Flavonol Glucoside and Antioxidant Enzyme Biosynthesis Affected by

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Flavonol Glucoside and Antioxidant Enzyme Biosynthesis Affected by Mycorrhizal Fungi in Various Cultivars of Onion (Allium cepa L.) Mohanna Mollavali, Saheb Ali Bolandnazar, Dietmar Schwarz, and Fariborz Zaare Nahandi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04791 • Publication Date (Web): 22 Dec 2015 Downloaded from http://pubs.acs.org on January 3, 2016

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Journal of Agricultural and Food Chemistry

Flavonol Glucoside and Antioxidant Enzyme Biosynthesis Affected by Mycorrhizal Fungi in Various Cultivars of Onion (Allium cepa L.)

Mohanna Mollavali1*, Saheb Ali Bolandnazar1, Dietmar Schwarz 2, Fariborz Zaare Nahandi1

1

Department of Horticulture, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

2

Leibniz Institute for Vegetable and Ornamental Crops. Theodor-Echtermeyer-Weg 1,

14979 Großbeeren, Germany

* Corresponding author: E-mail: [email protected].

1 ACS Paragon Plus Environment


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ABSTRACT

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The objective of this study was to investigate the impact of mycorrhizal symbiosis on qualitative

3

characteristics of onion (Allium cepa L.) For this reason, five onion cultivars with different scale

4

color and three different strains of arbuscular mycorrhizal fungi (Diversispora versiformis,

5

Rhizophagus intraradices, Funneliformis mosseae) were used. Red cultivars mainly ‘Red Azar

6

shahr’ showed highest content in vitamin C, flavonols and antioxidant enzymes. Mycorrhizal

7

inoculation increased total phenolic, pyruvic acid and vitamin C of onion plants. Considerable

8

increase was observed in quercetin-4′-O-monoglucoside and isorhamnetin-4′-O-monoglucoside

9

content in plants inoculated with Diversispora versiformis, but quercetin-3,4′-O-diglucoside was

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not significantly influenced. Analyses for phenylalanine ammonia-lyase (PAL) and antioxiodant

11

enzyme activities such as polyphenol oxidase (PPO), catalase (CAT) and peroxidase (POD)

12

revealed that all excep PPO were enhanced by mycorrhizal inoculation. Overall, these findings

13

suggested that mycorrhizal inoculation influence biosynthesis of flavonol glucosides

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antioxidant enzymes by increasing nutrient uptake or by induction of the plant defence system.

15

KEYWORDS: AMF, pyruvic acid, vitamin C, phenylalanine ammonia-lyase


and

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INTRODUCTION

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Antioxidant compounds scavenge free radicals and play an important role in human health with

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reducing the risk of chronic diseases including cancer and cardiovascular diseases. Grains, fruits

19

and vegetables are consider as natural sources for antioxidant compounds such as vitamin C,

20

vitamin E, carotenes, phenolic compounds (flavonoids and phenolic acids), phytate,

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phytoestrogens and nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids, and

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amines).1,2

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Onion (Allium cepa L.) plants are a rich source of three main groups of phytochemicals

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consisting of nonstructural and soluble carbohydrates, sulfur (S)-containing compounds and

25

different types of phenolics, mainly flavonols.3 Earlier reports revealed that phenolic compounds

26

can act as antioxidants due to their high tendency to chelate metals.4

27

Onion contain cosiderable amount of flavonoids, mainly Quercetin-4′-O-monoglucoside

28

(QMG) and quercetin- 3,4′-O-diglucoside (QDG) comprising up to 80% of the total flavonoid

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content.5

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Many studies report that frequent consumption of onion is associated with reduced risk of

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many forms of cancer, cardiovascular and neurological diseases, osteoporosis and cataract

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formation.6 Concerning the health benefits of onion plants, more attention should be paid to

33

promote their proven antioxidant properties. Genotypic, environmental factors, pre- and post-

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harvest treatments can affect onion yield and qualitative characteristics mentioned before.7,8

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Soil microorganisms can help plants to enhance their growth and quality. Among them are

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arbuscular mycorrhizal fungi (AMF) one of the most important soil microorganisms which

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associated with over 80% of terrestrial plant species. The fungi promote plant growth by 3 ACS Paragon Plus Environment


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enhancing nutrient uptake, mainly phosphorus and resistance or by biotic and abiotic stress

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tolerance. In return they obtain fixed carbon compounds from the host plants. During this

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mutualistic association, some metabolic changes in host plants roots occur as a plant defence

41

response.9 It has been reported that AMF can affect directly or indirectly in host plant roots in the

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production and accumulation of secondary metabolites, including antioxidants and phenolic

43

compounds.10,11

44

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) as a key enzyme in the flavonoid

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biosynthesis can be affected by genotype, plant developmental stage, various stresses caused by

46

a number of environmental factors and mineral nutrition.12 AMF have been implicated in

47

increased PAL activity in clonized clover roots13.

