Heteromeric geranylgeranyl diphosphate synthase contributes to

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Heteromeric geranylgeranyl diphosphate synthase contributes to carotenoid biosynthesis in ripening fruits of red pepper (Capsicum annuum var. conoides) Qiang Wang, Xing-Qi Huang, Tian-Jun Cao, Zhong Zhuang, Ran Wang, and Shan Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04052 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

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Journal of Agricultural and Food Chemistry

Heteromeric geranylgeranyl diphosphate synthase contributes to carotenoid biosynthesis in ripening fruits of red pepper (Capsicum annuum var. conoides)

Qiang Wanga, Xing-Qi Huanga, Tian-Jun Caoa, Zhong Zhuanga, Ran Wangb* and Shan Lua*

aState

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

University, Nanjing 210023, China bZhengzhou

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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ABSTRACT

2

Pepper (Capsicum annuum) fruits are a rich source of carotenoids. Geranylgeranyl

3

diphosphate (GGPP) is the precursor for carotenoid biosynthesis, and is produced by GGPP

4

synthase (GGPPS), which belongs to the prenyl transferase (PTS) family. In this study, we

5

identified from pepper genome a total of 8 PTS homologs. Our subcellular localization,

6

enzymatic activity, and expression level analyses proved that, among these homologs,

7

Capana04g000412 is the only functional GGPPS (CaGGPPS1) for carotenoid biosynthesis

8

in pepper fruits. We demonstrated that CaGGPPS1 interacts with a catalytically inactive

9

small subunit homolog protein CaSSUII, and such an interaction promotes CaGGPPS1

10

enzymatic activity. We also revealed a protein-protein interaction between CaSSUII and a

11

putative phytoene synthase, and repression of carotenoid accumulation by silencing

12

CaSSUII in pepper fruits. Taken together, our results suggest an essential contribution of

13

the CaGGPPS1/CaSSUII interaction to carotenoid biosynthesis in ripening pepper fruits.

14 15

KEY WORDS: Capsicum annuum, carotenoid, fruit, geranylgeranyl diphosphate synthase,

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red pepper, ripen, small subunit

2


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INTRODUCTION

18

Carotenoids are important terpenoid pigments widely distributed in higher plants, algae,

19

and certain groups of fungi and bacteria 1. Besides their light-harvesting and photo-

20

protection functions in photosynthetic organs, carotenoids also confer the distinct yellow,

21

orange and red colors in flowers and fruits, which are generally attractive to pollinators and

22

seed-dispersal animals 2. Moreover, carotenoids can be further cleaved into volatile

23

apocarotenoids, such as β-ionone, which are constituents of fragrances and aromas in

24

flowers and fruits as well 3. For human beings, carotenoids are also essential nutrients as

25

sources of provitamin A and serve as antioxidants to prevent aging-related diseases such

26

as macular degeneration 4.

27

Most fruits are rich in carotenoids. During ripening, metabolic pathways in these fruits

28

rewire to facilitate accumulation of carotenoids, accompanied by a decrease in chlorophyll

29

levels. As one of the most common vegetables and an important source of condiments,

30

tomato (Solanum lycopersicum) has been established as a model system for studying

31

different aspects of fruit ripening 5. Together with tomato in the Solanaceae family, pepper

32

(Capsicum annuum) fruits also accumulate a large amount of carotenoids, which vary with

33

different germplasms, environmental conditions and developmental stages, and display a

34

wide spectrum of colors ranging from white to deep red

35

carotenoid studies focused on the determination and quantitation of different carotenoid

36

compounds 7, 9. Although several enzymes participating in carotenoid biosynthesis, such as

37

phytoene synthase (PSY) and phytoene desaturase (PDS), have been cloned and

38

characterized, there is still very limited information on the molecular mechanisms

39

underlying carotenoid biosynthesis in pepper fruits 9-11.

6-8.

However, most pepper

3



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Carotenoid metabolism has been widely studied in different organisms, and also

41

extensively reviewed in recent years 1-2, 4. In all higher plants, carotenoid biosynthesis starts

42

from the methylerythritol 4-phosphate (MEP) pathway in plastids. With pyruvate and

43

glyceraldehyde 3-phosphate as substrates, the MEP pathway produces simultaneously the

44

five carbon (C5) isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate

45

(DMAPP) 12. Three molecules of IPP are condensed with one molecule of DMAPP into

46

geranylgeranyl diphosphate (GGPP, C20) by GGPP synthase (GGPPS). GGPP is not solely

47

used for carotenoid biosynthesis in plastids. It is also the immediate substrate for the

48

biosynthesis of the phytol side chain of chlorophylls and of diterpenoids such as

49

gibberellins 13. The allocation of GGPP among different downstream metabolic branches

50

largely determines the biosynthetic capability of each branch. For example, PSY is the

51

entry enzyme that directs metabolite flux into carotenoid biosynthesis by condensing two

52

molecules of GGPP into phytoene (C40) 14. When PSY was constitutively overexpressed

53

in tomato, transgenic plants showed a dwarfism phenotype, demonstrating the competition

54

of carotenoid and gibberellin biosynthetic branches for GGPP. This is an early evidence

55

reminding that the regulation of GGPP supply cannot be neglected. Till now, although

56

there has been a large body of studies demonstrating the regulation of carotenoid

57

biosynthesis by enzymes and transcription factors either in the upstream MEP pathway 15

58

or downstream steps beyond PSY

59

sequestration 18-19, the contribution of GGPPS is largely unknown.

