Biochemical and Molecular Factors Governing Peel-Color

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Biochemical and Molecular Factors Governing PeelColor Development in 'Ora' and 'Shani' Mandarins Livnat Goldenberg, Matat Zohar, Lina Kirshinbaum, Yossi Yaniv, Adi Doron-Faigenboim, Ron Porat, Nir Carmi, and Tal Isaacson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00669 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Journal of Agricultural and Food Chemistry

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Biochemical and Molecular Factors Governing PeelColor Development in 'Ora' and 'Shani' Mandarins Livnat Goldenberg,†,§ Matat Zohar,¥ Lina Kirshinbaum,¶ Yossi Yaniv,‡ Adi DoronFaigenboim, ⱡ Ron Porat†,* Nir Carmi‡ and Tal Isaacson¥ †

Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, P.O. Box

15159, Rishon LeZion 7505101, Israel. § Faculty

of Agricultural, Food and Environmental Quality Sciences, Hebrew University of

Jerusalem, Rehovot 76100, Israel. ¥

Newe Ya'ar Research Center, ARO, Ramat Yishay 30095, Israel.

¶

Biology and DNA Laboratory, Division of Identification and Forensic Science, Israel Police,

Jerusalem, Israel. ‡

Department of Fruit Tree Crops, ARO, The Volcani Center, P.O. Box 15159, Rishon LeZion

7505101, Israel. ⱡ

Department of Genomics and Bioinformatics, ARO, the Volcani Center, P.O. Box 15159,

Rishon LeZion 7505101, Israel.

Corresponding author:

Dr. Ron Porat Tel.:

972-3-9683617

Fax:

972-3-9683622

E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: To identify factors governing peel-color development in mandarins, we

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examined carotenoid content and composition and the expression of carotenoid-related

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genes during four stages of ripening (i.e., green, breaker, yellow and orange) in two

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varieties: ‘Ora’, which has orange fruit, and ‘Shani’, which has orange-reddish fruit. The

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two varieties had different carotenoid compositions and ‘Shani’ had a significantly higher

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level of total carotenoid pigments. ‘Shani’ was rich in the deep orange β-cryptoxanthin and

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the orange-reddish β-citraurin; whereas ‘Ora’ was rich in the orange violaxanthin. RNA-Seq

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analysis revealed significantly greater expression of the carotenoid-biosynthesis genes PSY,

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βLCY, βCHX and CCD4b, as well as MEP-pathway genes and several ethylene-biosynthesis

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and -signaling genes in ‘Shani’ fruit. In contrast, the expression levels of genes involved in

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the synthesis of α-branch carotenoids (i.e., εLCY and εCHX) and ZEP, which is involved in

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the formation of violaxanthin, were significantly higher in the ‘Ora’ fruit.

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KEYWORDS: mandarin, peel color, carotenoids, ripening, RNA-Seq.


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INTRODUCTION

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To enhance the visual attractiveness of mandarins, citrus breeders are interested in developing

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new varieties with peels that have a deep orange to reddish in color.1 The achievement of this

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objective requires a better understanding of the biochemical and molecular factors that govern

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the development of mandarin peel color and carotenoid composition.

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The color of mandarin peels is mainly controlled by the levels and configuration of

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their carotenoid pigments. Most of the genes of the carotenoid-biosynthesis pathway have

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been identified in plants2,3 and in citrus particularly (Figure 1).4,5 Earlier studies have

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classified citrus into three different groups based on their carotenoid profiles: the carotenoid-

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poor group includes grapefruits, pomelos, lemons and limes; the violaxanthin-abundant group

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is comprised mainly of oranges; and the β-cryptoxanthin-abundant group is comprised mainly

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of mandarins.6–9 The most abundant pigments identified in mandarins were violaxanthin, β-

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cryptoxanthin, and β-citraurin.4,10,11

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Mandarins have a profound orange to reddish color and a unique carotenoid profile.4,10 In

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addition to having large contents of pigments, mandarins own a unique mixture of carotenoid

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and apocarotenoid pigments, and are especially rich in β-cryptoxanthin and β-citraurin. β-

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cryptoxanthin imparts a deep orange color,6 and β-citraurin divulges a reddish color, even at

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low concentrations.12–14 The key gene controlling β-citraurin biosynthesis is carotenoid

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cleavage dioxygenase 4b (CCD4b) – it was recently cloned independently by two laboratories

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in Spain and Japan.13,14

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The differences in carotenoid profiles between mandarins and oranges were attributed

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to the following factors: (1) mandarins have greater gene expression levels of phytoene

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synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS) and the lycopene β-

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cyclase (βLCY) carotenoid biosynthetic genes; (2) mandarins have poorer gene expression

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levels of the β-ring hydroxylase (βCHX) and the zeaxanthin epoxidase (ZEP) xanthophyll-



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synthesis genes; (3) mandarins have greater gene expression levels of the 9-cis-

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epoxycarotenoid dioxygenase (NCED) violaxanthin catabolism gene; and (4) mandarins have

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greater gene expression levels of the CCD4b which is the key β-citraurin biosynthetic

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gene.9,11

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In commercial practice, the peel color of early-season citrus fruit is artificially

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enhanced through exposure to the ripening hormone ethylene.15 In Satsuma mandarins,

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ethylene treatment was reported to increase β-cryptoxanthin levels in the peel by 90% and

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more,16,17 and to induce the expression of the PSY, PDS, βLCY, βCHX and ZEP carotenoid

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biosynthesis genes.17,18 This indicates that ethylene plays a crucial role in the regulation of

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carotenoid biosynthesis in mandarins.