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Antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx),

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catalase (CAT), polyphenol oxidase (PPO) and peroxidase (POD) play an important role to

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protect plants from oxidative stresses. Thus, increasing production of reactive oxygen species

51

(ROS) during mycorrhizal symbiosis has been reported.14 Under stress condition, mycorrhizal

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inoculation can enhance tolerance of host plants by increasing or decreasing the activity of

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antioxidant enzyme such as SOD, CAT, POD and ascorbate peroxidase (APX).15,16

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Further investigations are needed to determine the effect of onion scale color on qualitative

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characteristics such as flavonol glucosides and antioxidant properties, despite previous studies.14-

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16

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quality. With e regard to AMF fungi impact on plant growth and nutrition, we hypothesized that

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AMF fungi can enhance the flavonol glucosides by induction of antioxidants and enzymes

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responsible for phenolic compounds biosynthesis. Therefore, the aim of this study was to

Moreover, limited information is available on the efficiency of different AMF strains on onion


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investigate how AMF could change and/or enhance quercetin glucoside compounds in different

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scale color of onion (Allium cepa L.).

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MATERIALS AND METHODS

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Plant Material and Mycorrhizal Inoculation. A pot experiment was carried out from

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May to October 2012 in the Agricultural Research Station of the University of Tabriz, Iran. The

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experiment was arranged as factorial based on a completely randomized block design with two

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factors and three replications. The first factor was consisted of four Iranian native genotypes of

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onion (‘Red Azar-shahr’, ‘White Kashan’, ‘Yellow Gholi Ghesse’, ‘Pink Horand’) and a

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commercial cultivar (‘Red Rosita’). Three different strains of mycorrhizal fungi (Diversispora

69

versiformis, Rhizophagus intraradices, G.Funneliformis mosseae) were constituting the second

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factor. Seeds were disinfected with sodium hypochlorite (1%) for 10 minutes and then sown in a

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sandy loam. The soil was autoclaved at 121 °C for 2 h. Fifty grams of AMF inoculum (a mixture

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of spores, hyphae, AM root fragment and soil) were mixed into one kg of soil.17 The control pots

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received the same amount of sterilized inoculum. Three plants from each treatment were sampled

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randomly every week for 50 days (from emergence to transplanting) to determine the incidence

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of root colonization. Nine weeks after sowing, three seedlings were transplanted to plastic pots

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(22 cm in diameter, 21 cm in depth and volume of 6 L). The mean temperatures during the

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experiments in the greenhouse were 26/18 °C day/night and the mean relative humidity was 50–

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70%. Onion plants were grown at 200 µmol m-2 s-1 light intensity.

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Harvest. Plants were harvested four months after transplanting when 80% of the onion

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leaves had fallen. Then, the roots were separated from the bulbs; one plant from each pot was

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used for determination of vitamin C, pyruvic acid and total phenolic content. The roots and the



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outer skin from the remaining plants were removed and then were divided into four wedge-

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shaped pieces by a longitudinal cutting. Two pieces of each bulb immediately were stored for

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determination of enzymes activity assay for a short period at -20 ºC.The other two pieces were

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rapidly frozen in liquid nitrogen and were freeze-dried for the analysis of flavonol glycosides

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concentration.

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Total Phenolic Content (TPC). TPC was determined using the Folin-Ciocalteu method

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described by Slinkard and Singleton,18 with gallic acid as standard. In brief, 0.1 mL of 85%

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methanolic onion extract was added to 1 mL of Folin-Ciocalteu 10% reagent and was shaken.

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After 6 min, 2 mL of sodium carbonate (7.5%) was added into the mixture and then was placed

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for 2 h in the dark. The absorbance was measured at 760 nm with an UV-VIS Spectrophotometer

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(Spekol 1500, Analytik Jena, Germany). The results were expressed as mg of gallic acid

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equivalents (GAE) per 100 g fresh weight.

