Variation in Key Flavonoid Biosynthetic Enzymes and Phytochemicals

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Variation in key flavonoid biosynthetic enzymes and phytochemicals in Rio Red grapefruit (Citrus paradisi Macf) during fruit development Priyanka R. Chaudhary, Haejeen Bang, Guddadarangavvanahally K. Jayaprakasha, and Bhimanagouda S. Patil J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02975 • Publication Date (Web): 03 Nov 2016 Downloaded from http://pubs.acs.org on November 8, 2016

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Journal of Agricultural and Food Chemistry

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Variation in key flavonoid biosynthetic enzymes and phytochemicals in Rio Red grapefruit (Citrus paradisi Macf) during fruit development

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Priyanka R. Chaudhary, Haejeen Bang, Guddadarangavvanahally K. Jayaprakasha*, and Bhimanagouda S. Patil*

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Vegetable and Fruit Improvement Center, Department of Horticultural Sciences,

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Texas A&M University, College Station, TX 77845, United States.

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8 9 10 11 12 13 14 15 16 17 18 19

*Corresponding authors

20 21 22 23 24 25 26 27 28 29 30 31

Bhimanagouda S. Patil, Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, 1500 Research Parkway, Suite# A120, College Station, TX-77845, USA Tel.: +1 979 862 4521; fax: +1 979 862 4522. E-mail address: [email protected] G.K. Jayaprakasha, Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, 1500 Research Parkway, Suite# A120, College Station, TX-77845, USA Tel.: +1 979 845 2743; fax: +1 979 862 4522.

ACS Paragon Plus Environment


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E-mail address: [email protected]

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______________________________________________________________________________

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ABSTRACT: In the current study, phytochemical contents and expression of genes involved in

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flavonoid biosynthesis in Rio Red grapefruit were studied at different developmental and

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maturity stages for the first time. Grapefruit were harvested in June, August, November, January,

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and April and analyzed for the levels of carotenoids, vitamin C, limonoids, flavonoids, and

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furocoumarins by HPLC. In addition, genes encoding for phenylalanine ammonia lyase (PAL),

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chalcone synthase (CHS), chalcone isomerase (CHI) and 1, 2-rhamnosyltransferase (2RT) were

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isolated and their expression in grapefruit juice vesicles was studied. Fruit maturity had

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significant influence on the expression of the genes, with PAL, CHS, and CHI having higher

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expression in immature fruits (June), while 2RT expression was higher in mature fruits

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(November and January). The levels of flavonoids (except naringin and poncirin), vitamin C and

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furocoumarins gradually decreased from June to April. Furthermore, limonin levels sharply

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decreased in January. Lycopene decreased while β-carotene gradually increased with fruit

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maturity. Naringin did not exactly follow the pattern of 2RT or of PAL, CHS, and CHI

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expression, indicating that the four genes may have complementary effects on the level of

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naringin. Nevertheless, out of the marketable fruit stages, early-season grapefruits harvested in

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November contained more beneficial phytochemicals as compare to mid- and late-season fruits

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harvested in January and April respectively.

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KEYWORDS: Citrus paradisi, flavonoids, phenylalanine ammonia lyase, chalcone synthase,

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chalcone isomerase, 1, 2-rhamnosyltransferase

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INTRODUCTION

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Plants produce several secondary metabolites to protect themselves from biotic and abiotic

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stresses. These secondary metabolites are classified on the basis of their structure, biosynthesis,

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and their functional groups. Flavonoids are class of secondary metabolites which occur

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ubiquitously in plants, and are classified in six different groups on the basis of their molecular

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structure: flavones, iso-flavones, flavonols, flavanones, anthocyanidins, and flavanols

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(catechins).1

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Citrus fruits are rich sources of flavanones, which are present in different fruit parts including

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rind (flavedo), albedo, juice sacs, lamella, and seeds.2 Flavanones are synthesized in response to

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different stresses. Naringin, narirutin, hesperidin, quercetin, eriocitrin, neohesperidin, didymin,

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poncirin are common flavanones reported in different citrus species. These compounds have

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been demonstrated for good antioxidant activity, chelating agents, and can benefit human health

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in different ways.3, 4 For instance, flavonoids inhibit DNA damage,5, 6 tumor development,7 and

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cell proliferation8 thus potentially acting as chemopreventative agents for different cancers.

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Flavonoids are biosynthesized through the phenylpropanoid pathway, which is a common

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pathway for tannins, flavonoids, lignins, stilbenes, and coumarins. Phenylalanine is the common

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precursor, marking as the first step in the phenylpropanoid pathway. Phenylalanine ammonia-

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lyase (PAL), the first enzyme involved in phenylpropanoid pathway, converts phenylalanine into

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cinnamate. Chalcone synthase (CHS), the first enzyme in flavonoid biosynthesis, converts 4-

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coumaroyl-CoA into naringenin chalcone, which is further converted into naringenin (flavanone)

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by chalcone isomerase (CHI).9 Naringenin is the main precursor for all flavanone aglycones

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which is further converted into naringenin 7-O-glucoside with help of 7-O-glucosyltransferase.

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The conversion

of

naringenin-7-O-glucoside into

naringin


is

catalyzed

by 1,

2-


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rhamnosyltransferase (2RT); while, 1, 6-rhamnosyltransferase (6RT) catalyzes the conversion of

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naringenin-7-O-glucoside to narirutin.10

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Several factors influence PAL expression in citrus fruits, including pathogen attack,11

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irradiation,12 ethylene and chilling injury.13, 14 Moriguchi et al. isolated CHS from Citrus sinensis

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(Osb.) and CHI from Citrus unshiu (Mark.), and studied their expression in citrus fruits during

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development.15,

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(Citrus paradisi, Macf.). Grapefruit also contains health promoting phytochemicals such as

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carotenoids, vitamin C, limonoids, and furocoumarins. The information regarding accumulation

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of phytochemicals in the pulp during fruit development and maturity is not been fully

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understood. In current study, phytochemicals such as vitamin C, carotenoids, limonoids and

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furocoumarins were examined by analyzing fruits harvested at different developmental and

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maturity stages. In the current study, we have isolated genes corresponding to PAL, CHS, CHI,

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and 2RT from immature grapefruits and studied their expression in juice vesicles of Rio Red

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grapefruit. This is the first study to isolate the main genes involved in flavanone biosynthesis

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from grapefruit and understanding the expression pattern during fruit development.

