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Effect of developmental stage and reduced UVB and low UV conditions on secondary plant metabolite profiles in pak choi (Brassica rapa spp. chinensis) Mandy Heinze, F Hanschen, Melanie Wiesner-Reinhold, Susanne Baldermann, Jan Graefe, Monika Schreiner, and Susanne Neugart J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03996 • Publication Date (Web): 03 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Journal of Agricultural and Food Chemistry

Graphical abstract

Effect of developmental stage and reduced UVB and low UV conditions on secondary metabolite profiles in pak choi (Brassica rapa spp. chinensis)

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Effect of developmental stage and reduced UVB and low UV conditions on secondary

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plant metabolite profiles in pak choi (Brassica rapa spp. chinensis)

3

Mandy

4

Baldermann1,2, Jan Gräfe1, Monika Schreiner1, Susanne Neugart1,3

5

Address

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1

7

Grossbeeren, Germany

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2

9

14558 Nuthetal, Germany

Heinze1,

Franziska

S.

Hanschen1,

Melanie

Susanne

Leibniz Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, 14979

Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116,

10

3

11

Avenue, 70118 New Orleans, LA, USA

Department of Biological Sciences, Loyola University New Orleans, 6363 St. Charles

12

13

14

Wiesner-Reinhold1,

Corresponding author: Dr. Susanne Neugart, [email protected]

1 Environment ACS Paragon Plus


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Abstract

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Pak choi (Brassica rapa ssp. chinensis) is rich in secondary metabolites and contains

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numerous antioxidants, including flavonoids and hydroxycinnamic acids as well as

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carotenoids and chlorophylls, along with glucosinolates that can be hydrolysed to

19

epithionitriles, nitriles, or isothiocyanates. Here, we investigate the effect of reduced exposure

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to ultraviolet B (UVB) and UV (reduced UVA and UVB) at four different developmental

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stages of pak choi. We found that both plant morphology and the secondary metabolite

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profiles were affected by reduced exposure to reduced UVB and low UV depending on the

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plant’s developmental stage. In detail, mature 15- and 30-leaf plants had higher

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concentrations of flavonoids and hydroxycinnamic acids as well as carotenoids and

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chlorophylls, whereas sprouts contained high concentrations of glucosinolates and their

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hydrolysis products. Dry weight and leaf area increased due to the reduced UVB and low UV.

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In 30-leaf plants, for flavonoids and hydroxycinnamic acids, less complex compounds were

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favoured, e.g. sinapic acid acylated kaempferol triglycoside instead of the corresponding

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tetraglycosides. Moreover, also in 30-leaf plants, zeaxanthin, a carotenoid linked to protection

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during photosynthesis, was increased under low UV conditions. Interestingly, most

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glucosinolates were not affected by reduced UVB and low UV conditions. However, this

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study underlines the importance of 4-(methylsulphinyl)butyl glucosinolate in response to

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exposure to UVA and UVB. Further, reduced UVB and low UV conditions resulted in higher

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concentrations of glucosinolate-derived nitriles. In conclusion, exposure to low doses of UVB

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and low UV from early to late developmental stages did not result in overall lower

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concentrations of secondary plant metabolites.

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Keywords: flavonoids, hydroxycinnamic acids, carotenoids, glucosinolates, nitriles, UVB

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reduction 2 Environment ACS Paragon Plus

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Introduction

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Pak choi (Brassica rapa ssp. chinensis) is a leafy vegetable that contains secondary plant

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metabolites such as carotenoids, chlorophylls and phenolic compounds, namely flavonoids

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and hydroxycinnamic acids, many of which have been reported to confer health-promoting

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effects

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anthocyanins. Moreover, pak choi also synthesizes comparatively high amounts of aliphatic

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glucosinolates 3. Upon cell disruption, glucosinolates are hydrolysed by the endogenous

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myrosinase and, among epithionitriles and nitriles, the health-promoting isothiocyanates are

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released 4.

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Plant development from sprouts to the mature plants is linked to an increase in fresh weight as

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well as lea area. For example, in tropical tree species, flavonoid concentrations decreased with

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leaf age

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hydrocycinnamic acids decreased during progressive developmental stages 6. In lime,

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chlorophyll and carotenoid concentrations were highest in middle-aged leaves 7. Finally, in

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the model plant Arabidopsis, the highest total glucosinolate concentrations were found in

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siliques, florescences and seeds sprouts

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choi cultivars

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conditions, thereby leading to inconclusive evidence of their direct effects on secondary plant

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metabolite formation.

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Ultraviolet (UV) radiation is a key environmental factor that affects plant development and is

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a natural part of solar radiation. Highly energetic shorter wavelengths of solar UV (UVB;

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290-315 nm, UVA 315-400 nm) can potentially induce a number of deleterious effects in

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plants, including disruption of the integrity and function of important macromolecules (DNA,

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proteins and lipids), oxidative damage, changes in plant biochemistry, partial inhibition of

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photosynthesis and growth reduction

1, 2

. The cultivar Amur mainly has red coloured leaves due to its ability to produce

5

, while in bilberry, the flavonoid glycoside concentrations increased, but

8

– a finding that was also observed in several pak

9

. Ontogenetic effects are often strongly influenced by environmental

10, 11

. Consequently, UVB has traditionally been



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considered as a stressor that can cause serious plant damage. However, recent studies have

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highlighted the regulatory properties of low, ecologically-relevant UVB levels that trigger

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distinct changes resulting in a structure-dependent accumulation of secondary plant

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metabolites

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that function as UV-shielding components and antioxidants

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secondary plant metabolites is rare; however, a previous study has shown that UVB also

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affects glucosinolates and carotenoids and that these changes can ultimately affect overall

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plant quality

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glycosides to UVB is dependent on the chemical structure of the compound as well as on the

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duration of UVB treatment and the plant’s adaptation time

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containing a catechol structure have also been reported to exhibit high antioxidant activity 17.

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Thus in Brassicales species, ambient doses of UVB radiation led to an enhanced quercetin to

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kaempferol ratio compared to plants that did not undergo exposure to UVB 18. In Arabidopsis,

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mainly quercetin glycoside concentration were enhanced by greater exposure to UVB

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radiation

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absorb light for photosynthesis. Xanthophylls are involved in light-dependent reactions and

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play a key role in photoprotection of the photosystems

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concentrations are decreased at higher UVB levels

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exposure to UVB was observed to lead to higher carotenoid concentrations

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enhanced UVA resulted in higher concentrations of β-carotene, neoxanthin and zeaxanthin as

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well as chlorophylls a and b 24. Only a limited number of glucosinolate compounds have been

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shown to respond to enhanced, but non-stressing, UVB radiation, with an increase in 4-

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(methylsulphinyl)butyl glucosinolate in broccoli and benzyl glucosinolate in nasturtium 25, 26.

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To date, many studies have focused on the effects of exposure to increased levels of UVB

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radiation on the plant's secondary metabolism, and consequently, their profile. However, it

12

. It can enhance concentrations of flavonoids and related phenolic compounds 13, 14

. The investigation of other

12

. Previous investigations on kale underline that the response of flavonoid

15, 16

. Phenolic compounds

19

. Plants possess protein-pigment complexes (chlorophylls and carotenoids) that

20

. Carotenoids and chlorophyll b

21, 22

. However, in rapeseed, enhanced


23

. In laurel,

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should be borne in mind that most greenhouse-grown plants are not exposed to high levels of

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UV radiation since widely used glass and plastic covers are not permeable to UVB, and only

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in small part, to UVA. The regulatory effects of this reduced exposure to UV, and

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consequently, changes in the biosynthesis of secondary plant metabolites have to date not

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been studied in detail. In corn crops, such as sorghum and wheat, reduced exposure to UVB

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and UVA radiation was found to result in higher growth, yield and photosynthetic pigment

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concentrations (cholorophyll and carotenoids) 27, 28. Moreover, in tomato fruits, lycopene was

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increased in flesh and peel as consequence of UVB decrease

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species, a structure-specific increase of flavonoid glycosides, but a decrease of phenolic acids

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was found after UVB exclusion, while total UV exclusion, including both UVA and UVB, led

29, 30

. Finally, in Brassicales

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to low concentrations of flavonoid glycosides and phenolic acids 18.

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We therefore hypothesize that the regulatory effects, and consequently, biosynthesis of

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secondary plant metabolites, due to reduced exposure to UVB and low UV, generate different

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changes at different developmental stages. These changes may also affect overall plant quality

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in terms of plant growth and development, aroma/smell, taste, colour and human health-

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promoting properties. Thus, in the present study, we investigated whether reduced exposure to

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UVB or low UV (reduced UVA and UVB) during growth of pak choi has an ontogenetic-

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dependent impact on (1) fresh and dry weight and leaf area as well as (2) different secondary

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plant metabolites, namely flavonoid glycosides and hydroxycinnamic acid derivatives,

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carotenoids and chlorophylls as well as glucosinolates, along with the formation of their

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respective breakdown products. This study provides deeper insights into the plant’s UV

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response occurring under controlled environmental conditions and will help to develop new

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and improved management strategies to achieve increased plant quality in key horticultural

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crops.

