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Anthocyanin accumulation and molecular analysis of correlated genes in purple kohlrabi (Brassica oleracea var. gongylodes L.) Yanjie Zhang, Zongli Hu, Mingku Zhu, Zhiguo Zhu, Zhijin Wang, Shibing Tian, and Guoping Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00473 • Publication Date (Web): 08 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Journal of Agricultural and Food Chemistry

Anthocyanin accumulation and molecular analysis of correlated genes in purple kohlrabi (Brassica oleracea var. gongylodes L.) Yanjie Zhang1, Zongli Hu1, Mingku Zhu1, Zhiguo Zhu, Zhijin Wang2, Shibing Tian2, Guoping Chen1* 1

Bioengineering College, Key Laboratory of Biorheological Science and Technology

(Chongqing University), Ministry of Education, Chongqing University, Campus B, Room 515, 174 Shapingba Main Street, Chongqing 400044, People’s Republic of China; 2

The Institute of Vegetable Research，Chongqing Academy of Agricultural Sciences,

401329 Chongqing , People’s Republic of China. * Corresponding author. Guoping Chen, Tel: 00862365112674; Fax: 0086 23 65112674; E-mail: [email protected].

ACS Paragon Plus Environment


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Abstract: Kohlrabi (Brassica oleracea var. gongylodes L.) is an important dietary

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vegetable cultivated and consumed widely for the round swollen stem. The purple

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kohlrabi shows abundant anthocyanin accumulation in the leaf and swollen stem.

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Here, different kinds of anthocyanins were separated and identified from the purple

5

kohlrabi cultivar (Kolibri) by high-performance liquid chromatography-electrospray

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ionization tandem mass spectrometry. In order to study the molecular mechanism of

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anthocyanin biosynthesis in purple kohlrabi, the expression of anthocyanin

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biosynthetic genes and regulatory genes in the purple kohlrabi and green cultivar

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(Winner) was examined by quantitative PCR. Compared with the colorless parts in the

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two cultivars, most of the anthocyanin biosynthetic genes and two transcription

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factors were drastically up-regulated in the purple tissues. To study the effects light

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shed on anthocyanin accumulation of kohlrabi, total anthocyanin contents and

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transcripts of associated genes were analyzed in sprouts of the both cultivars grown

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under light and dark conditions.

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Keywords Anthocyanin accumulation, Purple kohlrabi, structural genes，transcription

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factors, HPLC-ESI-MS/MS, Brassica oleracea var. gongylodes L.

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INTRODUCTION

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Anthocyanins，as an important subclass of flavonoids, is the main water-soluble

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pigments which are widely distributed among higher plants. The red, blue and purple

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colors found in plant tissues including flowers, leaves, fruits and roots are always

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attributed to the accumulation of this kind of vacuole pigments. Apart from the

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well-known physiological function of serving as pollinator and seed disperser

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attractant, anthocyanins also play essential roles in protecting plants against the

25

damages from UV radiation, coldness, drought stress and microbial agents(1-5). In

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addition, most plants synthesize anthocyanins as sunscreen which can absorb UV light

27

and serve as free radical scavengers to cancel out the damaging consequence of

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irradiation(6). Growing evidences indicate that regular intake of anthocyanins can

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reduce the risk of suffering from artherosclerosis and related diseases by inhibiting the

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low-density lipid oxidation(7). Besides, anthocyanins can also provide protection

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against cancer and other chronic illnesses (8-12). The health-promoting effects of

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anthocyanins are usually believed to be closely linked with the high antioxidant

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activities and the capacity of eliminating reactive oxygen species. Recent articles

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show that this kind of second metabolites is able to modulate signaling pathways in

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mammalian cell for the explanation of some beneficial biological effects (13, 14). As

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an important subclass of flavonoids, anthocyanins not only play important roles in the

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physiological plant processes of coloration and adaption to various environmental

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conditions, but also act as a health-promoting supplement for human diet.