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We recently explored the existence of a wide genetic diversity in fruit quality traits

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among 46 different mandarin varieties that exhibited a wide range of peel colors, from green

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to yellow, orange and red.19 In order to elucidate the biochemical and molecular factors

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governing peel-color development in mandarins, we evaluated carotenoid content and

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composition and the expression patterns of carotenoid-related genes during four ripening

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stages (i.e., green, breaker, yellow and orange) in two mandarin varieties with distinctive peel

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colors: 'Ora', which is typically orange, and 'Shani', which has a unique orange/reddish color

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(Figure 2A).

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MATERIALS AND METHODS Plant Material. Fruits of two mandarin varieties, ‘Ora’ and ‘Shani’ (C. reticulata

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Blanco), were obtained from the Israeli citrus breeding collection at the Agricultural Research

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Organization, the Volcani Center, Rishon LeZion, Israel. Fruits of each variety were

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harvested from the same experimental orchard, during the 2016-17 ripening season, at four

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ripening stages: green, breaker, yellow and full orange. The fruits’ outer peel tissue (flavedo)


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was separated by hand peeler, weighed, frozen in liquid nitrogen and kept at -80°C until

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further analysis.

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Color Measurements. Peel color was measured with a CR-310 Chroma Meter (Minolta,

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Tokyo, Japan). The results were expressed in terms of lightness (0–100), chroma (0–100) and

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hue angle (H°). Data points were means  SE of 10 fruits.

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Ethylene Measurements. Ethylene production was measured at the full-orange ripening

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stage. Mandarins were placed in 2-L sealed jars, each equipped with a septum, and the jars

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were kept sealed for 4 h before sampling. For each variety, we used three jars, which each

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contained five fruits. Headspace samples were taken with a 10-mL syringe and were injected

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into a gas chromatograph equipped with a Model 3300 flame ionization detector (GC-FID;

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Varian, Walnut Creek, CA, USA).

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Carotenoid Extraction. Peel pigments were extracted from 200 mg of frozen flavedo

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tissue for each replicate. The tissue was grinded and suspended in 4 mL of extraction

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solution (hexane:ethanol:acetone 2:1:1, v/v/v) including 0.1% (w/v) butylated

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hydroxytoluene (BHT) which was added right before extraction, and gently mixed for 5 min.

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For saponification, 1 mL of methanolic KOH 20% (w/v) was added and the samples were

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vortexed and then gently mixed for 2 h. Saponified carotenoids were extracted by adding 4

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mL of diethyl ether and 4 mL of an aqueous 12% (w/v) NaCl solution to each sample. The

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hypophases were collected, washed with water and collected again. Water residues were

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eliminated by the addition of sodium sulfate and filtration. The hypophases were then dried

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under a stream of N2 and the lipid extracts were re-dissolved in 200–600 μL acetone

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(depending on the color intensity of the sample) and 50-100 μL were injected to the HPLC.

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All procedures were conducted under dim light to avoid structural change and degradation

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of the carotenoids.



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High-Pressure Liquid Chromatography (HPLC) Analysis. Carotenoid analysis was

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performed according to Ronen et al.20 In more details: A Waters HPLC system equipped

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with a Waters 600 pump, a Waters PDA detector 996 and a Waters 717 plus auto-sampler

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(Milford, MA, USA) was used. A Spherisorb ODS2 C18 column (Waters; 5 µm, 4.6 × 250

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mm) coupled with a guard cartridge system (SecurityGuard™, Phenomenex, CA, USA),

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was used for carotenoid separation at room temperature (~24ºC). A gradient was applied at a

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constant flow of 1.6 mL min-1 with acetonitrile:water (9:1; v/v) and ethyl acetate as

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described in previous work.21 Spectra of eluted HPLC solvent within a wavelength range of

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250–600 nm were recorded and absorption peaks were analyzed using Empower software

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(Waters). The linear limit of detection was estimated to be between 10–20 ng and 1.5–2 μg,

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depending on the carotenoid compound. Carotenoids were identified by their absorption

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spectra and retention times, and in most cases by authentic standards. The β-carotene

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standard was obtained from Sigma-Aldrich, the β –citraurin and phytoene standards were

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obtained from CaroteNature (Switzerland) and β-cryptoxanthin and lutein from

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Extrasynthase (France). All carotenoid peaks were normalized22 to correct for their specific

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mass extinction coeffiecients23 in relation to β-carotene (= 1), using xanthophylls (1), β-

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cryptoxanthin (1.086), ζ-carotene (1.014), phytofluene (1.920) and phytoene (2.074). Total

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carotenoid content was determined based on β-carotene and β –citraurin calibration curves

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prepared with authentic standards. Total carotenoid is presented as μg g-1 Fresh Weight

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(FW).

