The Dark-Purple Tea Cultivar ‘Ziyan’ Accumulates a Large Amount ofDelphinidin-Related AnthocyaninsYun-Song Lai, Sha Li, Qian Tang,* Huan-Xiu Li, Shen-Xiang Chen, Pin-Wu Li, Jin-Yi Xu, Yan Xu,and Xiang Guo

College of Horticulture, Sichuan Agricultural University, 211 Huimin Road, Wenjiang District, Chengdu 611130, People’s Republic ofChina

*S Supporting Information

ABSTRACT: Recently, we developed a novel tea cultivar ‘Ziyan’ with distinct purple leaves. There was a significant correlationbetween leaf color and anthocyanin pigment content in the leaves. A distinct allocation of metabolic flow for B-ringtrihydroxylated anthocyanins and catechins in ‘Ziyan’ was observed. Delphinidin, cyanidin, and pelargonidin (88.15 mg/100 gFW in total) but no other anthocyanin pigments were detected in ‘Ziyan’, and delphinidin (70.76 mg/100 g FW) was particularlypredominant. An analysis of the catechin content in ‘Ziyan’ and eight other cultivars indicated that ‘Ziyan’ exhibits a preferencefor synthesizing B-ring trihydroxylated catechins (with a proportion of 74%). The full-length cDNA sequences of flavonoidpathway genes were isolated by RNA-Seq coupled with conventional TA cloning, and their expression patterns werecharacterized. Purple-leaved cultivars had lower amounts of total catechins, polyphenols, and water extract than ordinary non-anthocyanin cultivars but similar levels of caffeine. Because dark-purple-leaved Camellia species are rare in nature, this studyprovides new insights into the interplay between the accumulations of anthocyanins and other bioactive components in tealeaves.

KEYWORDS: purple tea, anthocyanin, catechin, gene expression, RNA-Seq

■ INTRODUCTION

Tea plants (Camellia sinensis (L.) O. Kuntze) are used toproduce the most widely consumed beverage. In 2012, theworld acreage of tea gardens increased to 4.07 million hectares,which produced 4.78 million tons of tea and contributed toU.S. $5.18 billion of world tea exports.1 Many laboratoryexperiments have demonstrated that tea has good preventiveeffects for multiple diseases, including cancer, metabolicsyndrome, cardiovascular disease, and neurodegenerativediseases.2 The common bioactive components of tea includefree amino acids (FAAs), caffeine, and catechins. For example,catechins exert their cancer-preventing activity by inhibitingenzyme activities and signaling pathways and through othermechanisms.3 In addition to the health benefits, teacomponents also impart the taste and aromatic characteristicsof tea.4

Currently, there is increasing interest in anthocyanin-rich teasworldwide. Anthocyanins are a group of pigments that existwidely in higher plants, thus rendering the plant kingdomcolorful. Moreover, anthocyanins are beneficial to human healthbecause they are powerful antioxidants and anti-inflammatoryagents.5 Because anthocyanins are water-soluble and tea is aparticularly popular beverage, drinking tea is a good mode ofdaily anthocyanin intake. However, most tea cultivars do notcontain abundant anthocyanins in their leaves. To the best ofour knowledge, only three popularly grown red- or violet-leavedcultivars exist in the world, and they are the Chinese cultivar‘Zijuan’,6 the Japanese cultivar ‘Sunrouge’,7 and the Kenyan teaTRFK306.8 A chemical component analysis of the tea madefrom these three cultivars has been reported,6,9,10 and the

nutraceutical properties of ‘Sunrouge’1−13 and TRFK3061−15

have been approved using mice or human cells.Both anthocyanins and catechins are synthesized in the

flavonoid biosynthetic pathway. Therefore, these two metabo-lites share some key catalytic steps.16 For example, phenyl-alanine ammonia-lyase (PAL) catalyzes the first reaction of thesecondary phenylpropanoid metabolism, and chalcone synthase(CHS) catalyzes the first reaction in the pathway specific forflavonoids including flavones, flavonols, proanthocyanidins,catechins, and anthocyanins. Flavonoid 3′-hydroxylase (F3′H)adds a hydroxyl group at the 3-position in the B-ring offlavonoids, and flavonoid 3′5′-hydroxylase (F3′5′H) adds ahydroxyl group at the 3- and 5-positions in the B-ring. Thisdifferential hydroxylation of the B-ring results in the majordifference between cyanidin and delphinidin and betweencatechin-type catechins including catechin (C), epicatechin(EC), catechin gallate (CG), epicatechin gallate (ECG), andgallocatechin-type catechins including gallocatechin (GC),epigallocatechin (EGC), gallocatechin gallate (GCG), andepigallocatechin gallate (EGCG). The biosynthetic pathwayfor catechins has been known for a long time, and the relatedgenes, such as leucoanthocyanidin reductase (LAR) andanthocyanidin reductase (ANR), have been characterized.17−19