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Anthocyanins are synthesized through a branch of phenylrpopanoid pathway and the



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genes that directly participate in the process of anthocyanin accumulation have been

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well studied in snapdragon (Antirrhinum majus), maize (Zea mays), Arabidopsis

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(Arabidopsis thaliana), petunia (Petunia hybrida), grape (Vitis vinifera L.) and blood

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Orange (Citrus sinensis L. Osbeck) in recent years (15-17). The pathway responsible

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for anthocyanin accumulation is showed in figure 1. The biochemical reaction that

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cinnamic acid is converted by phenylalanine ammonia-lyase (PAL) from

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phenylalanine represents the initial step, cinnamate 4-hydroxylase (C4H),

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4-coumaroyl:CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI),

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flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) anthocyanidin

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synthase (ANS) and flavonoid-5-glucosyltransferase (5-GT) catalyze the sequential

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reactions with the resulted metabolites as substrates in the following steps (18-20). It

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is worth to mention that B ring of the dihydrokaempferol (DHK) can be further

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hydroxylated by flavonoid 3′-hydroxylase (F3′H) to produce dihydroquercetin and

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leads to the production of the cyanidin-based anthocyanins (21). Colored

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anthocyanidins are formed as a result of the activity of ANS, but the immediate

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modification, largely by glycosylation is rather necessary for their stabilization (22,

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23). Then, these modified anthocyanidins will be transported into vacuolar from

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cytosol. As a glycosylated form of anthocyanidin, anthocyanins include 400

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molecules to the least extent and exhibit various colors depending on pH, metal

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cations, co-pigmentation, and modifications of the backbone (24).

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Anthocyanins, as the metabolites of the flavonoid pathway, are synthesized under

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the complicated regulation of diverse regulatory genes mainly at the transcriptional


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level (22, 23, 25). The flavonoid downstream pathway (from F3H to 5GT) of

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anthocyanin biosynthesis is regulated by several different families of regulatory genes

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including MYB transcriptional factors, bHLH transcriptional factors and WD40-like

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proteins (26). By analyzing mutants of Arabidopsis thaliana with abnormal levels of

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anthocyanins, and protein interaction assays, it has been proved that the transcription

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of anthocyanin biosynthetic genes is directly regulated by a transcriptional activation

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MYB-bHLH-WD40 complex (MBW) consist of R2R3 MYB, bHLH and WD40

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proteins (27-29). Recent studies show that variation in content of anthocyanins or

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tissue specificity in plants is governed mainly by the activity of the R2R3 MYB

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transcription factors in the MBW complex (30-36). However, the bHLH proteins

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always play essential roles in the synergistic regulation of anthocyanin accumulation

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(29, 37-39). In addition, heterogonous expression of bHLH genes individually really

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induced visible anthocyanin accumulation in host plant (40-42).

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As a biennial plant native to northern Europe, kohlrabi (Brassica oleracea var.

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gongylodes) belongs to the Brassicaceae family and is grown widely for the round

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swollen stem at the base of the plant. Extensive studies show that Cruciferous

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vegetables (including kohlrabi) supply human beings with healthy diet for the high

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levels of carotenoids, ascorbic acid and tocopherols contained in the edible parts (43,

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44). In addition to the natural antioxidants mentioned above, most of the antioxidative

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effect associated with food intake is largely due to the presence of phenolic

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compounds including anthocyanins, flavones, flavonols and so on (45). Compared

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with the green cultivars of kohlrabi, the purple cultivars which display abundant



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anthocyanin accumulation in the stems and leaves apparently attract more attention

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from consumers for the brilliant color and high levels of health-promoting ingredients.

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However, being a nutritionally Brassica vegetable worldwide, the molecular

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mechanisms underlying the biosynthesis of anthocyanins in purple kohlrabi still

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remain unknown. In this paper, the components of anthocyanin production in the

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purple kohlrabi cultivar (Kolibri) were characterized with high-performance liquid

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chromatography−electrospray

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(HPLC-ESI-MS/MS). In the next, the transcripts of anthocyanin biosynthetic and

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regulatory genes were analyzed by quantitative real-time polymerase chain reaction

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(qRT-PCR) in different tissues of the purple and green cultivars (Kolibri and Winner).

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Furthermore, the influence of light on the development and anthocyanin accumulation

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of kohlrabi sprouts at different stages were analyzed. The consistent increase of the

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anthocyanin biosynthetic genes with regulatory factors indicate that transcriptional

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activation of BoPAP2 and BoTT8 in a light independent manner mainly account for

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the up-regulation of anthocyanin structural genes and the onset of anthocyanin

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accumulation in purple kohlrabi. The results above enhanced our understanding about

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the mechanisms of anthocyanin biosynthesis in purple kohlrabi at both metabolic and

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molecular levels.

ionization

tandem

mass

spectrometry

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MATERIALS AND METHODS

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Chemicals and Solvents. Anthocyanin (cyanidin 3, 5-diglucoside) for external

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standards was purchased from Phytolab (Germany). High-performance liquid


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chromatography (HPLC)-grade formic acid and methanol (MeOH) were bought from

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Sigma. All the other solvents were provided from Aldrich (St. Louis, MO).