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Vitamin C and Pyruvic Acid Concentration. Vitamin C content of bulbs was

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measured using the 2, 6 dichlorophenol indophenol volumetric method.19

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Pyruvic acid concentration, as an indicator of pungency, was measured using the method

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described by Schwimmer and Weston.20 Onion juice from one-half of a bulb was filtered through

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two layers of cheesecloth and then centrifuged. Subsequently, 0.5 mL water and 0.5 mL of 0.125

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g L−1 DNPH in 2 M HCl were added to the 20 µL of extract. The samples were placed in a 37 ºC

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water bath for 10 min, then 2.5 mL of 0.6 M NaOH was added. Standards were prepared by

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adding 25–200 µL of 1 mM sodium pyruvic acid and the absorbance at 420 nm with the UV-VIS

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Spectrophotometer Spekol 1500 was measured.


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Flavonol Glycoside Analysis Using High-Performance Liquid Chromatography

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with Diode Array Detector (HPLC–DAD). The flavonoid profile of the three major flavonol

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glycosides (quercetin-4′-O-monoglucoside (QMG), quercetin-3,4′-O-diglucoside (QDG) and

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isorhamnetin-4′-O-monoglucoside, were analyzed. HPLC analysis of the flavonols glycosides

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was performed as described by Rohn et al.21 For the analysis of onion bulbs, 2.5 g of lyophilised,

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powdered onion samples were extracted with 50 mL of aqueous methanol (70%) for 30 min

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under continuous stirring. The extract were filtered (Whatman filter, Ø150 mm, 597½) and 4 mL

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of the filtrate was dried under a stream of nitrogen. After subsequent dilution with 2 mL of

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water, the exctract was loaded onto a solid phase extraction column (Chromabond PA, 6 mL, 500

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mg, Macherey–Nagel, Düren, Germany). The column was washed with 10 mL of water to

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remove sugars and further water soluble compounds. The flavonol glucosides were eluted with

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10 mL of a methanol/water/acetic acid mixture (90:5:5, v/v).This dilution was used for the LC–

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DAD analysis (Smartline series system from Knauer GmbH, Berlin, Germany). The Low

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Pressure Gradient consisted of a Smartline manager (5050 series), pump (1000 series),

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autosampler (3950 series) and diode array detector (2600 series). The system was controlled by

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ClarityChrom 3.0 software (Knauer GmbH, Berlin- Germany). A binary gradient system based

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on Riehle et al.22 with eluent (A) 0.1% formic acid in water, eluent (B) 0.1% formic acid in

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acetonitrile was carried out on a Luna® 5 µm C18 100 Å (150×3.00 mm) column equipped with

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a C18 security guard (4×3.00 mm), both from Phenomenex Inc. (Aschaffenburg, Germany).

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Gradient elution was used for methanolic SPE eluates: 5% B isocratic (0–2 min), 5–10% B (2–6

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min), 10–30% B (6–45 min), 30–95% B (45–55 min), 95% B isocratic (55–60 min), 95–5% B

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(60–65 min), and 5% isocratic (65–75 min). The flow rate was 0.6 mL/min and the column

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temperature was 21 °C. Simultaneous detection was performed at 280, 325 and 365 nm.



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Protein Quantification. Protein concentration was estimated using the Bradford

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method.23

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Reagent and Standard Solutions. Five-fold Coomassie Brilliant Blue (CBB) stock solution was

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prepared as Bradford reagent. 50 mg of CBB was mixed with 25 mL of methanol and 50 mL

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orthophosphoric acid in a dark bottle and kept in the refrigerator. A serial dilution series (0.002-

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0.01 mg/mL) of Bovine Serum Albumin (0.1 mg/mL) was made as standard solution.

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Sample Preparation. 1 g of fresh tissue was ground in 4 mL of extraction buffer (0.01 M

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potassium phosphate) and was then centrifuged at 18000 g at 4 °C for 10 min. The supernatant

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was used for protein and enzyme assay.

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100 µL of the protein extract was added to 160 µL of reagent and 740 µL of buffer and mixed.

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Formation of blue color was measured at the wavelength of 595 nm using the UV-VIS

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Spectrophotometer.

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Phenylalanine Ammonia-Lyase. PAL activity was measured with the method as

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described by Zucker.24 In brief, the rate of conversion of L-phenylalanine to trans-cinnamic acid

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was determined spectrophotometrically with the UV-VIS Spectrophotometer at 290 nm for

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approximately 5 minutes. The assay mixture contained L-phenylalanine (0.833 mM), Tris-HCl

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buffer (pH 8.5) and enzyme extract in a total volume of 3.0 mL.

