Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in

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Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in Ripening Red Pepper (Capsicum annuum var. conoides) Fruits Qiang Wang, Tian-Jun Cao, Hui Zheng, Chang-Fang Zhou, Zhong Wang, Ran Wang, and Shan Lu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Journal of Agricultural and Food Chemistry

Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in Ripening Red Pepper (Capsicum annuum var. conoides) Fruits

Qiang Wang,† Tian-Jun Cao,† Hui Zheng,† Chang-Fang Zhou,† Zhong Wang‡, Ran Wang*,‡ and Shan Lu*,†

†

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,

Nanjing University, Nanjing 210023, China ‡

Zhengzhou Tobacco Research Institute, Zhengzhou 450001, China

*

Corresponding

authors:

Shan

Lu,

[email protected];

Ran

Wang,

[email protected]

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

Carotenoids are essential phytonutrients for the human body. Higher plants usually

3

synthesize and accumulate carotenoids in their leaves, flowers, and fruits. Most

4

carotenoids have either two β-rings on both ends, or β- and ε-rings separately on two

5

ends of their molecules, and are synthesized from the acyclic lycopene as the precursor.

6

Lycopene β- and ε-cyclases (LCYB and LCYE, respectively), catalyze the β- and ε-

7

cyclization of lycopene, respectively, and regulate the metabolic flux from lycopene to

8

its downstream β,β- (by LCYB alone) and β,ε- (by LCYE and LCYB) branches. In this

9

study, we identified and characterized genes for two LCYBs (CaLCYB1 and

10

CaLCYB2), one LCYE (CaLCYE1), and a capsanthin/capsorubin synthase (CaCCS1)

11

which is also able to β- cyclize lycopene, from the red pepper (Capsicum annuum var.

12

conoides) genome. By quantifying transcript abundances of these genes and contents

13

of different carotenoid components in ripening fruits, we observed a correlation

14

between the induction of both CaLCYBs and the accumulation of carotenoids of the

15

β,β- branch during ripening. Although capsanthin was accumulated in ripen fruits, our

16

quantification demonstrated a strong induction of CaCCS1 at the breaker stage, together

17

with the simultaneous repression of CaLCYE1 and the decrease of lutein content,

18

suggesting the involvement of CaCCS1 in competing against CaLCYE1 for

19

synthesizing carotenoids of the β,β- branch. Our results provide important information

20

for future metabolic engineering studies to manipulate carotenoid biosynthesis and

21

accumulation in fruits.

22 23

KEYWORDS: Capsicum annuum, carotenoid, fruit, lycopene, lycopene cyclase,

24

metabolic flux, red pepper, ripen 2


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Introduction

26

Carotenoids are not only essential pigments for photosynthesis and photoprotection in

27

plants, but also essential phytonutrients for the human body.1 In higher plants,

28

carotenoids are exclusively synthesized in plastids. Starting from pyruvate and

29

glyceraldehyde 3-phosphate, the methylerythritol 4-phosphate (MEP) pathway

30

simultaneously produces the C5 isopentenyl diphosphate (IPP) and its isomer

31

dimethylallyl diphosphate (DMAPP).2 Three molecules of IPP are condensed with one

32

molecule of DMAPP into the C20 geranylgeranyl diphosphate (GGPP), which is shared

33

by the biosynthetic pathways for diterpenoids, carotenoids, and side chains of

34

tocopherols and chlorophylls, by GGPP synthase (GGPPS).3 Phytoene synthase (PSY)

35

catalyzes the condensation of two molecules of GGPP into the C40 phytoene and directs

36

the metabolic flux into carotenoid biosynthesis. Phytoene is then desaturated and

37

isomerized into lycopene (ψ,ψ-carotene) with an acyclic structure (Figure 1). The two

38

open ends of lycopene can be both β- cyclized, or be β- and ε- cyclized separately. Only

39

in very limited plant species, can lycopene be ε- cyclized on its both ends, such as the

40

biosynthesis of lactucaxanthin (ε,ε-carotene-3,3’-diol) in lettuce (Lactuca sativa).4

41

Carotenoids with two β-rings include mainly β-carotene (β,β-carotene) and its

42

oxygenated derivatives such as zeaxanthin, antheraxanthin, and violaxanthin, whereas

43

those with β- and ε-rings include mainly lutein (β,ε-carotene-3,3’-diol). In plants,

44

carotenoids of the β,β- and β,ε- branches beyond lycopene are involved in different

45

physiological and metabolic processes. For example, although β-carotene and lutein are

46

both major carotenoids for photosystems and light-harvesting complexes, they localize

47

in the inner and the peripheral light-harvesting antennae, respectively.1,5 Moreover, the

48

photoprotective role of lutein is mainly in the deactivation of triplet chlorophylls, while

49

that of zeaxanthin, antheraxanthin and violaxanthin is in the deactivation of excited 3



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singlet chlorophylls and non-photochemical quenching.6 In addition, plant hormones

51

abscisic acid and strigolactones are synthesized from β-carotene.7

52

Carotenoids of the β,β- and β,ε- branches are not evenly synthesized. The

53

cyclization of lycopene is catalyzed by lycopene β- and ε-cyclases (LCYB and LCYE,

54

respectively). Members of the LCYB and LCYE subfamilies share relatively high

55

sequence similarities but with different catalytic properties. In general, LCYEs can only

56

utilize lycopene as a substrate to cyclize its one open end, producing δ-carotene (ε,ψ-

57

carotene). One rare exception is the LCYE from lettuce that is able to cyclize lycopene

58

on both ends to produce ε-carotene (ε,ε-carotene).4 LCYBs can cyclize lycopene on its

59

one or both open ends to produce γ- (β,ψ-carotene) or β-carotene, respectively, or

60

cyclize the open end of δ-carotene to produce α-carotene (β,ε-carotene) (Figure 1). The

61

expression of LCYB and LCYE has been demonstrated to regulate the bifurcation of the

62

metabolic flux from lycopene to its downstream β,β- and β,ε- branches.8 For examples,

63

in tomato (Solanum lycopersicum) fruits that accumulate lycopene as the predominant

64

carotenoid constituent, the overexpression of either its own LCYB or exogenous LCYBs

65

from Pantoea stewartii, daffodil (Narcissus pseudonarcissus) and other plants all

66

resulted in a successful conversion from lycopene into β-carotene in transgenic fruits.9-

67

12

68

tubers and rapeseed seeds.13-14 However, when LCYB was silenced, contents of both β-

69

carotene and lutein were found to be down-regulated in tomato.15

The silencing of LCYE was found to increase the contents of β-carotene in potato

70

In pepper, carotenoids with both β,β- and β,ε- structures are synthesized in leaves,

71

with lutein as one of the major species.16 In fruits, β-carotene and zeaxanthin, which

72

both have the β,β-structure, are synthesized during the entire ripening process, whereas