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RNA Isolation, cDNA Library Construction and RNA-Seq. Total RNA was extracted

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according to the CTAB protocol24 and each combination of variety and ripening stage

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included three biological replicates, each containing flavedo tissues collected from two

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different fruits. The RNA samples were treated with DNase (Invitrogen; Carlsbad, CA, USA)

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according to the supplier’s instructions. RNA concentrations were determined with a


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NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and

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RNA purity and integrity were further verified with a Model 2100 Total RNA BioAnalyzer

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(Agilent Technologies; Santa Clara, CA, USA). Library preparation and sequencing were

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performed at The Center for Genomic Technologies, The Alexander Silbelman Institute of

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Life Sciences, The Hebrew University of Jerusalem, Israel. Twenty-four single-end RNA-Seq

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libraries were prepared using Illumina NextSeq 500 and Trueseq RNA protocols.

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Transcriptome Analysis. The resulting raw short-reads were subjected to a filtering and

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cleaning procedure. First, the SortMeRNA tool was used to filter out rRNA.25 Then, the

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FASTX Toolkit was used (http://hannonlab.cshl.edu/fastx_toolkit/ index.html, version

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0.0.13.2) to trim read-end nucleotides with quality scores < 30, using fastq_quality_trimmer,

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and remove reads with less than 70% base pairs with a quality score ≤ 30 using the

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fastq_quality_filter. The Bowtie 2, version 2.1 alignment tool was used to map the cleaned

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reads on the Citrus clementina v1.0 (clementine) genes extracted from Phytozome database

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(https://phytozome.jgi.doe.gov/pz/portal.html).

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The transcript quantification (the number of reads per gene) from the RNA-Seq data

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was determined using the Bowtie 2 aligner, version 2.126 and the expectation-maximization

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method (RSEM), by estimating maximum-likelihood expression levels.27 Differential

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expression analysis was done with the DESeq2 R package in the R environment.28 Genes that

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were more than two-fold differentially expressed with a false discovery rate (FDR) corrected

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statistical significance of no more than 0.05 were considered differentially expressed.29

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Following the Trinity protocol,30 expression normalization was conducted using the trimmed

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means of M-values (TMM), after calculating the fragments per feature kilobase per million

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reads mapped (FPKM).

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Statistical Analysis. One-way analysis of variance (ANOVA) was applied to the chroma-

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meter and carotenoid data using Microsoft Excel. Tukey’s HSD and Student’s t-test were used



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to identify differences in carotenoid content among the different ripening stages and varieties.

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Those tests were performed using JMP 13.0 software (SAS Institute; Cary, NC, USA).

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RESULTS Peel Color. The development of the peel color of ‘Ora’ and ‘Shani’ mandarin fruits

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during ripening is illustrated in Figure 2A. As can be seen in that figure, the two varieties

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were similar in color at the breaker stage. But later on, ‘Ora’ turned yellow and then

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orange; whereas ‘Shani’ turned orange and then became deep orange/reddish. Lightness

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and chroma (saturation) indices gradually increased during ripening in both varieties.

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‘Ora’ had significantly higher lightness values during all ripening stages and significantly

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higher chroma values at the green and orange stages (Figure 2B). In contrast, the observed

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hue-angle values gradually decreased during ripening in both varieties, but were

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significantly higher in ‘Ora’ at the yellow and orange stages. The hue angles of the ‘Ora'’

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and ‘Shani’ peels at full maturation were 59° and 42°, respectively (Figure 2B). The lower

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hue angle values of the ‘Shani’ fruits indicate that those fruits had a deeper orange/reddish

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color than the ‘Ora’ fruits.

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Carotenoid Content and Composition. HPLC analysis of carotenoid content and

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composition was performed on flavedo tissues from both varieties during the four ripening

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stages. Total carotenoid content was more or less similar in the two varieties during the

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first and second early-ripening stages. But, later on, total carotenoid content drastically

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increased in ‘Shani’, as compared with ‘Ora’ (Figure 3). At the full-ripening stage, the

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flavedos of the ‘Ora’ and ‘Shani’ fruits contained 136.8 and 375.3 μg g-1 FW carotenoids,

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respectively (Figure 3).