16-17,

or by non-enzymatic proteins for carotenoid

60

Previous studies have revealed that higher plants usually have a short chain

61

prenyltransferase (PTS) gene family with multiple members to encode putative enzymes

62

such as GGPPSs and their homologs, for example, geranyl diphosphate (GPP, C10) 4


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synthases (GPPSs) that catalyze the condensation of one molecule of each of IPP and

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DMAPP to produce GPP (C10) as a precursor for monoterpenoids biosynthesis 12. In this

65

family, there are also a group of small subunit proteins (SSUs) that share slightly lower

66

sequence similarities with GGPPSs than GPPSs do. Although SSUs are catalytically

67

inactive, some of them were found to modulate GGPPS enzymatic activities through

68

protein-protein interactions 20. For example, the ectopic expression of an SSU protein from

69

snapdragon in tobacco turned the product of the endogenous GGPPS from GGPP into GPP,

70

whereas in rice the interaction between SSU (OsGRP) and GGPPS (OsGGPPS1) makes

71

the biosynthesis of GGPP more specific and more efficient 21-22.

72

A few pieces of evidence showing the regulation of GGPP allocation among

73

downstream metabolic branches by protein-protein interactions were recently reported. In

74

Arabidopsis thaliana, twelve PTS homolog proteins were identified 13. Although seven of

75

them have plastidic localization, only AtGGPPS11 was found to be a hub enzyme that

76

predominantly contributes to supply GGPP for downstream metabolic processes, most

77

probably through its transient interactions with corresponding entry enzymes for each

78

branch 23-24. Such regulation by protein-protein interaction was also demonstrated in rice.

79

OsGRP was proved to recruit OsGGPPS1 from stroma to a protein complex in the

80

thylakoid membrane, where it produces GGPP specifically for chlorophyll biosynthesis 22.

81

In pepper, a GGPPS was proposed to be associated with PSY in plastids, and its coding

82

gene was found to be strongly induced during fruit ripening 9, 11. However, it is not clear if

83

pepper has multiple homologs for GGPPS and how GGPP supply is regulated for

84

carotenoid biosynthesis during fruit ripening.

85

Here, we report the specific interaction between one of red pepper GGPPSs 5



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(CaGGPPS1) and an SSU (CaSSUII) in plastids. Our results showed that such an

87

interaction is essential for carotenoid biosynthesis in ripening pepper fruits.

6


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MATERIALS AND METHODS

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Plant material and growth conditions

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

91

Technology Trading Co., Ltd. (Beijing, China), and grown in a greenhouse. The ripening

92

of fruits was divided into six stages, i.e., immature green (IG, ca. 15 DPA), mature green

93

(MG, ca. 30 DPA), breaker (B, ca. 35 DPA), first immature red (FIR, ca. 38 DPA), second

94

immature red (SIR, ca. 43 DPA) and mature red (MR, ca. 50 DPA), according to Jang et

95

al. 25. Leaves and fruit pericarps at different stages were collected, immediately frozen in

96

liquid nitrogen and stored at -80°C till use.

97 98

Pigment analysis

99

Pigments were extracted from pepper fruits as described 26. Prior to the extraction, trans-

100

β-apo-8′-carotenal (Sigma-Aldrich, St. Louis, MO) (1 mg mL-1 dissolved in ethyl acetate)

101

was added as an internal standard 27.

102

A Waters 2695 separation module and 2998 photodiode array detector (PDA) (Waters,

103

Milford, MA) were used for high-performance liquid chromatography (HPLC) analysis of

104

extracted pigments on a reverse-phase Spherisorb ODS2 column (5 μm, 4.6×250 mm)

105

(Waters) using a 37-min gradient of ethyl acetate (0 - 100 %) in acetonitrile-water-

106

triethylamine (9:1:0.01) at a flow rate of 1 mL min-1 at 30°C 28. The elution profile was

107

compared with previous reports under similar chromatographic conditions, and the

108

ultraviolet/visible spectrum of each constituent was compared with published authentic

109

data to further confirm the peak identity 29. At least three replicates were performed for

110

each sample. 7



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Homolog identification and sequence analysis

113

Sequences of pepper PTS homologs were obtained by BLAST search against the pepper

114

genome database (http://peppersequence.genomics.cn) using sequences of AtGGPPS11

115

and AtSSUI from A. thaliana and of OsGGPPS1 and OsGRP from rice as queries 30. The

116

sequence of the previously identified SSU protein of snapdragon (Antirrhinum majus) was

117

downloaded from GenBank 21. The full-length amino acid sequences were aligned by using

118

ClustalW

119

constructed by using MEGA7 with default settings 32.

31

and manually edited. The maximum-likelihood phylogenetic tree was

120

The sequence of pepper homologs for PSY was obtained by BLAST searching using

121

the sequence of the functionally characterized PSY from Arabidopsis (At5g17230) as a

122

query 24.

123 124

Gene cloning and expression analysis

125

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

126

manufacturer’s instruction. Total RNA (400 ng) was reverse transcribed using a

127

PrimeScript 1st Strand cDNA synthesis Kit (TaKaRa). Full-length cDNAs of all pepper

128

PTS homologs were amplified using the first strand cDNA as a template, cloned in pMD19,

129

and sequenced by GenScript (Nanjing, China). High-fidelity PrimeSTAR DNA

130

polymerase (TaKaRa) was used according to the manufacturer’s instruction. Transcript

131

abundance of each gene studied was determined by quantitative real-time PCR (qPCR) in

132

a Thermal Cycler Dice Real Time System TP800 (TaKaRa) using a ChamQ SYBR qPCR

133

Master Mix (Vazyme, Nanjing, China), following the manufacturers’ manuals, and 8


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calculated using the comparative CT method

135

quantified as a reference 25. At least three biological replicates, each with three replicates

136

were analyzed for each sample. All primers used in this study are listed in Table S1 of the

137

Supporting Information.