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However, these genes have not yet been isolated and studied in grapefruit

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MATERIALS AND METHODS

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Plant material. For cloning experiment, immature Rio Red grapefruits were harvested in June

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2012 from TAMUK-Citrus Center, Weslaco, Texas. For maturity study, fruits were harvested at

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different developmental stages at intervals of 75 days in June (Stage I, cell division), August

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(Stage II, cell expansion), November (Stage III, maturity, early season), January (Stage III,

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maturity, mid-season), and April (Stage III, maturity, late season) in 2012-2013 (Figure 1A).

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Fruits were harvested from three different trees from different blocks representing three


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replications. Four fruits from each trees were used for gene expression and phytochemical

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analysis. Juice samples for phytochemical analysis were prepared by blending individual peeled

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grapefruits and were stored at -80°C until further analysis.

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Chemicals. Reagent grade butylated hydroxytoluene (BHT), metaphosphoric acid, tris (2-

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carboxy ethyl) phosphine hydrochloride, L-ascorbic acid, lycopene, β-carotene, narirutin,

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naringin, neohesperidin, didymin, poncirin, limonin, 6’, 7’-dihydroxybergamottin (DHB),

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bergamottin and auraptene were procured from Sigma Aldrich Co. (St. Louis, MO, USA).

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Analytical grade solvents were obtained from Fisher Scientific Research (Pittsburgh, PA, USA).

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Vitamin C quantification. Vitamin C was extracted and quantified using liquid

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chromatography according to our optimized protocol.17 Each sample was analyzed three times

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and the total ascorbic acid contents were expressed as mg/100 mL juice.

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Carotenoid analysis. Extraction of carotenoids was performed according to a previously

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published method with slight modifications.18 Juice samples (10 g) were extracted using

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chloroform (15 mL) containing BHT (0.2%). An Agilent HPLC 1200 Series (Foster City, CA,

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USA) system consisting of a solvent degasser, quaternary pump, autosampler, column, oven, and

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diode array detector was used for quantification. A C-18, Gemini 5 µm column (250 mm × 4.6

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mm i.d.) with a guard cartridge was used (Phenomenex, Torrance, CA, USA). Elution was

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carried out using a gradient mobile phase of acetonitrile (A) and isopropyl alcohol (B).

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Carotenoids were detected at 450 nm and quantified using external standard calibration.

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Limonoids, flavonoids, and furocoumarins analysis.

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Sample preparation. Extraction was carried out according to previously published method

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with slight modification.18 Juice samples (10 g) were extracted ethyl acetate (15 mL) on a shaker



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for 3 hours. The organic layer was separated and the residue was extracted twice. All extracts

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were pooled and the solvent was evaporated to dryness. The dried residue was reconstituted with

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4 mL acetone, filtered using a 0.45 µm PTFE filter, and further analyzed for limonoids,

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flavonoids, and furocoumarins using HPLC.

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Quantification of limonoids and flavonoids using HPLC. Limonoids and flavonoids were

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quantified simultaneously using a Waters HPLC (Milford, MA, USA), spectra model with a

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PDA detector (2996) coupled with a binary HPLC pump 1525 and 717 plus auto sampler. The

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chromatographic separations were conducted on a C-18, Gemini 5 µm column (250 mm × 4.6

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mm i.d.) from Phenomenex (Torrance, CA, USA). Limonoids were detected at 210 nm and

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flavonoids were detected at 280 nm.18 The entire chromatographic separation was performed

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with a gradient mobile phase of 0.03 M phosphoric acid (A) and acetonitrile (B) with 1 mL/min

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flow rate. Each sample was injected three times.

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Quantification of furocoumarins using HPLC. Furocoumarins were analyzed using

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previously described method.18 Each sample was analyzed in triplicate and the results were

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expressed as µg/100 g fresh weight.

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RNA isolation and cDNA synthesis. Total RNA was isolated from juice vesicles of grapefruit

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with the RNeasy plant mini kit (Qiagen, Valencia, CA, USA). First-strand cDNAs were

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synthesized using the Advantage RT-for-PCR kit (Clontech, Mountain View, CA, USA)

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according to the manufacturer’s instructions.

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Gene cloning. SMARTer RACE cDNA amplification kit (Clontech, Mountain View, CA,

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USA) was used to synthesize PAL, CHS, CHI, and 2RT first strand cDNAs from immature Rio

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Red grapefruit. Forward and reverse degenerate primers were designed using available gene


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sequences through NCBI (http://www.ncbi.nlm.nih.gov/) from citrus species and other crops

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using Blockmaker and codehop software (Table S1).

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Advantage® 2 Polymerase (Clontech, CA, USA) was used for RACE cDNA amplification pcr.

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After purification, PCR products were cloned in the TOPO vector (Invitrogen, Carlsbad, CA,

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USA). Colonies were picked and amplified using same primers and Phusion® High-Fidelity

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DNA Polymerase (New England Biolabs, Ipswich, MA, USA), and then sequenced in both

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directions.

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(http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Full-length

sequences

were

High fidelity and proof reading

aligned

using

Clustal

W2

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Southern blot analysis. Genomic DNA was extracted from immature grapefruits using the

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CTAB method with slight modifications.19 Genomic DNA was digested to completion with the

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restriction enzymes EcoRI and HindIII for PAL and 2RT and with AvaI, BsaI and XbaI for CHS

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and CHI. The digested DNA was then subjected to southern blot analysis under high stringency.

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DIG-labeled probes were synthesized using DIG PCR Probe Synthesis kit (Roche Diagnostics

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Corp, Indianapolis, IN USA).