73

capsanthin of the β,β- branch is only synthesized in mature fruits, and lutein of the β,ε-

74

branch is usually only found in immature fruits.16-17 Variations in carotenoid 4


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constituents and their contents during ripening suggest a variable capability in

76

converting lycopene to its two downstream branches.16-17 The cloning of genes for

77

GGPPS and PSY for the biosynthesis of lycopene, as well as the gene for carotene β-

78

hydroxylase (CHYB) downstream of lycopene, from red pepper have previously been

79

reported.18 However, for the regulation of lycopene allocation, only one LCY gene has

80

been cloned from red pepper, and its cognate enzyme showed the capability of cyclizing

81

lycopene to β-carotene, as a typical LCYB.19 Moreover, a capsanthin/capsorubin

82

synthase (CCS) that catalyzes the biosynthesis of capsanthin and capsorubin from

83

antheraxanthin and violaxanthin, respectively, was also found to possess LCYB activity

84

in bacterial pigment complementation assay.20-22 LCYE, which is essential for the

85

biosynthesis of lutein, has not been identified from red pepper yet. It is still unclear if

86

red pepper has other LCY homologues and how these cyclases are regulated in ripening

87

pepper fruits.

88

Here, we report the cloning and functional characterization of two LCYBs, one

89

LCYE and one CCS, which probably represent a full repertoire of the lycopene

90

cyclization enzymes in red pepper. Our analysis demonstrated an antagonistic

91

accumulation of carotenoids of the two branches, manipulated by the repression of

92

LCYE and the induction of both LCYB and CCS.

93 94

Materials and methods

95

Plant Material and Growth Conditions

96

Seeds of red pepper (Capsicum annuum var. conoides) were purchased from Duoyouqi

97

Technology Trading (Beijing, China) and grown in the greenhouse (16 h light / 8 h dark,

98

28 ± 2 °C). The ripening of fruits was divided into six stages as previously described, 5



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that is, immature green (IG), mature green (MG), breaker (B), first immature red (FIR),

100

second immature red (SIR) and mature red (MR), at ca. 15, 30, 35, 38, 43 and 50 days

101

post anthesis (DPA), respectively.23-24 Pericarps of fruits at different ripening stages

102

were collected, immediately frozen in liquid nitrogen, and stored at -80 °C till further

103

use.

104 105

Pigment Analysis

106

Pigments were extracted from red pepper fruits according to previous reports.25 Prior

107

to the extraction, trans-β-apo-8′-carotenal (Sigma-Aldrich, St. Louis, MO, United

108

States) (1 mg mL−1 dissolved in ethyl acetate) was added as an internal standard.26

109

A Waters 2695 separation module equipped with a 2998 photodiode array detector

110

(Waters, Milford, MA, United States) was used for high-performance liquid

111

chromatography (HPLC) analysis on a reverse-phase Spherisorb ODS2 column (5 μm,

112

4.6 × 250 mm) (Waters) using a 37-min gradient of ethyl acetate (0-100%) in

113

acetonitrile-water-triethylamine (9:1:0.01) at a flow rate of 1 mL min−1 at 30 °C.27 The

114

elution profile was compared with previous reports under similar chromatographic

115

conditions, and the ultraviolet/visible spectrum of each constituent was also compared

116

with published authentic data to further confirm the peak identity.28 Content of each

117

carotenoid constituent was calibrated and calculated using its corresponding molar

118

extinction coefficient. At least three replicates were performed for each sample.

119 120

Homologue Identification and Sequence Analysis

121

Sequences of the red pepper LCY homologue genes were obtained by searching the 6


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pepper genome (https://solgenomics.net/) using the tblastn algorithm. Sequences of

123

functionally characterized AtLCYB (GenBank Accession No. AAB53337) and

124

AtLCYE (AAB53336) from Arabidopsis thaliana, and of SlLCYB (EF650013) and

125

SlLCYE (Y14387) from tomato were downloaded from GenBank and used as queries.

126

Because CCS was also reported to possess LCYB activity, sequences of the previously

127

identified CCSs from red pepper (Q42435) and Lilium lancifolium (JF304153) were

128

also used as queries.29 Sequence alignment was performed using ClustalW, and the

129

Maximum-likelihood phylogenetic tree was constructed with 1000 bootstrap replicates

130

using MEGA 7.30 Prediction of the subcellular localization of LCY homologues was

131

performed using ChloroP.31

132 133

Gene Cloning and Expression Analysis

134

Total RNA was isolated using RNAiso Plus Reagent (TaKaRa, Shiga, Japan) and

135

reverse transcribed using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa),

136

according to manufacturer’s instructions. Full-length open reading frames (ORFs) of

137

LCY homologues were amplified using the first strand cDNA as a template, cloned in

138

pMD19 (TaKaRa) by the in-fusion technology (TaKaRa), and sequenced by GenScript

139

(Nanjing, China). High-fidelity PrimeSTAR DNA polymerase (TaKaRa) was used

140

throughout this study for DNA amplification. Transcript abundance of each gene was

141

determined by quantitative real-time PCR (qRT-PCR) in a Thermal Cycler Dice Real

142

Time System TP800 (TaKaRa) using a ChamQ SYBR qRT-PCR Master Mix (Vazyme,

143

Nanjing, China), following the manufacturer’s manuals, and calculated using the

144

comparative CT method.32 A 20-μL reaction system contained 2 μL cDNA template, 0.4

145

μL of each forward and reverse primers (10 μmol L−1), 7.2 μL water and 10 μL qRT7



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PCR Master Mix. Standard cycling condition was 95 °C for 5 min, 40 cycles of 95 °C

147

for 10 sec and 60 °C for 30 sec, followed by 95 °C for 15 sec. Transcript abundance of

148

β-tubulin was determined as a reference.33 At least three biological replicates, each with

149

three replicates were analyzed for each sample. All primers used in this study are listed

150

in Table S1 of the Supporting Information.

151 152

Subcellular Localization Analysis

153

ORF of each LCY homologue gene was cloned into pCAMBIA1300 (CAMBIA,

154

Canberra, Australia) by the in-fusion technology for subcellular localization assay. The

155

expression cassette contains sequentially the enhanced Cauliflower Mosaic Virus

156

(CaMV) 35S promoter, synthetic 5’ and 3’ untranslated regions of Cowpea Mosaic

157

Virus RNA-2

158

5’-end of the gene for mCherry, and the Heat Shock Protein (HSP) terminator from A.