33.

Transcript abundance of β-tubulin was

138 139

Subcellular localization analysis

140

For subcellular localization study, full-length open reading frames (ORFs) of pepper PTS

141

homologs were amplified from the 1st strand cDNA pool, and subsequently cloned into the

142

BamH I site of pSAT4A-mCherry-N1 (ABRC, Columbus, OH) to generate the constructs

143

to express cognate proteins with C-terminal mCherry fusions 34. For each construct, 20 μg

144

of plasmid was used for subsequent transfection.

145

For bimolecular fluorescence complementation (BiFC) assay, ORFs of a pair of genes

146

of interest without stop codons were cloned into pSAT1A-cEYFP-N1 and pSAT4A-

147

nEYFP-N1, respectively. For each pair, 10 μg plasmid of each of the two constructs were

148

mixed and then used for subsequent transfection.

149

For transient expression, protoplasts were isolated from leaves and transformed as

150

described

151

protoplasts. The protoplasts were incubated in the dark at 22°C overnight before

152

microscopy observation.

35.

PEG-mediated transfection was performed using 200 μL of the ice-cold

153

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

154

system was used for fluorescence observation. The mCherry fluorescent was excited at 543

155

nm, recorded from 580 to 620 nm. The reconstituted EYFP fluorescent was excited at 488

156

nm, recorded from 500 to 530 nm. Chlorophyll auto-fluorescence was excited at 543 nm 9



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and recorded from 680 to 720 28. All figures show representative images from at least five

158

independent experiments.

159 160

Yeast two-hybrid (Y2H) assay

161

For yeast two-hybrid assay, the Matchmaker GAL4 two-hybrid system (Clontech,

162

Mountain View, CA) was used. For testing the interaction between CaGGPPS1 and

163

CaSSUII, the full-length ORF of CaGGPPS1 was fused downstream of the DNA-binding

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domain (BD) of pGBK-T7, and the full-length ORF of CaSSUII was fused downstream of

165

the activation domain (AD) of pGAD-T7. For testing the interaction of CaSSUII and

166

CaGGPPS1 with CaPSY3, ORF of CaSSUII or CaGGPPS1 was fused downstream of BD

167

and that of CaPSY3 was fused downstream AD. Empty vectors (EV) were used as negative

168

controls. The interaction between AtGGPPS11 and AtSSUII was used as a positive control.

169

Cells of the yeast strain AH109 were co-transformed with both pGBK-T7 and pGAD-T7

170

constructs and spotted on nonselective (-LW) plates for 3 days at 30°C. Transformants

171

were tested for protein-protein interactions by growing on selective (-LWAH) plates

172

containing 40 mg L-1 X-α-Gal.

173 174

Pigment complementation and enzymatic assay

175

For proteins that are imported into chloroplast via the ΔpH pathway, their N-terminal

176

transit peptides are usually to be removed after the import

177

pepper PTS homologs were predicted online by ChloroP and TargetP 38-39. ORFs of their

178

coding sequences beyond the processing sites were amplified and cloned into pET32b

179

(Merck Millipore, Darmstadt, Germany) for prokaryotic expression. The functionally

36-37.

The processing sites of

10


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characterized OsGGPPS1

181

served as a negative control. Each of the constructs was co-transformed into Escherichia

182

coli BL21(DE3) (Merck Millipore) with pAC-94N, which harbors all the genes from

183

Pantoea stewartii for β-carotene biosynthesis in bacteria except for GGPPS

184

Transformant colonies accumulating β-carotene were picked from Luria-Bertani plates.

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The inoculation and quantitation of β-carotene contents were performed as previously

186

reported 41. When CaSSUII was co-expressed to study its impact on CaGGPPS1 activity,

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ORF of CaSSUII beyond the processing position was cloned into pET28a (Merck

188

Millipore), and the construct was co-transformed with pET32b-CaGGPPS1 and pAC-94N.

189

Transformants were analyzed as above described.

22

was cloned as a positive control, whereas the empty vector

40.

190

To functionally characterize the enzymatic activity, ORFs of CaGGPPS1 and CaSSUII

191

beyond the processing position were cloned into pMAL-C5X (NEB, Ipswich, MA). Each

192

of the fusion proteins with a maltose binding protein (MBP) tag was prokaryotically

193

expressed in E. coli BL21(DE3) cells and purified according to the manufacturer’s manual.

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For in intro enzymatic assay, 0.5 μmol L-1 of each of the fusion proteins was used in a final

195

volume of 400 μL reaction system containing IPP (400 μmol L-1) and DMAPP (200 μmol

196

L-1) in assay buffer (100 mmol L-1 HEPES, 5 mmol L-1 MgCl2, 10 mmol L-1 KCl, pH 7.5).