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Plant materials and culture conditions. Kohlrabi (Brassica oleracea var.

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gongylodes L.) seeds of green cultivar (Winner) and purple cultivar (Kolibri) were

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obtained from Chongqing Academy of Agriculture Sciences. The samples used for

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HPLC-ESI-MS/MS analysis were collected from the cuticles of swollen stem from

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the two cultivars of kohlrabi which were grown in a greenhouse with a 16-h

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photoperiod at 22℃. In addition, the mature leaves and the cuticles and fleshes of

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swollen stem were used for total anthocyanin and qRT-PCR analysis. The light and

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darkness treated samples used for total anthocyanin and qRT-PCR analysis were

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collected from sprouts of green and purple cultivars which were generated by follow

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procedures. Kohlrabi seeds were surface-sterilized with 70% ethanol for 60 s and

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2.5% (v/v) bleach solution for 5 min, and rinsed six times in sterile water. These seeds

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were then placed on half-strength sterilized Murashige−Skoog medium (1/2 MS)

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solidified with 0.8% agar. The two cultivars were germinated in a growth chamber

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under light/dark (16/8 h) or dark conditions at 26 ℃ and approximately 60% humidity.

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Samples were harvested after 3, 6, 9, and 12 days, measured for their length and fresh

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weight, frozen in liquid nitrogen, and stored at −80 ℃ until other analysis.

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RNA Extraction and qRT-PCR Analysis. The samples of the two kohlrabi cultivars

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were grounded into powder in liquid nitrogen. Total RNA was isolated from various

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tissues for three biological repeats using RNAiso reagent according to the

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manufacturer’s instruction (Takara, Dalian, PRC). RNA samples (1 µg) were reverse



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transcripted into complementary DNA (cDNA) with an oligo(dT)20 primer and

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M-MLV

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manufacturer’s protocol. The synthesized cDNAs were diluted five times in H2O for

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qRT-PCR analysis. qRT-PCR was carried out using the CFX96TM Real-Time System

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(C1000 thermal cycler). All reactions were performed using the GoTaq qPCR Master

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Mix according to the manufacturer’s instructions. Reactions were performed in

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triplicate using 5 µL of Master Mix, 0.25µM of each primer, 1 µL of diluted cDNA

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and DNase-free water to a final volume of 10 µL. The PCR amplification was as

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follows: 1 cycle of 2 min at 95℃, 40 cycles of denaturation for 5 s at 95 ℃, annealing

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for 20 s at 60℃, and elongation for 15 s at 72℃. Amplification was followed by a

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melting curve analysis with continual fluorescence data acquisition during the 60 −

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95℃ melt. Melt curve analysis of qPCR samples revealed that there was only one

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product for each gene primer reaction. The primers used for qPCR analysis of

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kohlrabi were designed by Primer Premier 5 and listed in Supplementary Table 1

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(Supporting Information).

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specific amplifications. The gene expression was normalized to BoApr as a reference

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gene for kohlrabi. Values reported here were calculated from three biological repeats

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for each sample.

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Anthocyanin

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extraction of kohlrabi was carried out in the same way as described for radish (46).

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The extract was filtered through a 0.2 µm PTFE syringe filter. The samples were then

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analyzed by an Agilent Technologies 1200 Series HPLC (Agilent Technologies, Palo

reverse

transcriptase

Extraction

(Promega,

Madison,

WI)

following

the

The PCR products were sequenced to confirm the

and

HPLC−ESI−MS/MS


Analysis.

Anthocyanin

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Alto, CA), equipped with an Agilent 1200 HPLC variable wavelength detector. The

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results were analyzed by Agilent 1200 HPLC ChemStation software. The

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chromatographic separation was performed on a Zorbax Stablebond Analytical

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SB-C18 column (4.6 mm × 250 mm, 5µm, Agilent Technologies, Rising Sun, MD).

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The injection sling was 5 µL. Elution was performed using mobile phase A (aqueous

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2% formic acid solution) and mobile phase B (methanol). The detection was at 520

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nm, and the column oven temperature was set at 40℃. The flow rate was 0.6 mL/min.