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Detailed analysis of the carotenoid content and composition of the mandarin flavedos revealed the presence of 11 dominant carotenoids including carotenes (α-


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carotene, β- carotene, ζ-carotene, phytofluene and phytoene), xanthophylls (neoxanthin,

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violaxanthin, antheraxanthin, lutein, zeaxanthin and β-cryptoxanthin) and the

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apocarotenoid β-citraurin (Table 1). Examination of carotenoid profiles during ripening

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revealed a developmental shift in carotenoid production in both varieties, resulting in a

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gradual decrease in the levels of the α-branch carotenoids α-carotene and lutein, as well as

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gradual increases in the levels of the β-branch carotenoids violaxanthin and β-

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cryptoxanthin (Figure 1, Table 1).

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Detailed examination of carotenoid composition at full ripening (the orange stage)

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revealed significant differences between ‘Ora’ and ‘Shani’ (Figure 4). In both varieties,

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violaxanthin was the most common carotenoid. However, it accounted for 57.6% of the

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total carotenoid content of the ‘Ora’ flavedos, but only 43.7% of the total carotenoid

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content of the ‘Shani’ flavedos (Figure 4). The other two main compounds contributing to

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the difference in the carotenoid compositions of the two varieties were β-cryptoxanthin

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and β-citraurin, which are known to provide a deep orange-reddish color (Figure 4).14 The

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absolute levels of β-cryptoxanthin and β-citraurin were significantly higher in ‘Shani’ (as

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compared to ‘Ora’) at all ripening stages, and at full ripening were 5 and 8 times higher,

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respectively (Table 1). β-cryptoxanthin accounted for 5.9% and 11.2% of the total

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carotenoid content of ‘Ora’ and ‘Shani’, respectively, and β-citraurin accounted for 5.4%

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and 15.1% of the total carotenoid content of ‘Ora’ and ‘Shani’, respectively (Figure 4).

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Differentially Expressed Genes. In order to elucidate the molecular factors governing

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peel-color development in ‘Ora’ and ‘Shani’ mandarins, we performed RNA-Seq analysis of

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flavedo from both varieties during all four ripening stages. Each of the cDNA libraries

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yielded between 31.2 million and 46.2 million clean single-end reads, each 125 bp in length.

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About 85% of the cleaned reads could be mapped to the Citrus Clementina genome

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reference. In order to identify differentially expressed genes among the different time



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points, we performed pair-wise comparisons and identified 5,889 and 8,698 differentially

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expressed transcripts in ‘Ora’ and ‘Shani’, respectively, of which 4,774 transcripts were

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common to both varieties (Figure 5). It is worth noting that we detected a much larger

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amount of unique differentially expressed transcripts in ‘Shani’ (3,924) as compared with

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‘Ora’ (just 1,115 transcripts) (Figure 5). Elaborated clustering analysis of the different gene

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expression patterns of each variety during the different ripening stages is provided in

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Supplementary Figure 1.

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Further analysis of gene-expression patterns focused on three main pathways that

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affect carotenoid accumulation: (i) the carotenoid-biosynthesis pathway, (ii) the

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methylerythritol 4-phosphate (MEP) pathway, and (iii) ethylene biosynthesis and ethylene

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signaling. Overall, the expression patterns of more than 120 different genes were examined

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for possible differences between ‘Ora’ and ‘Shani’ during all ripening stages, with special

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emphasis on genes that are differentially expressed during the third (yellow) and fourth

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(orange) ripening stages.

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With regard to genes involved in carotenoid biosynthesis, the expression levels of

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PSY1 and CCD4b1 were significantly higher in ‘Shani’ than in ‘Ora’ at the full-maturation,

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orange stage and the expression patterns of βLCY and βCHX were significantly higher in

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‘Shani’ at the yellow and orange stages (Figure 6A). In contrast, the expression levels of the

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violaxanthin biosynthetic gene ZEP and the α-branch genes lycopene ε-cyclase (εLCY) and ε-

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carotene hydroxylase (εCHX) were significantly higher in ‘Ora’ than in ‘Shani’ at some stages

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(Figure 1, Figure 6B).

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Regarding the upstream MEP-pathway genes that provide the precursors for

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carotenoid biosynthesis, it was found that expression levels of most MEP-pathway genes [i.e.,

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DOXP synthase (DXS), DOXP reductase (DXR), CDP-ME kinase (CMK), HMB-PP reductase

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(HDR) and DMAPP isomerase (IDI)] were significantly higher in ‘Shani’ flavedos than in


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‘Ora’ flavedos over most of the ripening stages and particularly during the later yellow and

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orange stages (Figure 7).

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Regarding ethylene biosynthetic and signaling, the transcript levels of the ethylene

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biosynthetic gene ACC oxidase (ACO), the ethylene receptor 2 (ETR2) gene and the ethylene

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response factor 1 (ERF1) gene were significantly higher in ‘Shani’ than in ‘Ora’ (Figure 8A).

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The expression level of ACO was significantly higher at the breaker and orange stages, the

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expression level of ETR2 was significantly higher at the yellow and orange stages and the

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expression pattern of ERF1 was significantly higher in ‘Shani’ fruits, as compared with ‘Ora’

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fruits, at all ripening stages (Figure 8A). Furthermore, ethylene production levels in the ripe

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fruit (orange stage) were significantly and nearly four times higher for ‘Shani’ ripe fruits, as

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compared with ‘Ora’ fruits (Figure 8B).