197

The reaction was carried out at 28°C. After 2 h (Figure S1), the solution was mixed with

198

200 μL of 0.2 mol L-1 Tris-HCl (pH 9.5) containing bovine intestinal alkaline phosphatase

199

(20 mg mL-1, > 10 DEA units mg-1, Sigma-Aldrich) and 2 units of shrimp alkaline

200

phosphatase (1 unit μL-1; TaKaRa), and incubated overnight at 30°C to hydrolyze

201

diphosphate products into their corresponding alcohols. After enzymatic hydrolysis, phytol

202

was added to the reaction system at a final concentration of 62.5 μmol L-1 as an internal 11



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standard. The mixture was extracted by 400 μL hexane for three times. The hexane

204

fractions were combined and then concentrated to 100 μL under nitrogen. A 1 μL portion

205

of the hexane phase was analyzed using an Agilent 7890A GC (Agilent, Santa Clara, CA)

206

equipped with an HP-5MS capillary column and a 5977A MS detector (Agilent). The

207

following temperature program was applied: 40°C for 2 min, an increase of 4°C/min to

208

250°C, and 250°C for 2 min.

209

To assess the impact of CaSSUII on CaGGPPS1 activity, both proteins were added to

210

the reaction at 0.5 μmol L-1, and the assay was performed as above described. Parallel

211

reactions using each of these two proteins were performed for a comparison.

212 213

Virus-induced gene silencing (VIGS) of CaSSUII

214

To silence the expression of CaSSUII specifically in red pepper fruits, we used the VIGS

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strategy 42. The first 300bp of CaSSUII ORF that has very low sequence similarities with

216

other PTS homologs was cloned into pTRV2. As the negative control, a pTRV2-EYFP

217

vector that targets an absent EYFP sequence was used. The recombinant plasmids and

218

pTRV1 were introduced into the Agrobacterium tumefaciens strain GV3101 by

219

electroporation. Agrobacterium cultures were grown and harvested as described earlier.

220

The cell pellet was resuspended in 10 mM MES, 10 mM MgCl2 and 200 μM

221

acetosyringone to a final OD600 of 0.8. Agrobacterium cultures containing pTRV1 and

222

pTRV2-CaSSUII or pTRV2/EYFP were mixed at 1:1 ratio and agroinfiltrated into

223

pericarps of mature green pepper fruits. Fruits were collected 30 days post infection, frozen

224

in liquid nitrogen, and stored at -80°C for subsequent analyses.

225 12


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RESULTS

227

Changes in pigment contents during ripening

228

In this study, we divided the ripening process of red pepper fruits into six stages according

229

to Jang et al.

230

demonstrated distinct changes in chlorophyll and carotenoid levels. For chlorophylls, their

231

contents rised from 167.3 μg g-1 fresh weight (FW) in the immature green (IG) fruits to

232

271.6 μg g-1 FW in mature green (MG) fruits, and then declined to 161.0 μg g-1 FW when

233

fruits were in the breaker (B) stage (Figure 1). No chlorophyll was detectable in fruits

234

beyond the breaker stage (Figure 1). In contrast to chlorophylls, carotenoids showed a

235

constant accumulation in red pepper fruits during ripening, increasing from 29.3 μg g-1 FW

236

in fruits at the beginning (IG) to 303.3 μg g-1 FW in mature red (MR) fruits (Figure 1).

25.

By quantifying pigment levels in fruits at each stage, our results

237 238

Sequence analysis and subcellular localization of the GGPPS family

239

GGPPS belong to the PTS family and are usually encoded by multiple genes. From the

240

chili pepper genome, we identified a repertoire of 8 genes for PTS homologs and amplified

241

their full-length cDNA fragments (Table 1) 30. One of these homologs, Capana03g002322,

242

has a much shorter open reading frame (ORF) (414-bp) and is most probably a pseudogene.

243

The deduced sequence of its cognate peptide does not contain the aspartate-rich motif

244

which is critical for the enzymatic activity of GGPPS, nor the CXXXC domain for protein-

245

protein interaction. Thus we excluded this homolog from further analysis. Five homologs

246

encode peptides containing both the first and second aspartate-rich motifs (FARM and

247

SARM, respectively) (Figure 2A). From our phylogenetic analysis, these 5 homologs also

248

clustered with previously identified GGPPSs from Arabidopsis (AtGGPPS11) and rice 13



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(OsGGPPS1) (Figure 2B). Therefore, these 5 homologs are putative GGPPSs that might

250

possess the capability of synthesizing GGPP from IPP and DMAPP. Another two

251

homologs, Capana00g004199 and Capana09g002331, encode peptides that contain only

252

FARMs and are clustered with catalytically inactive SSUs in our phylogenetic analysis

253

(Table 1, Figure 2). Previous studies found that SSUs could be further classified into two

254

different types (SSUI and SSUII for type I and II, respectively) based on their sequence

255

similarities 20, 43-44. In our phylogenetic analysis, Capana00g004199 and Capana09g002331

256

were grouped with previously identified SSUI from snapdragon and SSUIIs from

257

Arabidopsis and rice, respectively, and were therefore named as CaSSUI and CaSSUII,

258

respectively (Figure 2B).

259

It was reported that GGPPS could either form a homodimer with another molecule of

260

itself, or heterodimer with one molecule of SSU. The CXXXC domain in the sequence was

261

found to be essential for such a protein-protein interaction 43. From 7 homolog proteins,

262

except Capana03g002322, at least one CXXXC domain was identified, whereas the two

263

SSU members both have two CXXXC domains (Figure 2A). This suggests the possibility

264

of the putative GGPPS homologs to form either homo- or heterodimer.