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Materials and Methods 5 Environment ACS Paragon Plus


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Experimental conditions and design

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To produce pak choi sprouts, 0.8 g pak choi seeds (Brassica rapa ssp. chinensis cv. Amur)

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(Carl Pabst, Samen & Saaten GmbH, Grossbeeren, Germany) were sowed in Ø 9 cm pots. For

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the later developmental stages, 3 seeds per pot were sowed and after germination singularized.

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Plants were grown in a phytochamber (Johnson Controls, Essen, Germany) under controlled

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conditions (humidity: 70%; temperature: 22°C/18°C day/night each 12 h; light intensity

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photosynthetic active radiation (PAR) 500 µmol m-² s-1 under: control (0.059 kJ m-2 h-1 UVB),

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reduced UVB (0.017 kJ m-2 h-1 UVB) and low UV (0.002 kJ m-2 h-1 UVB)) for a duration of

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up to 12 weeks. To modulate the UV spectra, filters of different transmission characteristics

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were used. The Autostat CT5 filter (Gabler Druck- und Werbetechnikbedarf GmbH, Bochum,

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Germany) was selected to reduce particular UVB radiation, while the Rosco filter

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(Avantgarde Beleuchtungs- und Bühnentechnik GmbH, Berlin, Germany) was chosen to

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further lower UVB and UVA radiation since this filter is nearly non-permeable to UVA and

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UVB radiation. A polyethylene filter (Etra OY, Helsinki, Finland) was used as the control that

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is permeable to both UVA and UVB radiation, thus reflecting ambient UV conditions (Tab.

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1). Side plants were grown in the study, but excluded from the analysis of the effects on

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morphology and secondary plant metabolite profiles. Pak choi plants were harvested at four

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developmental stages, namely as sprouts and 5-leaf, 15-leaf, and mature 30-leaf plants. Per

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UV treatment (control, reduced UVB and low UV), four biological replicates consisting of

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three pooled plants (for the sprouts several plants per pot) were included. Fresh weight and

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dry weight were monitored. From 5-leaf plants onwards, the number of leaves was recorded,

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and the leaf area was investigated by imaging software (ImageJ). Leaf blades were removed

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for chemical analysis of secondary plant metabolites. For the direct analysis of glucosinolate

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breakdown products from fresh leaf material at the harvest day, 300 mg of mixed leaves per

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biological replicate were used, 0.5 ml of distilled water was added and the sample was then


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homogenized using a Retsch mixer mill (MM400 Retsch GmbH, Haan, Germany). For the

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analysis of other secondary plant metabolites, leaves without a middle rib from one plant were

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taken per biological replicate and frozen immediately in liquid nitrogen. For the analysis of

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carotenoids and chlorophylls, the samples were homogenized using the mixer mill cooled

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with liquid nitrogen and kept at -80°C. For the chemical analysis of glucosinolates and

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phenolic compounds (flavonoids and hydroxycinnamic acid derivatives), the samples were

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lyophilized for a minimum of 72 hours, and subsequently, ground to a fine powder.

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Extraction and chemical analysis of secondary plant metabolites

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Flavonoid glycosides and hydroxycinnamic acid derivatives

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To analyse the phenolic compounds, 20 mg lyophilized pak choi sample were extracted with

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60% methanol (per analysis, Carl Roth GmbH, Karlsruhe, Germany) as according to Neugart,

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et al.

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derivatives, an HPLC series 1100 (Agilent Technologies, Waldbronn, Germany) was used

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according to Neugart, et al. 32.

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Carotenoids and chlorophylls

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The

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methanol:tetrahydrofuran (1:1 / v:v, HPLC grade, Carl Roth GmbH, Karlsruhe, Germany).

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Therefore, the appropriate amount of frozen sample was weighed into a glass vial and the

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extraction solvent added before the sample thawed. Qualitative and quantitative analyses were

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performed by means of an UHPLC-DAD-QToF instrument (Agilent Technologies,

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Waldbronn, Germany) equipped with a long flow cell using external calibration standard

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curves 33, 34.

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Glucosinolates

31

. For the quantitative analysis of flavonoid glycosides and hydroxycinnamic acid

pigments

of

5

mg

fresh

material

were


extracted

completely

using


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Glucosinolate concentration was determined as desulpho-glucosinolates according to Witzel,

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et al. 35. Briefly, 20 mg powdered samples were extracted with 70% methanol (LC-MS grade,

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Th. Geyer GmbH & Co. KG, Renningen, Germany) and analysed using a 1290 Infinity

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UHPLC-DAD (Agilent Technologies, Waldbronn, Germany). Desulpho-glucosinolate

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concentration was calculated by the peak area relative to the area of the internal standard of

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desulphated 2-propenyl glucosinolate (HPLC grade, Carl Roth GmbH, Karlsruhe, Germany).

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Glucosinolate hydrolysis products

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After homogenization of plant tissues as described above, samples were left for at least 30

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min at room temperature, in order to achieve complete hydrolysis of the present

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glucosinolates. After which, the glucosinolate hydrolysis products were extracted as described

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in Hanschen et al. 36. Briefly, hydrolysis products were extracted twice using 2 mL methylene

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chloride (GC Ultra Grade, Carl Roth GmbH, Karlsruhe, Germany). After adding the internal

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standard benzonitrile (0.2 µmol, ≥ 99.9%, Sigma-Aldrich Chemie GmbH, Steinheim,

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Germany), extracts were dried using NaSO4 (≥ 99%, VWR International GmbH, Darmstadt,

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Germany), concentrated to 300 µL under N2 and subjected to GC-MS analysis using an

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Agilent 7890A Series GC System with an Agilent 5975C inert XL MSD. Analytes were

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analysed and quantified as previously reported 35.

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Statistics

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All data were statistically analysed by a two-factorial ANOVA. Fisher’s F test was performed

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to assess the main effect of plant developmental stage and UV treatment and their interactions

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followed by a comparison of the factor levels using Tukey’s HSD test. For not significant

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interactions, the means of levels of main factors were separated, while for significant

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interactions, all combinations of the factor levels were compared. Residuals were tested for

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Gaussian distribution using the Kolmogorov-Smirnov test. All tests were conducted at a


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significance level of 5%. Calculations were performed using StatisticaTM for WindowsTM

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(version 9.0, Statsoft Inc., Tulsa, Okla.).

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Results

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Effects of reduced UVB and low UV on fresh and dry weight as well as leaf area at different

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plant developmental stages

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In comparison to the control plants, pak choi sprouts had higher fresh weight under reduced

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UVB conditions, whereas under low UV conditions, fresh weight decreased. However, dry

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weight was not affected by either UV treatment in the sprouts. During plant development,

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fresh weight of pak choi plants increased from approximately 1 g per plant (5-leaf plants) to

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approximately 180 g per plant (30-leaf plants) and was not affected by either UV treatment. In

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general, the plant dry weight was approximately 7% of fresh weight in 5- and 15-leaf plants

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and increased to 15-20% of fresh weight in 30-leaf plants. The reduced UVB and low UV

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resulted in higher dry weights in 30-leaf plants. The leaf area increased with developmental

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stage from 38 cm² per plant (5-leaf plants) to approximately 950 cm² per plant (30-leaf

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plants). Low UV resulted in larger leaf areas in the 5- and 30-leaf pak choi plants compared to

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reduced UVB plants (Fig. 1).

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Effects of plant developmental stage and reduced UVB and low UV on flavonoid glycosides

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and hydroxycinnamic acid derivatives

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In pak choi, a high number of structurally different flavonoid glycosides and

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hydroxycinnamic acid derivatives were identified. Kaempferol glycosides (Tab. 2) are the

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main flavonoids in pak choi and increased with ongoing plant development. A detailed look

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showed that the non-acylated di- and triglycosides had the highest concentrations in the 30-

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leaf pak choi plants. The monoacylated kaempferol triglycosides showed a structure-

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dependent response to reduced UVB and low UV regarding the hydroxycinnamic acids that 9 Environment ACS Paragon Plus


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were acylated. While the coumaric acid and caffeic acid acylated kaempferol triglycosides

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had the highest concentrations in 5-leaf pak choi plants, ferulic acid, hydroxyferulic acid and

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sinapic acid acylated triglycosides had the highest concentrations in 30-leaf plants. The

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sinapic acid acylated tetraglycoside was highest in pak choi sprouts and then rapidly

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decreased with ongoing plant development. Interactions of developmental stage and UV

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treatment were only found in high complex monoacylated kaempferol tri- and tetraglycosides,

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namely

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hydroxyferuloyl-sophoroside-7-O-glucoside,

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glucoside, and kaempferol-3-O-sinapoyl-sophoroside-7-O-diglucoside, clearly highlighting

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that the effect of reduced UVB and low UV varied at different developmental stages. In the

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triglycosides, the highest concentrations were measured in 30-leaf plants with reduced UVB

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(1.9- to 2.2-fold increase compared to the control), whereas the tetraglycoside, kaempferol-3-

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O-sinapoyl-sophoroside-7-O-diglucoside, had the highest concentrations in sprouts with

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reduced UVB with a 1.4-fold increase compared to the control plants. For quercetin

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glycosides, the non-acylated di- and triglycosides of quercetin as well as the non-acylated

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isorhamnetin monoglycoside, the highest concentrations occurred in 30-leaf pak choi plants

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independent of either UV treatment (Tab. 3). The monoacylated quercetin triglycosides

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responded structure-dependently to the different plant development stages analysed and this

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was found to be based on the hydroxycinnamic acid acylated. Coumaric acid and caffeic acid

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acylated quercetin triglycosides were concentrated in 5-leaf pak choi plants compared to other

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developmental stage, whereas ferulic acid acylated triglycoside were predominant in 30-leaf

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plants. Quercetin-3-O-glucoside-7-O-glucoside and isorhamnetin-3-O-glucoside were both

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affected by plant developmental stage and UV treatment and were highest in 30-leaf plants

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with reduced UVB (1.5- and 1.7-fold increase, respectively, compared to the control), while

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in all other developmental stages, either UV treatment had no effect. The anthocyanins,

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namely cyanidin-3-O-disinapoyl-diglucoside-5-O-glucoside, cyanidin-3-O-sinapoyl, feruloyl-

kaempferol-3-O-feruloyl-sophoroside-7-O-glucoside,

kaempferol-3-O-

kaempferol-3-O-sinapoyl-sophoroside-7-O-


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diglucoside-5-O-glucoside and cyanidin-3-O-diferuloyl-diglucoside-5-O-glucoside, were

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affected only by developmental stage and were highest in 30-leaf plants (Tab. 4).