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DISCUSSION

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The color of mandarin peel is predominantly determined by its carotenoid content and

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composition.4,10 Therefore, the development of new varieties with an attractive deep

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orange/reddish color requires a better understanding of the unique carotenoid composition of

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orange/reddish fruit and the molecular factors that control color development.5 In the present

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study, we explored carotenoid content and composition, as well as the expression patterns of

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genes related to carotenoid biosynthesis and regulation, in two mandarin varieties that differ

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in their peel color: ‘Ora’ has an orange peel and ‘Shani’ has a deep orange/reddish peel

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(Figure 2).

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The key findings of the present study regarding the observed differences in carotenoid

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content and composition between ‘Ora’ and ‘Shani’ are as follows: 1) the flavedos of ‘Shani’

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fruits have a significantly higher level of total carotenoid pigments and 2) the flavedos of

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‘Shani’ fruits have a different carotenoid composition than the flavedos of ‘Ora’ fruits and are



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richer in the deep orange β-cryptoxanthin and the orange-reddish β-citraurin pigments than

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the flavedo of ‘Ora’ fruits, which is richer in the orange violaxanthin pigment (Figures 3-4).

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As so, the 'Shani' variety can be designated as β-cryptoxantin-abundant, whereas ‘Ora’ can be

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designated as violaxanthin-abundant.6,9 Higher carotenoid content and a composition favoring

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β-cryptoxantin and β-citraurin on the expense of violaxanthin are probably the main reasons

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for the markedly higher intensity of peel color in ripe ‘Shani’ flavedo in comparison to ripe

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‘Ora’ flavedo.

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Transcriptional regulation of carotenoid biosynthesis genes was previously shown to

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be a key factor determining carotenoid biosynthesis in many fruit such as tomato20,31,32 ,

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pepper33 and citrus9 . Our results suggest that the different carotenoid profiles of ‘Ora’ and

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‘Shani’ flavedos might be due to different patterns of gene expression, where elevated

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transcription of PSY and of the β-branch carotenoid-biosynthetic genes βLCY and βCHX in

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‘Shani’, during fruit ripening (Figure 6), leads to increased production of β-branch carotenoid

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and higher total carotenoid in comparison to ‘Ora’ (Figure 1). Similar association between

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gene expression and elevated levels of β-cryptoxantin were shown in the citrus cultivar

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‘Seinannohikari’.34 The decreased expression levels of the εLCY and εCHX in ‘Shani’ (Figure

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6) could explain the observed reduction in the α-branch carotenoid lutein during ripening

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(Figure 1). The difference in β-cryptoxantin accumulation between the two mandarin varieties

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is similar, to some extent, to the difference between flavedo of oranges (low β-cryptoxantin )

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and mandarins (high- β-cryptoxantin). Kato et al.11 suggested that the high β-cryptoxantin

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accumulation in mandarins, in contrast to oranges, is a result of up regulation of carotenoid

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biosynthesis genes upstream to β-cryptoxantin and down regulation of genes down stream to

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β-cryptoxantin.11 In a similar way, the lower expression of ZEP (Figure 6), responsible for

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violaxanthin biosynthesis, in ‘Shani’ in comparison to ‘Ora’, could explain the accumulation

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of β-cryptoxantin in ‘Shani’, while in ‘Ora’ the biosynthetic flux proceeds to violaxanthin. In


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addition, the higher expression of CCD4b, responsible for the production of β-citraurin13,14

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can explain the higher accumulation of β-citraurin in ‘Shani’ in comparison to ‘Ora’ in the

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flavedo of the ripe fruit (Figure 1).

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Carotenoids are derived from isoprenoids which are synthesized by the MEP pathway

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.35 Regulation of transcription of genes encoding the enzymes of the MEP pathway was

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shown to influence carotenoid biosynthesis.36,37 The elevated transcription levels of the Mep-

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pathway genes in ‘Shani’ in comparison to ‘Ora’ (Figure 7) suggest that in ‘Shani’ the

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pathway is more active providing higher amounts of precursors to the carotenoid biosynthesis

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pathway, and thus contributing to the higher total amount of carotenoids in flavedos of the

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‘Shani’ variety.