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Real-time PCR. Quantitative real-time PCR was performed on a CFX96 Real Time System

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(Bio-Rad, Hercules, CA, USA), using the SSO Advanced SYBR Green Supermix (Bio-Rad,

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USA). Reaction mix and conditions followed the manufacturer’s instructions. The protocol for

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all genes analyzed consisted of 30 s at 95°C for pre-incubation, then 40 cycles of 5 s 95°C for

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denaturation, 30 s at 61°C for annealing, and 10 s at 72°C for extension. Fluorescence intensity

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data were acquired during the extension time. For expression measurements, we used the Bio-

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Rad CFX manager Software release (Bio-Rad, USA) and calculated expression levels relative to

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values of a reference sample using the Relative Expression Software Tool. Normalization was

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performed using the expression levels of the Actin gene. For all genes analyzed, the reference



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sample was the expression value obtained in juice sacs of immature fruit harvested in June.

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Results are the average of three replicates.

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Statistical analysis. One way analysis of variance (ANOVA) was performed using PASW

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Statistics 18 software (SPSS Inc.). A general linear model was used to test significant differences

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and means were compared using Tukey’s HSD test at 5% probability level. The results were

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expressed as means ± SE.

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RESULTS AND DISCUSSION

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Vitamin C quantification. Vitamin C is one of the most important and abundant anti-oxidants

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present in grapefruit. Several studies have reported its health benefits. In the present study,

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vitamin C levels decreased in Rio Red grapefruit as the fruits matured (Figure 1B). Immature

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fruits harvested in June had significantly higher levels than the mature fruits, with nearly two-

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fold higher vitamin C contents, compared to the other harvests. Fruits attain maximum size and

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maturity by November and start ripening. Vitamin C levels were maintained during maturation

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from November to April. Higher levels of dehydroascorbic acid (DHA) were observed in

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immature fruits harvested in June (25.64 mg/ 100 mL juice). However, very low levels (< 2

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mg/100 mL juice) of DHA were detected as the fruits developed and matured from August to

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April (Figure 1B). In the present study, fresh juice was used for analysis to avoid any

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degradation during freeze-drying. The water content in mature fruits was higher than in

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immature fruits, which can lead to lower concentrations of vitamin C due to dilution. However,

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even after calculating the dry-weight equivalent, immature fruits showed higher vitamin C than

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mature fruits. A previous study in Valencia orange and lemon showed similar decreases in

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vitamin C per gram as the fruits matured.2 Similar results were reported in four citrus species

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(mandarin, clementine, orange and grapefruit) where decrease in ascorbic acid and DHA levels


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were observed in pulp as the fruits developed and matured, with highest levels found in fruits

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harvested in June.20 Unlike in the current study, where the vitamin C content remained stable in

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mature fruits, our previous study showed mature Rio Red grapefruits from November (early

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season) had higher vitamin C than in April.21 Vitamin C levels in fruits are affected by several

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factors including de novo biosynthesis, degradation and recycling of monodehydroascorbate and

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dehydroascorbate to ascorbic acid.20

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Carotenoids analysis. Citrus fruits contain more number of carotenoids than any other fruit

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crop. However, only three species of citrus accumulate lycopene and β-carotene in pulp, namely

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orange mutants such as Hong Anliu and Cara Cara, red-fleshed grapefruits, and pummelo. In the

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current study, lycopene and β-carotene showed different accumulation patterns in the juice

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vesicles (Figure 1B). Both carotenoids were significantly low in fruits harvested in June.

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Lycopene levels sharply increased in immature fruits harvested in August and then gradually

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decreased with maturity from November to April. By contrast, β-carotene levels gradually

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increased until mid-season (January) and then significantly decreased in late-season fruits

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(April). Lycopene content was significantly higher in August and November fruits, while it was

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lowest in immature fruits harvested in June. On other hand, β-carotene was higher in January

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and lower in fruits harvested in June and August. Lycopene levels were higher than β –carotene

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levels throughout the study. Lycopene β-cyclase is a key regulatory enzyme that catalyzes

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conversion of lycopene into β-carotene22 and lycopene accumulation in grapefruit has been

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proposed to be linked to a lower expression and functionality of β-cyclase enzymes.23 There was

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rapid accumulation of lycopene in immature fruits from June to August. During maturation,

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lycopene degraded gradually while β-carotene accumulated during maturation in early- and mid-

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season fruits (November to January) but later decreased in late-season fruits (April). Previous



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studies in grapefruit have reported decrease in lycopene during different harvest seasons as the

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fruits ripened.21, 24 In a recent study in Yuzu (Citrus junos Sieb ex Tanaka), Kjool (Citrus unshiu

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Marcow), and Dangyooja (Citrus grandis Osbeck), similar trends were observed, with lycopene

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decreasing and β-carotene increasing with maturity.2

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Limonoids analysis. Limonin is one of the bitter component of grapefruit and generally

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decreases with fruit maturity, thus reducing the bitterness. In current study, limonin was detected

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in fruits at all stages of maturity (Figure 1B). Limonin levels increased significantly in immature

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fruits from June to August, but decreased markedly from November as the fruits matured. The

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levels of limonin were found to be significantly higher in immature and early-season fruits

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(November), as compared to mid- (January) and late-season (April) harvest. For example, in

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mid-season fruits, limonin was detected only in few samples. Similarly, nomilin was detected

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only in the immature fruits (June and August, data not shown). Hasegawa et. al. studied the

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biosynthesis and accumulation of limonin in citrus and reported that both immature and mature

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fruits synthesize limonin from nomilin, obacuanone, and obacunoate.25 Non-bitter limonin

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monolactone (namely limonoate A- ring lactone) is reported to be converted to bitter dilactone

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limonin when it comes in contact with citrus juice.26, 27 Limonoate A- ring lactone is reported to

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decrease as the fruits matured in Navel oranges.28

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Furocoumarins quantification. Furocoumarins and coumarins are phenylpropanoids present

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in grapefruit pulp and peel.29 In the current study, we detected two furocoumarins in grapefruit