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Hydroxycinnamic acid mono- and diglycosides showed interactions of plant developmental

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stage and type of UV treatment (Tab. 5). In detail, while the hydroxycinnamic acid

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monoglycosides had high concentrations in sprouts at reduced UVB approximately 2.3- to

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2.4-fold higher than the control, the hydroxycinnamic acid diglycosides were highest at the

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30-leaf plant stage and had a 1.3- to 1.5-fold increase under reduced UVB conditions

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compared to the control. In contrast to the hydroxycinnamic acid, mono- and diglycosides, the

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hydroxycinnamic acid quinic acid derivatives were concentrated in 30-leaf plants without

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interactions to UV treatment (Tab. 6). For the hydroxycinnamic acid malates, there was a

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developmental stage effect, which was dependent on the chemical structure of the compound.

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While coumaroyl-malate and caffeoyl-malate were highest in 5-leaf plants, feruloyl-malate

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and hydroxyferuloyl-malate were highly concentrated in 30-leaf plants without an effect of

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the UV treatment. However, the main compound of pak choi, sinapoyl-malate, showed

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interactions of developmental stage and UV treatment with high concentrations in sprouts

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under low UV of 1.4-fold higher compared to control and in 30-leaf plants independent of the

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UV treatment used.

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Effects of plant developmental stage and reduced UVB and low UV on carotenoids and

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chlorophylls

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In pak choi, lutein was the predominant carotenoid found, followed by zeaxanthin. With

257

regard to the influence of ontogenic effects, carotenoids were affected by the developmental

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stage (Tab. 7) and showed the highest concentrations in 15-leaf plants (β-carotene,

259

neoxanthin, violaxanthin) and/or 30-leaf plants (lutein, zeaxanthin). Additionally, zeaxanthin

260

was susceptible to UV treatment in the 30-leaf pak choi, i.e.low UV resulted in 1.4-fold

261

higher zeaxanthin concentrations than reduced UVB alone. Finally, the concentration of the 11 Environment ACS Paragon Plus


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chlorophylls a and b was impacted by the developmental stage and increased in 15- or 30-leaf

263

plants, but was not influenced by reduced UVB and low UV (Tab. 8).

264

Effects of plant developmental stage and reduced UVB and low UV on glucosinolates and

265

their hydrolysis products

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In pak choi, aliphatic glucosinolates, comprising mainly alkenyl glucosinolates (Tab. 9, S1) as

267

well as indolic and aromatic glucosinolates, were present (Tab. 10, S2) with 3But

268

glucosinolate being most abundant. The concentration of aliphatic glucosinolates was affected

269

by the developmental stage and their ontogenetic response was distinctly dependent on their

270

chemical structure. For example, while the short-chain sulphur-containing aliphatic

271

glucosinolates 4MSOB and 4MTB decreased from sprouts to the 30-leaf plants, for long-

272

chain sulphur-containing 5MSOP, a decrease was first observed (sprouts to 5-leaf plants)

273

followed by an increase with ongoing plant development (15- to 30-leaf plants). The

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concentration of 4MSOB increased by 1.4-fold at reduced UVB treatment in sprouts, but not

275

in older developmental stages. In contrast, alkenyl glucosinolates (3But, 4Pent, 2OH3But,

276

2OH4Pent) had the lowest concentrations in 5-leaf plants and were significantly higher in 30-

277

leaf plants. In addition, 3But was not only highly concentrated in pak choi sprouts, but also in

278

30-leaf plants. The aromatic and indolic glucosinolates were also affected by the

279

developmental stage, but not by either type of UV treatment. While the indolic precursor

280

glucosinolate I3M showed highest concentrations in sprouts and decreased during plant

281

development, the methoxylated and hydroxylated derivatives of I3M, such as 1MOI3M,

282

4OHI3M and 4MOI3M, were found in low concentrations in 5-leaf plants followed by a

283

marked increase in concentration in 30-leaf plants. The same ontogenetic pattern was also

284

observed for the aromatic glucosinolate 2PE.

285

After autolysis of pak choi’s glucosinolates, mainly epithionitriles (EPTs, Tab. 11, S3), but

286

also nitriles (cyanides: CNs, Tab. 12, S4) and small amounts of isothiocyanates (ITCs, Tab. 12 Environment ACS Paragon Plus

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13, S5) were formed. All hydrolysis products were affected by the developmental stage. In

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pak choi, the glucosinolate hydrolysis products from 3But glucosinolate [4-pentenenitrile

289

(3But-CN), 1-cyano-3,4-epithiobutane (CETB) and 3-butenyl ITC (3But-ITC)] were usually

290

most abundant in 30-leaf plants. Moreover, the hydrolysis products of 4Pent glucosinolate [1-

291

cyano-4,5-epithiopentane (CETPent) and 4-pentenyl ITC (4Pent-ITC)] were also dominant.

292

Next to them, further CNs, deriving from the glucosinolates 4MTB, 5MTP, 4MSOB, I3M or

293

2PE [5-(methylthio)pentanenitrile (4MTB-CN), 6-(methylthio)hexanenitrile (5MTP-CN), 5-

294

(methylsulfinyl)pentanenitrile (4MSOB-CN), 6-(methylsulfinyl)hexanenitrile (5MSOP-CN),

295

indole-3-acetonitrile (IAN), 3-phenylpropanenitrile (2PE-CN)], EPTs from the glucosinolates

296

2OH3But and 2OH4Pent [1-cyano-2-hydroxy-3,4-epithiobutane (CHETB), 1-cyano-2-

297

hydroxy-4,5-epithiopentane

298

(methylthio)pentyl ITC (5MTP-ITC) from 5MTP, 5-vinyl-1,3-oxazolidine-2-thione (OZT)

299

from 3But, 2-phenylethyl ITC (2PE-ITC) from 2PE] were identified and quantified.

300

In all four developmental stages studied, EPTs were the main hydrolysis products detected

301

and accounted for 72-94% of total glucosinolate breakdown products. CNs were the second

302

most abundant hydrolysis products in hydrolysed sprouts (17-27% of hydrolysis products). In

303

contrast, ITCs were found only in very low amounts and accounted only for 0.6-3.6% of total

304

hydrolysis products at all four developmental stages. 3But-CN and 2PE-CN were affected by

305

the developmental stage (Tab. 12). While 3But-CN was mainly formed in hydrolysed sprouts

306

and 30-leaf plants, 2PE-CN dominated in 15-leaf plants, respectively. With regard to the two

307

different UV treatments of pak choi during four different developmental stages, formation of

308

CNs, but not ITCs or EPTs, was affected. In detail, three out of four sulphur-containing CNs,

309

namely 4MTB-CN, 4MSOB-CN and 5MSOP-CN, were not only affected by developmental

310

stage, since their formation was high in sprouts but low at the 5- or 15-leaf stages before

311

increasing at the 30-leaf stage, but also increased by 1.8- (4MTB-CN, 5-MTP-CN) to 8.9-fold

(CHETPent)]

and

ITCs


or

derivatives

thereof

[5-


Page 16 of 39

312

at reduced UVB in sprouts. Of interest is that the same trend was also found for IAN and that

313

all these CNs were most abundant at the sprout developmental stage.

314

Discussion

315

Effect of reduced UVB and low UV on fresh and dry weight as well as leaf area at different

316

plant developmental stages

317

With regard to morphological changes of the pak choi plants, they are in accordance with the

318

literature where additional UVB exposure results in smaller but thicker leaves and lower

319

weight 14, 37.