159

thaliana.34-35 For the negative control, the fragment for the LCY-mCherry fusion protein

160

was substituted with the gene for enhanced yellow fluorescent protein (EYFP) (Figure

161

S1 of the Supporting Information). Each construct was introduced into Agrobacterium

162

tumefaciens strain GV3101. Leaves of Nicotiana benthamiana were infiltrated with a

163

mixture of equal amounts of Agrobacterium cells harboring the constructs for transient

164

expression of individual LCY-mCherry protein and EYFP together.36

flanking the coding region of the LCY homologue fused in frame to the

165

A FluoView FV1000 (Olympus, Tokyo, Japan) laser scanning confocal microscopy

166

system was used for fluorescence observation. The mCherry fluorescent was excited

167

with 543 nm laser and recorded from 580 to 620 nm. The EYFP fluorescent was excited

168

with 488 nm laser and the emitted light was recorded from 500 to 530 nm. For

169

chlorophyll auto-fluorescence observation, 543 nm laser excitation and 680 to 720 nm 8


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recording range were used.27

171 172

Functional Characterization of Red Pepper LCYs

173

The pAC-LYC plasmid that carries genes for GGPP synthase (CrtE), PSY (CrtB) and

174

phytoene desaturase (CrtI) to facilitate the biosynthesis of lycopene in Escherichia coli

175

was a gift from Dr. Cunningham.37 For pigment complementation assay, full-length

176

ORF of each gene was amplified and cloned into pMAL-C5X (NEB, Ipswich, MA,

177

United States) by the in-fusion technology for prokaryotic expression. AtLCYB and

178

AtLCYE were used as positive controls, and the empty pMAL-C5X vector was used as

179

a negative control. Each of the expression vectors was cotransformed into E. coli

180

TOP10 cells with pAC-LYC. Transformed colonies were screened on Luria-Bertani

181

(LB) plates containing 34 μg mL−1 chloramphenicol and 100 μg mL−1 carbenicillin. To

182

study the collaboration of LCYB and LCYE in cyclizing lycopene, we substituted the

183

coding region for β-lactamase (for carbenicillin resistance) in pMAL-C5X with that for

184

aminoglycoside phosphotransferase (for kanamycin resistance) to generate pMAL-

185

C5X-Kan. LCYB and LCYE were cloned into pMAL-C5X and pMAL-C5X-Kan,

186

respectively, and simultaneously transformed into E. coli TOP10 cells harboring pAC-

187

LYC. Transformed colonies were screened on LB plates containing 34 μg mL−1

188

chloramphenicol, 100 μg mL−1 carbenicillin and 50 μg mL−1 kanamycin. Cell

189

inoculation, pigment extraction and quantification were performed as previously

190

reported.38

191 192

Results

193

Variation in Carotenoid Profile during Red Pepper Fruit Ripening 9



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From our HPLC analysis, no lycopene was detected in fruits at any ripening stage,

195

demonstrating an efficient cyclization of lycopene to carotenoids of the two

196

downstream branches (Figure 2). Fruits at immature green (IG) and mature green (MG)

197

stages accumulated carotenoids of both β,β- and β,ε- branches, with neoxanthin (26.76%

198

- 29.34%), violaxanthin (28.57% - 34.21%), β-carotene (11.16% - 11.70%) and lutein

199

(27.87% - 30.40%) as major species (Figure 2 A,B). From the breaker stage, fruits

200

began to accumulate more carotenoids of the β,β- branch, such as capsanthin (38.48%

201

- 50.21%), zeaxanthin (19.85% - 26.33%) and violaxanthin (10.33% - 16.41%) (Figure

202

2 C-F).

203

Although different combinations of carotenoids sharing the β,β- structure were

204

identified in fruits throughout the ripening process, lutein was the only constituent of

205

the β,ε- branch in fruits. In IG fruits, the content of lutein was 4.89 μg g−1 fresh weight

206

(FW), whereas that of total carotenoids in the β,β- branch was 12.65 μg g−1 FW (Figure

207

3 A). With the ripening of fruits, the amounts of lutein and total carotenoids of the β,β-

208

branch increased to 7.68 and 18.53 μg g−1 FW, respectively, at the MG stage (Figure 3

209

A). A drastic change of the carotenoid profile was observed at the breaker stage (Figure

210

2 C). While the total amount of carotenoids of the β,β- branch raised rapidly to 116.80

211

μg g−1 FW, which was 6.3-fold of its MG stage level, the content of lutein was lowered

212

to 2.84 μg g−1 FW, about only 0.37-fold of its MG stage level (Figure 3 A). Beyond the

213

breaker stage, carotenoids of the β,β- branch further accumulated to a final level of

214

318.55 μg g−1 FW at the MR stage, whereas lutein was only detectable at trace amounts

215

at the breaker and the first immature stages (FIR) stages (Figure 2 C-F).

216

Because it costs one molecule of lycopene to synthesize each molecule of the

217

carotenoid constituents in β,β- or β,ε-branches, we not only quantified the contents of

218

different carotenoids, but also compared their molar values. The combined molar value 10


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of all carotenoids in each branch, therefore, represents the metabolic flux from lycopene

220

into the corresponding branch, and the ratio between the molar values of the two

221

branches indicates the allocation of the metabolic flux. From our calculation, the β,ε-

222

/β,β- ratios were 0.41 and 0.55 at the IG and MG stages, respectively, showing that

223

more lycopene (from 29.1% to 35.5%) was directed to the β,ε- branch (lutein) during

224

the transition from IG to MG stage (Figure 3 B). However, this ratio immediately

225

decreased to 0.03 at the breaker stage, and then close to zero at the FIR stage,

226

demonstrating an overwhelming activity of β,β- cyclization.

227 228

Gene Cloning and Sequence Analysis

229

To figure out the molecular mechanism underlying the variation in carotenoid profile

230

during fruit ripening, we searched the red pepper genome for all sequences sharing

231

significant similarities with known LCY genes. Our homologous BLAST identified 8

232

LCY homologues, together with one CCS (Table 1). For five of the LCY homologues,

233

their ORFs were found to be very short (429-603 bp), and their transcripts could not be

234

detected by qRT-PCR in fruits at any of the ripening stages. Therefore, we excluded

235

these 5 homologues as pseudogenes from our further studies.

236

We then compared the deduced amino acid sequences of the four red pepper

237

homologues with LCYB and LCYE from Arabidopsis and tomato, LCYE from lettuce,

238

and CCS from Lilium lancifolium. Our alignment revealed that all these homologues

239

share the conserved cyclase motifs, transmembrane helixes, and the dinucleotide-

240

binding domain, supporting that there are putative members of the LCY family (Figure

241

4).22,39-40 We further performed phylogenetic analysis of these homologues and more

242

previously identified LCYs and CCSs. Two of the three LCY homologues 11



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(Capana05g000023 and Capana10g002320) were clustered with known LCYBs,

244

whereas the rest LCY homologue (Capana09g000177) belonged to the LCYE clade

245

(Figure 5). The CCS homologue (Capana06g000615) stayed with the characterized

246

CCS from L. lancifolium as an independent clan within the LCYB clade (Figure 5). The

247

overall sequence identities inside the LCYB and LCYE clades were above 31.8% and

248

42.8%, respectively, whereas CaLCYE1 showed 32.4% and 32.5% sequence identities

249

with CaLCYB1 and CaLCYB2, respectively. Therefore, we named Capana05g000023,

250

Capana10g002320, and Capana09g000177 as CaLCYB1, CaLCYB2, and CaLCYE1,

251

respectively. The amino acid sequence of Capana06g000615 was the same with the

252

previously reported red pepper CCS (Q42435),29 and has higher sequence identities

253

with CaLCYB1 and CaLCYB2 (52.1% and 52.0%, respectively) than with CaLCYE1

254

(32.0%). We named it as CaCCS1 in this study.