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Polyphenol oxidase. PPO activity was determined following the method described by

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Mayer et al.25 The reaction mixture consisted of 100 µL of the enzyme extract and 1400 µL of

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0.01 M sodium phosphate buffer (pH=7). In extremis, 500 µL substrate (1 M pyrocatechol) was

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added to the mixture to start the reaction. The changes in absorbance at 420 nm were recorded at

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10 s intervals for 5 min with 3 replications.


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Peroxidase. POD activity was estimated by measuring the color development at 470 nm

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with spectrophotometer Spekol model 1500 during the formation of tetraguaiacol.26 The assay

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mixture comprised; 1000 µL of 0.1 M sodium phosphate buffer (pH=7), 500 µL guaiacol, 200 µL

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of the enzyme extract and 300 µL of H202.

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Catalase. Decomposition of H2O2 was assayed followed spectrophotometrically by the

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decrease in absorbance at 240 nm.26 3 mL of reaction mixture in quartz cuvette contained; 2300

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µL of phosphate buffer 25 mM (pH=7), 200 µL of enzyme extract and 500 µL of H202.

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Statistical Analysis. Data were analyzed according to experimental design and means

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were compared by the Duncan’s multiple range test. Multivariate analysis of variance

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(MANOVA) was applied to evaluate the treatments effect with a significance level of P≤0.01.

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All statistical analyses were carried out by using SPSSsoftware package (v. 18.0, SPSS, IBM,

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USA).

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RESULTS

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Pyruvic Acid, Reducing Sugar, Vitamin C and Total Phenolic Content.

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Mycorrhizal inoculation and cultivar affected pyruvic acid, reducing sugar, vitamin C and TPC,

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significantly (Table 1).

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The interaction between cultivar and mycorrhizal inoculation on reducing sugar and vitamin

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C content was significant. Interestingly, AMF colonization of ‘Red Azar shar’, ‘Yellow Gholi

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ghesse’ and ‘Red Rosita’ cultivars, increased reducing sugars up to 2-2.5 fold compared with the

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non AMF treatments, most with Rhizophagus intraradices. As shown in Figure 2, the lowest

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vitamin C content among all cultivars was observed in non-inoculated plants from ‘White

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Kashan’ and ‘Pink Horand’. A significant difference between the AMF and non AMF 9 ACS Paragon Plus Environment


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inoculation was found for vitamin C in all cultivars studied. The highest vitamin C content was

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achieved in plants of‘Red Azar shahr’ inoculated with Rhizophagus intraradices (15.27±2.04

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mg/100g FW) followed by ‘Gholi ghesse’ and ‘Red Rosita’(14.87±1.2 and 14.81±0.89 mg/100g

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FW) (Figures 1,2). A significant variation for pyruvic acid and TPC, was observed among the

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cultivars. As expected, red cultivars (‘Rosita’ and ‘Azar shahr’) had the highest pyruvic acid

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(8.28±0.85 and 8.22±0.94 µmol/g FW) and TPC (62.81±6.38 and 60.37±4.69 mg gallic acid/g

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FW). Notably, a significant increase was found between application of mycorrhizal fungi and

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levels of pyruvic acid and TPC. Differences between mycorrhizal fungi strains were significant

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and Diversispora versiformis was the most effective strain to improve concentration of pyruvic

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acid and TPC in the bulb by 41.1% and 34%, compared with the non-inoculated

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treatments(Table 3).

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Flavonol Glycosides Content. In the present study we identified three major flavonol

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compound consisting of QDG, QMG and Isorhamnetin- 4'-glucoside. No interactions were found

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between cultivars and mycorrhizal inoculation on flavonol glycosides content. The concentration

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of flavonol glycosides significantly differed among the cultivars (Table 1). Flavonol glycoside

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content for all substances in ‘was 2-3 fold higher in ‘Red Azar shahr’ than in the other cultivars

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studied (QDG, 84.8±0.28; QMG, 31.32±1.61; isorhamnetin 4'-glucoside, 3.29±0.41 µmol/g). As

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expected, non AMF plants of white ‘Kashan’ and pink ‘Horand’ contained the lowest content of

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flavonol glycosides (QDG, 28.3±1.24, 27.51±2.95; QMG, 7.33±0.63, 7.22±0.24; isorhamnetin

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4'-glucoside, 2.21±0.4, 1.59±0.22 µmol/g)( (Table 4).