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pulp, 6, 7-dihydroxybergamottin (DHB) and bergamottin; we also detected one coumarin,

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auraptene (Figure 1B). All three compounds were significantly higher in immature fruits and

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gradually decreased as the fruit matured. Immature fruits harvested in June had significantly

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higher DHB (~5-fold), bergamottin (~ 4-fold) and auraptene (~ 4-fold) than the fruits harvested


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in August. Furthermore, DHB and bergamottin were significantly higher in August than in the

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remaining three harvests. DHB levels decreased nearly 75-fold from the earliest harvest in June

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(1253.9 µg/g) to the late-season harvest in April (16.5 µg/g), bergamottin decreased 15-fold from

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June (366.5 µg/g) to April (24.8 µg/g), and auraptene decreased 6-fold from June (51.9 µg/g) to

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April (8.3 µg/g). Furocoumarins are phytoalexins, which protect from pests and diseases. A

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previous study in Rio Red grapefruit reported similar trends, where DHB and bergamottin

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decreased from early season (November) to late season (May).30 Auraptene is a prenylated

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coumarin found in higher concentrations in citrus (grapefruit) peel and peel oil as compared to

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juice sacs; auraptene also has anti-tumor properties.31 In Chinotto (Citrus × myrtifolia Raf.)

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furocoumarins, namely bergapten and epoxybegamottin, also decreased with fruit ripening.32

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Quantification of flavonoids. The levels of different flavanones in the fruits at different

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stages were measured (Figure 2). Grapefruit contains mainly flavanones, which include

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rutinosides and neohesperidoses. Rutinosides are tasteless; while, neohesperidoses impart bitter

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taste. In grapefruit, naringin, poncirin, and neohesperidin are neohesperidoses; while, narirutin

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and didymin are rutinosides. Narirutin, didymin and neohesperidin levels were several fold

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higher in the pulp of immature grapefruits collected in June, relative to the levels in the pulp of

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fruits collected at other time points. The levels of these compounds decreased significantly in

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immature fruits collected in August, and in mature fruits.

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The accumulation of naringin and poncirin in grapefruit pulp differs from other flavanones

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described above. Naringin and poncirin levels in fruit pulp gradually increased until November,

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but declined sharply during maturity from January. The grapefruit flavonoid profile is distinct

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from that of other citrus species, as grapefruit predominantly contains naringin, in higher

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concentrations than in other citrus species.33 Citrus fruits containing naringin are also reported to



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contain neohesperidin, which could be related to the biosynthetic pathway (Figure 3). In the

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present study, naringin was the most abundant flavanone and accounted for more than 50% of

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total flavonoids (Figure 2). Similar results were previously reported by Hagen et al in Ruby Red

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grapefruit, where decreases in flavanone glycosides including narirutin, naringin, neohesperidin,

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didymin and poncirin were observed with fruit maturity.34 Previous studies have suggested that

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naringin is higher in young tissues as compared to mature tissues in grapefruit.35 In yuzu citrus,

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both hesperidin and naringin are present and their levels decreased in pulp and increased in peel

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with fruit maturity.36 Another study in Satsuma mandarin reported decrease in flavanones with

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fruit maturity, with hesperidin and narirutin being the major flavonoids detected.16

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Naringin is the major characteristic flavanone present in bitter tasting citrus fruits such as

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grapefruit and pummelo and is not found in other non-bitter citrus species such as sweet orange

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and mandarin. Previous studies have focused on characterization of 6RT leading to biosynthesis

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of non-bitter rutinosides such as narirutin and hesperidin in other citrus varieties.10 However, not

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much attention has been given to temporal expression of 2RT in bitter citrus species such as

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grapefruit. Therefore, to further understand the different biochemical steps involved in

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production of the bitter component, genes involved in naringin biosynthesis were cloned and

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their expression during fruit development all the way to maturity was studied.

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Sequence analysis of PAL, CHS, CHI, and 2RT. Four main genes involved in naringin

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biosynthesis were selected based on their regulatory role. PAL catalyzes the first committed step

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in phenylpropanoid pathway by deaminating L-phenylalanine to trans-cinnamic acid (Figure 3).

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CHS synthase condenses three malonyl Co-A molecules with one molecule of 4-coumaroyl Co-

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A to synthesize naringenin chalcone, which is further isomerized to naringenin with help of CHI.

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Naringenin is glucosylated at seventh position by 7-O-glucosyltransferase and further


Page 13 of 35


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glucosylated to form either bitter naringin by 2RT or into tasteless narirutin by 6RT. We selected

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to study the biosynthesis pathway of naringin, it being highest in content among flavanones and

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imparting bitter taste which is distinct to grapefruit and pummelo fruits. PCR products of PAL,

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CHS, CHI, and 2RT were sequenced individually after extraction, purification and cloning. The

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open reading frames for PAL, CHS, CHI, and 2RT were 1467, 1203, 669, and 1359 bp,

288

respectively. Multiple copies of PAL, CHS and CHI were detected having differences in

289

nucleotide and deduced amino acid sequences (Figure 4-5). Total 13 colonies were picked and

290

sequenced for PAL, out of which PAL1 had four colonies, PAL2 had one colony and PAL3 had

291

three colonies. While only partial sequences were obtained from other colonies (data not

292

included). Three amino acid differences were observed among PAL1, PAL2 and PAL3 (Figure

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4A). PAL1 and PAL2 have three nucleotide differences and one amino acid difference. While,

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PAL3 has fifteen nucleotide differences and two amino acid differences with PAL1 and PAL2.

295

The deduced amino acid sequence of grapefruit PAL (Table S2) showed significant sequence

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identity to Citrus clementina × reticulata FPAL1 (CAB42793.1) (96%) and FPal2

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(CAB42794.1)

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(AEX32790.1) (89%), Oryza sativa (NP_001047481.1) (73%) and Prunus salicina

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(AFP24940.1) (86%). The grapefruit PAL sequence has higher amino acid sequence similarity to

300

FPAL2 than FPAL1 of Citrus clementina × reticulata.