320

Effects of plant developmental stage and reduced UVB and low UV on flavonoid glycosides

321

and hydroxycinnamic acid derivatives

322

For the flavonoid glycosides and hydroxycinnamic acid derivatives, the response to reduced

323

exposure to reduced UVB and low UV is highly chemical structure-specific for individual

324

compounds. For example, acylated flavonoid glycosides and hydroxycinnamic acid

325

derivatives, including coumaric acid and caffeic acid, both acids produced at the beginning of

326

flavonoid biosynthetic pathway, were more concentrated in sprouts. In contrast, acylated

327

flavonoid glycosides and hydroxycinnamic acid derivatives, including ferulic acid,

328

hydroxyferulic acid and sinapic acid, acids produced later on in the biosynthesis, as well as

329

highly complex non-acylated flavonoid glycosides and anthocyanins, were in higher

330

concentrations in 30-leaf plants. Quercetin and kaempferol glycosides acylated with caffeic

331

acid are known for their high antioxidant activity and UV-shielding potential

332

present study, the highest concentrations of most hydroxycinnamic acid derivatives, including

333

sinapoyl-malate, the main hydroxycinnamic acid derivatives of pak choi 1, were quantified in

334

mature 30-leaf plants. Of interest is that older plants produce greater amounts of ROS

335

thereby demanding higher concentrations of potent antioxidants, such as chlorogenic acids, 14 Environment ACS Paragon Plus

16, 17

. In the

38

,

Page 17 of 39


336

for the protection of their DNA. 39. Additionally, anthocyanins can accumulate with increased

337

plants age to protect photosystem II against excessive visible light

338

in the present study. Moreover, higher UVB radiation levels are also associated with

339

increasing ROS production as well as the intracellular accumulation of antioxidants such as

340

flavonoids and other phenolics. Consequently, reduced UVB and low UV would be expected

341

to lower the concentrations of flavonoids and other phenolics in pak choi. However, in the

342

present study, a differentiated, structure-specific response was found for the kaempferol and

343

quercetin glycosides of pak choi. Further, the structure-specific response to reduced UVB and

344

low UV was less dependent on the acylated hydroxycinnamic acid in kaempferol glycosides

345

than on the glycosylation pattern as mainly kaempferol triglycosides were increased with

346

reduced UVB in 30-leaf plants. Harbaum-Piayda et al. 41 found in pak choi that total flavonoid

347

concentration, mainly represented by monoacylated kaempferol triglycosides, increased with

348

exposure to additional UVB at 22 C, especially kaempferol-3-O-caffeoyl-diglucoside-7-O-

349

glucoside (containing a catechol structure). In kale treated with subsequent doses of UVB,

350

caffeic acid and hydroxyferulic acid monoacylated kaempferol triglycosides (containing a

351

catechol structure) were increased with exposure to higher UVB radiation

352

compounds containing a catechol structure have a higher antioxidant activity than those

353

without a catechol structure, and are therefore of high interest for human nutrition 17, 42. In the

354

present study, the response to reduced UVB and low UV was less dependent on the acylated

355

hydroxycinnamic acid in kaempferol glycosides than on the glycosylation pattern since

356

mainly kaempferol triglycosides were increased with reduced UVB in 30-leaf plants. The

357

accumulation of specific flavonoid glycosides appears to be an intrinsic part of the UVB

358

response, with expression of several UDP-glucosyltransferases (UDPgtfp) being directly

359

controlled by UVB 8. Glycosylation has also been reported to decrease the antioxidant activity

360

of flavonoids as well as to affect accumulation, stability and solubility of flavonoids

361

Interestingly, with subsequent UVB doses on kale, the response of acylated kaempferol 15 Environment ACS Paragon Plus

40

which was not the case

16

. Phenolic

43, 44

.


Page 18 of 39

362

glycosides was diverse and depended on the number of glucose moieties in the 7-O position

363

16

364

kaempferol triglycosides, which had the highest concentrations in 30-leaf plants with reduced

365

UVB, whereas the sinapic acid monoacylated kaempferol tetraglycoside kaempferol-3-O-

366

sinapoyl-sophoroside-7-O-diglucoside showed the highest concentrations in sprouts with

367

reduced UVB. In kale, kaempferol triglycosides were shown to have a slightly higher

368

antioxidant activity compared to the kaempferol tetraglycosides 45. This consequently leads us

369

to the conclusion that the pak choi examined in this study needed higher antioxidants while

370

aging and under reduced UVB. In pak choi and canola, the highest increase after treatment

371

with additional UVB was found for the non-acylated kaempferol-3-O-diglucoside-7-O-

372

glucoside

373

kaempferol-3-O-diglucoside-7-O-glucoside

374

were not affected by reduced UVB in 30-leaf plants. Currently, it is not yet clear why

375

kaempferol-3-O-diglucoside-7-O-glucoside and quercetin-3-O-diglucoside-7-O-glucosid were

376

not affected with reduced UVB since in kale, a reduction of sugar moieties in non-acylated

377

quercetin glycosides did not necessarily lead to higher antioxidant activity

378

anthocyanins are induced with higher UVB doses

379

anthocyanin concentrations were not influenced by the different UV treatments, which imply

380

that the plants did not suffer from abiotic stress causing e.g. ROS. Moreover, in pak choi,

381

there is a high variation of hydroxycinnamic acid derivatives. The main hydroxycinnamic acid

382

derivative of pak choi is sinapoyl-malate 1 which is known for its high shielding effect against

383

UV radiation 48. However, in the present study, the highest concentration of sinapoyl-malate

384

was found in sprouts exposed to low UV. As for the flavonoid glycosides, the glycosylation

385

pattern seems to be most important. For example, the enhancement of hydroxycinnamic acid

386

derivatives was reported to correlate with higher UV radiation due to their function as UV-

387

shielding and antioxidant activity

. Data presented here also underline this suggestion as the sinapic acid monoacylated

41, 46

. This finding is in contrast to the present study in which the non-acylated and

quercetin-3-O-diglucoside-7-O-glucoside

17

. Generally,

47

. However, in the present study,

49

. However, such an enhancement was not found in the


Page 19 of 39


388

present study. Consequently, further studies on the effects of reduced exposure to UV and the

389

subsequent effects on the biosynthesis of phenolic compounds are needed.

390

Effects of plant developmental stage and reduced UVB and low UV on carotenoids and

391

chlorophylls

392

Carotenoids and chlorophylls are part of the photosystem and are required for light-harvesting

393

as well as photoprotection.50 Generally, carotenoids and chlorophylls are at higher

394

concentrations in middle aged leaves compared to earlier developmental stages 7. This finding

395

is consistent with the accumulation of carotenoids and chlorophylls measured in the pak choi

396

plants during this study.

397

UVB light can increase or decrease carotenoid and chlorophyll concentrations. However, the

398

response is strongly species-specific and time-dependent, e.g. in tomato fruit, carotenoid

399

concentrations increased after UV exposure

400

carotenoid concentrations seem to be affected by other UV-shielding compounds such as

401

anthocyanins. In green lettuce, an increase in lutein and zeaxanthin was observed after UVB

402

exposure, whereas, in a red lettuce variety, an opposite trend was found

403

Amur, both carotenoids and anthocyanins can be found and under low UV conditions, the

404

zeaxanthin concentration increased in 30-leaf plants. Zeaxanthin, which is accumulated under

405

high light conditions in general, is involved in the light xanthophyll-cycle reaction, and high

406

concentrations of zeaxanthin are linked to higher resistance in Arabidopsis to photooxidative

407

stress by light protective and antioxidant mechanisms

408

increases under low UV, thereby increasing the area for photosynthesis concomitantly. This

409

finding is consistent with our results that changes in chlorophyll concentrations were only

410

found in response to different developmental changes, but not to either UV treatment.

411

Therefore, it can be assumed that changes in the photosystem stoichiometry did not occur

22, 29, 30

. It is interesting to note that changes in

51

. In pak choi cv.

64, 65


. In our study, the leaf area


Page 20 of 39

412

under our experimental conditions. Such long-term responses have however been reported as

413

consequences of an imbalanced excitation of the photosystems 52.

414

To date, the accumulation of xanthophylls in response to reduced UVB light has not been

415

sufficiently studied and further research is needed to better understand how and why

416

vegetables respond in species-specific manner in respect to developmental stage and genetic

417

background to modulated light conditions.

418

Effects of plant developmental stage and reduced UVB and low UV on glucosinolates and

419

their hydrolysis products

420

The glucosinolates were either high in sprouts or in 30-leaf plants with shifts from sulphur-

421

containing methylthioalkyl and methylsulphinylalkyl glucosinolates to (hydroxy)alkenyl

422

glucosinolates. A high content of glucosinolates in sprouts of several cultivars of pak choi has

423

previously been determined 3. The shift of sulphur-containing alkyl to alkenyl glucosinolates

424

was reported to be dependent on a functional AOP2 homologue to Arabidopsis in pak choi

425

53

426

discussed as antioxidants in response to UV exposure. An increment in 4MSOB

427

concentrations with enhanced UVB exposure has been reported previously in broccoli 26. As

428

broccoli does not contain a functional allele of AOP2, it therefore accumulates 4MSOB

429

glucosinolate due to enhanced UVB exposure. In our study, reduced UVB did not have an

430

effect on 4MSOB concentrations in mature 30-leaf plants, but interestingly, increased

431

4MSOB concentrations were observed in sprouts. Previously, treatment of broccoli with

432

enhanced narrow banded UVA at the wavelength 365 nm resulted in lower 4MSOB

433

concentrations, but showed higher 4MSOB concentrations in plants when treated with UVA

434

at 385 nm wavelengths 54. However, in pak choi, 4MSOB concentrations increased at low UV

435

treatment. Therefore, it can be concluded that low and high UVB and UVA also have an

3,

, which might be influenced by plant developmental stage. In general, glucosinolates are not


Page 21 of 39


436

impact on the glucosinolate concentrations. However, further research will be needed to

437

differentiate between the effects of UVA and UVB on glucosinolate concentrations.