Anthocyanin-rich flowers are common in the Camellia genus,20

and several reports on the transcriptome have provided someinformation on the expression of anthocyanin genes.21−23

Received: August 18, 2015Revised: March 16, 2016Accepted: March 19, 2016

Article

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© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b04036J. Agric. Food Chem. XXXX, XXX, XXX−XXX

However, these previous studies did not demonstrate asituation in which tea leaves accumulate large amounts ofanthocyanins other than catechins.We previously found a tea tree with dark purple young

shoots at an altitude of approximate 1500 m in Sichuanprovince, China. A novel purple-leaved cultivar ‘Ziyan’ was thendeveloped (Figure 1). This cultivar is more purple than any

other reported violet cultivars according to the pictures, and itis definitely more purple than ‘Zijuan’, a well-known violet-leaved tea cultivar, under the same cultivation conditions. Inthis study, we analyzed anthocyanins and major teacomponents, including catechins, free amino acids (FAAs),and caffeine. The flavonoid pathway genes in ‘Ziyan’ wereisolated, and their expression patterns were profiled todemonstrate the association of gene expression and metaboliteaccumulation.

■ MATERIALS AND METHODSPlant Materials. All of the tea plants used in this study were

cultivated at the Yizhichun Tea Research Foundation in Muchuancounty (Sichuan province, China). A medium level of fertilizer wasapplied: 300 kg/ha urea in the spring, 150 kg/ha urea after the springand summer crop, 1500 kg/ha cake fertilizer, and 750 kg/hacompound fertilizer (N:P:K, 2:1:1) after the autumn crop. All of thesamples in the following experiments were collected in autumn unlessotherwise specified. Young tender shoots of one bud and two leaves(B2L) were plucked in spring and autumn, and they were processedinto green tea or simply steamed into dry samples for the catechin andFAA analyses. B2L samples were collected to measure the anthocyanincontent. The second top leaves of B2L were used to isolate total RNAfor gene isolation and RNA-Seq, and they were also used forcomparing the gene expression between different cultivars. The first,third, and fifth top leaves of one bud and six leaves (B6L) werecollected to profile the expression of anthocyanin genes during leafmaturation.Color Measurement. Color was measured by a CM-2600d

spectrophotometer (Konica Minolta, Japan). The instrument used aD65 illuminant and a 45°/normal illuminating and viewing geometry,and the color space/colorimetric data of L*, a*, and b* were readimmediately. The chroma and hue angle (h°) were calculated asfollows:

= +

* > * > ° = * *

* < ° = ° + ∗ *

* > * < ° = ° + * *

∗ ∗a b

a b h b a

a h b a

a b h b a

chroma ( )

if 0 and 0, (arctan( / ))

if 0, 180 (arctan( / ))

if 0 and 0, 360 (arctan( / ))

2 2

degrees

degrees

degrees

Anthocyanin Measurement by HPLC. The fresh shoot samplesfor anthocyanin measurement were prepared according to the method

described by Kerio et al.8 with small modifications. In brief, thepigment solutions extracted by 50% methanol (v/v) were concen-trated using a Rotavapor R-300 (Buchi, Switzerland) and filtered bymembrane filters (0.45 μM). Reverse-phase solid-phase extraction(RP-SPE) using a Sep-Pak C18 cartridge (Waters, USA) was used topurify the anthocyanin pigments. The column was previously activatedwith methanol followed by 0.01% aqueous HCl (v/v). Anthocyaninswere adsorbed onto the column, and sugars, acids, and other water-soluble compounds were removed by washing the cartridge twice with0.01% aqueous HCl (v/v). Less polar polyphenolics were subsequentlyeluted with 2 volumes of ethyl acetate. The anthocyanins were theneluted with methanol containing 0.01% HCl (v/v).

The acid hydrolysis HPLC method was adopted to simplify theanthocyanin profile.24 In the acid hydrolysis of anthocyanins, 0.2 mLof an anthocyanin solution was mixed with 1 mL of 2 N HCl in asealed 1.5 mL tube. The tube was placed in a preheated dry bath, andthe hydrolyzed samples were immediately cooled in an ice bath. AnAgilent 1260 HPLC system (Agilent, USA) and ZORBAX SB-C18column (Agilent, USA) were used for the HPLC measurement. Theeluents were mobile phase A (water/acetonitrile/formic acid; 87:3:10,v/v/v) and mobile phase B (100% acetonitrile). The chromatographicconditions were as follows: 0 min, 0% B; 5 min, 5% B; 10 min, 14% B;and 30 min, 30% B. The absorbance was detected at 520 nm by anAgilent VWD detector. Cyanidin-3-O-glucoside, cyanidin chloride,delphinidin chloride, pelargonidin chloride, malvidin chloride, andpeonidin chloride reference standards were purchased fromChromaDex (USA).