265

In higher plants, the entire metabolic pathway from the MEP pathway to carotenoid

266

biosynthesis occurs exclusively in plastids. Therefore, a functional GGPPS belonging to

267

this pathway should also localize in the plastid. To identify the subcellular localization of

268

the 5 putative GGPPS homolog proteins and the 2 CaSSUs, we fused each of them to the

269

N-terminal of mCherry and transiently expressed the corresponding fusion protein in

270

protoplasts. Our results demonstrated that two of the five putative GGPPSs

271

(Capana04g000412 and Capana05g000800) have chloroplast localization, whereas the 14


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other three have either cytosolic or mitochondrial localizations (Figure 3). Both SSUs have

273

chloroplast localization (Figure 3).

274 275

Expression of PTS homolog genes during ripening

276

We then quantified the expression of all PTS homolog genes in leaves and fruits at different

277

ripening stages. Our results showed that transcripts of both Capana00g002450 and

278

Capana00g002451 were barely detectable in all samples. Capana00g002452,

279

Capana05g000800 and CaSSUI were expressed at relatively lower levels in both leaves

280

and fruits. Transcript abundances of both Capana04g000412 and CaSSUII reached their

281

highest levels in mature red fruits, but were much lower in leaves and mature green fruits

282

(Figure 4, Figure S2 of the Supporting Information). Taken together, among PTS homologs,

283

Capana04g000412 and CaSSUII are predominantly expressed in ripening fruits.

284 285

Functional characterization of red pepper GGPPS

286

To assess the capability of these homolog proteins in producing GGPP, we performed

287

pigment complementation assay by transforming E. coli BL21(DE3) cells with a

288

combination of pAC-94N and a construct harboring each of the cognate homolog genes.

289

The plasmid pAC-94N contains genes for phytoene synthase (crtB), phytoene desaturase

290

(crtI) and lycopene cyclase (crtY) from Pantoea stewartii (Figure 5A). When a functional

291

GGPPS is co-expressed in bacterial cells containing pAC-94N, the pathway is

292

complemented, and β-carotene is synthesized, turning bacterial colonies into orange. In our

293

study, by quantifying the production of β-carotene in cells transformed with each of the

294

combinations, we found that only Capana04g000412 could complement the pathway and 15



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resulted in a significant accumulation of β-carotene (Figure 5B). Although

296

Capana05g000800 also showed a chloroplast localization, we could not detect its

297

enzymatic activity in producing GGPP in this assay.

298

To further confirm the production of GGPP by Capana04g000412, we performed in

299

vitro assay using IPP and DMAPP as substrates. After the reaction, we hydrolyzed the

300

diphosphate product by phosphatases to convert it into corresponding alcohol, which was

301

then detected by gas chromatography-mass spectrometry (GC-MS). From our assay, only

302

one product, which has identical retention time and mass spectrum with those of

303

geranylgeraniol, was detected (Figure 5C,D,E). This suggests that GGPP is the only

304

product of Capana04g000412 with IPP and DMAPP as substrates.

305

Taken together our gene expression, subcellular localization, and enzymatic activity

306

analyses, we concluded that Capana04g000412 is the only functionally active enzyme that

307

produces GGPP in chloroplasts of red pepper fruits. We, therefore, name it CaGGPPS1.

308 309

Protein-protein interactions for carotenoid biosynthesis

310

Because CaGGPPS1 and CaSSUII were both highly expressed in fruits and CaGGPPS1 is

311

the major enzyme that synthesizes GGPP in pepper fruit plastids, we performed a Y2H

312

assay to determine the possible CaGGPPS1/CaGGPPS1 or CaGGPPS1/CaSSUII

313

interactions. On selective medium containing X-α-gal, only yeast cells expressing both

314

CaGGPPS1 and CaSSUII were able to grow and turn colonies into blue, demonstrating an

315

in vivo interaction between these two proteins (Figure 6A). In a parallel assay, CaGGPPS1

316

did not seem to interact with itself (Figure 6A). We further fused CaGGPPS1 and CaSSUII

317

to the N- and C-halves of the EYFP protein, respectively, and transiently expressed these 16


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two fusion proteins in protoplasts. The reconstituted EYFP signal of CaGGPPS1-nEYFP

319

and CaSSUII-cEYFP proteins was clearly observed in chloroplasts of transfected

320

protoplasts (Figure 6B). When CaGGPPS1 was fused to both N- and C-halves of EYFP,

321

the reconstituted EYFP signal was barely detectable in transfected protoplasts. Therefore,

322

we concluded the plastidic interaction between CaGGPPS1 and CaSSUII in planta.

323

We further performed pigment complementation assay to figure out the impact of

324

CaSSUII on CaGGPPS1 activity. Our quantitation revealed that E. coli cells expressing

325

both CaGGPPS1 and CaSSUII accumulated a higher level of β-carotene than those

326

expressing CaGGPPS1 alone (Figure 6C). By in vitro enzymatic activity assay and GC-

327

MS analysis, we identified that a mixture of equal amounts of CaGGPPS1 and CaSSUII

328

also catalyzed the biosynthesis of GGPP as the sole product, albeit at a relatively higher

329

level, compared with the reaction catalyzed by CaGGPPS1 alone (Figure 6D). Our analysis

330

did not find any additional product in the reaction with CaGGPPS1 and CaSSUII.

331

Therefore, CaSSUII promotes the catalytic efficiency of CaGGPPS1 without altering its

332

specificity.