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Together, these data demonstrate that in the flavedo of ‘Shani’ variety, there seems to

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be a high metabolic flux toward the biosynthesis of the orange/reddish pigments β-

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cryptoxanthin and β-citraurin; whereas in ‘Ora’, there seems to be an attenuated metabolic

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flux toward the biosynthesis of violaxanthin. It is unclear why the transcription of the

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described sets of genes is so different between ‘Shani’ and ‘Ora’. Ethylene is known to induce

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carotenoid biosynthesis in citrus fruit.16–18,38,39 Our finding that the transcription of ethylene

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biosynthesis and ethylene signaling genes is higher in ‘Shani’ in comparison to ‘Ora’ (Figure

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8A), in combination with the detection of much higher levels of ethylene in ’Shani’ fruit than

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in ‘Ora’ fruit, suggest that in ‘Shani’ fruit the ripening process is more intensive, leading to

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higher ethylene biosynthesis and more rapid biosynthesis of carotenoid precursors and

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carotenoid compounds. The larger number of genes that are differentially expressed during

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ripening in ‘Shani’ fruit as compared with ‘Ora’ (5,889 and 8,698 differentially expressed

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transcripts respectively), further supports this hypothesis. It is interesting to note that the shift

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in carotenoid composition toward higher levels of β-cryptoxanthin and β-citraurin in peels of



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‘Shani’ in comparison to ‘Ora’ is similar to results obtained by application of ethylene to

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peels of Gannan Newhall navel orange39 and Satsuma mandarin.17

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Overall, our findings suggest that the development of orange/reddish peel color in

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‘Shani’ mandarins does not result solely from the accumulation of any specific carotenoid

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compound or from the enhanced expression of any specific gene, but rather is the cumulative

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result of various changes in the expression patterns of genes related to carotenoid metabolism,

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the MEP pathway and, presumably, also ethylene regulation.

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FUNDING

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This research was supported by Research Grant No. 203-1058-18 from the Chief Scientist of

300

the Israel Ministry of Agriculture & Rural Development.

301 302

DESCRIPTION OF SUPPORTING INFORMATION

303

Supplementary Table 1 – GenBank accession numbers and annotations of all of the

304

differentially expressed transcripts mentioned in this study.

305

Supplementary Figure 1 - Clustering analysis of DEGs in 'Ora' and 'Shani' varieties during the

306

green, breaker, yellow and orange stages of ripening.

307


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1 418

FIGURE LEGENDS

419

Figure 1. The carotenoid-biosynthesis pathway. GGPP, Geranygeranyl pyrophosphate; PSY,

420

phytoene synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene

421

desaturase; CRITSO, carotenoid isomerase; β-LCY, lycopene β-cyclase; ε-LCY, lycopene ε-

422

cyclase; β-CHX, β-ring carotene hydroxylase; ε-CHX, ε-ring carotene hydroxylase; ZEP,

423

zeaxanthin epoxidase; CCD4, carotenoid cleavage dioxygenase 4. The schematic diagram was

424

adapted from Alquézar et al. 4 with modifications.

425 426

Figure 2. Peel color of ‘Ora’ and ‘Shani’ fruits at the green, breaker, yellow and orange

427

stages of ripening. A) Photographs of ‘Ora’ and ‘Shani’ mandarin fruits at the green, breaker,

428

yellow and orange stages of ripening. B) Lightness (0–100), Chroma (0–100) and Hue angles

429

(0° represents a red color, 45° represents orange, 90° represents yellow and 120° represents

430

green) of ‘Ora’ and ‘Shani’ peels at the green, breaker, yellow and orange stages of ripening.

431

Data are means  SE of 5 fruits and an asterisk indicates a significant difference (P  0.05)

432

between ‘Ora’ and ‘Shani’ fruits at the same stage of ripening.

433 434

Figure 3. Total carotenoid contents of flavedo of ‘Ora’ and ‘Shani’ fruits at the green,

435

breaker, yellow and orange stages of ripening. Data are means  SE of 3 fruits. Different

436

capital letters indicate a significant difference among the ‘Ora’ fruits at the different ripening

437

stages. Different lower-case letters indicate a significant difference among the ‘Shani’ fruits at

438

the different ripening stages. An asterisk indicates a significant difference (P  0.05) between

439

‘Ora’ and ‘Shani’ fruits at a particular ripening stage.

440 441

Figure 4. Carotenoid composition in flavedo of ‘Ora’ and ‘Shani’ fruits at the orange (ripe)

442

stage of ripening. Numbers indicate the percent if each carotenoid of the total carotenoid

1 ACS Paragon Plus Environment

Page 21 of 32


2 443

content for each variety. Data points are means of 3 fruits. β-xanthopylls – neoxanthin,

444

antheraxanthin and zeaxanthin. Carotenes – α, β and ζ carotene, phytofluene and phytoene.

445 446

Figure 5. Venn diagram illustration of differentially expressed genes (DEGs) in flavedo of

447

‘Ora’ and ‘Shani’ during the green, breaker, yellow and orange stages of ripening. The

448

diagram displays the number of DEGs specific to ‘Ora’, the number of DEGs common to

449

‘Ora’ and ‘Shani’ and the number of DEGs specific to ‘Shani’.

450 451

Figure 6. Carotenoid-biosynthesis genes differentially expressed in flavedo of ‘Ora’ and

452

‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. A) Genes highly

453

expressed in ‘Shani’. B) Genes highly expressed in ‘Ora’. The results are expressed as

454

fragments per feature kilobase per million reads mapped (FPKM). An asterisk indicates a

455

significant differences (FDR  0.05) between ‘Ora’ and ‘Shani’ fruits at a particular ripening

456

stage, according to the DESeq2 analysis.