Determination of Anthocyanin Content by the pH Differ-ential Method. Fresh leaves were ground in liquid nitrogen and thenimmersed in a 1% HCl/methanol solution for 24 h at 4 °C. Theanthocyanin concentration of the extract solution was determined bythe pH differential method as previously described.25 The followingtwo buffer systems were used: a 0.025 M potassium chloride (KCl)buffer at pH 1.0 and a 0.4 M sodium acetate (NaC2H3O2) buffer at pH4.5. The anthocyanin sample (150 μL) was separately mixed with 750μL of KCl buffer and NaC2H3O2 buffer. After equilibration for 50 min,the absorbances at 524 and 700 nm were determined. Theanthocyanin-specific absorbance was determined as follows:

= − − −A A A A A( ) ( )524nm 700nm pH 1.0 524nm 700nm pH 4.5

The anthocyanin content in the fresh leaves was then determinedusing the following formula: anthocyanin pigment (mg/100 g) = (A ×MW × DF × V × 100)/(FW × ε × 1), where A is the absorbancedifference in the two pH solutions, MW is the molecular weight ofanthocyanin (465.5), DF is the dilution factor, V is the volume ofextract solution (6 mL), FW is the fresh weight, and ε is the cyanidin-3-glucoside molar absorbance (29000).

Determination of Total Content of Polyphenols, Caffeine,and Water Extracts. The total polyphenol content was measured bya spectrophotometer according to the modified Folin−Ciocalteumethod with reference to Chinese National Standard GB/T 8313-2008. The caffeine content was determined by using Chinese NationalStandard GB/T 8312-2002. The water extract content was determinedby using Chinese National Standard GB/T8305-2002.

Catechin Determination by HPLC. Catechins were analyzed bythe HPLC method according to ISO 14502-2-2005E. The HPLC wasrun on an Agilent 1260 (Agilent, USA), and a Phenomenex phenyl-hexyl column was used (Phenomenex Inc., Torrance CA, USA).Mobile phase A contained 9% acetonitrile and 2% acetic acid with 20μg/mL EDTA, and mobile phase B contained 80% acetonitrile and 2%acetic acid with 20 μg/mL EDTA. The binary gradient elutionconditions were as follows: 0% mobile phase B for 10 min, a lineargradient to 32% mobile phase B in 15 min, and then holding for 10min. The GC, EGC, C, EC, EGCG, GCG, ECG, and CG referencestandards were purchased from Sigma-Aldrich (UK) to generatecalibration curves. The HPLC conditions were as follows: flow rate of1 mL/min, column temperature of 35 °C, and detection wavelength of278 nm.

FAA Determination. The FAA profile was analyzed by HPLC(Waters 600) with an AccQ Tag column (3.9 mm × 150 mm) and a

Figure 1. Mother tree of ‘Ziyan’ (a) and a 3-year-old clone tree (b).

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fluorescence detector after derivatization following the manufacturer’sprotocol. The AccQ•Tag Reagent Kit was purchased from Waters(Milford, MA, USA).Gene Fragment Isolation. Total RNA was extracted from tea

leaves using KingFisher mL magnetic particle processors (ThermoScientific, USA) and a MagPure Universal RNA KF Kit (Magen,China). First-strand cDNA was synthesized using the TranscriptorFirst Strand cDNA Synthesis Kit (Roche, Germany). Degenerateprimers or ordinary primers were used to amplify the correspondinggene fragments from cDNA libraries (Supporting Information, TableS1).The DNA fragments were then inserted into pBLUE-T using theSimple Quick Ligation Kit (Aidlab, China) by the TA cloning methodand sent to be sequenced.Gene Expression Analysis. cDNA was synthesized using the

PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). qRT-PCR was conducted using FastStart Universal SYBR Green Master(Roche, Germany) and CFX96 Real-Time PCR detection systems(Bio-Rad, USA). A 20 μL reaction solution contained 0.2 μM each ofgene-specific primers and 2 μL of diluted cDNA. The PCR reactionconditions were as follows: preheating at 95 °C for 10 min and 40cycles at 94 °C for 5 s and at 58 °C for 30 s.Statistical Analysis. All of the experiments were performed in

triplicate unless described otherwise in the footnotes. The data weresubjected to an analysis of variance, and the significant difference testwas performed using Duncan’s multiple range test of IBM SPSSStatistics version 19.