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The gradient program is described as follows: 0–2 min, 10–20% B; 2–40 min, 20

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–55% B; 40–41 min, 55–80% B; 41–45 min, 80% B; 45–50 min, 80–10% B;

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50–55 min, 10% B. Quantification of the different anthocyanins was based on peak

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areas and calculated as equivalents of the standard compounds. All contents were

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expressed as milligrams per grams dry weight. Low-resolution electrospray mass

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spectrometry was performed with a solariX ion trap mass spectrometer (Bruker

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Daltoniks, Billerica, MA). The experimental conditions were as follows: ESI interface,

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nebulizer, 50 psi; dry gas, 15.0 psi; dry temperature, 320 °C; MS/MS, scan from m/z

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200 to 1500; ion trap, scan from m/z 200 to 1500; source accumulation, 50 ms; ion

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accumulation Time, 300 ms; flight time to acquisition cell, 1 ms; smart parameter

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setting (SPS), compound stability, 50%; trap drive level, 60%.

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Total Anthocyanin Analysis. Spectrophotometric differential pH method was used

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for the total anthocyanin measurement of kohlrabi following the procedure of Yuan et

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al. with slightly modification (25). The protocols are described as follows. Frozen

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samples (100 mg) were crushed into powder in liquid nitrogen, and extracted



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separately with 2 mL of pH 1.0 buffer and 2 mL of pH 4.5 buffer. In addition, pH 1.0

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buffer contains 50 mM KCl and 150 mM HCl, while pH 4.5 buffer contains 400 mM

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sodium acetate and 240 mM HCl. The mixtures were centrifuged at 14,000g for 15

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min at 4℃. The supernatants were gathered for measurement of absorbance at 510 nm.

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The amount of total anthocyanin was calculated according to the equation which

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follows:

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Amount (mg g-1 FW) = (A1－A2)×484.8/24.825×dilution factor:

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The A1 represents the absorbance of supernatants gathered from pH 1.0 buffer

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solution at 510 nm，while the A2 represents the other. 484.8 represents the molecular

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mass of cyaniding-3-glucoside chloride, while 24,825 reflects its molar absorptivity at

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510 nm. The total anthocyanin of sample was analyzed in triplicate.

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Statistical Analyses. SPSS, version 17.0 (SPSS Inc., Chicago, IL) was used for the

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data analysis. One-way analysis of variance (ANOVA) followed by pair wise

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comparisons was performed with posthoc Tukey’s honestly significant different (HSD)

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test, with significance set at p < 0.05 and p < 0.01.

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RESULTS AND DISCUSSION

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Phenotypic characterization of Kolibri and Winner.

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To study the biosynthesis of pigments in kohlrabi, the purple cultivar Kolibri and

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green cultivar Winner were chosen to study. Visual inspection of the kohlrabi

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cultivars showed that Kolibri displays more purple pigments than Winner (Figure 2 A

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and Figure 1 S in the Supporting Information). The pigments extracted from purple


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tissues of Kolibri share the same spectral properties with anthocyanin standards

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(cyanidin 3, 5-diglucoside) and is conformed as anthocyanins in the following

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HPLC-ESI-MS/MS analysis (Figure 2 C). Compared with Winner, most of the organs

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of Kolibri contain high amount of anthocyanins (Figure 2 B). During all the

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developmental stages of vegetative growth, the Kolibri synthesizes and accumulates

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anthocyanins constantly (Figure 1 S in the supporting information). Meanwhile, the

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young sprouts of Winner only show faint-purple color at the very beginning of

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germination, but turn into green immediately as the elongation of hypocotyls. During

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the initial weeks of growth, all the leaves of Winner turn into solid green, while those

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of Kolibri display dark purple pigments in the veins of leaves and pale purple color in

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the mesophyll tissue (Figure 2 A and Figure 1 S in the supporting information). The

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purple pigments of Kolibri plants, especially in the round stems, become intense

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during the development. After 2 months of cultivation, the Winner possesses green

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swollen stems, while Kolibri displays dark purple pigments at the skin of the swollen

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stems. The intense accumulation of anthocyanins in the purple cultivar indicates a

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high ability to synthesize and accumulate anthocyanins. The total anthocyanin content

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of skin of the purple swollen stem is about 0.63mg per gram in fresh weight, while

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little is detected in the corresponding tissue of Winner. These results show that the

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drastic differences in anthocyanin accumulation arise from cultivar and genetic

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specificity.

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Identification and quantitative analyses of anthocyanins in purple kohlrabi.

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A total of 6 major anthocyanins were separated and characterized from the cuticle



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extracts of the purple swollen stem with the method of HPLC-ESI-MS/MS (Figure 2

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C). In order to verify the identity of anthocyanins in Kolibri, fragmentation patterns of

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MS/MS (m/z) corresponding to the compounds emerged in HPLC profiles were

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analyzed according to the information of radical groups reported previously (21).