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Total 16 colonies were picked and sequenced for CHS out of which CHS1 had five colonies,

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CHS2 had four colonies and remaining other isoforms had single colony each. In CHS, copies

303

from CHS1 to CHS4 have only nucleotide differences and no amino acid differences (Figure

304

4B). CHS3 (partial cds) obtained from gDNA has an intron between two exons, which is most

305

common form of alternate splicing in plants 37 and is observed in CHS genes from other crops.38-

(99-99%),

Arabidopsis

thaliana

(AAC18870.1)


(85%),

Vitis

vinifera


Page 14 of 35

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40

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deletion of ten nucleotides, while CHS5 has deletion of two nucleotides leading to difference in

308

amino acids at the end of the sequence. This deletion is not observed in Citrus maxima

309

(ACX37403.1) or Citrus sinensis (BAA81663.1; BAA81664.1). Further, there is single amino

310

acid substitution in CHS6, CHS7 and CHS8 while double amino acid substitution in CHS9.

311

Comparison of deduced amino acid sequence of grapefruit CHS indicated 99% identity with the

312

CHS from Citrus maxima cultivar Feng wei (ACX37403.1), which is female parent of grapefruit

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(Table S2). On other hand, grapefruit CHS had higher amino acid sequence similarity with

314

CitCHS2 (BAA81664.1) (~98%) as compared to CitCHS1 (BAA81663.1) (~87%) of its male

315

parent crop, Citrus sinensis. Identity of grapefruit CHS isoforms is > 80% with Arabidopsis

316

thaliana (NP_196897.1), Vitis vinifera (AAB72091.1) and Oryza sativa (BAB39764.1).

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Total 24 colonies were picked and sequenced for CHI out of which CHI1A had ten colonies,

318

CHI3A had nine colonies, while CHI1B, CHI1C, CHI2, CHI3B and CHI4 had one colony each.

319

In CHI copies, CHI1 A, B and C have only nucleotide differences and no differences in amino

320

acid sequence. CHI1 (A,B,C) deduced amino acid sequences have complete similarity with

321

Citrus sinensis (BAA36552.1) (Table S2). CHI1A nucleotide sequence (ORF) has complete

322

similarity with Citrus sinensis nucleotide sequence (AB011794.1). CHI2 has just one amino acid

323

and two nucleotide differences with CHI1 and Citrus sinensis sequence (Figure 5A). While,

324

deduced amino acid sequence and nucleotide sequence (ORF) of CHI3 has complete similarity

325

with Citrus maxima cultivar Feng wei (ADB92596.1). CHI4 has one amino acid and three

326

nucleotide differences with CHI3 and Citrus maxima sequence. Total there are six amino acid

327

differences between CHI1 and CHI3 same as the differences between sequences of Citrus

328

sinensis (AB011794.1) and Citrus maxima (ADB92596.1). Grapefruit is a cross between Citrus

While, copies from CHS6 to CHS9 have differences in amino acid sequences. CHS1 has


Page 15 of 35


329

maxima and Citrus sinensis and therefore, CHI copies having exact similarities with both species

330

are found in the grapefruit genome. Furthermore, grapefruit CHI isoforms showed 68% identity

331

with Arabidiopsis thaliana (AAA32766.1), Vitis vinifera, Oryza sativa (AAM13448.1) and

332

Phaseolus vulgaris (CAA78763.1) (Table S2).

333

Total 12 colonies were picked and sequenced for 2RT. Grapefruit 2RT deduced amino acid

334

sequence showed 100% similarity with Citrus maxima (AAL06646.2), while it had one amino

335

acid and nucleotide difference with 2RT of Citrus maxima cultivar Fenghuangyou

336

(ACX70154.1) (Figure 5B, Table S2).

337

Southern blot analysis. To determine the copy number of these genes in grapefruit, we

338

performed Southern blot analysis on genomic DNA (Figure 6). This analysis showed four major

339

bands for PAL under HindIII, suggesting that PAL also has a multi-gene family in citrus. In

340

tomato 26 copies of PAL gene were isolated in diploid genome, with the high number of copies

341

present due to duplication; however, most of them were inactive due to gene silencing.41 Several

342

multiple isoforms help to extend regulatory flexibility under different developmental and stress

343

conditions

344

In CHS two major bands were detected with AvaI restriction enzyme while just one band was

345

seen under XbaI (weak band) and BsaI (strong band), indicating two copies in the genome. Only

346

one major band was detected for CHI under XbaI and indicating a single gene encoding CHI in

347

grapefruit. In 2RT single strong band was seen under EcorI while two bands were seen under

348

HindIII restriction enzyme. However, in current study multiple copies of PAL, CHS and CHI

349

were identified after cloning and sequencing. PAL, CHS, and CHI have been reported to be

350

multigene families in grape (Vitis vinifera),42 bilberry (Vaccinium myrtillus),43 tomato (Solanum



351

lycopersicum),41 and petunia (Petunia hybrida)39, 44. Multiple copies of genes are common and

352

can be attributed to their duplication and redundancy40 or their activation under different stress

353

conditions and in different tissues.45 Less number of bands as compared to clones identified can

354

be due to only one restriction site present and therefore at least two and possibly more copies of

355

CHS and CHI genes can be present in grapefruit.

Page 16 of 35

356

Flavonoid gene expression. To understand molecular mechanism underlying flavanone

357

composition in grapefruit, expression of PAL, CHS, CHI, and 2RT genes was measured during

358

fruit development and maturation (Figure 7). In general, expression of PAL, CHS, and CHI was

359

significantly higher in pulp of immature grapefruit harvested in June as compared to the mature,

360

early-, mid-, and late-season fruits harvested in November, January, and April respectively. In

361

contrast, expression of 2RT was significantly higher in early- and mid-season fruits compared to

362

immature and late-season fruits. The key step where the flavanones are converted into either

363

tasteless rutinosides is catalyzed by 6RT or into bitter neohesperidoses is catalyzed by 2RT. In a

364

recent study, Chen et al. studied expression of both 2RT and 6RT in sweet orange tissues (C.