■ RESULTSNovel Cultivar ‘Ziyan’ Shows a Dark Purple Color. The

leaf color of the dark-purple-leaved cultivar ‘Ziyan’, the purple-leaved cultivar ‘Zijuan’, and green-leaved cultivars ‘Fudingdabai’and ‘Anjibaicha’ was measured by a spectrophotometer andexpressed by L*a*b* color space values (Table 1). Basically,

violet cultivars showed higher a* value and much lower L* andb* values than the other cultivars. These differences resulted inmuch lower chroma and h° values for the violet tea leaves. Theh° value of ‘Ziyan’ and ‘Zijuan’ indicated a red-purple color, andthe h° value of ‘Anjibaicha’ and ‘Fudingdabai’ indicated ayellow-green color in the L*a*b* color space. When the twoviolet cultivars were compared, ‘Ziyan’ showed lower L*,chroma, and h° values, indicating a darker purple color.Twenty-seven seedlings derived from the natural crossing of

‘Ziyan’ were obtained in 2012. The anthocyanin content of B2Lsamples ranged from 1.61 to 93.73 mg/100 g FW, thus showinga color from green to purple. To investigate the possible

correlation between pigments and color scales, the leaves ofthese plants were subjected to both color measurement andanthocyanin measurement. A significant and negative correla-tion was found between the anthocyanin content and the L*,b*, chroma, and h° values. The h° value (R2 = 0.511) is the bestindicator among these parameters of anthocyanin content(Figure 2).

The Dark-Purple Color of ‘Ziyan’ Is Attributed toDelphinidin-Related Pigments. The anthocyanin extractsolution was first subjected to acid hydrolysis, and then thereaction solution was separated and detected by the HPLCsystem. A representative HPLC chromatogram of the ‘Ziyan’B2L sample is presented in Figure 3. Delphinidin, cyanidin, andpelargonidin were detected in both the ‘Ziyan’ and ‘Zijuan’cultivars (Table 2) but not in the green ‘Fudingdabai’ cultivar.For both cultivars, delphinidin was the predominant pigment,much higher than the other two pigments, and only a traceamount of pelargonidin was detected. In terms of the totalamount, the B2L and B3L samples of ‘Ziyan’ contained 26.4and 47.6% higher anthocyanidin contents than those of ‘Zijuan’,respectively, and this was definitely attributed to delphinidin. Asimilarly higher amount of total monomeric anthocyanin in‘Ziyan’ was also detected by the pH-differential method.

Color and Pigment Changes during Leaf Develop-ment. Younger leaves of ‘Ziyan’ have a purple color, but theolder leaves have a green color. One shoot usually develops fiveto seven leaves if the shoot is not plucked in a crop season. Tostudy the relationship between leaf maturation and anthocyaninaccumulation, we compared the second top leaf of the B2L−

Table 1. Color Measurements of Tea Leavesa

sample L* a* b* chroma h°

ZJ-B2L2 24.44 0.99 1.19 1.55 50.19FD-B2L2 38.16 −9.40 25.39 27.08 110.31AJ-B2L2 70.70 0.01 31.49 31.49 89.98ZY-B2L2 22.88 1.25 0.78 1.48 31.98ZY-B3L2 23.48 1.67 0.83 1.87 26.24ZY-B4L2 23.43 1.13 0.98 1.50 40.84ZY-B5L2 23.99 1.35 1.31 1.89 55.12ZY-B6L1 24.31 1.47 0.25 1.49 9.68ZY-B6L2 23.56 1.65 1.19 2.03 35.87ZY-B6L3 25.89 1.52 2.30 2.76 56.56ZY-B6L4 27.64 1.06 3.76 3.91 74.23ZY-B6L5 29.23 −0.55 4.94 4.97 96.40ZY-B6L6 29.70 −1.47 5.85 6.03 104.08

aB2L2, the second top leaf of one bud with two leaves (B2L); ZJ,Zijuan; FD, Fudingdabai; AJ, Anjibaicha; ZY, Ziyan. Each datumrepresents the mean value of 10 leaves.

Figure 2. Purple color of naturally occurring hybrid plants of ‘Ziyan’ isascribed to anthocyanin pigments: (a) correlation analyses betweenanthocyanin concentration (A524) and color space parameters (L*,b*, chroma, and h°); (b) a* value and b* value distribution of eachmeasured sample. The color from green to purple indicates theanthocyanin concentration (A524) from low to high.

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B5L samples and the leaves from the second top leaf to thesixth top leaf of the B6L samples.The second top leaf of B3L had the lowest hue angle value,

and the second top leaf of B5L had the highest hue angle value(Table 1), indicating that the anthocyanin production capabilityof young leaves correlated with the branch developmentalphase. In the B6L branches, the younger leaves had higher a*values but lower L*, b*, chroma, and h° values (Table 1). Thetop second and third leaves contained the highest total amountof anthocyanidins (Table 2). Along with the increase in leaf age,the total amount of anthocyanins decreased gradually. The totalamount in the sixth leaf was only 64.8% of that in the secondleaf, and this decrease was attributed to all three anthocyanidincomponents.Gene Expression Analysis of Anthocyanin-Related