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Mycorrhizal inoculation compared with the non AMF plants significantly affected on QMG

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up to2.5 fold and isorhamnetin-4'-glucoside content by 1.5 fold in the bulbs. Whereas, AMF

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inoculation had no significant effect on QDG content (Table 1). Diversispora versiformis was the 10 ACS Paragon Plus Environment

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most effective AMF strain and increased the content of QMG in the bulbs up to 25.02±0.82,

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however, differences between AMF strains on isorhamnetin-4'-glucoside was not significant

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(Table 4).

196 197

PAL, PPO, POD and CAT Enzyme Specific Activity. Significant variation of enzyme activity was observed with mycorrhizal inoculation for all cultivars studied (Table 2).

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The most pronounced effect of AMF inoculation on PAL, POD and CAT enzymes activity

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was found with Diversispora versiformis followed by Rhizophagus intraradices inoculation

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(Figures 3, 5 and 6). AMF inoculation of onion plants was notable in ‘Red Azar shahr’ and

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markedly increased with Diversispora versiformis the specific activity of PAL, POD and CAT

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up to 0.794±0.007, 536.38±4.47 and 12.77±0.38 U/mg protein, respectively. ‘White Kashan’ and

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‘Pink Horand’ without AMF inoculation received the lowest enzymes activity (Figures 3, 5 and

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6). In contrast, PPO activity was diminished when bulbs were inoculated with AMF. Plants

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inoculated with Diversispora versiformis showed the lowest value of PPO activity among all

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AMF treatments. Furthermore, the highest PPO activity was observed in control plants of ‘Red

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Rosita’

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respectively)(Figure 4).

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DISCUSSION

and

‘Yellow

Gholi

ghesse’

(0.61±0.003

and

0.588±0.005

U/mg

protein,

210

Pyruvic Acid, Total Phenolic Content, Reducing Sugar and Vitamin C. In onions,

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organosulfur compounds are responsible for the taste and flavor which are synthesized from a

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common precursor, the (S)-alk(en)yl-cysteine sulfoxides. An obvious trend was observed

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between pyruvic acid levels and the scale color. Previous studies showed that onion cultivars

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with different scale color are different in their organosulfur and phenolic compounds.27,28 11 ACS Paragon Plus Environment


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Phenotypic differences such as shape, size, color and bulb diameter caused qualitative variation

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in different genotypes.29 A positive correlation between darker pigments of bulbs and higher

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pungency in the order as follows: white< red< yellow.30 In our study, the pyruvic acid level was

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in a similar range as reported by Gallina et al.31

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Soil microorganisms, plant nutrition and sulfur availability affect flavor intensity of onions.20 In

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the present study AMF inoculation increased enzymatically produced pyruvic acid and this

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increase has been demonstrated to be due to improving sulfur and nitrogen nutrition by AMF

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inoculation (data not shown).32,33

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Higher TPC in red cultivars compared with the other colored cultivars studied concur with the

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finding by Lachman et al.34 The increased TPC in AMF inoculated plants can be due to

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induction of defense mechanisms as a result of the symbiosis. Previous studies showed that

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mycorrhizal inoculation increased phenolics concentration and antioxidant activity in host

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plants.35,36 A positive linear correlation was observed between total phenolic content and

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antioxidant activity (r2 =0.524**) in dependent of the cultivars tested (data not shown). A similar

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correlation has been reported between TPC and antioxidants in onions by Cheng et al.37

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In mutualistic symbiosis of AMF and cultivated plants, water and nutrient uptake, especially

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phosphorus, is improved for the host plant and in exchange, the fungi demand organic carbon for

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their growth.38 This association help mycorrhizal plants to grow better compared with non-

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mycorrhizal plants, with higher leaf areas and consequently, higher photosynthetic rates.