365

sinensis).10 Despite having higher expression of 2RT, its functional inability resulted in very low

366

levels of naringin, poncirin for detection in fruit tissues of ‘Anliu’ and ‘Honganliu’ sweet

367

oranges. Therefore, higher expression of 2RT does not warranty higher levels of naringin. In

368

current study, PAL, CHS and CHI have higher activity in early developmental stages which may

369

lead to higher accumulation of precursor naringenin in young fruitlets. In later maturity stages,

370

2RT expression is higher which can be linked to higher content of naringin and poncirin in

371

November. However, in January even though the expression of 2RT is high, no transient increase

372

in naringin levels is observed which can be attributed to less availability of naringenin due to low

373

expression of the upstream genes – PAL, CHS and CHI. Further, PAL and CHS are key flux


Page 17 of 35


374

regulating steps in flavonoid pathway and their low expression leads to reduced levels of

375

downstream compounds. PAL is the first step and main flux point for phenylpropanoid pathway

376

and flavonoid synthesis,46 and is also rate limiting flux point for other phenylpropanoid

377

secondary metabolites such as chlorogenic acid.47 On the other hand, CHS is the first committed

378

step in flavonoid synthesis and drives the carbon flux to the flavonoid pathway. In strawberry,

379

antisense CHS reduced the anthocyanins accumulation and increased accumulation of other

380

phenylpropanoid products by redirecting the flux into branch pathways.48 On other hand, even

381

though PAL, CHS and CHI levels are high in June and August, it is not corresponding to higher

382

accumulation of naringin in August, which may be due to low expression of 2RT in August.

383

Therefore, 2RT expression can be the bottle neck for naringin accumulation. Overall it can be

384

concluded that naringin biosynthesis is regulated by availability of precursor compound

385

naringenin and thus indirectly on PAL, CHS and CHI.

386

Furthermore, in red fleshed citrus fruits carotenoid and flavanone pathway are reported to be

387

interrelated. Red fleshed citrus fruits are reported to contain higher levels of flavonoids in

388

immature stages which gradually decreased as fruits matured.10 In our previous study white

389

grapefruit and pummelo varieties had higher naringin content as compared to red/pink fleshed

390

grapefruit varieties.49 Carotenoid pathway may have influence on flavonoid biosynthesis and

391

needs to be further investigated.

392

This is the first study in grapefruit (Citrus paradisi) to report the isolation of genes

393

corresponding to PAL, CHS, CHI, and 2RT, which encode enzymes from the flavonoid

394

biosynthetic pathway, and to examine their expression in different tissues of grapefruit. Relative

395

mRNA levels of genes encoding upstream enzymes, namely PAL, CHS, and CHI, were higher in

396

immature fruits, whereas 2RT, which encodes a downstream enzyme, had higher mRNA levels in



Page 18 of 35

397

mature grapefruits. Most of the flavonoids, vitamin C, furocoumarins, limonin, and lycopene had

398

higher levels in immature fruits, but decreased as fruits ripened. Overall, most of the

399

phytochemicals decreased with fruit development and maturity. Fruits harvested in November

400

have higher content amongst the marketable stages (January and April).

401

402

Supporting Information Available: Supplementary Table S1 and Table S2. This material is

403

available free of charge via the Internet at http://pubs.acs.org.

404 405

406

This study was supported by United States Department of Agriculture grant of Designing Foods

407

for Health through the Vegetable & Fruit Improvement Center 2010-34402-20875 and State

408

funding 2013-121277 VFIC-TX state appropriation. Authors also thank Dr. S. Manjunath,

409

Monsanto Company for critical reviewing the manuscript.

410

The authors declare no competing financial interest.

SUPPORTING INFORMATION

ACKNOWLEDGEMENTS

411 412

References

413

1.

Res. 1998, 18, 1995-2018.

414 415

Peterson, J.; Dwyer, J., Flavonoids: Dietary occurrence and biochemical activity. Nutr.

2.

Abeysinghe, D. C.; Li, X.; Sun, C.; Zhang, W.; Zhou, C.; Chen, K., Bioactive compounds

416

and antioxidant capacities in different edible tissues of citrus fruit of four species. Food

417

Chem. 2007, 104, 1338-1344.


Page 19 of 35

418


3.

Pharmacol. 1996, 38, 151-163.

419 420

Korkina, L. G.; Afanas'ev, I. B., Antioxidant and chelating properties of flavonoids. Adv.

4.

Tripoli, E.; Guardia, M. L.; Giammanco, S.; Majo, D. D.; Giammanco, M., Citrus

421

flavonoids: Molecular structure, biological activity and nutritional properties: A review.

422

Food Chem. 2007, 104, 466-479.

423

5.

Biol. 1994, 26, 771-774.

424 425

6.

Stapleton, A. E.; Walbot, V., Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage. Plant Physiol. 1994, 105, 881-889.

426 427

Kootstra, A., Protection from UV-B-induced DNA damage by flavonoids. Plant Mol.

7.

So, F. V.; Guthrie, N.; Chambers, A. F.; Moussa, M.; Carroll, K. K., Inhibition of human

428

breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and

429

citrus juices. Nutr. Cancer 1996, 26, 167-181.

430

8.

colon cancer cells. Cancer Lett. 1996, 110, 41-48.

431 432

9.

Winkel-Shirley, B., Flavonoid Biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001, 126, 485-493.

433 434

Kuo, S.-M., Antiproliferative potency of structurally distinct dietary flavonoids on human

10.

Chen, J.; Zhang, H.; Pang, Y.; Cheng, Y.; Deng, X.; Xu, J., Comparative study of

435

flavonoid production in lycopene-accumulated and blonde-flesh sweet oranges (Citrus

436

sinensis) during fruit development. Food Chem. 2015, 184, 238-246.

437

11.