255 256

Subcellular Location of CaLCYs

257

In higher plants, carotenoids are exclusively synthesized in plastids. Our online analysis

258

using ChloroP suggested that CaLCYB1, CaLCYB2, CaLCYE1, and CaCCS1 all

259

localized in plastids. To verify this prediction, we fused each protein to the N-terminal

260

of mCherry and transiently expressed the fusion proteins in tobacco leaves. The

261

mCherry signals of all fusion proteins merged with chlorophyll autofluorescence

262

perfectly, and were not overlapped with the cytosolic EYFP signals from the negative

263

controls, confirming the plastidic localization of all these proteins (Figure 6).

264 265

Functional Characterization of CaLCYs

266

To determine the catalytic properties of the putative CaLCYs, a bacterial pigment 12


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complementation system was used.37 The E. coli cells harboring the plasmid pAC-LCY

268

was able to produce lycopene, which facilitates our qualitative assessment of CaLCYs

269

(Figure 7 E).4,38 By HPLC analysis, β-carotene was detected in the assays with

270

CaLCYB1, CaLCYB2, or AtLCYB (as a positive control) (Figure 7 A). With CaLCYE1,

271

we identified the production of both monocyclic δ-carotene and bicyclic ε-carotene

272

(Figure 7 C). These results proved our categorization of these three proteins as two

273

LCYBs and one LCYE by phylogenetic analysis (Figure 5). However, different from

274

AtLCYE that produces predominantly δ-carotene in the complementation assay,

275

CaLCYE1 produces slightly more ε-carotene than δ-carotene (Figure 7 A, C). Our assay

276

with CaCCS1 also resulted in an accumulation of β-carotene, confirming the previous

277

report that this enzyme was able to β-cyclize lycopene (Figure 7 B). Neither δ- nor ε-

278

carotene was detected as a product of CaCCS1 (Figure 7 B). When CaLCYB1 or

279

CaLCYB2 was coexpressed with CaLCYE1 in E. coli cells harboring pAC-LYC, a blend

280

of δ-，ε-，α- and β-carotenes was detected in the products (Figure 7 D).

281 282

Expression Patterns of CaLCYs and CaCCS1

283

We then determined the transcript abundances of CaLCYs and CaCCS1 in ripening

284

fruits. Both CaLCYB1 and CaLCYB2 had higher expression levels than CaLCYE1 in

285

any of the ripening stages (Figure 8 A, B). Although CaLCYB1 and CaLCYB2 showed

286

similar variations in their gene expression during ripening, CaLCYB1 was expressed at

287

higher levels than CaLCYB2 after the initial IG stage (Figure 8 A). Different from

288

CaLCYE1 of which the transcript abundance started to decline from the breaker stage,

289

both CaLCYB genes reached their peak values until the FIR stage (Figure 8 A, B).

290

Transcripts of CaCCS1 were barely detectable in green fruits. However, its highest 13



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expression level was found immediately at the breaker stage, when fruits started turning

292

red (Figure 8 C).

293 294

Discussion

295

The variation in pigment profile is usually a distinct characteristic of ripening fruits.41-

296

43

297

fruits, red pepper accumulates a group of cyclized carotenoids, especially capsanthin,

298

in its fruits. This makes red pepper an ideal organism for studying the regulation of

299

carotenoid biosynthesis at the lycopene branching point and beyond during fruit

300

ripening.

Different from tomato that accumulates lycopene as the major carotenoid in ripen

301

The availability of a full genome sequence enables the elucidation of detailed

302

contributions of enzymes and their homologues in metabolic regulation.3,24,44-45 In

303

addition to CaLCYB1 and CaCCS1 that have been previously reported,19-21 our blast

304

search of the red pepper genome identified two novel homologue genes encoding

305

CaLCYB2 and CaLCYE1. These four enzymes might account for all the cyclization

306

process of lycopene in red pepper.

307

From our results, both carotenoid profile and gene expression pattern showed

308

distinct changes at the breaker stage. When the content of lutein was lowered to 0.37-

309

fold of its MG level, the combined content of all carotenoids of the β,β- branch

310

increased to 6.3-fold of the corresponding MG level. This variation agrees with the

311

simultaneous down-regulation of CaLCYE1 and up-regulation of both CaLCYBs and

312

CaCCS1. The possible involvement of CaCCS1 in this regulation is supported by the

313

fact that, although CCSs are able to catalyze the reactions from zeaxanthin or

314

violaxanthin to capsanthin or capsorubin, respectively, they still retain the enzymatic 14


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activity of β-cyclizing lycopene. These two reactions indeed share a similar catalytic

316

mechanism.46-47 Moreover, the overexpression of CaCCS1 in tobacco leaves was found

317

to enhance the accumulation of β,β- branch carotenoids, and its silencing in detached

318

pepper fruits resulted in a decrease in carotenoid content.18,48 These transgenic studies

319

both demonstrated that CaCCS1 β-cyclizes lycopene in planta, in addition to its unique

320

function in the synthesis of capsanthin/capsorubin in ripe fruits. Our phylogenetic

321

analysis also indicates the specialization of CCSs from other LCYBs in land plants,

322

after the divergence between LCYB and LCYE subfamilies. Although the detailed

323

contributions of CaLCYBs and CaCCS1 are largely unknown, the drastic induction of

324

CaCCS1 expression to its peak level, which was much higher than those of both

325

CaLCYBs, at the breaker stage suggests its overwhelming involvement in competing

326

against CaLCYE1 for synthesizing carotenoids of the β,β- branch.