333

Previous studies found pepper GGPPS in a protein complex containing PSY, which

334

catalyzes the condensation of GGPP into phytoene for carotenoid biosynthesis. We

335

searched pepper genome for genes encoding PSY homologs. Four different transcript

336

species were identified and named as CaPSY1-4 (Table 1). Our quantitation demonstrated

337

that one of these homologs, CaPSY3, was highly expressed in pepper fruits at stages from

338

the breaker to mature red, suggesting its specific involvement in carotenoid biosynthesis in

339

ripening pepper fruits (Figure 7A, Figure S2). We then performed a separate Y2H assay to

340

assess the interactions of CaGGPPS1 and CaSSUII with CaPSY3. Yeast cells expressing 17



Page 18 of 43

341

CaPSY3 with either CaGGPPS1 or CaSSUII could grow on selective plates containing X-

342

α-gal and turned colonies blue, suggesting a positive interaction between CaPSY3 and the

343

other two proteins (Figure 7B).

344 345

VIGS of CaSSUII

346

Our results demonstrated that CaSSUII interacts with both CaGGPPS1 and CaPSY3. To

347

address the essentiality of CaSSUII in carotenoid biosynthesis in pepper fruits, we

348

repressed CaSSUII expression by VIGS in fruits at the MG stage. After 30 days, the SSUII-

349

VIGS fruits showed a distinct greener phenotype (Figure 8A). Our pigment quantitation

350

confirmed a significantly lower level of carotenoids in CaSSUII-VIGS fruits than the

351

controls (Figure 8B). By qPCR, we found a significantly repressed expression of CaSSUII

352

in CaSSUII-VIGS fruits, compared with the controls (Figure 8C). However, transcript

353

abundance of CaGGPPS1 showed an insignificant decrease by the silencing of CaSSUII,

354

which might suggest a feedback regulation of CaGGPPS1 (Figure 8D). Transcript

355

abundance of CaPSY3 was largely unchanged in CaSSUII-VIGS fruits (Figure 8E).

356 357

DISCUSSION

358

In higher plants, GGPPSs and their homologs catalyze the biosynthesis of products with

359

different chain-lengths, such as GPP and geranylfarnesyl diphosphate (GFPP, C25), belong

360

to the PTS family with multiple members

361

similarities, it is difficult to predict the catalytic specificity of each of the members from

362

its deduced protein sequence. Moreover, these homologs have different subcellular

363

localizations in the cytosol, plastids, mitochondria, etc. to facilitate terpenoid biosynthesis

13, 22, 24, 45.

Because of the high sequence

18


Page 19 of 43


364

in separate compartments. Therefore, it is critical to analyze both catalytic specificity and

365

subcellular localization, so that the function of a GGPPS homolog could be rationalized.

366

In this work, we identified from pepper genome 8 genes for PTS homologs, which

367

include 1 pseudogene, 5 putative enzymes, and 2 SSUs. We found that only one of them,

368

CaGGPPS1 could catalyze the condensation of IPP and DMAPP into GGPP, which is a

369

characteristic property of genuine GGPPS, and possesses a plastid localization. Although

370

we could not fully exclude the contribution of other putative enzymes to produce GGPP,

371

considering significant lower transcript abundances of other candidates, we concluded that

372

CaGGPPS1 is responsible for carotenoid biosynthesis in ripening pepper fruits.

373

CaGGPPS1 has the highest sequence identity with the previously reported chili pepper

374

GGPPS, with only a few amino acid residues substituted 9, 46. These two enzymes are most

375

probably orthologs in different varieties. In a previous report, chili pepper GGPPS was

376

identified from affinity chromatography to exist as a homodimer with a native molecular

377

weight of ~74 ± 2 kDa

378

interaction between two molecules of CaGGPPS1, We demonstrated the interaction

379

between CaGGPPS1 and CaSSUII instead. Our sequence analysis revealed that the

380

molecular weights for CaGGPPS1 and CaSSUII are 40.2 kDa and 36.8 kDa, respectively.

381

Therefore, it is also possible that the larger size observed for chili pepper GGPPS in the

382

previous report was from a GGPPS/SSUII heterodimer. Moreover, from our results,

383

although CaSSUII is catalytically inactive as other SSUs are, its interaction with

384

CaGGPPS1 promotes the efficiency of CaGGPPS1 in synthesizing GGPP as a sole product.

385

This is similar to the finding of OsGRP in rice, and further demonstrates that SSUII

386

proteins have the functions to enhance the enzymatic activity of GGPPS and to recruit

11.

In this study, although we could not detect a protein-protein

19



Page 20 of 43

387

GGPPS to different downstream branches, both through protein-protein interaction 22. It is

388

interesting that a recent report, however, found that an SSUII protein from Arabidopsis is

389

able to modify the product of AtGGPPS11 into GPP, similar to the function of SSUI

390

members

391

would help to decipher the functional diversity of SSUII members through evolution.

44.

Further analysis with more GGPPS-SSU pairs from different plant species

392

A surprise to us is the interaction of CaPSY3 with both CaGGPPS1 and CaSSUII. This

393

probably explains why CaGGPPS1 was found in a protein complex for carotenoid

394

biosynthesis containing PSY and other enzymes such as IPP isomerase (IPI) 11. It is likely

395

that CaSSUII not only promotes the enzymatic activity of CaGGPPS1, but also helps to

396

bridge the interaction between CaGGPPS1 and the protein complex for carotenoid

397

biosynthesis. However, we did not observe a distinct signal in chloroplasts demonstrating

398

such an interaction in planta by BiFC assay, suggesting that the interaction between

399

CaPSY3 and CaGGPPS1 might be either transient or specific to chromoplasts. Detailed

400

sub-organelle localization analysis of related enzyme proteins will help to figure this out.

