume of 20 µl. To remove RNA

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complementary to the cDNA, nucleic acid samples were incubated with 1 µl of RNase H (NEB,

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Pickering, ON, Canada) at 37 °C for 20 min. The cDNA concentration in the RT mix was

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measured using Qubit ssDNA Assay kit (Invitrogen, Burlington, ON, Canada) fluorometric

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quantitation according to the manufacturer’s protocol. The cDNA was diluted to 2 ng µl-1 with

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DEPC-H2O and used as templates for gene expression analysis.

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2.6 Gene expression analysis by RT-qPCR

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Gene expression was analyzed with real-time quantitative PCR (RT-qPCR) using the

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selected specific primers listed in Table 1. Primers for seven structural flavonoid genes and the

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transcript regulator, FaMYB1, were designed on the sequences reported by Almeida et al.6;

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FaPAL, a key gene of the phenylpropanoid pathway, was referred to Pombo et al. 30; FaASR, a

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stress response gene, was obtained according to Ayub et al.

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reference gene. The specificity of the primers was validated in silico using primer BLAST

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analysis (http://www.ncbi.nlm.nih.gov/Blast.cgi) and then according to the melting curve

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obtained from RT-qPCR as described below. RT-qPCR was performed using the MxPro 3000P

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qPCR system (Agilent, Cedar Creek, TX, USA) with a QuantiTect SYBR Green PCR kit

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(Qiagen, Mississauga, ON, Canada). PCR assays were performed in the following reaction

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mixture: 10 ng of cDNA, 300 nM of each primer, and 10 µl of 2× SYBR green PCR master mix

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in a total volume of 20 µl. PCR reactions were conducted under the following conditions: 15 min

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at 90 °C; 45 cycles at 94 °C for 15 s, 52 °C for 30 s and 72 °C for 30 s. Melting curve analysis

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was performed at 95 °C for 60 s, at 65 °C for 30 s, and at 95 °C for 30 s. All the qPCR reactions

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were normalized by the comparative (2-∆∆CT) method using the CT value corresponding to F-

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Actin. No-template controls and melting curve analyses were included for each gene and PCR

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; and actin was used as the

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reaction. Three PCR runs were carried out for each cDNA and gene to serve as technical

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replicates.

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2.7 Statistical analysis

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The experiment used a completely randomized design with three replicates (9 plants per

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replicate) of four treatment groups (control, UV-C low, UV-C middle and UV-C high). Data

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were expressed as mean ± standard deviation (SD) and evaluated by one-way analysis of

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variance (ANOVA) followed by the least significant difference (LSD) test at p < 0.05 when

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necessary, to separate the means. All statistical analyses were performed using the SAS software

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package (SAS Institute Inc., Cary, NC, USA) and the graphs were plotted using GraphPad Prism

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6.01 (GraphPad Software Inc., San Diego, CA).

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3. Results

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3.1 Effect of preharvest UV-C treatments on flavonoid content of ripe strawberry fruit

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As shown in Figure 2, three anthocyanins, namely pelargonidin 3-glucoside/rutinoside and

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cyanidin 3-glucoside; four flavonols, specifically glucoside and glucuronide of quercetin and

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kaempferol; and a non-flavonoid, ellagic acid, were identified as the main polyphenolic

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compounds extracted from strawberry cv. Albion. Pelargonidin 3-glucoside was the dominant

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anthocyanin with a content ten times higher than that of the other anthocyanins, followed by the

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flavonol quercetin 3-glucuronide (Figure 2). In the present study, the concentrations of the

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individual phenolic compounds were all significantly (p < 0.05) influenced by preharvest UV-C

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treatments. The fruit subjected to the low- and middle-doses of UV-C irradiation showed

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significantly (p < 0.05) higher accumulation of these polyphenols relative to the high dose group

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and the control (Figure 3). In the extracts from low- and middle-dose groups, the content of

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cyanidin 3-glucoside, was approximately 43 % higher than in the other two groups (Figure 3A).