Ballester, A. R.; Izquierdo, A.; Lafuente, M. T.; González-Candelas, L., Biochemical and

438

molecular characterization of induced resistance against Penicillium digitatum in citrus

439

fruit. Postharvest Biol. Technol. 2010, 56, 31-38.



440

12.

Oufedjikh, H.; Mahrouz, M.; Amiot, M. J.; Lacroix, M., Effect of γ-irradiation on

441

phenolic compounds and phenylalanine ammonia-lyase activity during storage in relation

442

to peel injury from peel of Citrus clementina Hort. Ex. Tanaka. J. Agric. Food Chem.

443

2000, 48, 559-565.

444

13.

Lafuente, M. T.; Zacarias, L.; Martínez-Téllez, M. A.; Sanchez-Ballesta, M. T.; Dupille,

445

E., Phenylalanine ammonia-lyase as related to ethylene in the development of chilling

446

symptoms during cold storage of citrus fruits. J. Agric. Food Chem. 2001, 49, 6020-6025.

447

14.

Lafuente, M. T.; Zacarias, L.; Martı́nez-Téllez, M. A.; Sanchez-Ballesta, M. T.; Granell,

448

A., Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by

449

fruit age in citrus. Postharvest Biol. Technol. 2003, 29, 309-318.

450

15.

Moriguchi, T.; Kita, M.; Tomono, Y.; Endo-Inagaki, T.; Omura, M., One type of

451

chalcone synthase gene expressed during embryogenesis regulates the flavonoid

452

accumulation in citrus cell cultures. Plant Cell Physiol. 1999, 40, 651-655.

453

16.

Moriguchi, T.; Kita, M.; Tomono, Y.; Endo-Inagaki, T.; Omura, M., Gene expression in

454

flavonoid biosynthesis: Correlation with flavonoid accumulation in developing citrus

455

fruit. Physiol. Plant 2001, 111, 66-74.

456

17.

Chebrolu, K. K.; Jayaprakasha, G. K.; Yoo, K. S.; Jifon, J. L.; Patil, B. S., An improved

457

sample preparation method for quantification of ascorbic acid and dehydroascorbic acid

458

by HPLC. LWT - Food Sci. Technol. 2012, 47, 443-449.

459

18.

Chaudhary, P.; Jayaprakasha, G. K.; Porat, R.; Patil, B. S., Degreening and postharvest

460

storage influences ‘Star Ruby’ grapefruit (Citrus paradisi Macf.) bioactive compounds.

461

Food Chem. 2012, 135, 1667-1675.


Page 20 of 35

Page 21 of 35

462


19.

Nucleic Acids Res. 1980, 8, 4321-4326.

463 464

Murray, M. G.; Thompson, W. F., Rapid isolation of high molecular weight plant DNA.

20.

Alós, E.; Rodrigo, M. J.; Zacarías, L., Differential transcriptional regulation of l-ascorbic

465

acid content in peel and pulp of citrus fruits during development and maturation. Planta

466

2014, 239, 1113-1128.

467

21.

Patil, B. S.; Vanamala, J.; Hallman, G., Irradiation and storage influence on bioactive

468

components and quality of early and late season ‘Rio Red’ grapefruit (Citrus paradisi

469

Macf.). Postharvest Biol. Technol. 2004, 34, 53-64.

470

22.

Zhang, J.-c.; Zhou, W.-j.; Xu, Q.; Tao, N.-g.; Ye, J.-l.; Guo, F.; Xu, J.; Deng, X.-x., Two

471

lycopene β-cyclases genes from sweet orange (Citrus sinensis L. Osbeck) encode

472

enzymes with different functional efficiency during the conversion of lycopene-to-

473

provitamin A. J. Integr. Agric. 2013, 12, 1731-1747.

474

23.

Alquézar, B.; Zacarías, L.; Rodrigo, M. J., Molecular and functional characterization of a

475

novel chromoplast-specific lycopene β-cyclase from Citrus and its relation to lycopene

476

accumulation. J. Exp. Bot. 2009, 60, 1783-1797.

477

24.

2000, 48, 1507-1511.

478 479

25.

26.

484

Maier, V. P.; Beverly, G. D., Limonin monolactone, the nonbitter precursor responsible for delayed bitterness in certain citrus juices. J. Food Sci. 1968, 33, 488-492.

482 483

Hasegawa, S.; Herman, Z.; Orme, E.; Ou, P., Biosynthesis of limonoids in Citrus: Sites and translocation. Phytochem. 1986, 25, 2783-2785.

480 481

Lee, H. S., Objective measurement of red grapefruit juice color. J. Agric. Food Chem.

27.

Maier, V. P.; Margileth, D. A., Limonoic acid A-ring lactone, a new limonin derivative in Citrus. Phytochem. 1969, 8, 243-248.



485

28.

Hasegawa, S.; Ou, P.; Fong, C. H.; Herman, Z.; Coggins, C. W.; Atkin, D. R., Changes in

486

the limonoate A-ring lactone and limonin 17-.beta.-D-glucopyranoside content of navel

487

oranges during fruit growth and maturation. J. Agric. Food Chem. 1991, 39, 262-265.

488

29.

Dugrand, A.; Olry, A.; Duval, T.; Hehn, A.; Froelicher, Y.; Bourgaud, F., Coumarin and

489

furanocoumarin quantitation in citrus peel via ultraperformance liquid chromatography

490

coupled with mass spectrometry (UPLC-MS). J. Agric. Food Chem. 2013, 61, 10677-

491

10684.

492

30.

Girennavar, B.; Jayaprakasha, G. K.; Patil, B. S., Influence of pre- and post-harvest

493

factors and processing on the levels of furocoumarins in grapefruits (Citrus paradisi

494

Macfed.). Food Chem. 2008, 111, 387-392.

495

31.

Murakami A; Kuki W; Takahashi Y; Yonei H; Nakamura Y; Ohto Y; Ohigashi H; K., K.,

496

Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoylphorbol-13-acetate-induced

497

tumor promotion in ICR mouse skin, possibly through suppression of superoxide

498

generation in leukocytes. Jpn. J. Cancer Res. 1997, 88, 443-452.