438

The concentrations of glucosinolate hydrolysis products differed at different developmental

439

stages based on the enzymatic breakdown process and on the precursor glucosinolate

440

concentration. Due to a higher abundance of the precursor glucosinolate, EPTs and ITCs were

441

highest in mature 30-leaf plants. The formation of mainly EPTs from alkenyl glucosinolates

442

has been reported previously for pak choi 36 and is due to the presence of the epithiospecifier

443

protein, which interacts in the degradation of the aglycon formed by myrosinase enzyme and

444

results in the release of EPTs from alkenyl glucosinolates 55. From glucosinolates devoid of a

445

terminal double bond in the side chain, this epithiospecifier protein favours the formation of

446

CNs 56. Therefore, by modulating the activity of epithiospecifier protein, it should be possible

447

to increase concentrations of health-promoting ITCs. In an earlier study in broccoli sprouts,

448

epithiospecifier protein transcripts were shown to be decreased due to moderate UVB doses

449

(0.6 kJ m-2 d-1). However, it still remains unclear whether this UVB treatment affected the

450

hydrolysis of glucosinolates in that study

451

products of the present study, no effect was observed for either EPTs or ITCs under the UV

452

treatments, while CN formation was enhanced under reduced UVB in sprouts. In Arabidopsis

453

and other Brassicales species, several nitrile specifier proteins were identified that favour the

454

formation of simple CNs instead of EPTs or ITCs. Thus, the presence and change in nitrile

455

specifier protein activity could explain the results observed in the present study56,

456

Nevertheless, whether nitrile specifier proteins are also present in pak choi still needs to be

457

clarified.

458

In summary, in pak choi changes in plant morphology and secondary plant metabolites are

459

more affected by developmental stage and less by exposure to reduced UVB and low UV.

460

Higher concentrations of flavonoids and hydroxycinnamic acids as well as carotenoids and

26

.With regard to the glucosinolate hydrolysis


57

.


461

chlorophylls are found in older plants, whereas sprouts are a source of glucosinolates and their

462

hydrolysis products. A regulatory effect of reduced UVB and low UV conditions on the

463

biosynthesis of secondary plant metabolites was found. Dry weight and leaf area increased

464

due to reduced UVB and low UV. Flavonoids and hydroxycinnamic acids respond in

465

chemical structure-dependent manner and less complex compounds are favored. Therefore, it

466

can be assumed that the antioxidant activity is not negatively affected by exposure to reduced

467

UVB and low UV. For carotenoids, an interaction of morphological changes (increase of leaf

468

area) and activation of photosynthesis can be deduced as an effect of reduced UVB and low

469

UV. However, this study underlines the importance of the glucosinolate 4MSOB in response

470

to UVA and UVB exposure, even though its function is not clear yet. To our knowledge, this

471

is the first report on the direct effects of UVA and UVB on glucosinolate hydrolysis products

472

in pak choi at different developmental stages. Interestingly, CN concentrations increased,

473

whereas both EPTs and ITCs were not affected by reduced UVB. This finding points to the

474

presence of not yet identified CN specifier proteins in B. rapa. Importantly, there is currently

475

no evidence that either UVA or UVB treatment will modulate glucosinolate hydrolysis to

476

increased formation of health-promoting ITCs. Consequently, daily exposure to low UVB

477

doses of 0.059 kJ m-2 h-1 UVB is sufficient to modify the secondary metabolism of plants.

478

However, an induction of secondary plant metabolites with higher antioxidant activity can be

479

expected with higher doses of UVB. Thus, this knowledge might lead to the production of

480

health-promoting vegetables in the future. Further experiments are needed to specify the

481

optimal daily exposure to precise UVB doses to promote the production of health-promoting

482

secondary plant metabolites.

483

484

Acknowledgements


Page 22 of 39

Page 23 of 39


485

We thank Antje Bamberg, Marcus Fricke, Andrea Jankowsky, Annett Platalla and Angela

486

Schröter for technical support.

487

Appendix A. Supplementary data

488

489

References

490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526

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44. Bowles, D.; Lim, E. K.; Poppenberger, B.; Vaistij, F. E., Glycosyltransferases of lipophilic small molecules. Annu Rev Plant Biol 2006, 57, 567-597. 45. Fiol, M.; Adermann, S.; Neugart, S.; Rohn, S.; Mügge, C.; Schreiner, M.; Krumbein, A.; Kroh, L. W., Highly glycosylated and acylated flavonols isolated from kale (Brassica oleracea var. sabellica) — Structure–antioxidant activity relationship. Food Research International 2012, 47, 80-89. 46. Olsson, L. C.; Veit, M.; Weissenbock, G.; Bornman, J. F., Differential flavonoid response to enhanced UV-B radiation in Brassica napus. Phytochemistry 1998, 49, 10211028. 47. Cominelli, E.; Gusmaroli, G.; Allegra, D.; Galbiati, M.; Wade, H. K.; Jenkins, G. I.; Tonelli, C., Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. Journal of Plant Physiology 2008, 165, 886-894. 48. Dean, J. C.; Kusaka, R.; Walsh, P. S.; Allais, F.; Zwier, T. S., Plant sunscreens in the UV-B: Ultraviolet spectroscopy of jet-cooled sinapoyl malate, sinapic acid, and sinapate ester derivatives. Journal of the American Chemical Society 2014, 136, 14780-14795. 49. Edreva, A., The importance of non-photosynthetic pigments and cinnamic acid derivatives in photoprotection. Agriculture Ecosystems & Environment 2005, 106, 135-146. 50. Cazzonelli, C. I., Goldacre Review: Carotenoids in nature: insights from plants and beyond. Funct. Plant Biol. 2011, 38, 833-847. 51. Caldwell, C. R.; Britz, S. J., Effect of supplemental ultraviolet radiation on the carotenoid and chlorophyll composition of green house-grown leaf lettuce (Lactuca sativa L.) cultivars. Journal of Food Composition and Analysis 2006, 19, 637-644. 52. Moejes, F. W.; Matuszynska, A.; Adhikari, K.; Bassi, R.; Cariti, F.; Cogne, G.; Dikaios, I.; Falciatore, A.; Finazzi, G.; Flori, S.; Goldschmidt-Clermont, M.; Magni, S.; Maguire, J.; Le Monnier, A.; Muller, K.; Poolman, M.; Singh, D.; Spelberg, S.; Stella, G. R.; Succurro, A.; Taddei, L.; Urbain, B.; Villanova, V.; Zabke, C.; Ebenhoh, O., A systems-wide understanding of photosynthetic acclimation in algae and higher plants. J. Exp. Bot. 2017, 68, 2667-2681. 53. Neal, C. S.; Fredericks, D. P.; Griffiths, C. A.; Neale, A. D., The characterisation of AOP2: A gene associated with the biosynthesis of aliphatic alkenyl glucosinolates in Arabidopsis thaliana. BMC Plant Biol 2010, 10, 170. 54. Rechner, O.; Neugart, S.; Schreiner, M.; Wu, S.; Poehling, H. M., Different narrowband light ranges alter plant secondary metabolism and plant defense response to aphids. Journal of Chemical Ecology 2016, 42, 989-1003. 55. Burow, M.; Wittstock, U., Regulation and function of specifier proteins in plants. Phytochem Rev 2009, 8, 87-99. 56. Burow, M.; Markert, J.; Gershenzon, J.; Wittstock, U., Comparative biochemical characterization of nitrile-forming proteins from plants and insects that alter myrosinasecatalysed hydrolysis of glucosinolates. FEBS Journal 2006, 273, 2432-2446. 57. Kuchernig, J. C.; Burow, M.; Wittstock, U., Evolution of specifier proteins in glucosinolate-containing plants. BMC Evolutionary Biology 2012, 12, 127.

669


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670 671

Figure 1. Fresh weight (A), dry weight (B) and leaf area (C) of pak choi influenced by

672

ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ

673

m-² h-1 UVB; low UV: 0.002 kJ m-² h-1 UVB) at four different developmental stages. For

674

sprouts, all plants from one single pot were harvested. For 5- to 30-leaf plants, one plant per

675

pot was harvested. Different letters represent significant differences (p ≤ 0.05 by Tukey’s

676

HSD test) (n=3, mean ± standard error).