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Quercetin 3-glucuronide, for which the content was 75 % and 56.4 % higher in the middle and

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low doses, respectively, compared to the control, was the phenolic compound influenced to the

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greatest extent by prehavest UV-C (Figure 3D). Although quercetin 3-glucoside was the least

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enhanced compound, its content was nonetheless increased by 25 % in samples of fruit irradiated

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with supplemental UV-C light every two or three days (Figure 3F). Total anthocyanin content

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(Figure 3 A, B, C) was increased by 43.8 % and 43.5 % in the low- and middle-dose groups,

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respectively, when compared to the control. For flavonols (Figure4, D, E, F), the total

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anthocyanin content was 35.4 % and 50.3 % higher in these two groups, respectively, relative to

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the control. Ellagic acid (Figure 3G) was also significantly stimulated in samples subjected to

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middle dose irradiation. In the middle dose group, at 29.66 µg g-1, ellagic acid was increased by

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72.5 % relative to the control, while the content in the low dose group was increased by 57.9 %.

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In the fruit treated with the high UV-C dose, no significant change was found in the contents of

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these phenolics relative to the control (Figure 3).

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3.2 Effect of preharvest UV-C treatments on PAL and TAL activities

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PAL activity was significantly (p < 0.05) stimulated in all fruit subjected to UV-C

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irradiation, showing increases of 28.9 %, 13.0 % and 26.7 % in the low, middle and high dose

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groups, respectively, compared to the control (Figure 4 A). Significant (p < 0.05) stimulation of

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TAL by preharvest UV-C treatment was also found in the middle and high UV-C dose groups,

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with 41.0 % and 46.2 % higher activity (Figure 4B). However, TAL activity in fruit treated with

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the low dose of UV-C light showed no obvious alteration (Figure 4B).

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3.3 Effect of preharvest UV-C treatment on the expression of flavonoid biosynthesis pathway

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genes

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The FaPAL transcript and eight structural genes of the flavonoid pathway were investigated

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in ripe fruit developed while subjected to different preharvest UV-C radiation doses as shown in

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Figure 5. Prehavest UV-C significantly (p < 0.05) increased the expression of FaPAL, especially

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in the middle (4.9×) and high (4.2×) dose groups, which had significantly higher expression

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levels than the low group (2.1×) (Figure 5A).

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The expression level of transcripts FaCHS1 (Figure 5B), FaCHI (Figure 5C) and FaFHT

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(Figure 5D) showed similar patterns of change among the different UV-C treatments, with the

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highest expression level found in the low dose UV-C treated group, which showed significant

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increases of 15×, 82× and 3× relative to the control, respectively (p < 0.05). These three genes

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were also affected by the middle dose treatment, with respective increments of 1.8×, 3.3× and

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1.6×, although only the change in FaCHI was significant (p < 0.05). The expression of two genes,

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FaDFR (Figure 5E) and FaFLS (Figure 5F), was significantly increased in the fruit that received

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the middle (2.7× and 1.4×) and high (2.9× and 1.7×) dose treatments. However, the highest

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increase in FaDFR expression (22×) relative to the control was found in the low dose group. No

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significant change in FaANS was observed in the treated samples compared to the control. The

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expression level of FaFGT was similar to that for FaFLS, with significant stimulation found in

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the middle dose (27×) and high dose (19×) groups.

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3.4 Effect of preharvest UV-C treatment on FaMYB1 and FaASR transcription

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The relative expression of a flavonoid gene transcription factor, FaMYB1, and of the ABA-

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stress-ripening (ASR) transcription factor, FaASR, were also quantified in this study (Figure 6).

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The level of FaMYB1 transcripts was slightly up-regulated (p < 0.05, around 1.5×) in the low-

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and middle-dose groups, while that of fruit in the high UV-C group showed no obvious change

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(Figure 6A) compared to the control. The expression of FaASR (Figure 6 B) was greatly (p