327

Although lutein is a major carotenoid species in leaves and fruits at early ripening

328

stages, to the best of our knowledge, this is the first report of the cloning and

329

characterization of an LCYE in red pepper for its production. However, in our pigment

330

complementation assay, CaLCYE1 demonstrated its activity of cyclizing both open

331

ends of lycopene to produce ε-carotene in E. coli. This was similar to the results using

332

LCYEs from Arabidopsis, rice, and maize, while no carotenoids with ε,ε- structure were

333

identified in these plants.40,49-51 It is possible that the cytosolic environment of E. coli

334

for the complementation assay is different from the plastidic environment in planta in

335

the availability of protein folding machinery, cofactors, membrane systems, etc.40,49,51

336

Moreover, a 6-amino acid motif in the LCYE from lettuce was demonstrated to

337

determine the capabilities of LCYE to form dimers and to cyclize on one or two ends

338

of lycopene, suggesting that dimerization might be a key for LCYE to catalyze on both

339

ends.4,8 15



Page 16 of 46

340

Taking together, in this work, we cloned and functionally characterized three LCYs

341

and one CCS from red pepper, and reported the correspondence between the variation

342

in their gene expression and the accumulation of carotenoids in the β,β- branch in

343

ripening fruits. Our results illustrate the regulation of carotenoid biosynthesis at its first

344

bifurcation step and provide new insights into the manipulation of carotenoid

345

biosynthesis for the nutritional enhancement of food crops.

346 347

Funding

348

The work was supported by the National Natural Science Foundation of China (NSFC,

349

nos. 31770331, 90817002).

350 351

ACKNOWLEDGMENT

352

We thank Zhong Zhuang for the help with confocal observation.

353 354

ABBREVIATIONS

355

CaMV Cauliflower Mosaic Virus

356

CHYB carotene β-hydroxylase

357

CCS capsanthin/capsorubin synthase

358

DMAPP dimethylallyl diphosphate

359

DPA day post anthesis

360

EYFP Enhanced Yellow Fluorescent Protein 16


Page 17 of 46


361

FW fresh weight

362

GGPP geranylgeranyl diphosphate

363

GGPPS GGPP synthase

364

GPP geranyl diphosphate

365

HPLC high-performance liquid chromatography

366

HSP Heat Shock Protein

367

IPP isopentenyl diphosphate

368

LCY lycopene synthase

369

LCYB lycopene β-cyclase

370

LCYE lycopene ε-cyclase

371

MEP methylerythritol 4-phosphate

372

ORF open reading frame

373

PDA photodiode array detector

374

PSY phytoene synthase

375

qRT-PCR quantitative real-time PCR

376 377

Supporting Information

378

Supplemental Figure S1. Structure of the vector used for the transient expression of

379

fusion proteins in tobacco leaves.

380

Supplemental Table S1. Primers used in this study. 17



Page 18 of 46

381

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24


Page 25 of 46


FIGURE CAPTIONS Figure 1. Carotenoid metabolism beyond the lycopene branching point. Carotenoids sharing the β,β- and β,ε- structures are grouped in separate boxes. Abbreviations are: GGPP, geranylgeranyl diphosphate; LCYB and LCYE, lycopene βand ε-cyclases, respectively; CHYB and CHYE, carotene β- and ε-hydroxylases, respectively; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NSY, neoxanthin synthase; CCS, capsanthin/capsorubin synthase.

Figure 2. Variation in carotenoid profile in ripening red pepper fruits. Pigments were extracted from fruits at different ripening stages and separated by HPLC. Contents of lutein (Lut), neoxanthin (Neo), violaxanthin (Vio), β-carotene (β-Car), βcryptoxanthin (β-Cry), capsanthin (Cap), antheraxanthin (Ant), zeaxanthin (Zea) in each sample were quantified. IS, internal standard. Data are means ± SEM, n = 5.

Figure 3. Quantification of carotenoids of the β,β- and β,ε- branches in ripening red pepper fruits. (A) Contents of total carotenoids of the β,β- and β,ε- branches. (B) Molar ratios between total carotenoids of the β,ε- and β,β- branches. The molar value was calculated by dividing the content of each carotenoid constitute by its corresponding relative molecular weight. Carotenoids of the β,β- branch included β-carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, and capsanthin. Lutein was the only component in the β,ε- branch. Data are means ± SEM, n = 3.

Figure 4. Alignment of sequences of LCY homologues. Deduced amino acid sequences of Capana05g00023 (CaLCYB1), Capana10g002320 25



Page 26 of 46

(CaLCYB2), Capana06g000615 (CaCCS1) and Capana09g000177 (CaLCYE1) were aligned with sequences of functionally characterized LCYB and LCYE from Arabidopsis (AtLCYB and AtLCYE) and tomato (SlLCYB1, SlLCYB2 and SlLCYE), CCS from Lilium lancifolium (LlCCS) and LCYE from lettuce (LsLCYE). The conserved di-nucleotide binding domain, cyclase motifs, and transmembrane (TM) helixes are indicated.

Figure 5. Phylogenetic analysis of the lycopene cyclase family. Lycopene cyclase sequences used for constructing the Maximum-likelihood tree were from the cyanobacterium Synechococcus elongatus (SyneLCY, GenBank Accession No. CAA52677), the red alga Bangia fuscopurpurea (BfLCYB1, KX943552), the green algae Ostreococcus lucimarinus (OlLCYB, XP_001422489; OlLCYE, XP_001422490) and Dunaliella salina (DsLCYB1, ACA34344; DsLCYB2, ANY98896), the liverwort Marchantia polymorpha (MpLCYB, AB794089; MpLCYE, AB794090), the dicots Solanum lycopersicum (SlLCYB1, EF650013; SlLCYB2, AF254793; SlLCYE, Y14387), Lactuca sativa (LsLCYE, AF321538), Arabidopsis thaliana (AtLCYB, AAB53337;

AtLCYE,

AAB53336),

and

Nicotiana

tabaccum

(NtLCYB,

NP_001311716), and the monocots Lilium lancifolium (LlCCS, JF304153) and Oryza sativa

(OsLCYB,

BAD16478.1;

OsLCYE,

NP_001043410),

together

with

Capana05g000023 (CaLCYB1), Capana10g002320 (CaLCYB2), Capana06g000615 (CaCCS1) and Capana09g000177 (CaLCYE1) identified from red pepper in this study. Values displayed at the nodes indicate the percentage consensus support as calculated using a bootstrapping test with 1,000 replications. The scale bar indicates 20% sequence divergence.

26


Page 27 of 46


Figure 6. Subcellular localization of red pepper LCY homologue proteins. Each of the proteins was fused to the N-terminus of mCherry and transiently expressed in tobacco leaves. Enhanced yellow fluorescent protein (EYFP) alone was expressed as a negative control to indicate a cytosolic localization. Representative images observed under mCherry, chlorophyll (Chl) and EYFP channels and the merged signals are shown. All figures show representative images from at least five independent experiments. Scale bars = 20 μm.

Figure 7. Functional characterization of red pepper LCYs. (A-D). Pigment complementation assay in the E. coli cells harboring pAC-LYC and constructs to express different LCYs. Carotenoids were extracted from the bacterial cells expressing LCYB (AtLCYB) and LCYE (AtLCYE) from Arabidopsis thaliana as positive controls and empty vector as a negative control (A), CaLCYB1, CaLCYB2 and CaCCS1 (B), CaLCYE1 (C), and CaLCYE1 together with CaLCYB1 or CaLCYB2 (D), and separated by HPLC. (E). pAC-LYC contains genes for geranylgeranyl diphosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) to facilitate the biosynthesis of lycopene in E. coli. (F). The absorption spectrum of each peak recorded by the photodiode array detector.