Table 1. Spectra of UVA and UVB at the applied ultraviolet radiation treatments

Control1 2

Reduced UVB 3

UVB [W m-²]

UVB [kJ m-2 h-1]

UVA [W m-²]

UVA [kJ m-2 h-1]

UVB [%]

UVA [%]

0.0164

0.059

57

205

100

100

0.0049

0.017

50

181

29.8

88.3

3

3.6

1.6

Low UV 0.0006 0.002 1 Polyethene filter, 2Autostat CT5 filter, 3Rosco filter

1

26


Page 28 of 39

Page 29 of 39


Table 2. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on selected kaempferol glycosides (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each kaempferol glycoside(n=4, mean ± standard error). K: kaempferol; glc: glucose; soph: sophorose; cou: coumaroyl; caf: caffeoyl; fer: feruloyl; hfer: hydroxyferuloyl; sin: sinapoyl Developmental stage

UV treatment

Sprouts

Control Reduced UVB Low UV Control Reduced UVB Low UV Control Reduced UVB Low UV Control Reduced UVB Low UV

5-leaf plants

15-leaf plants

30-leaf plants

Main effect Developmental stage

UV treatment

Developmental stage UV treatment Developmental stage x UV treatment

Kaempferol glycosides

Sprouts

178.4±3.8 169.8±9.5 227.1±16.5 209.1±47.6 170.1±48.3 261.7±30.7 183.1±14.6 187.5±23.5 186.7±33.2 323.1±37.7 542.2±67.9 495.2±38.8

A A A A A A A A A AB C BC

k-3-O-glc7-O-glc

k-3-Odiglc7-O-glc

24.9±2.1 29.1±2.7 32.4±2.6 32.4±7.7 38.2±8.4 63.7±12.2 110.0±9.9 113.0±12.8 107.0±22.5 137.0±9.6 162.5±4.0 189.4±45.1

8.0±0.1 8.2±0.2 7.8±0.5 7.4±1.0 6.8±1.8 8.6±0.9 8.9±0.7 9.0±0.3 8.9±0.5 17.1±1.5 37.8±10.5 26.7±3.9

A

k-3-Ocousoph7-O-glc 7.0±0.2 4.8±0.3 10.4±1.4 11.5±2.0 11.0±3.0 12.0±1.5 2.5±0.2 2.6±0.2 3.4±0.1 4.6±0.6 4.9±0.5 3.1±0.4

A

k-3-Ocaf-soph7-O-glc

k-3-Ofer-soph7-O-glc

35.1±1.7 19.7±2.1 75.8±7.9 84.8±22.6 56.8±17.7 85.9±12.2 8.4±0.2 11.2±3.5 12.3±2.8 36.6±8.4 73.9±20.2 74.5±13.3

12.0±0.5 9.0±0.7 15.1±0.8 19.3±4.9 16.3±5.2 24.8±3.7 17.4±2.4 18.8±1.0 20.6±2.0 37.8±9.1 80.8±18.4 63.6±7.0

B

A A B

7.4±0.8 11.5±1.2 2.8±0.2 4.2±0.3

5-leafs 15-leafs 30-leafs

28.8±1.6 44.8±6.5 110.0±8.4 163.0±15.4

Control Reduced UVB Low UV

223.4±20.6 267.4±45.4 292.7±33.9

76.1±13.0 85.7±14.7 98.1±19.1

10.4±1.1 15.5±4.1 13.0±2.2

A A A

6.4±1.0 5.8±1.1 7.2±1.1

***

***

***

***

***

***

*

ns

*

ns

*

ns

*

ns

ns

ns

ns

*

C A A

k-3-Osin-soph7-O-glc

BC AB AB AB A AB A A A CD E D

44.3±1.2 42.5±3.5 47.5±2.9 34.5±7.3 26.4±9.3 39.2±7.7 27.0±3.1 25.6±5.5 26.9±6.5 54.3±18.3 120.0±19.2 90.1±13.1

AB AB AB A A A A A A B C BC

k-3-Osinsoph7-O-diglc 30.8±1.5 43.8±2.2 33.0±2.6 9.9±1.9 10.5±4.3 15.4±2.4 6.7±1.2 5.3±1.1 5.4±1.0 8.5±1.2 11.2±1.6 11.0±3.2

A

191.8±9.6 213.6±25.1 185.8±13.0 453.5±38.6

A B C

8.0±0.2 7.6±0.7 8.9±0.3 27.2±4.3

A A A A A A A A A AB C BC

k-3-Ohfersoph7-O-glc 16.5±2.8 12.6±0.3 5.1±0.4 9.1±2.0 4.1±1.2 12.1±1.9 2.2±0.2 2.0±0.3 2.1±0.3 27.1±5.3 51.0±4.5 36.8±1.2

43.5±7.6 75.8±10.3 10.7±1.4 61.7±9.4

C B BC

12.0±0.8 20.1±2.6 18.9±1.1 60.7±8.5

11.4±1.7 8.4±1.3 2.1±0.2 38.3±3.6

44.8±1.6 33.4±4.5 26.5±2.7 88.2±12.1

35.9±2.1 12.0±1.7 5.8±0.6 10.3±1.2

41.2±8.9 40.4±9.0 62.2±8.7

A A A

21.6±3.5 31.2±8.6 31.0±5.3

13.7±2.8 17.4±5.2 14.0±3.6

40.0±5.2 53.7±11.2 50.9±7.2

14.0±2.6 17.7±4.1 16.2±2.9

***

***

***

ns

ns

ns

***

*

*

27


B C BC A A A A A A A A A


* significant at p ≤ 0.05; ** significant at p ≤ 0.01; *** significant at p ≤ 0.005; ns, not significant

28


Page 30 of 39

Page 31 of 39


Table 3. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on selected quercetin glycosides (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each quercetin glycoside(n=4, mean ± standard error). q: quercetin; i: isorhamnetin; glc: glucose; soph: sophorose; cou: coumaroyl; caf: caffeoyl; fer: feruloyl Developmental stage

UV treatment

Sprouts


5-leaf plants

15-leaves plants

30-leaf plants


UV treatment

Sprouts

Quercetin glycosides 72.2±7.6 52.2±2.5 107.7±13.7 119.8±23.7 162.5±43.1 156.3±21.2 59.7±6.2 65.2±10.5 66.8±11.5 72.8±0.9 92.8±6.1 100.4±8.3

Q-3-O-glc7-O-glc 7.0±0.7 3.1±0.3 4.4±0.7 5.2±0.7 5.1±1.7 6.4±1.8 5.5±0.4 5.9±0.6 6.2±0.9 9.8±0.5 14.9±0.2 12.1±1.2

AB A A A A AB AB AB AB BC D CD

A

5-leaves 15-leaves 30-leaves

77.4±8.4 146.2±17.1 63.9±5.1 88.7±4.7


81.1±8.2 93.2±14.9 107.8±10.5

B A A

Q-3-Ocou-soph7-O-D-glc 5.4±0.3 3.7±0.3 6.6±1.4 8.4±1.7 15.3±4.6 12.3±0.8 4.4±0.1 4.1±0.2 4.2±0.4 7.2±0.5 9.5±0.6 8.2±0.6

Q-3-O-diglc7-O-glc 8.4±0.6 12.5±0.7 8.8±1.0 11.6±2.6 13.5±3.4 16.1±4.2 14.6±1.7 16.4±2.5 15.0±4.4 22.7±1.9 29.5±3.4 30.8±4.8

A 4.8±0.6 5.5±0.8 5.9±0.4 12.3±0.7

9.9±0.7 13.7±1.9 15.3±1.6 27.7±2.2

6.9±0.5 7.3±1.2 7.3±0.9

14.3±1.6 18.0±2.1 17.7±2.7

Q-3-Ocaf-soph7-O-D-glc 46.6±6.5 28.3±1.9 82.2±11.1 90.7±20.2 124.1±32.9 114.4±14.0 26.0±3.4 31.0±7.7 30.0±6.8 19.3±2.2 25.4±1.5 33.4±8.2

A 5.2±0.6 12.0±1.7 4.2±0.2 8.3±0.4

A A B

6.3±0.6 8.2±1.6 7.8±0.9

Developmental *** *** *** stage UV treatment ns ns ns Developmental ns *** ns stage x UV treatment * significant at p ≤ 0.05; ** significant at p ≤ 0.01; *** significant at p ≤ 0.005; ns, not significant

B AB AB

Q-3-O-soph7-O-fer-diglc 4.8±0.5 4.6±0.4 5.6±0.3 3.9±0.5 4.6±1.4 7.1±0.5 9.3±1.3 7.7±0.8 11.5±2.0 13.8±2.1 13.4±1.6 15.9±4.0

A 52.4±7.8 109.7±13.1 29.0±3.3 26.0±3.1 45.6±8.7 52.2±13.1 65.0±10.2

B A A

I-3-O-glc 5.6±0.6 5.1±0.5 5.9±0.4 55.5±19.5 24.2±7.1 71.5±7.8 77.6±12.9 101.4±14.8 113.3±17.7 238.0±45.5 488.1±34.1 421.4±31.1

A 5.0±0.3 5.2±0.6 9.5±0.9 14.4±1.5 8.0±1.2 7.6±1.1 10.0±1.4

A B C

5.5±0.3 50.4±8.9 97.4±9.1 382.5±37.4 94.2±25.2 154.7±51.3 153.0±42.0

***

***

***

***

ns

ns

ns

***

ns

ns

ns

***

29


A A A AB AB AB AB AB AB C D D


Page 32 of 39

Table 4. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on selected hydroxycinnamic acid monoglycosides and hydroxycinnamic acid diglycosides (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each hydroxycinnamic acid (HCA) glycoside (n=4, mean ± standard error). Developmental stage

UV treatment

Sprouts


335.5±13.9 269.4±14.2 389.2±28.0 345.7±45.5 345.1±88.8 444.8±31.2 369.0±20.7 343.4±39.8 380.3±47.1 494.7±13.9 559.6±17.9 596.0±6.3

Sprouts

331.4±18.0

A


378.5±34.5 364.2±20.2 550.1±14.5


386.2±20.4 379.4±35.8 452.5±26.4

5-leaf plants

15-leaf plants

30-leaf plants


UV treatment


HCA

***

*

Caffeoylglycoside 21.2±3.4 27.3±3.1 16.7±0.8 17.6±5.6 8.6±2.9 20.0±3.0 4.9±0.4 4.6±0.2 4.7±0.3 8.3±0.6 10.9±0.7 13.5±2.0