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Consequently, 6 new kinds of modified cyanidin were identified as the major

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anthocyanins in the purple cultivar (Table 1). However, it is strange that pelargonidin

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based anthocyanins were not detected in the purple kohlrabi. Furthermore, all the

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anthocyanin modifications in Kolibri were found to be glycosylated cyanidin at the

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C5 position of anthocyanidins. Meanwhile, acylation at the C3 position of

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anthocyanidins seems like a common modification in this study.

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Total anthocyanin content was found to be 3.02 mg/g of dry weight for swollen

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stem skin of Kolibri with the application of HPLC, while there was no trace amount

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of anthocyanins detected in the corresponding tissue in Winner (Table 1). The content

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of anthocyanins in Kolibri is similar to those found in the head tissues of red cabbage

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reported before (25). Moreover, the anthocyanin, cyanidin 3-(caffeoyl) p-coumaroyl

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(sinapoyl) diglucoside-5-glucoside, shows the highest level (2.08 mg/g dry weight) in

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the stem skin of Kolibri (Table 1).

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Transcriptional analysis of anthocyanin biosynthetic and regulatory genes in the

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both kohlrabi cultivars.

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In order to investigate the mechanisms underlying the anthocyanin accumulation in

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purple kohlrabi, the transcripts of anthocyanin biosynthetic enzymes and regulatory

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genes were examined in the leaves, skins and flesh of the two cultivars by qPCR. The


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expression of anthocyanin biosynthetic genes BoPAL, BoC4H, BoCHS, BoCHI,

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RsF3H, BoF3′H, BoDFR, BoANS and Bo5GT are shown in Figure 3. Compared with

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the pigment less tissues, most of the anthocyanin pathway genes were significantly

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up-regulated in the purple leaves and stem skins of Kolibri.

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expression of anthocyanin structural genes from BoF3H was drastically increased in

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the purple kohlrabi in comparison with the green cultivar. In the cuticles of swollen

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stem, the purple kohlrabi displayed increased expression of nearly all anthocyanin

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pathway genes except BoCHI and BoC4H. In consistent with pigment production, the

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higher folds of expression changes happened in skins of swollen stem, while the lower

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folds of expression changes happened in leaves. Among the up-regulated genes,

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BoF3H exhibited the highest folds of increase (1000 folds at least) in both the leaves

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and skins of swollen stems. As the results showed in figure 2, trace amount of all the

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anthocyanin structural genes were detected by qPCR in different organs of Winner

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with the absence of visible anthocyanin production. These results indicate that large

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amount of transcripts of structural genes is a prerequisite for abundant anthocyanin

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accumulation. Moreover, the constant up regulation of anthocyanin biosynthetic genes

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in certain colorful organs and/or tissues rich of anthocyanins was also found in

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red-fleshed apple, purple eggplant, purple tomato, red pear and pap1-D Arabidopsis

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(30, 32, 34-36). Collectively, the purple kohlrabi shares similar mechanisms of

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transcriptional regulation in mediating anthocyanin accumulation with those high

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anthocyanin content plants mentioned above.

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In the leaves, the

Here, it is worth discussing the absence of pelargonidin based pigments we have



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referred in the anthocyanin profile analysis of the purple kohlrabi. Firstly, the fact that

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the biosynthetic pathway for anthocyanin accumulation is intact should be admitted

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for the apparent anthocyanin accumulation in certain tissues in Kolibri. In addition,

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the substrate specificity of the BoDFR and BoANS is also relatively broad (47). Then,

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the question seems to become more confused. However, the higher activity of BoF3′H

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due to the dramatically increased transcripts in purple kohlrabi might provide a

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reasonable answer to this question (Figure 3). In the anthocyanin biosynthetic

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pathway showed in figure 1, most of the dihydrokaempferol are applied to produce

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dihydroquercetin with two hydroxyl group. Consequently, it is no doubt that most of

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the final products should be cyanindin based anthocyanins. By the same token, the

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common glycosylated cyanidin at the C5 position of anthocyanidins in Kolibri might

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probably due to the increased expression of Bo5GT in purple stem skins.