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Improved phosphorus uptake in inoculated plants plays an important role during the breakdown

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of carbohydrates and in starch metabolism. Increasing reducing sugars in red cultivars inoculated

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with Rhizophagus intraradices can be ascribed by higher photosynthetic rates and phosphorus


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concentration in symbiosis with this strain(data not shown). This result is in agreement with

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previous findings reporting higher of reducing sugar in content in AMF inoculated plants.39

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In the present study vitamin C content affected by both cultivar and AMF inoculation. It has

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been reported that vitamin C level varies between different onion cultivars and that red and

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yellow cultivars have higher vitamin C content than others.40 Mycorrhizal symbiosis resulted in

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an enhanced water and nutrient uptake and consequently in an increase of biomass, soluble

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solids, vitamin C and other qualitative characteristics caused by an increased root surface area.41

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Flavonol Glycosides Content. A significant difference of QDG, QMG and

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isorhamnetin-4'-glucoside concentrations were observed among the cultivars studied. Previous

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reports have demonstrated significant differences of flavonol content between different onion

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cultivars and reported that red cultivars had higher flavonol content followed by yellow cultivars.

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Also white cultivars lower concentration of these compounds compared with other cultivars,

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although even 10 fold lower.42,43 Flavonoids are compounds responsible for onion pigmentation.

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Quercetin and anthocyanin in red/purple cultivars are the main flavonoid compounds in onions.

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The main differences between onion cultivars with different scale color caused by activation or

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diactivation of genes involved in anthocyanin synthesis pathway. White onion cultivars have low

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amount of quercetin derivatives which is due to the reduced transcription of the chalcone

254

synthase gene in white onions.44 In all cultivars QDG levels was 3-4 fold higher than QMG what

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confirms with the result of Beesk et al.45 Chromatographic analysis of three major flavonol

256

glucosides revealed that generally all flavonol glucosides concentrations were higher in AMF

257

inoculated plants than non AMF ones. Remarkably, QDG content increased in inoculated plants

258

by Diversispora versiformis compared with other AMF strains and control treatment. Different

259

AMF species have different efficiency to enhance secondary metabolites in the host plants. 13 ACS Paragon Plus Environment


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Mycorrhizal inoculation can increase antioxidant activity as defense response during AMF

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symbiosis by increasing flavonoid content in inoculated plants. Increasing flavonol glucosides in

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mycorrhizal plants is in agreement with earlier studies of Guo et al.33 and Perner et al.35 due to

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phytochemicals formation and in response to attack by fungal pathogens.

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PAL, PPO, POD and CAT Enzymes Specific Activity.

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A higher PAL activity of Diversispora versiformis inoculated ‘Red Azar shahr’ plants can be

266

explained by a higher root colonization of this cultivar compared with the other cultivars (data

267

not shown). Flavonols are assumed potent ROS-scavenging compounds. PAL activity as a key

268

enzyme in the flavonoid biosynthesis pathway depends on some factors, such as genotype, plant

269

development stage, organ, light, temperature, disease and mineral nutrition.14,46 Flavonoids can

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act as signal molecules during mycorrhizal symbiosis. A possible mechanism is morphological

271

similarity between mycorrhizal fungi structure and some pathogens. Moreover, regarding the

272

effect of mycorrhizal fungi on increasing mineral nutrients, PAL activity can increase in

273

mycorrhizal plants. It was reported that mycorrhizal colonization caused induction of PAL

274

activity in inoculated plants.47 Increased accumulation of PAL and chalcone synthase enzymes in

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the colonized root cells of Phaseolus vulgaris has been reported by Kristopher and Anderson.48

276

Largely induced POD and CAT activity of inoculated ‘Red Azar shahr’ plants with

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Diversispora versiformis can be justified with a high flavonol content in the cultivar and

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flavonol’s potential to scavenge free radicals formed in plant cells. It should be reminded that

279

AMF symbiosis had an important role to protect host plants against biotic and abiotic stresses by

280

nutritional and non-nutritional factors.11 Production of activated oxygen species is one of the


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plant responses to pathogenic infection.15 Increasing expression of some genes related to plant

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defence response, has been reported in cells containing arbuscules.49

283

In contrast to the POD and CAT activities, PPO activity was affected inversly by AMF

284

colonization. This adverse relationship has been demonstrated to be attributedto the role of

285

onion extract as a natural inhibitor of the PPO activity to prevent or reduction of enzymatic

286

browning in vegetables.50 One possible mechanism can be the induction of endogenous

287

inhibitor/regulator of PPO in onion plants by AMF symbiosis. Another possible mechanism is

288

organosulfur compounds presence in onions particularly, thiosulfinates and (S)-alk(en)yl-

289

cysteine sulfoxides, which are responsible for inhibitory of PPO activity.51 It appears

290

mycorrhizal inoculation reduces PPO activity by enhancing organosulfur compounds, measured

291

as pyruvic acid in this experiment.