Figure 8. Transcript abundances of CaLCYs and CaCCS1 in ripening fruits. Transcript abundances of CaLCYBs (A), CaLCYE1 (B) and CaCCS1 (C) in red pepper fruits at different ripening stages were quantified by qRT-PCR. The expression level of β-tubulin was determined as a reference. Data are means ± SEM, n = 5.

27



Page 28 of 46

TABLE Table 1. List of Red Pepper Homologue Genes for Lycopene Cyclases. locus

ORF length (bp)

Capana05g000023 / CaLCYB1

1497

Capana10g002320 / CaLCYB2

1671

Capana00g002014

603

Capana07g001071 (pseudogene) Capana09g000177 / CaLCYE1 (pseudogene) Capana12g001558

429

Capana12g001560 (pseudogene) Capana12g001589 (pseudogene) Capana06g000615 / CaCCS1 (pseudogene)

555

1578 477 525 1497

28


Page 29 of 46


FIGURES Figure 1.

29



Page 30 of 46

Figure 2.

30


Page 31 of 46


Figure 3.

31



Page 32 of 46

Figure 4.

32


Page 33 of 46


Figure 5.

33



Page 34 of 46

Figure 6.

34


Page 35 of 46


Figure 7.

35



Page 36 of 46

Figure 8.

36


Page 37 of 46


TOC graphic

37



Page 38 of 46

OPP

GGPP lycopene

γ-carotene

LCYB

LCYE δ-carotene

LCYB

β,β- branch

LCYB

β,ε- branch

β-carotene

α-carotene

CHYB

CHYB/E OH

HO

β-cryptoxanthin HO

zeaxanthin

HO

ZEP

HO

CHYB

OH

VDE

OH

O

antheraxanthin ZEP HO

VDE

OHCCS

NSY

violaxanthin

OH O OH

HO O

CCS

lutein

O OH

C HO

neoxanthin

OH

O

capsanthin

OH

OH O OH

abscisic acid


HO O

capsorubin

Page 39 of 46


Immature Green

20 Neo

10

Mature Green

Neo

Vio

β-Car

Neo

Cap Ant

eo Vi o C ap An t Lu t Ze β- a C r β- y C ar

N 50 40

β-Car

Lut Zea

30 20 10

20

80

First Immature Red

IS Vio

60

Zea

Cap Ant

β-Cry

Neo

β-Car

Vio

IS

Cap


20

Second Immature Red

25

100

Zea

80 β-Car

Ant

β-Cry

Neo


20

25

200

IS β-Car

Zea Ant

Neo 15 20 Retention time (min)

150 100

β-Cry 25


50 0


Vio

10

20

N

15 Cap

40

40 0

10

60

60

N

Mature Red

20

N

15

60

80

40

0

10

40

25

N

15


0

10

20

0

25

Vio

40

20

2 eo Vi o C ap An t Lu t Ze β- a C r β- y C ar

20

IS

10

20

4

N

15

Breaker

20

80

6

0 10

30

60

µg g-1 FW

8 Lut

10

40

2

10

µg g-1 FW

Absorbance (440nm) Absorbance (440nm) Absorbance (440nm)

25

IS

20

0 100

Absorbance (440nm)

F

20

30

0

E

15

40

0

D

β-Car

4

0 10

0

C

Lut

µg g-1 FW

Absorbance (440nm)

50

Vio

µg g-1 FW

6

0

B

8

IS

30

µg g-1 FW

40

µg g-1 FW

Absorbance (440nm)

A


Figure 3.

Total carotenoids (µg g-1 FW)

A

400 300 200 100 0

Molar ratio (β,ε−/β,β−)

B

β,ε-branch β,β-branch

IG

MG

B

FIR

SIR

MR

IG

MG

B

FIR

SIR

MR

0.6

0.4

0.2

0


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Figure 4.

Capana05g000023 Capana10g002320 AtLCYB SlLCYB1 SlLCYB2 LlCCS Capana06g000615 AtLCYE SlLCYE LsLCYE Capana09g000177

------------MDTLLRTPNN--------LEFL--- HGFGVKVSAFSSVKSQKFGAKKFCEGLGS------------------------MDIWFKTPNN--------LEFLQP-FYGFSVKGSTFSSVKTQKFGFRNFCGNWGRGVCVRPLWYGCSP ------------MDTLLKTPNK--------LDFFIPQFHGFERLCSNNPYHSRVRLGVKKRAIKIVS------------------------MDTLLKTPNN--------LEFLNP-HHGFAVKASTFRSEKHHNFGSRKFCETLG-------------------------MEALLKPFPS--------LLLSSPTPHRSIFQQNPSFLSPTTKKKSRKCLLRNKSSK-------------------------------------------------MSTLQLPALLTAGELRHPSR----RTKCS-----------------------METLLKPFPS--------PLLSIPTPNMYSFKHNSTFPNPTKQKDSRKFHYRNKSST----------MECVGARNFAA-MAVSTFPSWS---CRRKFPVVKRYSYRNIRFGLCSVRASGGGSSGSESCVAVRE-------------MECVGVQNVGA-MAVLTRPRLN---RWSGGELCQE---KSIFLAYEQYESKCNSSSGSDSCVVDKE-------------MECFGARNMTATMAVFTCPRFTDCNIRHKFSLLKQ--RRFTNLSASSSLRQIKCSAKSDRCVVDKQGI-----------MECIGAGKFGA-MAVFTRPRLK---EIVRKRVMPR---RKQCLWPINMQVKCSSS-GSESCVVDKE--------------

43 59 47 45 49 25 49 62 59 66 58


------------------------------------------RSVCVKASSSALLELVPETKKENLDFELPMYD----PS DPVYSGSFSTSGFPLGVCVKGGTFNSEKPQKFGFREVGGNWGRGVCVKASSSTLLDLVPETKKENLDFELPMYD----PS ---------------------------------------------SVVSGSAALLDLVPETKKENLDFELPLYD----TS ------------------------------------------RSVCVKGSSSALLELVPETKKENLDFELPMYD----PS -------------------------------------------------LFCSFLDLAPTSKPESLDVNISWVD----PN -------------------------------------------------SLRSFLDLTPVSKPEPLTIDIPYHD----PS -------------------------------------------------HFCSFLDLAPTSKPESLDVNISWVD----TD ---------------------------------------DFADEEDFVKAGGSEILFVQMQQNKDMDEQSKLVDKLPPIS ---------------------------------------DFADEEDYIKAGGSQLVFVQMQQKKDMDQQSKLSDELRQIS ---------------------------------------SVADEEDYVKAGGSELFFVQMQRTKSMESQSKLSEKLAQIP ---------------------------------------DFADEEDYIKAGGSQLVFVQMQQKKDMDQQSKLSDKLRQIS