401

However, it is still unknown when and how this complex is assembled and how it is

402

regulated as a whole. Moreover, although IPI was found in this complex

403

function in the conversion between IPP and DMAPP is required for the biosynthesis of

404

both GPP and GGPP in plastids 2, 23, the involvement of IDI in the complex specifically for

405

carotenoid biosynthesis via GGPP needs to be carefully confirmed to uncover the

406

regulation of metabolic regulation between monoterpenoids and carotenoids in ripening

407

fruits.

11,

because its

408 409

ABBREVIATIONS 20


Page 21 of 43


410

Activation domain (AD)

411

Bimolecular fluorescence complementation (BiFC)

412

Dimethylallyl diphosphate (DMAPP)

413

DNA-binding domain (BD)

414

Empty vectors (EV)

415

First aspartate-rich motif (FARM)

416

Fresh weight (FW)

417

Gas chromatography-mass spectrometry (GC-MS)

418

Geranyl diphosphate (GPP)

419

Geranylfarnesyl diphosphate (GFPP)

420

Geranylgeranyl diphosphate (GGPP)

421

GGPP synthase (GGPPS)

422

GPP synthase (GPPS)

423

High-performance liquid chromatography (HPLC)

424

IPP isomerase (IPI)

425

Isopentenyl diphosphate (IPP)

426

Maltose binding protein (MBP)

427

Methylerythritol 4-phosphate (MEP)

428

Open reading frame (ORF)

429

Photodiode array detector (PDA)

430

Phytoene desaturase (PDS)

431

Phytoene synthase (PSY)

432

Prenyl transferase (PTS) 21



433

Quantitative real-time PCR (qPCR)

434

Second aspartate-rich motif (SARM)

435

Small subunit (SSU)

436

Virus-induced gene silencing (VIGS)

437

Yeast two-hybrid (Y2H)

Page 22 of 43

438 439

ACKNOWLEDGMENTS

440

The work was supported by the State Key Basic Research and Development Plan of China

441

(No. 2013CB127004), and the National Natural Science Foundation of China (NSFC, Nos.

442

31770331, 90817002). The authors wish to thank Zhong Zhuang for the help with confocal

443

observation, and to thank Yi-Dan Zhang and Jin-Di Rui for the technical assistance.

444 445

SUPPORTING INFORMATION

446

Supplemental Figure S1. GGPP production in our in vitro enzymatic activity assay with

447

different reaction time.

448

Supplemental Figure S2. Expression of red pepper GGPPS and PSY homolog genes at

449

low levels.

450

Supplemental Table S1. Primers used in this study.

22


Page 23 of 43


451

FIGURE CAPTIONS

452

Figure 1. Chlorophyll and carotenoid contents in red pepper fruits.

453

Pigments were extracted from pericarps at immature green (IG), mature green (MG),

454

breaker (B), first immature red (FIR), second immature red (SIR) and mature red (MR)

455

stages, and analyzed by HPLC. Data represent means ± SEM, n = 3.

456 457

Figure 2. Sequence analysis of red pepper PTS homologs.

458

(A). Alignment of sequences of red pepper PTS homologs with previously characterized

459

GGPPSs from Arabidopsis (AtGGPPS11) and rice (OsGGPPS), and SSUs from

460

Arabidopsis (AtSSUII), rice (OsGRP) and snapdragon (AmSSUI). Conserved FARM and

461

SARM motifs are in red boxes, and the CXXXC domains are in blue boxes. Truncation

462

positions for heterologous expression of homolog proteins in Escherichia coli for pigment

463

complementation assays are indicated by an upper triangle.

464

(B). Phylogenetic analysis of sequences in (A). The maximum likelihood phylogenetic tree

465

was constructed using MEGA7 with default settings. The bootstrap values are labeled

466

beside the branches, and the scale bar indicates 20% amino acid sequence divergence.

467 468

Figure 3. Subcellular localization of red pepper PTS homolog proteins.

469

Each of the proteins was fused to the N-terminus of mCherry and expressed in mesophyll

470

protoplasts. Representative images observed under bright field (Bright), chlorophyll (Chl)

471

and mCherry channels and the merged signals are shown. mCherry alone was expressed as

472

a negative control to show its localization. Scale bars = 10 μm.

473 23



Page 24 of 43

474

Figure 4. Expression of Ca04g000412 and CaSSUII during red pepper fruit ripening.

475

Transcript abundances of each gene at different ripening stages were quantified by qRT-

476

PCR and normalized against the abundance of β-tubulin. Data are means ± SEM, n = 3.

477 478

Figure 5. Catalytic property of red pepper PTS members.

479

(A). pAC-94N contains Pantoea stewartii carotenoid biosynthetic genes for phytoene

480

synthase (crtB), phytoene desaturase (crtI) and lycopene β-cyclase (crtY). When co-

481

expressed with a gene for GGPPS (crtE), the pathway is complemented, and the

482

transformed BL21(DE3) cells are able to produce and accumulate β-carotene.

483

(B). Complementation assay for detecting GGPPS activity of the red pepper PTS members.

484

Each of the tested genes was cloned into the pET-32b vector and co-transformed with pAC-

485

94N into Escherichia coli BL21(DE3) cells. Empty vector (EV) and functionally

486

characterized OsGGPPS1 from rice were used as negative and positive controls,

487

respectively. β-Carotene produced in transformed cells were measured by absorbance at

488

440 nm. Data are means ± SEM, n = 3. **P < 0.01, Student’s t test.