457 458

Figure 7. MEP-pathway genes differentially expressed in flavedo of ‘Ora’ and ‘Shani’ fruits

459

at the green, breaker, yellow and orange stages of ripening. The results are expressed as

460

fragments per feature kilobase per million reads mapped (FPKM). An asterisk indicates a

461

significant difference (FDR  0.05) between ‘Ora’ and ‘Shani’ fruits at the same ripening

462

stage, according to the DESeq2 analysis.

463 464

Figure 8. Ripening regulation in flavedo of ‘Ora’ and ‘Shani’ mandarins. A) Ethylene-

465

biosynthesis and ethylene-signaling genes differentially expressed in flavedo of ‘Ora’ and

466

‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. Results are

467

expressed as fragments per feature kilobase per million reads mapped (FPKM). An asterisk

468

indicates a significant difference (P  0.05) between ‘Ora’ and ‘Shani’ fruits at the same 2 ACS Paragon Plus Environment


Page 22 of 32

3 469

ripening stage, according to the DESeq2 analysis. B) Ethylene production of ‘Ora’ and

470

‘Shani’ at the orange ripening stage. Data are means ± SE of 3 replications, each containing 5

471

fruit. An asterisk indicates a significant difference (P  0.05) between ‘Ora’ and ‘Shani’

472

fruits.

473


Page 23 of 32


4

Table 1. Carotenoid content of flavedo in ‘Ora’ and ‘Shani’ mandarinthe at s green, breaker, yellow and orange stages of ripening. Carotenoid content (μg g-1) Green