Genes. Full-length cDNA sequences of flavonoid-related genesin ‘Ziyan’ were identified by a combination of a PCR-basedmethod and RNA-Seq (Supporting Information). There weremultiple gene members for CHS, CHI, F3H, F3′H, F3′5′H,ANS, and ANR. In most cases, only one gene member waspredominantly expressed, for example, F3H1, F3′H1, andANR1. The transcription levels of late anthocyanin pathwaygenes (F3′5′H, DFR, and ANS) were much lower than those ofearly anthocyanin pathway genes (Figure 4a). This togetherwith the low expressions of F3′H, F3′5′H, and ANS in green-leaved cultivars implies that late anthocyanin genes are decisivein anthocyanin accumulation. When the two violet cultivarswere compared, the transcriptional levels of CHI and ANS werehigher in ‘Zijuan’, whereas the other genes had higher levels in‘Ziyan’ (Figure 4b). The higher expression level of the key

genes, especially F3′5′H, may account for the higher level ofdelphinidin-based pigment. In both purple cultivars, thetranscription levels of all of the anthocyanin genes decreasedsharply during leaf maturation (Figure 4b). In the third andfourth leaves, the transcription levels of these genes becamealmost undetectable. This explains why only tender tea leavesaccumulate anthocyanins and the pigment expression is quitetransient. On the basis of the above results, it is probably theactivities of F3′5′H and ANS that determine the delphinidinphenotype in Camellia plants. We then investigated thetranscription levels of these two genes in the naturally occurringhybrids of ‘Ziyan’ (Figure 5). Although no correlation ofanthocyanin phenotype and gene expression was observed, it isclear that hybrid plants that express these two genes accumulatehigh amounts of anthocyanins (>30 mg/100 g FW).

Free Amino Acids (FAAs). Major biochemical componentswere analyzed in the steamed dry sample and/or the green teasample. In contrast with the steamed dry sample, green tea wasmade after standard complex tea processing. The amount andcomposition of FAAs were analyzed by the HPLC method(Table 3). In terms of the steamed dry sample, the totalamount of FAAs in ‘Ziyan’ was 12.3% lower than that in‘Fudingdabai’ but 62.4% higher than that in ‘Zijuan’. After greentea processing, ‘Ziyan’ still contained a moderate amount ofFAAs. ‘Ziyan’ also showed higher or comparable levels of Asp,Glu, Arg, and threonine, the most four important FAAs. Asp,Glu, Arg, and threonine especially determine green tea qualitybecause they have a high coefficient of correlation (0.787−0.876) with green tea taste. These results indicate theanthocyanin accumulation in ‘Ziyan’ did not decrease theprocessed tea quality in terms of FAAs.

Polyphenols, Water Extract, Caffeine, and Catechins.Polyphenols, water extract, and caffeine were analyzed in 11green tea samples (Table 4). ‘Ziyan’ and ‘Zijuan’ had loweramounts of polyphenols and water extract but not caffeine. It islikely that the abnormal anthocyanin accumulation decreasesother metabolites’ accumulation.The catechin contents among ‘Ziyan’, ‘Zijuan’, and

‘Fudingdabai’ were compared in spring and autumn (Table5). There was no significant difference between the threecultivars in the levels of total catechins in the spring. Basically,more catechins are accumulated in the autumn; the totalcatechin level of ‘Ziyan’ was significantly lower than that of‘Zijuan’. EGC, GCG, ECG, and EGCG are the major catechincomponents, accounting for >74% in all of the samples.Moreover, we noted that the level of gallocatechin-type (G-type, GC, EGC, GCG, and EGCG) catechins was especially

Figure 3. HPLC chromatogram of the pigments extracted from freshB2L samples of ‘Ziyan’.

Table 2. Anthocyanidin Contents (mg/100 g, Means ± SD) in Fresh Tea Leaves Detected by HPLC and the pH-DifferentialMethoda

HPLC method

sample delphinidin cyanidin pelargonidin total content pH method

ZY-B2L 70.76 ± 3.42a 14.99 ± 0.69c 2.39 ± 0.33ab 88.15 ± 4.40a 52.34 ± 4.94aZJ-B2L 46.74 ± 2.60c 20.21 ± 0.88a 2.77 ± 0.43a 69.72 ± 3.83b 36.99 ± 5.21bcZY-B3L 68.90 ± 2.05a 15.82 ± 0.55c 2.25 ± 0.31abc 86.96 ± 2.60a 41.83 ± 3.71bZJ-B3L 38.96 ± 3.30d 17.90 ± 0.67b 2.05 ± 0.52abc 58.91 ± 3.60c 36.82 ± 1.91bcZY-B4L 57.86 ± 6.90b 12.57 ± 1.11d 2.62 ± 0.71a 73.05 ± 8.71b 31.85 ± 2.44cZY-B5L 49.17 ± 7.53c 9.41 ± 0.66e 1.64 ± 0.13bc 60.21 ± 8.28c 24.55 ± 2.51cZY-B6L 47.37 ± 1.18c 8.20 ± 0.13e 1.51 ± 0.19c 57.08 ± 0.11c 22.67 ± 1.91c

aB2L-B6L, one bud with 2−6 leaves; ZJ, Zijuan; ZY, Ziyan. The different letters following the numbers indicate significant differences betweendifferent samples (p < 0.05).