CD D BCD BCD AB BCD A A A AB ABC ABC

Feruloylglycoside 7.5±1.3 17.9±0.9 7.1±0.3 8.2±1.5 5.7±1.4 12.1±0.8 7.0±0.2 6.7±0.6 6.3±0.3 13.7±1.0 18.2±1.5 17.5±1.1

AB CD A AB A BC A A A C D D

Sinapoylglycoside 40.0±6.2 92.3±2.4 21.6±1.6 19.1±3.6 11.3±2.9 25.0±2.4 12.9±0.9 12.2±1.2 12.5±1.3 29.3±3.4 37.7±2.3 25.1±7.1

D E ABC AB A ABCD AB AB AB BCD CD BCD

Disinapoyldiglycoside 13.7±0.5 12.9±1.3 12.9±1.3 11.2±1.5 9.7±2.7 15.7±2.1 14.7±1.9 14.9±0.9 16.5±1.3 26.1±2.8 37.4±1.4 27.7±3.4

A A A A A A A A AB BC D C

Sinapoylferuloyldiglycoside 7.0±0.3 6.4±0.2 7.4±0.5 8.9±1.2 8.4±2.3 12.5±1.5 9.8±0.6 10.1±0.7 10.4±0.6 18.4±1.4 28.4±0.9 22.7±1.6

AB A AB AB AB B AB AB AB C D C

Trisinapoyldiglyciside 11.9±0.5 11.3±1.0 11.3±1.0 9.1±1.0 8.3±2.3 12.1±1.3 11.3±0.7 10.9±0.8 11.3±0.9 20.2±1.3 26.5±1.6 24.5±1.6

A A A A A A A A A B C BC

Disinapoylferuloyldiglyciside 6.8±0.3 6.4±0.2 7.4±0.6 7.7±1.0 7.7±2.0 10.8±1.1 9.7±0.5 10.0±0.6 10.0±0.6 16.5±0.7 22.0±0.9 20.4±1.2

10.8±1.6 8.7±1.0 6.6±0.2 16.4±0.9

51.3±9.3 18.4±2.3 12.5±0.6 30.7±2.9

13.2±0.6

6.9±0.2

11.5±0.5

6.9±0.2

A A B

21.7±1.9 15.4±2.6 4.7±0.1 10.9±0.9

12.2±1.4 15.4±0.8 30.4±2.1

10.0±1.0 10.1±0.3 23.2±1.4

9.9±1.0 11.2±0.4 23.7±1.1

8.8±0.9 9.9±0.3 19.6±0.8

A A A

13.0±2.3 12.9±2.4 13.7±1.7

9.1 ±0.9 12.7±1.6 10.7±1.2

25.3±3.2 38.4±8.6 21.1±2.2

16.4±1.7 18.7±2.9 18.2±1.8

11.0±1.2 13.3±2.3 13.3±1.6

13.1±1.2 14.3±2.0 14.8±1.6

10.2±1.0 11.5±1.7 12.2±1.3

***

***

***

***

***

***

***

ns *

*** ***

*** ***

ns

**

ns

*

**

***

*

*

ns

30


A A A A A A A A A B C BC

Page 33 of 39



31



Page 34 of 39

Table 5. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on selected hydroxycinnamic acid quinic acid and hydroxycinnamic acid malates (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each hydroxycinnamic acid quinic acid derivative and hydroxycinnamic acid malate (n=4, mean ± standard error). Developmental stage

UV treatment

Sprouts

Control Reduced UVB Low UVB Control Reduced UVB Low UVB Control Reduced UVB Low UVB Control Reduced UVB Low UVB

5-leaf plants

15-leafplants

30-leaf plants

Main effect DS

UV treatment

Caffeoylquinic acid 3.2±0.1 3.0±0.1 3.4±0.3 6.9±0.8 9.0±3.4 12.5±3.5 18.2±2.2 17.9±4.4 20.1±5.2 19.0±1.8 24.3±1.6 19.4±3.2

sprouts 5-leaves 15-leaves 30-leaves

3.2±0.1 9.5±1.7 18.7±2.2 20.9±1.4

Control Reduced UVB Low UVB

11.8±1.9 13.5±2.5 13.9±2.3

Feruloylquinic acid 4.1±0.2 4.0±0.1 4.7±0.4 4.8±0.5 5.4±1.5 7.1±1.0 8.4±0.5 8.9±0.6 9.0±0.7 11.0±0.4 13.7±0.7 17.2±3.8

A B C C

4.3±0.2 5.8±0.6 8.8±0.3 14.0±1.4 7.1±0.7 8.0±1.0 9.5±1.5

A A B C

Coumaroylmalate 6.2±0.5 2.4±0.2 11.6±1.6 15.1±2.5 20.8±6.0 18.8±1.6 4.9±0.7 5.4±0.3 5.8±0.4 4.1±0.2 5.0±0.4 55.2±50.8

Caffeoylmalate 21.4±3.1 3.0±0.1 24.9±3.6 35.0±8.6 45.0±15.5 31.1±1.9 14.5±1.3 13.6±2.5 14.4±2.5 8.7±0.5 12.1±1.0 16.2±4.0

6.7±1.3 18.2±2.1 5.4±0.3 21.4±16.9

16.4±3.2 37.1±5.6 14.2±1.1 12.3±1.6

7.6±1.3 8.4±2.3 22.9±12.4

19.9±3.3 18.4±5.4 21.7±2.2

Developmental ns *** *** stage UV treatment ns * ns Developmental ns ns ns stage x UV treatment * significant at p ≤ 0.05; ** significant at p ≤ 0.01; *** significant at p ≤ 0.005; ns, not significant

HydroxyFeruloylmalate 8.4±0.7 4.1±0.2 7.5±0.7 7.5±0.8 5.3±1.3 7.1±0.2 9.3±0.5 8.0±1.0 8.6±1.3 12.9±0.8 16.0±0.6 44.6±28.1

Feruloylmalate 24.6±3.1 9.0±0.8 36.8±2.8 49.4±9.6 58.0±14.1 73.5±7.1 72.6±4.2 76.3±2.5 93.5±3.4 97.4±7.5 91.1±3.2 100.7±28.0

A B A A

23.5±3.7 60.3±6.3 80.8±3.3 96.4±8.9 61.0±7.6 58.6±8.6 76.1±9.1

A B BC C

6.7±0.6 6.6±0.5 8.6±0.5 24.5±9.5

Sinapoylmalate 148.0±14.6 60.0±10.1 203.2±15.0 130.8±21.2 127.2±33.5 168.6±17.4 149.2±7.3 121.8±27.2 135.4±29.9 175.6±7.6 179.9±8.3 155.1±45.3

A A A B

137.1±19.1 142.2±14.2 135.5±12.8 170.2±14.5

9.5±0.6 8.4±1.3 17.0±7.5

150.9±7.5 122.2±14.9 165.6±14.6

***

***

*

ns

ns ns

ns ns

ns ns

* *

32


AB A B AB AB AB AB AB AB B B B

Page 35 of 39


Table 6. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on carotenoids (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each carotenoid (n=4, mean±standard error). Developmental stage Sprouts

5-leaf plants

15-leaf plants

30-leaf plants


UV treatment

UV treatment Control Reduced UVB Low UV Control Reduced UVB Low UV Control Reduced UVB Low UV Control Reduced UVB Low UV

Sprouts

Carotenoids 169.1±9.8 177.8±9.4 174.2±4.6 263.4±9.2 243.1±15.9 271.2±12.1 279.1±29.5 335.8±11.7 327.8±24.0 321.8±22.6 273.5±34.5 364.8±33.2

Lutein 59.2±3.9 67.0±4.2 64.3±2.4 103.6±3.7 96.0±7.0 105.8±5.9 117.2±13.4 136.2±3.9 134.8±11.1 133.9±10.7 113.9±15.5 152.4±14.4

A


173.7±4.5 259.2±7.5 314.2±14.2 320.0±19.5


258.4±16.9 257.6±19.9 284.5±24.1

B C C

ß-Carotin 24.8±1.7 27.0±1.7 27.5±0.8 46.0±0.9 40.5±5.3 47.7±1.6 48.3±5.7 61.2±3.4 55.7±4.7 44.2±4.9 41.9±5.4 52.7±3.5

A 63.5±2.1 101.8±3.2 129.4±6.0 133.4±8.6 103.5±8.2 103.3±8.9 114.3±11.1

B C C

Zeaxanthin 52.6±2.2 51.6±2.0 48.7±2.4 49.8±1.5 45.7±0.4 56.3±2.5 41.9±2.4 52.3±2.5 59.5±3.8 82.3±4.5 64.5±7.0 91.0±8.0

AB AB AB AB AB AB A AB AB CD BC D

Violaxanthin 23.5±1.8 22.6±1.2 23.7±1.4 42.5±4.8 42.2±2.7 41.0±3.2 45.1±5.4 55.5±1.9 48.6±2.1 43.8±2.4 38.0±5.2 48.3±5.6

A 26.4±0.9 44.7±1.9 55.1±2.9 46.3±2.8 40.8±2.9 42.6±3.1 45.9±3.6

Developmental *** *** *** stage UV treatment ns ns ns Developmental stage x UV ns ns ns treatment * significant at p ≤ 0.05; ** significant at p ≤ 0.01; *** significant at p ≤ 0.005; ns, not significant