292

In conclusion, our data confirm that red cultivars have higher amount of pyruvic acid (a

293

pungency indicator), vitamin C and antioxidant enzyme activities as a result of their high

294

flavonoid content. Increased antioxidant enzyme activity indicated that mycorrhizal inoculation

295

may act as biotic stress and induce activities of antioxidant enzymes by increasing nutrient and in

296

consequence enhanced flavonol content or by activating certain defence responses. Generally,

297

application of Diversispora versiformis caused an increase in total phenolics, flavonol

298

glucosides, PAL and antioxidant enzymes activities. Diversispora versiformis was an efficient

299

fungal strain in onion plants that enhanced their flavonoid and antioxidant enzymes activities.

300

However, more detailed studies needs to clarify the exact role of mycorrhizal fungi symbiosis on

301

increasing the antioxidant activity and regulation of plant defensive response.

302

ABBREVIATIONS USED



Page 16 of 34

303

AMF, Arbuscular mycorrhizal fungi; QMG, quercetin-4′-O-monoglucoside; QDG, quercetin-

304

3,4′-O-diglucoside; PAL, phenylalanine ammonia-lyase; PPO, polyphenol oxidase; CAT,

305

catalase; POD, peroxidase; TPC, total phenolic content; HPLC–DAD, high-performance liquid

306

chromatograph with diode array detector; ACSOs, (S)-alk(en)yl-cysteine sulfoxides

307

ACKNOWLEDGMENTS

308

We would like to thank Prof. Sascha Rohn and Dr. Peer Riehle, Institute of Food Chemistry,

309

University of Hamburg, who provided HPLC-DAD analysis. Prof. Aliasgharzad, Department of

310

Soil Science, University of Tabriz, is acknowledged for kindly providing AMF inocula and

311

constructive suggestions in this work.

312

Funding

313

This research was supported by the Ministry of Science, Research and Technology of Iran

314

(MSRT).

315

Notes

316

The authors declare no competing financial interest.

317

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318

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444


FIGURE CAPTIONS Figure 1. Changes in reducing sugar content in different onion cultivars affected by mycorrhizal fungi Figure 2. Changes in vitamin C content in different onion cultivars affected by mycorrhizal fungi Figure 3. Changes in PAL specific activity in different onion cultivars affected by mycorrhizal fungi Figure 4. Changes in PPO specific activity in different onion cultivars affected by mycorrhizal fungi Figure 5. Changes in POD specific activity in different onion cultivars affected by mycorrhizal fungi Figure 6. Changes in CAT specific activity in different onion cultivars affected by mycorrhizal fungi



Page 24 of 34

Table 1. Pyruvate, Reducing Sugars, Vitamin C and Flavonols of Onion Affected by Cultivar and Mycorrhizal Inoculationa Mean square Source of

d.f Pyruvate Reducing Vitamin

variation

sugars

C

Total

QDG1)

QMG2)

phenolic

Isorhamnetin4′-Omonoglucoside

Cultivar (C)

4

38.67*

5.24*

82.39*

1021.68* 7231.64*

1255.72*

4.45*

AMF (M)

3

15.93*

9.79*

60.69*

659.06*

1054.77ns

576.71*

2.97*

C×M

12

8.72ns

2.27*

9.76*

106.92ns

644.32ns

239.41ns

1.65ns

Error

40

5.02

0.09

1.62

70.51

696.79

228.71

1.01

a

*significantly different at P≤0.05. 1)quercetin-3,4′-O-diglucoside. 2)quercetin-4′-O-monoglucoside


Page 25 of 34


Table 2. Antioxidant Enzyme Activity of Onion Affected by Cultivar and Mycorrhizal Inoculationa Mean square d.f

Protein

PPO1)

POD2)

CAT3)

PAL4)

Cultivar (C)

4

65.15*

0.04*

91465.77*

15.68*

0.09*

AMF (M)

3

2.96*

0.10*

98633.58*

9.02*

0.25*

C×M

12

20.06*

0.02*

95411.55*

12.38*

0.02*

Error

40

0.30

0.00

181.42

0.05

0.00

Source of variation

a

* significantly different at P≤0.05. 1) polyphenol oxidase 2) peroxidase 3)catalase 4) phenylalanine ammonia-lyase



Page 26 of 34

Table 3. Effect of Cultivar and Mycorrhizal Inoculation on Pyruvate and Total Phenolic Content of Onion Bulbsa