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higher than that of catechin-type (C-type, C, EC, CG, andECG) catechins in ‘Ziyan,’ and the ratio of C-type to G-typewas very stable despite the seasonal change of the total catechinand constituents. To verify that ‘Ziyan’ prefers to synthesize B-ring trihydroxylated catechins, we analyzed the constituents innine cultivars (Table 6). As a result, the ratio of C-type to G-type in ‘Ziyan’ was significantly lower than that of the eightother cultivars including ‘Zijuan’. The green tea (unaerated)processing did not affect the catechin constituent, whereas theblack tea (aerated) processing did (data not shown).

■ DISCUSSION

‘Ziyan’ is a new cultivar that has a more intense purple colorthan ‘Zijuan’, a widely known violet cultivar in China, under thesame cultivation conditions in Sichuan province, China. In thisstudy, we quantified the color difference between these twocultivars and showed that ‘Ziyan’ leaves are more red/purplethan ‘Zijuan’ in terms of hue value in the color wheel. In thisstudy, the hue angle (h°) value correlated negatively with theanthocyanin content. This comparison provided a simple wayto measure the amount of anthocyanin in the tea leaves. The

Figure 4. Transcription of flavonoids genes was quantified by FPKM (fragments per kilobase of transcript per million mapped reads) using theRNA-Seq data set (a) and qRT-PCR (b). (a) The transcription level in log2(FPKM) is represented by colors ranging from low (green) to high (red)in the entire pathway. (b) The relative transcription level modified by CsRuBP expression is represented by colors ranging from low (green) to high(red) for each gene. F3′5′H1 and F3′5′H2 were amplified by the same primers in qRT-PCR, as were ANR1 and ANR2. Each datum is the mean ofthree biological repeats. FD, ‘Fudingdabai’; ZJ, ‘Zijuan’; ZY, ‘Ziyan’; B6L1-B6L5, 1st−5th leaves of B6L shoot.

Figure 5. Transcription levels of F3′5′H and ANS genes in hybrid plants. CsRuBP was used to normalize the expression of the genes under identicalconditions. Vertical bars show the standard error of the means of three biological repeats.

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total amount of the three anthocyanidins in two-leaf shoots of‘Ziyan’ was 88.15 mg/100 g FW. This number was comparablein anthocyanin content with anthocyanin-rich vegetables andfruits, such as grapes (27−120 mg/100 g FW) and red radishes(100 mg/100 g FW).26 These results indicated that ‘Ziyan’ teacan be a major source of anthocyanin intake. Previous studieshave reported the total anthocyanin content in ‘Zijuan’ greentea to be 29.14 mg/g6 and the total anthocyanin content in‘Sunrouge’ dry leaves to be 0.86−2.17 mg/g.7 It is difficult tocompare the anthocyanin contents between these papersbecause the plant cultivation conditions, sample preparation,and anthocyanin detection methods are different. However,‘Ziyan’ appeared more violet than the other cultivars and

accumulated much more anthocyanin than ‘Zijuan’ in ourstudy.The higher total amount of anthocyanin in ‘Ziyan’ compared

with ‘Zijuan’ was totally due to delphinidin-related pigments. Itis known that anthocyanin molecules are complex, but they canbe divided into several groups according to the types of theanthocyanidin aglycones. Anthocyanidins can result from acid-mediated anthocyanin hydrolysis.24 We used this strategy toidentify the composition of anthocyanins by the HPLCmethod. Delphinidin, cyanidin, and pelargonidin were identi-fied using five authentic anthocyanidin standards (Table 2).Delphinidin was the major pigment, and it was accumulatedmuch more in ‘Ziyan’ than in ‘Zijuan’. Petunidin was notassayed in this study. Nevertheless, petunidin is probably notaccumulated in ‘Ziyan’ because its peak in a reasonable eluentorder of standards did not appear (Figure 3). The anthocyanin