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To verify whether any of the regulatory genes controlling the transcription of

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anthocyanin structural genes were up-regulated in Kolibri, the transcripts of some

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vital anthocyanin biosynthesis regulatory orthologous genes of Arabidopsis, BoPAP1,

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BoPAP2, BoMYB113, BoMYB114, BoTT8 and BoTTG1 were examined. In

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Arabidopsis, four R2R3 MYB genes (AtPAP1, AtPAP2, AtMYB113 and AtMYB114)

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are known to regulate the biosynthesis of anthocyanins directly, while bHLH protein

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AtTT8 and AtTTG1 play coordinate roles in the formation of transcriptional regulation

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complex (MBW) (26). Thence, four MYB genes which shared high similarities with

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anthocyanin biosynthesis activator of Arabidopsis were cloned from the kohlrabi and

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designated as BoPAP1, BoPAP2, BoMYB113 and BoMYB114. Similarly, BoTT8 and


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BoTTG1, as the orthologous genes of AtTT8 and AtTTG1 respectively, were also

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cloned. As the results shown in Fig. 4, BoPAP2 and BoTT8 were the only two

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regulatory genes greatly up-regulated in the tissues rich of anthocyanins. In the leaves,

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the expression levels of BoPAP2 and BoTT8 in Kolibri were about 990- and 6.8-folds

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higher than those in Winner respectively. In the skins of swollen stem, the expression

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levels of BoPAP2 and BoTT8 in Kolibri were about 452- and 57.7-folds higher than

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those in the green cultivar respectively.

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In the process of activating anthocyanin biosynthetic genes, the MYB and bHLH

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transcription factors make different contribution among plant species. In the

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anthocyanin biosynthesis regulatory complex of MBW, R2R3 MYB transcription

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factors regulate anthocyanin biosynthesis specifically (48). However, the functions of

293

bHLH transcription factors and WD proteins are rather pleiotropic (26). In addition,

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the increased expression of both MYB and bHLH is necessary for transcriptional

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activation of anthocyanin biosynthetic genes in petunia, cauliflower and Arabidopsis

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(26, 31, 49). Consequently, the dramatically increase of the pigment production and

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anthocyanin structural genes in purple kohlrabi should be due to the coordinated

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transcriptional activation of BoPAP2 and BoTT8.

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Light sheds evident influence on the growth and anthocyanin accumulation of

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kohlrabi sprouts.

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To study the effects of light on development and pigment production of kohlrabi,

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sprouts of the both cultivars grown under light and dark conditions were used as

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materials. The length, fresh weight and total pigment contents of sprouts were



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examined every 3 days until 12 days after sowing (DAS). As the results figure 5

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shows, the treated sprouts of Kolibri and Winner displayed completely different

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phenotypes in production of anthocyanins. Kolibri showed apparent anthocyann

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accumulation during all the stages of development, no matter of lightness or darkness.

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However, the pigment intensity in sprouts of Kolibri cultured in light is higher than

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that cultured in darkness. This phenomenon suggests that the production of pigments

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is not totally independent of lightness. On the contrary, only tiny amount of pigments

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were detected in the sprouts of Winner grown under lightness and the production of

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pigments is totally dependent on lightness (Figure 5 A and B). Whereas, these two

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kohlrabi cultivars shared the similar trends of growth (Figure 5 C and D). The length

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increased with time during the entire process of experiments.

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in darkness showed much higher speed of elongation than cultured in lightness. The

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lengths of sprouts grown under the dark condition were 2 folds higher than those

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under light condition after cultured in medium for 9 days. In addition, the fresh weight

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cultured for 12 days under dark condition did not enhance evidently in comparison

319

with 9 day old sprouts. That is to say, the maximum biomass for kohlrabi sprouts

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cultured under darkness reached the top at around 9 days. These results are

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concordant with the findings of buckwheat sprouts in a previous report (50).

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Expression profiles of anthocyanin biosynthetic and regulatory genes in sprouts

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of the two kohlrabi cultivars grown in light and dark conditions.

The sprouts cultured

324

To investigate the molecular mechanism of anthocyanin accumulation under light

325

and dark conditions, the expression profiles of anthocyanin pathway and regulatory


Page 16 of 37

Page 17 of 37


326

genes in the four groups of materials gathered at different stages were examined. The

327

expression patterns of BoPAL, BoC4H, BoCHS, BoCHI, BoF3H, BoF3′H, BoDFR,

328

BoANS, and Bo5GT are shown in Figure 6. During the different development stages in

329

the light treated sprouts of Kolibri, all the anthocyanin structural genes exhibited the

330

top expression level at 3 DAS. The expression patterns agree well with the amounts of

331

pigment production in the corresponding tissues (Figure 5 B). Compared with the

332

purple sprouts treated in darkness at 3 DAS, the expression of BoPAL, BoC4H, BoCHI,