77 135 78 79 76 52 76 103 100 107 99


K-GVVVDLAVVGGGPAGLAVAQQVSEAGLSVCSIDPNPKLIWPNNYGVWVDEFEAMDLLDCLDATWSGATVYIDDNTTKD K-GVVVDLAVVGGGPAGLAVAQQVSEAGLSVCSIDPSPKLIWPNNYGVWVDEFEAMDLLDCLDATWSGAVVYVDDDRTKN K-SQVVDLAIVGGGPAGLAVAQQVSEAGLSVCSIDPSPKLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYVDEGVKKD K-GVVVDLAVVGGGPAGLAVAQQVSEAGLSVCSIDPNPKLIWPNNYGVWVDEFEAMDLLDCLDATWSGAAVYIDDNTAKD SNRAQFDVIIIGAGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPNNYGVWVDEFENLGLENCLDHKWPMTCVHINDNKTKY S-AHRYDAAIIGCGPAGLRLAECAAARGLRVCCIDPAPLSPWPNNYGAWLDELHPLGLASIFDHIWPTATIAIDGDNIKH LDGAEFDVIIIGTGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPNNYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKY IGDGALDHVVIGCGPAGLALAAESAKLGLKVGLIG--PDLPFTNNYGVWEDEFNDLGLQKCIEHVWRETIVYLDDDKPIT AGQTVLDLVVIGCGPAGLALAAESAKLGLNVGLVG--PDLPFTNNYGVWEDEFKDLGLQACIEHVWRDTIVYLDDDEPIL IGNCILDLVVIGCGPAGLALAAESAKLGLNVGLIG--PDLPFTNNYGVWQDEFIGLGLEGCIEHSWKDTLVYLDDADPIR SGQTVLDLVVIGCGPAGLALAAESAKLGLNVGLVG--PDLPFTNNYGVWEDEFKDLGLQACIEHVWQDTIVYLDDADPIL Di-nucleotide binding domain

156 214 157 158 156 131 156 181 178 185 177


LNRPYGRVNRKQLKSKMMQKCILNGVKFHQAKVIKVIHEESK-SMLICNDGITIQATVVLDATGFSR-SLVQYDKPYN-P LDRPYGRVNRKQLKSKMMQKCILNGVKFHQAKVIKAIHEEAK-SMLICSDGVTIQAKVVLDATGFSR-CLVQYDKPYN-P LSRPYGRVNRKQLKSKMLQKCITNGVKFHQSKVTNVVHEEAN-STVVCSDGVKIQASVVLDATGFSR-CLVQYDKPYN-P LHRPYGRVNRKQLKSKMMQKCIMNGVKFHQAKVIKVIHEESK-SMLICNDGITIQATVVLDATGFSR-SLVQYDKPYN-P LGRPYGRVSRKKLKLKLLNSCVENRVKFYKAKVWKVEHEEFE-SSIVCDDGKKIRGSLVVDASGFAS-DFIEYDRPRN-H LSRPYGRVNRSSLKTLLLENCTTTGVRFHPSKAWNIEHEELR-SSVSCSDGSAVTASLVIDAGGFST-PFIEYDRPRNRR LDRPYGRVSRKKLKLKLLNSCVENRVKFYKAKVLKVKHEEFE-SSIVCDDGRKISGSLIVDASGYAS-DFIEYDKPRN-H IGRAYGRVSRRLLHEELLRRCVESGVSYLSSKVDSITEASDGLRLVACDDNNVIPCRLATVASGAASGKLLQYEVGGPRV IGRAYGRVSRHFLHEELLKRCVEAGVLYLNSKVDRIVEATNGQSLVECEGDVVIPCRFVTVASGAASGKFLQYELGSPRV IGRAYGRVHRDLLHEELLRRCVESGVSYLSSKVERITEAPNGYSLIECEGNITIPCRLATVASGAASGKFLEYELGGPRV IGRAYGRVSRHLLHEELLKRCVEAGVLYLNSKVDRIVEASSGHSLVECEGDVVIPCRFVTVASGAASGKFLQYELGGPRV

233 291 234 235 233 209 233 261 258 265 257


GYQVAYGILAEVEEHPFDVNKMVFMDWRDSHLKNNVELKERNSRIPTFLYAMPFSSNRIFLEETSLVARPGLGMDDIQER GYQVAYGILAEVEEHPFDTSKMLFMDWRDSHLNNSIELKERNRKVPTFLYAMPFSSNRIFLEETSLVARPGLRMDDIQER GYQVAYGIVAEVDGHPFDVDKMVFMDWRDKHLDSYPELKERNSKIPTFLYAMPFSSNRIFLEETSLVARPGLRMEDIQER GYQVAYGILAEVEEHPFDVNKMVFMDWRDSHLKNNTDLKERNSRIPTFLYAMPFSSNRIFLEETSLVARPGLRIDDIQER GYQIAHGVLVEVDNHPFDLDKMVLMDWRDSHLGNEPYLRVNNAKEPTFLYAMPFDRDLVFLEETSLVSRPVLSYMEVKRR GYQIAHGILAEVNRHPFDLNQMLLMDWSDAHLDNEPHLRAHNAAIPTFLYAMPFNENLVFLEETSLVGRPVLDYSEVKKR GYQVAHGILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDRNLVFLEETSLVSRPMLSYMEVKRR CVQTAYGVEVEVENSPYDPDQMVFMDYRDYTNEK---VRSLEAEYPTFLYAMPMTKSRLFFEETCLASKDVMPFDLLKTK SVQTAYGVEVEVDNNPFDPSLMVFMDYRDYLRHD---AQSLEAKYPTFLYAMPMSPTRVFFEETCLASKDAMPFDLLKKK CVQTAYGIEVEVENNPYDPDLMVFMDYRDFSKHK---PESLEAKYPTFLYVMAMSPTKIFFEETCLASREAMPFNLLKSK SVQTAYGVEVEVDNNPYDPSLMVFMDYRDYVRHD---VQSLEAKYPTFLYAMPMSPTRVFFEETCLASKDAMPFDLLKKK Cyclase motif