499

32.

Barreca, D.; Bellocco, E.; Caristi, C.; Leuzzi, U.; Gattuso, G., Flavonoid composition and

500

antioxidant activity of juices from chinotto (Citrus × myrtifolia Raf.) fruits at different

501

ripening stages. J. Agric. Food Chem. 2010, 58, 3031-3036.

502

33.

Nogata, Y.; Sakamoto, K.; Shiratsuchi, H.; Ishii, T.; Yano, M.; Ohta, H., Flavonoid

503

composition of fruit tissues of citrus species. Biosci. Biotechnol. Biochem. 2006, 70, 178-

504

192.

505

34.

Hagen, R. E.; Dunlap, W. J.; Wender, S. H., Seasonal variation of naringin and certain

506

other flavanone glycosides in juice sacs of Texas Ruby Red grapefruit. J. Food Sci. 1966,

507

31, 542-547.


Page 22 of 35

Page 23 of 35

508


35.

Jourdan, P. S.; McIntosh, C. A.; Mansell, R. L., Naringin levels in citrus tissues: II.

509

quantitative distribution of naringin in Citrus paradisi MacFad. Plant Physiol. 1985, 77,

510

903-908.

511

36.

Yoo, K. M.; Lee, K. W.; Park, J. B.; Lee, H. J.; Hwang, I. K., Variation in major

512

antioxidants and total antioxidant activity of yuzu (Citrus junos Sieb ex Tanaka) during

513

maturation and between cultivars. J. Agric. Food Chem. 2004, 52, 5907-5913.

514

37.

Bioinform Biol. Insights 2015, 9, 47-52.

515 516

Zhang, C.; Yang, H.; Yang, H., Evolutionary character of alternative splicing in plants.

38.

Han, Y.; Ding, T.; Su, B.; Jiang, H., Genome-wide identification, characterization and

517

expression analysis of the chalcone synthase family in maize. Int. J. Mol. Sci. 2016, 17,

518

161-176.

519

39.

Koes, R. E.; Spelt, C. E.; van den Elzen, P. J. M.; Mol, J. N. M., Cloning and molecular

520

characterization of the chalcone synthase multigene family of Petunia hybrida. Gene

521

1989, 81, 245-257.

522

40.

multigene family in the morning glory genome Plant Mol. Biol. 2000, 42, 79-92.

523 524

Durbin, M. L.; McCaig, B.; Clegg, M. T., Molecular evolution of the chalcone synthase

41.

Chang, A.; Lim, M.-H.; Lee, S.-W.; Robb, E. J.; Nazar, R. N., Tomato phenylalanine

525

ammonia-lyase gene family, highly redundant but strongly underutilized. J. Biol. Chem.

526

2008, 283, 33591-33601.

527

42.

Sparvoli, F.; Martin, C.; Scienza, A.; Gavazzi, G.; Tonelli, C., Cloning and molecular

528

analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis

529

vinifera L.). Plant Mol. Biol. 1994, 24, 743-755.



530

43.

Jaakola, L.; Määttä, K.; Pirttilä, A. M.; Törrönen, R.; Kärenlampi, S.; Hohtola, A.,

531

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,

532

proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiol.

533

2002, 130, 729-739.

534

44.

van Tunen, A. J.; Koes, R. E.; Spelt, C. E.; van der Krol, A. R.; Stuitje, A. R.; Mol, J. N.,

535

Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida:

536

coordinate, light-regulated and differential expression of flavonoid genes. EMBO J. 1988,

537

7, 1257-1263.

538

45.

Koes, R. E.; Spelt, C. E.; Reif, H. J.; van den Elzen, P. J. M.; Veltkamp, E.; Mol, J. N.

539

M., Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone

540

synthase multigene family. Nucleic Acids Res. 1986, 14, 5229-5239.

541

46.

Liu, R.; Xu, S.; Li, J.; Hu, Y.; Lin, Z., Expression profile of a PAL gene from Astragalus

542

membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis.

543

Plant Cell Rep. 2006, 25, 705-710.

544

47.

Howles, P. A.; Sewalt, V.; Paiva, N. L.; Elkind, Y.; Bate, N. J.; Lamb, C.; Dixon, R. A.,

545

Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals

546

control points for flux into phenylpropanoid biosynthesis. Plant Physiol. 1996, 112,

547

1617-1624.

548

48.

Lunkenbein, S.; Coiner, H.; de Vos, C. H. R.; Schaart, J. G.; Boone, M. J.; Krens, F. A.;

549

Schwab, W.; Salentijn, E. M. J., Molecular characterization of a stable antisense chalcone

550

synthase phenotype in strawberry (Fragaria × ananassa). J. Agric. Food Chem. 2006,

551

54, 2145-2153.


Page 24 of 35

Page 25 of 35

552


49.

Girennavar, B.; Jayaprakasha, G. K.; Jifon, J. L.; Patil, B. S., Variation of bioactive

553

furocoumarins and flavonoids in different varieties of grapefruits and pummelo. Eur.

554

Food Res. Technol. 2008, 226, 1269-1275.

555



556

Page 26 of 35

Figure Captions

557

Figure 1 A. Grapefruits harvested at different developmental and maturity stages. B. Changes in

558

vitamin C, limonoids (limonin), carotenoids (lycopene, and β-carotene), and furocoumarins (6,7-

559

dihydroxybergamottin, bergamottin and auraptene) in pulp during grapefruit development and

560

maturation from June to April. Data represent means ± S.E. of three replications, each replication

561

containing three samples. Means with different letters indicate significant differences at each

562

time period (P< 0.05).

563 564

Figure 2. Variation in flavonoid levels in grapefruit pulp during development and maturation

565

from June to April. Data represent means ± S.E. of three replications, each replication containing

566

four samples. Means with different letters indicate significant differences at each time period (P

Variation in Key Flavonoid Biosynthetic Enzymes and Phytochemicals

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