333

BoF3H, BoF3′H, BoDFR, BoANS and Bo5GT were all slightly raised in the purple

334

sprouts treated in lightness. In the same development stage, the expression of BoCHS

335

in light treated purple sprouts was significantly higher (8.2 folds) than that of purple

336

sprouts under darkness. These results suggest that the expression of BoCHS in purple

337

kohlrabi sprouts is strictly light dependent. Therefore, it is reasonable that the contents

338

of anthocyanins in light treated Kolibri sprouts are higher than those in dark treated

339

sprouts during all the developmental stages. Combined with expression analysis of

340

anthocyanin biosynthetic genes at other developmental stages in purple sprouts, it can

341

be conclude that light enhances the existing production of anthocyanins by

342

strengthening the expression of structural genes, especially BoCHS, at mRNA level.

343

As we have mentioned that trace amount of anthocyanins was detected in the light

344

treated sprouts of Winner, it is not astonishing that the transcripts of most of

345

anthocyanin pathway genes in the corresponding samples were significant higher than

346

that in dark treated sprouts.

347

Expression profiles of anthocyanin biosynthesis regulatory genes in sprouts of the



348

two kohlrabi cultivars grown in light and dark conditions are showed in figure 7. In

349

the light treated sprouts of Kolibri at the stage of 3DAS, the expression levels of

350

BoPAP1, BoPAP2 and BoTT8 were about 11-, 6- and 101- folds higher than those in

351

the light treated green cultivar, respectively. It seems that the anthocyanin structural

352

genes are coordinated regulated by BoPAP1, BoPAP2 and BoTT8 in sprouts of Kolibri

353

under light. Furthermore, BoPAP1 was significantly raised at transcriptional level

354

under light in purple sprouts during all the stages of development and was about 23-

355

folds higher than in the dark treated sprouts at 3 DAS. However, light did not shed

356

significant influence on the transcription of BoPAP2 and BoTT8 in purple sprouts. In

357

conclusion, the anthocyanin accumulation and up-regulation of structural genes in the

358

purple kohlrabi sprouts is mainly due to the transcriptional activation of BoPAP2 and

359

BoTT8. In addition, BoPAP1 is the major regulatory gene responsible for the enhanced

360

production of pigments in light treated sprouts.

361

MYB genes associated with the regulation of anthocyanin accumulation are

362

characterized by a conserved DNA-binding domain including two imperfect repeat

363

(R2R3) with a specific motif for the interaction with bHLH domain of bHLH proteins

364

in plant kingdom. The four MYB genes cloned from kohlrabi exhibited high sequence

365

similarities among each other. The putative proteins contain conserved R2R3 MYB

366

domains and belong to the same subgroup 10 of MYB proteins as described by Allan

367

et al. in anthocyanin production regulation (48). In purple swollen stem of Kolibri, the

368

up-regulation of BoTT8 and BoPAP2 probably account for the accumulation of

369

anthocyanins by transcriptional activating the structural genes. Transcriptional factors


Page 18 of 37

Page 19 of 37


370

of MYB or bHLH have been reported to be responsible for pigment production in

371

many plant species. In certain anthocyanin-accumulating plants (such as red-fleshed

372

apple, purple sweet potato, in pap1-D Arabidopsis, and maize), the anthocyanin

373

accumulation has been found to be due to an activation of a MYB transcriptional

374

factor(30, 36, 51-53). On the contrary, white-skinned grape arise from the mutations

375

in a R2R3 MYB protein (54). Besides, heterologous expression of bHLH

376

transcriptional factors alters pigment production in the transgenic tomato (40-42, 55).

377

In the tissues rich of anthocyanins in Kolibri, both BoPAP2 and BoTT8 genes were

378

constitutively up-regulated. In the light treated sprouts of Kolibri, BoPAP1 was

379

up-regulated in large folds. Thence, a model illustrating that the transcriptional

380

regulation complex consist of MYB, bHLH and other proteins (BoTTG1) which

381

regulates anthocyanin accumulation coordinatedly was presented in figure 8. In the

382

model, BoTT8 and BoPAP2 promotes kohlrabi colouration in a light independent

383

manner, while the light induced transcriptioa factor BoPAP1 (indicated in dashed

384

cycle) enhanced the existing production of pigments in the light treated sprouts of

385

Kolibri. In summary, the eye-catching purple kohlrabi not only supply human beings

386

with healthy diet for the high levels of carotenoids, ascorbic acid and tocopherols

387

contained in the edible parts, but also with large amounts of natural antioxidants

388

anthocyanins. The elucidations of anthocyanin accumulation at molecular and

389

metabolic levels in purple kohlrabi provide an important basis for the breeding of new

390

kohlrabi cultivars with more excellent agronomic characters.