Table 3. Concentrations (mg/g) of Free Amino Acidsa

green tea steamed dry sample

component ZY ZJ FD ZY ZJ FD

Asp 8.47a 7.08b 4.43c 6.39a 4.17c 5.22bGlu 5.91b 3.95c 10.31a 8.84a 3.85c 8.23bAsn 2.98b 0.96c 3.31a 0.65a 3.29b 0.51aSer 2.32a 0.65c 1.81b 1.23b 1.22b 1.38aGln 0.37b 0.00c 0.73a 0.41b 0.35c 0.60aHis 5.70a 3.27b 0.00c 3.98b 2.99c 7.06aArg 1.23b 0.73c 7.27a 3.41b 1.67c 11.93aThr 1.03a 1.04a 0.89b 0.88a 0.73b 0.60cAla 0.88c 1.26a 1.04b 0.94a 0.50c 0.86bGABA 0.79b 0.37c 1.37a 0.62b 0.82a 0.51cPro 0.90b 0.74c 1.45a 0.45b 0.81a 0.37cthreonine 30.67b 26.36c 36.68a 42.61a 20.49b 43.84aTyr 0.42b 0.56a 0.57a 0.27b 0.58a 0.19cVal 0.94a 0.86b 0.78c 0.26b 0.79a 0.17cLys 0.95b 0.75c 1.18a 0.40b 1.25a 0.40bIle 0.71a 0.53c 0.58b 0.22b 0.45a 0.14cLeu 0.81b 0.57c 0.89a 0.29b 0.76a 0.17cPhe 0.81a 0.83a 0.84a 0.25b 0.99a 0.20cMet 1.75b 1.85b 4.90a 4.81b 1.66c 5.24a

total 67.64b 52.37c 79.04a 76.88b 47.35c 87.62aaZY, Ziyan; ZJ, Zijuan; FD, Fudingdabai. The different letters following the numbers indicate significant differences between the cultivars in the samesample type (p < 0.05).

Table 4. Major Biochemical Components of Tea in GreenTea Samplesa

tea sample polyphenols (%) water extract (%) caffeine (%)

tea 1 16.71b 48.97abc 4.16bctea 2 16.73b 49.16bcd 4.21bctea 3 16.82b 50.69a 4.31btea 4 16.54b 49.27ab 4.08cdtea 5 13.17d 45.92abcd 3.70etea 6 13.34d 47.33abc 3.78etea 7 15.12c 47.84abc 3.81etea 8 15.27c 48.86abc 4.01dFD 19.25a 45.72abcd 4.55aZJ 11.03e 38.64d 3.72eZY 10.88e 40.35cd 3.97d

CV (%) 17.47 8.22 6.66aCV, coefficient of variance. FD, Fudingdabai; ZJ, Zijuan; ZY, Ziyan.The different letters following the numbers indicate significantdifferences between the cultivars (p < 0.05).

Table 5. Contents (%) of Catechins in Steamed DrySamplesa

2014 spring 2014 autumn

component ZY ZJ FD ZY ZJ FD

C 0.53b 0.84a 0.19c 0.12b 0.33a 0.33aGC 0.30b 0.37a 0.34ab 0.09c 0.24a 0.17bEC 0.50c 0.87a 0.69b 0.33c 1.09a 0.75bEGC 0.71b 0.76ab 0.86a 1.22b 1.63a 1.28bCG 0.43a 0.29b 0.15c 0.61a 0.56b 0.42cGCG 1.54a 0.99b 1.65a 0.16b 0.33a 0.08cECG 1.15b 1.79a 1.86a 1.39b 2.77a 1.68bEGCG 4.60a 3.32b 4.39a 7.56a 5.37c 6.20b

total 9.76a 9.23a 10.13a 11.48b 12.32a 10.91baZY, Ziyan; ZJ, Zijuan; FD, Fudingdabai. The different lettersfollowing the numbers indicate significant differences between thethree cultivars in one season (p < 0.05).

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components of ‘Zijuan’ in this study corresponded well withprevious papers. Using the HPLC-ESI-MS method, a total of 14anthocyanin molecules have been detected in ‘Zijuan’ fresh leafextract.27 However, all of these pigments can be reduced todelphinidin, cyanidin, or pelargonidin. For the Japanese‘Sunrouge’ cultivar, only delphinidin- and cyanidin-relatedanthocyanins have been detected.9 In contrast with theseAsian cultivars, a Kenyan violet tea TRFK306 accumulatespeonidin and malvidin in addition to the three pigmentsdetected in this study.8 In TRFK306, malvidin (rather thandelphinidin) is the major component, and the amount ofpelargonidin is considerably higher compared with otherpigments. This large difference in anthocyanin constituentsbetween the Asian violet cultivars and the African cultivarsuggests a geographical influence. ‘Ziyan’ and ‘Zijuan’ are bothfound in southwestern China, and these species are probablymore primitive than the Kenyan violet tea. Li et al.20 analyzedthe anthocyanin composition of red flower petals in 33 wildspecies using 25 authentic anthocyanins, and their chemicaltaxonomic analysis suggests that southwestern China is thepresumed center of origin of the red-flowered Camellia species.When wild plants are cultivated, mutation of the anthocyanincomposition is suggested to be in the following order:delphinidin → cyanidin → pelargonidin. Delphinidin-typeanthocyanins versus other anthocyanins display a violet color;however, high concentrations of cyanidin-type anthocyaninsalso make plant organs appear violet to some extent. Therefore,utilization of the wild germplasm in southwestern China ismore effective in developing real violet tea cultivars. Our studysupports such a hypothesis.‘Ziyan’ prefers to accumulate G-type catechins, and the

