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

1, 2

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

4

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

15

Zisheng Luo

16

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

18

ACS Paragon Plus Environment


Highlights

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

22

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

23

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

24

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

25 26 27

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

28


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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

32

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

35

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

36

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

37

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

38

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

39

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

40

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

43

UV-C; RT-qPCR.



44

1. Introduction

45

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

46

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

47

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

48

and increasing fruit postharvest quality and shelf life

49

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

50

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

1, 2

. Furthermore, epidemiologic studies

51

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

52

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

54

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

55

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

56

genetic, biochemical and molecular levels

57

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

58

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

60

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

61

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

66

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

69

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

70

(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.

80

13

12

. Postharvest treatments such as the application of exogenous plant

, water-deficit stress stimulation

11

and modified storage conditions

14

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

89

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


17

. Postharvest UV-C

and tomato

18

. However,


90

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

92

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

99

the different UV-C doses.

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2. Materials and methods

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2.1 Plant material

27

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

133

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

134

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


24

at a dose


135

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,

149

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)

154

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

155

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

160

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.

28

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-



181

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

190

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

200

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,

206

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

207

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

208

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

209

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

210

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

213

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

214

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

215

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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


31

; and actin was used as the


227

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

228

replicates.

229

2.7 Statistical analysis

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

231

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

232

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

233

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

234

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

235

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

236

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

these phenolics relative to the control (Figure 3).

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

genes



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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

Preharvest Ultraviolet C Irradiation Increased the Level of Polyphenol

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