313 371 314 315 313 289 313 338 335 342 334


MVARLSHLGIKVKSIEEDEHCVIPMGGPLPVLPQRVVGIGGTAGMVHPSTGYMVARTLAAAPVVANAIIQYLSSER---MVARLNHLGIKVKSIEEDERCVIPMGGPLPVIPQRVVGIGGTAGMVHPSTGYMVARTLAAAPVVADAIIQYLGSEK---MAARLKHLGINVKRIEEDERCVIPMGGPLPVLPQRVVGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVRYLGSPS---MVARLNHLGIKVKSIEEDEHCLIPMGGPLPVLPQRVVGIGGTAGMVHPSTGYMVARTLAAAPVVANAIIQYLGSER---MVARLRHLGIKVKSVIEEEKCVIPMGGPLPRIPQNVMAIGGNSGIVHPSTGYMVARSMALAPVLAEAIVEGLGSTR---MVARLRHLGIKVERVLEEEKCLFPMGGPLPRMPQRVMGYGGAGGMVHPSSGYQIARALALAPELAEAMVECLGSTR---MVARLRHLGIKVRSVLEEEKCVITMGGPLPRIPQNVMAIGGTSGIVHPSSGYMVARSMALAPVLAEAIVESLGSTR---LMLRLDTLGIRILKTYEEEWSYIPVGGSLPNTEQKNLAFGAAASMVHPATGYSVVRSLSEAPKYASVIAEILREETTKQI LMLRLNTLGVRIKEIYEEEWSYIPVGGSLPNTEQKTLAFGAAASMVHPATGYSVVRSLSEAPKCASVLANILRQHYSKNM LMSRLKAMGIRITRTYEEEWSYIPVGGSLPNTEQKNLAFGAAASMVHPATGYSVVRSLSEAPNYAAVIAKILRQDQSKEM LMLRLDTLGVRIKEIYEEEWSYIPVGGSLPNTEQKTLAFGAAASMVHPATGYSVVRSLSEAPKCASVLANILRQNHIKNM Cyclase motif TM helix

389 447 390 391 389 365 389 418 415 422 414


SHS--GDELSAAVWKDLWPIERRRQREFFCFGMDILLKLDLPATRRFFDAFFDLEPRYWHGFLSSRLFLPELIVFGLSLF NHL--GDELSTSVWKDLWPIERRRQREFFCFGMDILLKLDLSATRRFFDAFFDLEPRYWHGFLSSRLFLPELMFFGLSLF SNSLRGDQLSAEVWRDLWPIERRRQREFFCFGMDILLKLDLDATRRFFDAFFDLQPHYWHGFLSSRLFLPELLVFGLSLF SHS--GNELSTAVWKDLWPIERRRQREFFCFGMDILLKLDLPATRRFFDAFFDLEPRYWHGFLSSRLFLPELIVFGLSLF MIR--GSQLYHRVWNGLWPLDRRCVRECYSFGMETLLKLDLKGTRRLFDAFFDLDPKYWQGFLSSRLSVKELGLLSLCLF MIT--GKSMNCKVWGSLWPAGRRWEREYYCFGMETLLSLDLKQTRRFFDAFFNLEPRYWHGFMSSRLSITELAQLSLSLF MIR--GSQLYHRVWNGLWPSDRRRVRECYCFGMETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLF NSN-----ISRQAWDTLWPPERKRQRAFFLFGLALIVQFDTEGIRSFFRTFFRLPKWMWQGFLGSTLTSGDLVLFALYMF LTSSSIPSISTQAWNTLWPQERKRQRSFFLFGLALILQLDIEGIRSFFRAFFRVPKWMWQGFLGSSLSSADLMLFAFYMF ISLGKYTNISKQAWETLWPLERKRQRAFFLFGLSHIVLMDLEGTRTFFRTFFRLPKWMWWGFLGSSLSSTDLIIFALYMF LTSSSTPSISTQAWNTLWPQERKRQRSFFLFGLALILQLDIEGIRSFFRAFFRVPKWMWQGFLGSSLSSADLMLFAFYMF TM helix SHASNTSRLEIMTKGTLPLVHMINNLLQDKE 498 SHASNTSRIEIMTKGTLPLVTMINNLLRDAE 556 SHASNTSRLEIMTKGTVPLAKMINNLVQDRD 501 SHASNTSRFEIMTKGTVPLVNMINNLLQDKE 500 GHGSNMTRLDIVTKCPLPLVRLIGNLAIESL 498 AHASWKSRVDVVTKCPLPLARMIGNLALQAI 474 GHASNLARLDIVTKCTVPLVKLLGNLAIESL 498 VISPNNLRKGLINHLISDPTGATMIKTYLKV 524 IIAPNDMRKGLIRHLLSDPTGATLIRTYLTF 526 VIAPHSLRMELVRHLLSDPTGATMVKAYLTI 533 IIAPNDMRKGLIKHLLSDPTGATMIRTYVTF 525

467 525 470 469 467 443 467 493 495 502 494




Figure 5.

0.2

CCS

48

LCYB LCYE

99 SlLCYB1 99 Capana05g000023/CaLCYB1 NtLCYB 75 75 Capana10g002320/CaLCYB2 90 AtLCYB 100 OsLCYB MpLCYB 99 LlCCS SlLCYB2 97 100 Capana06g000615/CaCCS1 100 OlLCYB DsLCYB1 80 DsLCYB2 100 BfLCYB1 OlLCYE MpLCYE 100 100 OsLCYE 100 ZmLCYE LsLCYE 100 AtLCYE 59 SlLCYE 74 100 Capana09g000177/CaLCYE1 SyneLCY


Page 42 of 46

Page 43 of 46


Figure 6.

EYFP

Chl

mCherry

Merged

CaLCYB1

CaLCYB2

CaLCYE1

CaCCS1



A

E

40 AtLCYB

β

30

DMAPP CrtE

EV GGPP

20

9-cis-β

L ɛ

10

CrtB Phytoene CrtI

0 40 β

Absorbance at 440nm(AU)

IPP

AtLCYE

pAC-LYC


δ

B

Page 44 of 46

Lycopene

CaCCS1 CaLCYB1

30

CaLCYB2

20

F

9-cis-β

lycopene (19.5 min)

δ-carotene (20.5 min)

ɛ-carotene (22.6 min)

α-carotene (22.0 min)

β-carotene (22.5 min)

9-cis-β-carotene (22.8 min)

10

C 1500 Absorbance at 440nm(AU)

CaLCYE1

100

δ

ɛ-carotene and its isoforms

50

0


D 100

CaLCYB1+CaLCYE1

δ

80

CaLCYB2+CaLCYE1 β

60 L

40

ɛ

α

9-cis-β

20 0

18

20

22 Retention time (min)

24

26

300

400

500 300 400 Wavelength (nm)


500

Page 45 of 46


Figure 8.

A 1.5

B 0.025

CaLCYB1 CaLCYB2

C CaLCYE1

300

CaCCS1

0.015

0.010

0.005


R M

SI R

B

FI R

G

IG

R M

SI R

B

FI R

G

IG

M

R M

SI R

FI R

B

G M

100

0

0

0

200

M

0.5

Relative expression level


1.0

IG


0.020


TOC graphic 73x29mm (600 x 600 DPI)


Page 46 of 46

Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in

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