allocation of the metabolic flow for G-type and C-typecatechins was stable between seasons. Such a preference withrespect to catechins is consistent with anthocyanins.Anthocyanin genes are involved with not only anthocyaninbiosynthesis but also catechin biosynthesis in tea leaves. Thetranscription of almost all of the anthocyanin-related genes inthe ‘Fudingdabai’ green cultivar corroborates this. The F3′5′Hgene is necessary for delphinidin and G-type catechins but notfor cyanidin and C-type catechins. The higher proportion ofdelphinidin and G-type catechins in ‘Ziyan’ is likely to bedependent on the higher transcription level of F3′5′H (Figure5).Because anthocyanins and catechins share their biosynthesis

pathway, there is potential competition between these two

compounds. The total amount of catechins, including C, GC,CG, GCG, EC, EG, ECG’ and EGCG, in violet tea cultivars wassignificantly lower than in ordinary cultivars (Table 6). Theaccumulation of a high amount of anthocyanins in ‘Ziyan’ didnot reduce the catechin accumulation compared with ‘Zijuan,’probably due to the higher transcription level of CHS, whichcontrols the metabolic flow as the first key enzyme of theflavonoid pathway. In addition to catechins, the water extractand polyphenols were also decreased in the violet cultivars.These results indicate competition between anthocyanins andother metabolites. Interestingly, Kerio et al.8,10 arrived at acontrary conclusion in the study of Kenyan purple tea. Teasmade from Kenyan purple-leaved cultivars have similar levels ofcatechins (including only C, EC, EG, ECG, and EGCG) andpolyphenols and lower caffeine, but when compared amongpurple-leaved cultivars there is a negative correlation betweenanthocyanins and total catechins. We obtained results differentfrom these previous papers, which may be due to thedifferences in the germplasm. Therefore, a wide comparisonbetween natural, anthocyanin-rich tea germplasms under thesame cultivation conditions is necessary to thoroughly revealthe effect of abnormal anthocyanin accumulation on othermajor tea components.In our study, we quantified the color presentation, profiled

the anthocyanin accumulation during leaf development, andcharacterized the anthocyanin components of ‘Ziyan’, the dark-purple-leaved tea cultivar. Full-length genes in the flavonoidpathway were isolated and used to quantify their expressionlevels and characterize the expression patterns. These resultsprovided a better understanding of the anthocyanin phenotypeof ‘Ziyan’. Moreover, we analyzed catechins and other major teabiochemical components in a small number of tea cultivars andthen compared violet-leaved cultivars with ordinary, green-leaved cultivars. These studies demonstrated the effect of highamounts of anthocyanin on the metabolite allocation in tealeaves.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jafc.5b04036.

Identification of full-length flavonoid pathway genes andmultiple sequence alignment (PDF)

Table 6. Analysis of C- and G-Type Catechins in Cultivarsa

green tea steamed dry sample

cultivar total catechin (%) C/Gb total catechin (%) C/Gb leaf color

Zijuan 8.70e 0.664a 9.23c 0.696a purpleChuannong no. 2 10.74cd 0.489b 11.39b 0.479b greenChuannong no. 6 13.32b 0.424c 13.29a 0.423c greenHuangjingui 16.07a 0.415d 14.08a 0.411cd greenFudingdabai 9.62de 0.429cd 10.13c 0.400de yellow-whiteTieguanyin 15.20a 0.459bc 12.21b 0.390e greenJinguanyin 13.40b 0.356e 11.88b 0.389e greenChuannong no. 9 10.94c 0.350e 11.79b 0.384e greenZiyan 10.41cd 0.242f 9.76c 0.364f dark purple

CV (%) 22.58 27.11 13.94 23.40aCV, coefficient of variance. The different letters following the numbers indicate significant differences between the cultivars (p < 0.05). bC/G, theratio of C-type catechins with G-type catechins.

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■ AUTHOR INFORMATIONCorresponding Author*(Q.T.) Phone: +86-28-8629-1748. E-mail: tangqi2008@126.com.Author ContributionsThis work was supported by the Sichuan Provincial KeyTechnology R&D Program (Grant 2014NZ0105).NotesThe authors declare no competing financial interest.

■ ABBREVIATIONS USEDB2L, one bud and two leaves; FAA, free amino acid; FW, freshweight; quantitative real time (qRT)-PCR; C, catechin; GC,gallocatechin; EC, epicatechin; EGC, epigallocatechin; CG,catechin gallate; GCG, gallocatechin gallate; ECG, epicatechingallate; EGCG, epigallocatechin gallate

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