Treatments

Cultivar

Mycorrhiza

a

Pyruvate

Total phenolic (mg

(µmol/g FW)

galic acid/g FW)

Yellow gholi Ghesse

8.07±0.82a

50.99±5.90b*

White Kashan

4.34±0.63b

41.65±3.29c

Red Rosita

8.28±0.85a

62.81±6.38a

Pink Horand

5.75±0.47b

45.25±4.47bc

Red Azar-shahr

8.22±0.94a

60.37±4.69a

Non-inoculated

5.42±0.52b

43.24±4.74c

Funneliformis mosseae

7.51±0.87a

56.37±2.75ab

Rhizophagus intraradices

7.14±0.76a

51.27±7.98b

Diversispora versiformis

7.65±0.71a

57.97±4.75a

Means followed by non-similar letters are significantly different at P≤ 0.05 according to Duncan’s

multiple range test. Values are means±SD (n=3).


Page 27 of 34


Table 4. Effect of Cultivar and Mycorrhizal Inoculation on Flavonols Content of Onion Bulbsa Quercetin-3,4'-

Treatments

glucoside (µmol/g)

Cultivar

Quercetin-4'-

Isorhamnetin-4'-

glucoside

glucoside

(µmol/g)

(µmol/g)

Gholi Ghesse

31.69 ±2.67b *

13.88±1.14bc*

White Kashan

28.30±1.24b

7.33±0.63c

2.21±0.4b

Red Rosita

49.01±0.81b

22.29±1.46ab

2.33±0.27b

Pink Horand

27.51±2.95b

7.22±0.24c

1.59±0.22b

Red Azar-shahr

84.80±0.28a

31.32±1.61a

3.29±0.41a

Mycorrhizae Non-inoculated

34.24±0.66a

10.87±0.76b

1.71±0.15b


44.44±0.79a

17.55±2.24ab

2.57±0.27a


48.97±1.21a

14.04±0.92ab

2.55±0.45a


52.93±1.15a

25.02±0.82a

2.66±0.24a

a

2.43±0.23b*

Means followed by non-similar letters are significantly different at P≤ 0.05S according to Duncan’s

multiple range test. Values are means±SD (n=3).



Page 28 of 34

9 8

a

a b

b

7 Reducing sugar(%)

control c

6 c-f

5

cd cde e-h fgh hi gh

4

c-f

d-g

fghe-h

ij

fgh


j

j


3 2


1 0 Gholi ghesse

Kashan

Rosita

Horrand


Azar shahr

Page 29 of 34


18 16 Vitamin C (mg/100gr FW)

abc

a

a

a

ab

ab

14

abc

bcd

cd

de

12

def efg

10

cde Funneliformis mosseae

fgh

efg

g 8

gh

control

gh h

h Rhizophagus intraradices

6 4 2


0 Gholi ghesse

kashan

Rosita

Horrand

Azar shahr



Page 30 of 34

1

PAL activity (U/mg protein)

0.9

a control

0.8

b

0.7

c d

0.6 e

ef

0.5

0.3

fg

ij

0.4

j

hi

j

ef gh

gh


j Rhizophagus intraradices

k

k

l

l

0.2

ef

0.1


0 Gholi ghesse

kashan

Rosita

Horrand

Azar shahr


Page 31 of 34


PPO activity (U/mg protein)

0.7 0.6

a

ab cd

bc cd

d

0.5 0.4

control

bc d g

e f

cde f

h

i

kl

k 0.3


j

ij l


0.2 0.1


0 Gholi ghesse

kashan

Rosita

Horrand

Azar shahr



Page 32 of 34

POD activity (U/mg protein)

700 a 600

control b

500


400 300

cd d

200

e fg

100

g

cd cd

cd

e

f

e

c

c

c

f

g

f

e Diversispora versiformis

0 Gholi ghesse

kashan

Rosita

Horrand


Azar shahr


Page 33 of 34


16 a

CAT activity (U/mg protein)

14

control 12 10

c de

8

de ef

hi

b

c

i hi

ghi

e

d

d de gh

fg de

ef

de

6

Funneliformis mosseae Rhizophagus intraradices

4 Diversispora versiformis

2 0 Gholi ghesse

kashan

Rosita

Horrand

Azar shahr



TOC Graphic Mycorrhizal colonization changes secondary metabolites of onion plants


Page 34 of 34

Flavonol Glucoside and Antioxidant Enzyme Biosynthesis Affected by

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