Breaker

Yellow

Orange

Ora

Shani

Ora

Shani

Ora

Shani

Ora

Shani

neoxanthin

5.9 ± 1.6 A

9.1 ± 2.0 a

2.4 ± 0.5 A

3.1 ± 0.8 a

3.6 ± 0.9 A

5.8 ± 1.3 a

4.3 ± 1.3 A

10.3 ± 4.6 a

violaxanthin

25.1 ± 4.6 B

35.8 ± 6.7 b

29.2 ± 6.0 B

20.3 ± 3.2 b

31.4 ± 9.4 B

78.8 ± 11.6 A

164.2 ± 42.4 a

6.2 ± 1.0 *b

1.8 ± 0.9 B

4.0 ± 0.5 b

2.4 ± 1.2 B

21.3 ± 4.5 *b

7.4 ± 1.2 A

56.5 ± 13.8 *a

5.0 ± 1.6 A

8.3 ± 0.9 a

4.0 ± 1.3 A

3.2 ± 0.5 a

7.9 ± 2.7 A

9.0 ± 1.4 a

10.3 ± 2.2 A

19.5 ± 7.2 a

15.5 ± 3.2 A

18.0 ± 3.9 a

3.6 ± 2.1 B

9.9 ± 3.6 a

6.5 ± 3.0 AB

12.0 ± 6.3 a

4.5 ± 0.6 AB

10.0 ± 1.6 *a

zeaxanthin

2.6 ± 0.7 A

2.6 ± 0.3 a

1.1 ± 0.3 A

2.3 ± 0.6 a

2.5 ± 1.3 A

3.4 ± 1.5 a

1.7 ± 0.1 A

3.7 ± 0.7 a

β-cryptoxanthin

1.6 ± 0.3 B

6.7 ± 1.0 *a

2.0 ± 0.6 B

5.6 ± 1.7 a

2.4 ± 0.3 B

27.8 ± 9.0 *a

8.1 ± 1.4 A

42.1 ± 13.7 *a

α-carotene

4.5 ± 0.7 A

4.9 ± 1.0 a

tr B

2.1 ± 0.7 ab

1.3 ± 0.2 B

nd *b

1.6 ± 0.1 B

nd *b

β-carotene

6.7 ± 1.3 A

8.5 ± 1.6 a

1.5 ± 0.5 B

2.8 ± 1.0 b

1.7 ± 0.3 B

2.6 ± 0.5 b

1.7 ± 0.0 B

3.5 ± 0.7 ab

nd C

nd b

tr C

tr ab

1.0 ± 0.0 B

2.7 ± 1.1 ab

1.6 ± 0.1 A

3.5 ± 0.8 a

phytofluene

1.8 ± 0.6 A

1.9 ± 0.5 a

1.3 ± 0.2 A

1.7 ± 0.9 a

1.6 ± 0.1 A

5.7 ± 2.0 a

2.2 ± 0.2 A

7.2 ± 1.1 *a

phytoene

2.9 ± 1.0 A

3.8 ± 0.7 a

1.9 ± 0.5 A

3.1 ± 1.8 a

2.4 ± 0.5 A

9.6 ± 4.4 a

2.1 ± 1.0 A

9.3 ± 1.3 *a

others

4.0 ± 1.5 B

7.1 ± 1.3 b

4.7 ± 1.8 AB

7.5 ± 2.8 b

3.6 ± 0.8 B

30.4 ± 3.8 *a

12.6 ± 2.7 A

45.6 ± 5.5 *a

Compound

β-citraurin

nd B

antheraxanthin lutein

ζ-carotene

81.6 ± 12.5 *ab

Data are means  SE of 3 fruits. Different capital letters indicate a significant difference in ‘Ora’ among different ripening stages, different lower-case letters indicate significant 4 difference in ‘Shani’ among different ripening stages, and an asterisk indicates a significant differences (P  0.05) between ‘Ora’ and ‘Shani’ at the same ripening stage. nd – ACS Paragon Plus Environment not detected; tr – traces.


Page 24 of 32

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FIGURES

Figure 1. The carotenoid-biosynthesis pathway. GGPP, Geranygeranyl pyrophosphate; PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRITSO, carotenoid isomerase; β-LCY, lycopene β-cyclase; ε-LCY, lycopene εcyclase; β-CHX, β-ring carotene hydroxylase; ε-CHX, ε-ring carotene hydroxylase; ZEP, zeaxanthin epoxidase; CCD4, carotenoid cleavage dioxygenase 4. The schematic diagram was adapted from Alquézar et al.4 with modifications.5 ACS Paragon Plus Environment

Page 25 of 32


6

Figure 2. Peel color of ‘Ora’ and ‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. A) Photographs of ‘Ora’ and ‘Shani’ mandarin fruits at the green, breaker, yellow and orange stages of ripening. B) Lightness (0–100), Chroma (0–100) and Hue angles (0° represents a red color, 45° represents orange, 90° represents yellow and 120° represents green) of ‘Ora’ and ‘Shani’ peels at the green, breaker, yellow and orange stages of ripening. Data are means ± SE of 5 fruits and an asterisk indicates a significant difference (P ≤ 0.05) between ‘Ora’ and ‘Shani’ fruits at the same stage of 6 ripening. ACS Paragon Plus Environment


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7

Figure 3. Total carotenoid contents of flavedo of ‘Ora’ and ‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. Data are means  SE of 3 fruits. Different capital letters indicate a significant difference among the ‘Ora’ fruits at the different ripening stages. Different lower-case letters indicate a significant difference among the ‘Shani’ fruits at the different ripening stages. An asterisk indicates a significant difference (P  0.05) between ‘Ora’ and ‘Shani’ fruits at a particular ripening stage.


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Figure 4. Carotenoid composition in flavedo of ‘Ora’ and ‘Shani’ fruits at the orange (ripe) stage of ripening. Numbers indicate the percent if each carotenoid of the total carotenoid content for each variety. Data points are means of 3 fruits. β-xanthopylls – neoxanthin, antheraxanthin and zeaxanthin. Carotenes – α, β and ζ carotene, phytofluene and phytoene.



Page 28 of 32

9

Figure 5. Venn diagram illustration of differentially expressed genes (DEGs) in flavedo of ‘Ora’ and ‘Shani’ during the green, breaker, yellow and orange stages of ripening. The diagram displays the number of DEGs specific to ‘Ora’, the number of DEGs common to ‘Ora’ and ‘Shani’ and the number of DEGs specific to ‘Shani’.


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Figure 6. Carotenoid-biosynthesis genes differentially expressed in flavedo of ‘Ora’ and ‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. A) Genes highly expressed in ‘Shani’ B) Genes highly expressed in ‘Ora’. The results are expressed as fragments per feature kilobase per million reads mapped (FPKM). An asterisk indicates a significant differences (FDR  0.05) between ‘Ora’ and ‘Shani’ fruits at a particular ripening stage, according to the DESeq2 analysis.



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11

Figure 7. MEP-pathway genes differentially expressed in flavedo of ‘Ora’ and ‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. The results are expressed as fragments per feature kilobase per million reads mapped (FPKM). An asterisk indicates a significant difference (FDR  0.05) between ‘Ora’ and ‘Shani’ fruits at the same ripening stage, according to the DESeq2 analysis.


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Figure 8. Ripening regulation in flavedo of ‘Ora’ and ‘Shani’ mandarins. A) Ethylene-biosynthesis and ethylene-signaling genes differentially expressed in flavedo of ‘Ora’ and ‘Shani’ fruits at the green, breaker, yellow and orange stages of ripening. Results are expressed as fragments per feature kilobase per million reads mapped (FPKM). An asterisk indicates a significant difference (FDR  0.05) between ‘Ora’ and ‘Shani’ fruits at the same ripening stage, according to the DESeq2 analysis. B) Ethylene production of ‘Ora’ and ‘Shani’ at the orange ripening stage. Data are means ± SE of 3 replications, each containing 5 fruit. An asterisk indicates a significant difference (P  0.05) between ‘Ora’ and ‘Shani’ fruits.



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