B C B

Neoxanthin 8.9±0.7 9.7±0.7 10.1±0.6 21.6±0.7 18.8±2.2 20.4±1.0 26.6±3.5 30.6±1.0 29.3±2.7 17.7±1.3 15.2±2.2 20.4±2.3

A 51.0±1.2 50.6±1.6 51.2±2.7 79.3±4.8

23.3±0.8 41.9±1.9 49.7±2.2 43.3±2.7

56.6±4.1 53.5±4.0 63.9±5.4

38.7±2.8 39.6±3.0 40.4±3.5

B C BC

A 9.6±0.4 20.3±0.8 28.8±1.5 17.8±1.2 18.7±1.8 18.6±1.2 20.0±2.2

***

***

***

***

ns

ns

***

ns

ns

33


B C B


Page 36 of 39

Table 7. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on total and aliphatic glucosinolates (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each glucosinolate (n=4, mean ± standard error). GS, glucosinolates; 4MTB, 4-(methylthio)butyl GS; 4MSOB, 4-(methylsulfinyl)butyl GS, 5MSOP, 5(methylsulfinyl)pentyl GS; 3But, 3-butenyl GS; 4Pent, 4-pentenyl GS; 2OH3But, 2-hydroxy-3-butenyl GS; 2OH4Pent, 2-hydroxy-4-pentenyl GS Developmental stage Sprouts

5-leaf plants

15-leaf plants

30-leaf plants



Sprouts 5-leaves 15-leaves 30-leaves

UV treatment

Developmental stage UV treatment Developmental


Total GS 468.1±14.609

4MTB 38.9±2.957

4MSOB 84.2±6.703

C

5MSOP 21.3±1.384

3But 238.7±12.778

4Pent 20.8±1.776

2OH3But 28.1±1.167

2OH4Pent 0.0±0.000

566.0±26.812 574.6±56.267 139.3±35.847

52.4±5.484 50.4±6.733 0.0±0.000

88.6±2.822 116.9±15.011 17.3±2.262

C D AB

24.9±0.310 24.9±2.923 3.4±0.914

297.2±16.057 280.0±34.853 65.4±18.584

26.7±2.294 23.7±1.665 30.3±10.835

32.6±4.410 41.1±1.123 8.8±1.724

0.0±0.000 0.0±0.000 0.7±0.249

89.4±26.278 293.5±93.977 332.9±59.475

0.0±0.000 0.0±0.000 0.0±0.000

22.9±5.603 66.2±29.904 2.7±0.435

AB B A

1.7±0.494 7.7±2.437 10.8±2.613

33.2±10.421 100.8±37.827 147.0±27.345

13.5±4.716 62.1±19.038 105.0±23.664

5.8±1.797 22.1±9.315 31.4±4.248

0.2±0.055 5.1±2.392 6.3±0.705

262.6±90.685 256.9±82.966 685.1±123.473

0.3±0.311 1.6±0.868 0.0±0.000

1.8±0.582 2.4±0.550 0.0±0.000

A A A

7.8±2.580 6.3±1.909 21.4±2.602

113.2±42.822 105.2±36.062 347.8±39.705

88.5±37.788 87.7±32.881 191.5±51.677

9.9±1.463 22.1±6.455 81.1±32.013

6.1±0.996 7.8±0.509 6.1±2.728

756.3±142.504 715.0±180.543

0.0±0.000 0.0±0.000

0.0±0.000 0.0±0.000

A A

13.9±5.348 15.6±5.072

323.2±38.509 319.8±86.305

285.3±75.891 243.8±64.470

83.6±29.445 91.2±39.617

9.3±3.202 9.3±2.545

536.2±24.181 174.0±40.848 284.1±42.467 718.7±79.201

A A A B

47.2±3.295 0.0±0.000 0.6±0.350 0.0±0.000

B A A A

96.5±6.661 35.4±11.318 2.3±0.300 0.0±0.000

23.6±1.105 4.2±1.104 8.3±1.371 16.9±2.544

C A A B

271.9±14.262 66.4±15.510 121.7±19.562 330.2±31.134

B A A B

23.7±1.247 35.3±9.089 93.7±16.874 240.2±35.749

A A A B

33.9±2.158 12.2±3.606 21.1±3.561 85.3±17.786

A A A B

0.0±0.000 1.9±0.979 6.7±0.460 8.2±1.553

406.3±60.423

9.7±4.400

26.0±8.974

14.2±2.154

199.7±29.651

86.9±21.947

37.3±9.992

3.2±0.987

418.5±77.276 459.9±70.889

13.1±5.973 13.0±5.780

28.3±9.389 46.3±14.611

12.1±2.581 13.6±2.419

191.693±34.341 201.4±35.061

103.5±33.893 104.3±27.331

32.9±10.418 44.1±11.757

3.9±1.269 5.5±1.211

***

***

***

***

***

***

***

***

ns ns

ns ns

*** *

ns ns

ns ns

ns ns

ns ns

ns ns

34


A A B B

Page 37 of 39


stage x UV treatment * significant at p ≤ 0.05; ** significant at p ≤ 0.01; *** significant at p ≤ 0.005; ns, not significant

35



Page 38 of 39

Table 8. Influence of developmental stage and ultraviolet radiation treatment (UVB control: 0.059 kJ m-² h-1 UVB; reduced UVB: 0.017 kJ m-² h-1 UVB; low UV: 0.002 kJ m-²h-1 UVB) on nitriles (µg g-1 fresh weight) in pak choi. Different letters represent significant differences (p ≤ 0.05 by Tukey’s HSD test) for each nitrile (n=4, mean±standard error). 4MTB-CN, 5-(methylthio)pentylnitrile, 5MTP-CN, 6-(methylthio)hexylnitrile; 4MSOB-CN, 5-(methylsulfinyl)pentylnitrile; 5 MSOPCN, 6-(methylsulfinyl)hexylnitrile; 3But-CN, 4pentenenitrile; IAN, indole-3-acetonitrile; 2PE-CN, 3-phenylpropanenitrile. In order to illustrate from which glucosinolate the nitrile derived, abbreviation of nitriles are usually based on the cyanide (CN) name. Developmental stage Sprouts

5-leaf plants

15-leaf plants

30-leaf plants


UV treatment



Sprouts

Total nitriles 25.5±3.261 52.2±8.370 33.1±1.844 3.1±1.182 3.9±2.217 2.3±0.676 10.4±1.624 10.8±1.769 8.3±1.216 14.4±4.235 12.2±4.828 9.5±2.280

4MTB-CN BC D CD A A A AB AB AB ABC AB AB

10.3±1.334 19.0±1.648 15.6±1.780 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.2±0.197 0.0±0.000

5MTP-CN B C C A A A A A A A A A

4MSOB-CN

1.1±0.132 2.1±0.198 1.6±0.364 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 1.5±0.731 2.6±2.528 0.2±0.124


36.9±4.371 3.1±0.809 9.8±0.874 12.0±2.143

15.0±1.363 0.0±0.000 0.0±0.000 0.1±0.066

1.6±0.177 0.0±0.000 0.0±0.000 1.4±0.846


13.4±2.449 19.8±5.395 13.3±3.124

2.6±1.192 4.8±2.153 3.9±1.788

0.6±0.237 1.2±0.643 0.4±0.190

1.1±0.270 9.0±2.721 2.6±0.480 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000

A A A A

5MSOP-CN A B A A A A A A A A A A

0.0±0.000 3.9±1.145 0.6±0.123 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000

3But-CN A B A A A A A A A A A A

IAN

8.5±1.400 8.9±2.460 9.0±1.036 0.4±0.162 0.1±0.118 0.4±0.211 2.8±0.467 1.9±0.576 2.7±0.807 9.1±2.346 7.9±1.944 6.5±2.100

4.2±1.363 0.0±0.000 0.0±0.000 0.0±0.000

1.5±0.617 0.0±0.000 0.0±0.000 0.0±0.000

8.8±0.912 0.3±0.097 2.5±0.353 7.8±1.158

0.3±0.133 2.3±1.178 0.6±0.308

0.0±0.000 1.0±0.501 0.1±0.072

5.2±1.135 4.7±1.208 4.7±1.029

2.3±0.458 5.1±0.740 1.8±0.616 0.4±0.096 0.9±0.401 0.3±0.069 0.9±0.060 0.4±0.165 0.4±0.080 0.3±0.054 0.2±0.027 0.3±0.040

B A A B

2PE-CN B C AB A AB A AB A A A A A

2.3±0.459 4.2±0.894 2.0±0.272 2.3±0.971 2.9±1.876 1.6±0.448 6.7±1.166 8.5±1.245 5.3±0.936 3.6±1.275 1.4±0.347 2.4±0.491

3.0±0.547 0.5±0.150 0.6±0.087 0.2±0.026

2.8±0.430 2.3±0.668 6.8±0.707 2.5±0.504

1.0±0.230 1.7±0.552 0.7±0.214

3.7±0.651 4.2±0.876 2.8±0.455

***

***

*

***

***

***

***

***

ns

*

ns

*

***

ns

***

ns

*

***

ns

***

***

ns

***

ns

36


A A B A

Page 39 of 39



37


Effects of Developmental Stages and Reduced ... - ACS Publications

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