489

(C) and (D). Gas chromatograms of the production of GGPP from in vitro enzyme activity

490

assay using Capana00g002452, Capana04g000412, CaSSUII and a mixture of

491

Capana04g000412 and CaSSUII at equal molecular ratio (C) and of a mixture of authentic

492

geraniol (GOH), farnesol (FOH) and geranylgeraniol (GGOH) (D) under selected ion

493

monitoring (SIM) mode (m/z 69). The produced GGPP was hydrolyzed to GGOH for

494

separation and detection. Phytol was added as an internal standard.

495

(E). Mass spectra comparison of Capana04g000412 product at 46.797 min under full scan

496

mode (m/z 40-500) with the authentic GGOH. 24


Page 25 of 43


497 498

Figure 6. Interaction between CaGGPPS1 and CaSSUII.

499

(A). Pair-wise yeast two-hybrid assay of the interactions. CaGGPPS1 and CaSSUII were

500

fused with the DNA binding domain (BD) of pGBK-T7 and the activation domain (AD)

501

of pGAD-T7, respectively. Yeast cells expressing both proteins were spotted on non-

502

selective medium (-LW), and a ten-fold serial dilutions were spotted on selective medium

503

supplied with X-α-Gal (-LWAH + X-α-Gal). Interactions between empty vectors (EV) and

504

between AtGGPPS11 and AtSSUII were used as negative and positive controls,

505

respectively.

506

(B). BiFC detection for the interactions between CaGGPPS1 and CaSSUII proteins in

507

mesophyll protoplasts. Co-expressed fusion proteins are indicated. Fluorescence of

508

reconstructed EYFP is shown in the “EYFP” panel. Scale bars = 10 μm.

509

(C). Complementation assay for detecting the impact of CaSSUII on the enzymatic activity

510

of CaGGPPS1. Escherichia coli BL21(DE3) cells harboring pAC-94N were co-

511

transformed with pET-32a-CaGGPPS1, or pET-28b-CaSSUII, or both. β-Carotene

512

produced in transformed cells was quantified by measuring the absorbance at 440 nm. Data

513

are means ± SEM, n = 3. **P < 0.01, Duncan’s Multiple Range test.

514

(D). In vitro enzyme activity assay to identify the effect of CaSSUII on CaGGPPS1. The

515

GGOH content in the reaction catalyzed by a mixture of CaGGPPS1 and CaSSUII at equal

516

molecular ratio was normalized by that in the control group using CaGGPPS1 alone at the

517

same concentration. Data are means ± SEM, n = 3.*P < 0.05, Student’s t test.

518 519

Figure 7. Protein-protein interactions of CaPSY3. 25



Page 26 of 43

520

(A). Quantification of CaPSY3 expression level during pepper fruit ripening. Transcript

521

abundances were quantified by qRT-PCR and normalized against that of β-tubulin. Data

522

are means ± SEM, n = 3.

523

(B). Pair-wise yeast two-hybrid assay of the interactions. CaGGPPS1 and CaSSUII were

524

fused with the DNA binding domain (BD) of pGBK-T7 and CaPSY3 was fused with the

525

activation domain (AD) of pGAD-T7. The empty vector pGBK-T7 was used as a negative

526

control, and the interaction between AtGGPPS11 and AtSSUII was used as a positive

527

control. Yeast cells expressing both proteins were spotted on non-selective medium (-LW),

528

and a ten-fold serial dilutions were spotted on selective medium supplied with X-α-Gal (-

529

LWAH + X-α-Gal).

530 531

Figure 8. Virus-induced gene silencing of CaSSUII in red pepper fruits.

532

(A). Representative fruits with CaSSUII silenced by VIGS. Fruits infected by pTRV2-

533

EYFP were used as a control.

534

(B). Total carotenoid contents in pericarp tissue around the injection site after 30-day

535

growth. Data are means ± SEM, n = 3. *P < 0.05, Student’s t test.

536

(C-E) Expression levels of CaSSUII, CaGGPPS1, and CaPSY3 in pericarp tissues around

537

the injection sites of CaSSUII-VIGS and EYFP-VIGS fruits. Transcript abundances were

538

compared with that of β-tubulin. Data are means ± SEM, n = 3. *P < 0.05, Student’s t test.

539

26


Page 27 of 43


540

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cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry

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40. Cunningham, F. X., Jr.; Gantt, E., A portfolio of plasmids for identification and

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682

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683

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685

TABLE

686

Table 1. List of red pepper homolog genes for prenyl transferases and phytoene

687

synthases. Locus

ORF

Capana00g002450

Capana00g002452

1116 Length 867 (bp) 1107

Capana03g002322

414

Capana04g000412 / CaGGPPS1

1110

Capana05g000800

1098

Capana00g004199 / CaSSUI

996

Capana09g002331 / CaSSUII

1011

Capana01g001510/ CaPSY1

1152


1326


1260


342

Capana00g002451

688

34


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689

FIGURES

690

Figure 1.

691

35



692

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Figure 2.

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36


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694


Figure 3.

695

37



696

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Figure 4.

697

698

38


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699


Figure 5.

700

39



701

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Figure 6.

702

40


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703


Figure 7.

704

41



705

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Figure 8.

706

42


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TOC graphic

708

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Heteromeric geranylgeranyl diphosphate synthase contributes to

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