391



392

Abbreviation used

393

PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL,

394

4-coumarateCoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H,

395

flavone 3-hydroxylase; F3'H, flavonoid 3',-hydroxylase; DFR, dihydroflavonol

396

reductase; ANS, anthocyanidin synthase; 5-GT, flavonoid-5-glucosyltransferase; GST,

397

glutathione S-transferase; DHK, dihydrokaempferol; HPLC, high-performance liquid

398

chromatography; ESI-MS/MS, elctrospray ionization tandem mass spectrometry;

399

qRT-PCR, quantitative real-time PCR; DAS, days after sowing.

400 401

Acknowledgment

402

This work was supported by National Natural Science Foundation of China (nos.

403

30871709, 30600044, 31171968) and Technology System of National Bulk Vegetable

404

Industry--Eggplant Breeding Position (CARS-25-A-06).

405

Supporting Information description

406

A table of primers used for qPCR analysis of anthocyanin biosynthetic genes and

407

associated regulatory genes in kohlrabi, a figure depicting phenotypic characterization

408

of Kolibri and Winner during all the developmental stages of vegetative growth.

409 410 411 412 413 414 415 416 417

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550 551 552 553 554 555 556 557 558

53. Espley, R. V.; Brendolise, C.; Chagne, D.; Kutty-Amma, S.; Green, S.; Volz, R.; Putterill, J.;

559

FIGURE CAPTIONS

560

Figure 1. Schematic representation of the biosynthetic pathway of the anthocyanins.

561

The names of the compounds in boxes are indicated. The enzyme names are PAL,

562

phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarateCoA

563

ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone

564

3-hydroxylase; F3'H, flavanone 3'-hydroxylase; DFR, dihydroflavonol 4-reductase;

565

ANS, anthocyanidin synthase; 5-GT, flavonoid-5-glucosyltransferase; PAT, putative

566

anthocyanin transporter.

567

Figure 2. Anthocyanin analysis of different kohlrabi cultivars. (A) Photographs of the

568

different tissues of two kohlrabi cultivars (Kolibri on the left and Winner on the right)

569

used in this study. (B) Total anthocyanin content analysis of leaves, stem flesh and

570

skins in the two kohlrabi cultivars. PS (Purple skin of swollen stem); GS (Green skin

571

of swollen stem); PS (Flesh of swollen stem in purple cultivar); GS (Flesh of swollen

572

stem in green cultivar); PL (Purple leave); GL (Green leave). Error bars represent the

573

standard error of the mean (n = 3). (C) HPLC profiles of anthocyanins extracted from

574

the skins of the purple swollen stem. Peak numbers refer to the anthocyanins are listed

575

in Table 1. Structures and major cleavage of cyanidin 3-(caffeoyl) p-coumaroyl

576

(sinapoyl) diglucoside-5-glucoside in reference to peak 6 is framed in box.

Schouten, H. J.; Gardiner, S. E.; Hellens, R. P.; Allan, A. C., Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 2009, 21, 168-83. 54. Walker, A. R.; Lee, E.; Bogs, J.; McDavid, D. A.; Thomas, M. R.; Robinson, S. P., White grapes arose through the mutation of two similar and adjacent regulatory genes. The Plant journal : for cell and molecular biology 2007, 49, 772-85. 55. Albert, N. W.; Lewis, D. H.; Zhang, H.; Irving, L. J.; Jameson, P. E.; Davies, K. M., Light-induced vegetative anthocyanin pigmentation in Petunia. Journal of experimental botany 2009, 60, 2191-202.


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577

Figure 3. Expression analysis of anthocyanin biosynthetic genes in different tissues of

578

the two kohlrabi cultivars. PS (Purple skin of swollen stem); GS (Green skin of

579

swollen stem); PS (Flesh of swollen stem in purple cultivar); GS (Flesh of swollen

580

stem in green cultivar); PL (Purple leave); GL (Green leave). Error bars represent the

581

standard error of the mean (n = 3). Error bars represent the standard error of the mean

582

(n = 3). Statistical significance of the differences between samples was calculated

583

with ANOVA by paired-group comparisons. Different letters in uppercase indicate

584

significance at P < 0.01. Different letters in lowercase indicate significance at P

Brassica oleracea var. gongylodes L. - American Chemical Society

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