Plant Physiology Preview. Published on June 8, 2015, as … · 2015. 6. 8. · 23 Research Paper 24 A comprehensive analysis of chromoplast differentiation reveals complex protein

Running Title 1

Proteomic profiling of chromoplast differentiation 2

Corresponding Author 4

Professor Xiuxin Deng 5

Key Laboratory of Horticultural Plant Biology of Ministry of Education Huazhong 6

Agricultural University Wuhan 430070 PR China 7

Tel +86-27 87286906 8

email xxdengmailhzaueducn 9

Plant Physiology Preview Published on June 8 2015 as DOI101104pp1500645

Research Paper 23

A comprehensive analysis of chromoplast differentiation reveals complex protein 24

changes associated with plastoglobule biogenesis and remodelling of protein systems in 25

orange flesh1 26

Yunliu Zeng1 Jiabin Du1 Lun Wang1 Zhiyong Pan1 Qiang Xu1 Shunyuan Xiao 12 and 27

Xiuxin Deng1 28

1Key Laboratory of Horticultural Plant Biology (Ministry of Education) College of 29

Horticulture and Forestry Science Huazhong Agricultural University Wuhan 430070 PR 30

China 31

2Institute for Bioscience and Biotechnology Research University of Maryland Rockville 32

MD USA 33

Author for correspondence E-mail xxdengmailhzaueducn 34

Footnotes 36

This work was supported by the National 973 project of China (No 2011CB100601) and 37

NSFC (No 31330066 and 31221062) 38

Correspondence Author 39

Professor Xiuxin Deng email xxdengmailhzaueducn 40

Key Laboratory of Horticultural Plant Biology (Ministry of Education) College of 41

China 43

Abstract (250 words) 48

Globular and crystalloid chromoplasts were observed to be region-specifically formed in 49

orange flesh (Citrus sinensis Osbeck) and converted from amyloplasts during fruit maturation 50

which was associated with the composition of specific carotenoids and expression of 51

carotenogenic genes Subsequent iTRAQ-based quantitative proteomic analyses of purified 52

plastids from the flesh during chromoplast differentiation and senescence identified 1386 53

putative plastid-localized proteins 1016 of which were quantified by spectral counting The 54

iTRAQ values reflecting the expression abundance of three identified proteins were validated 55

by immunoblotting Based on iTRAQ data chromoplastogenesis appeared to be associated 56

with three major protein expression patterns (1) marked decrease in abundance of the proteins 57

participating in the translation machinery through ribosome assembly (2) increase in 58

abundance of the proteins involved in terpenoid biosynthesis (including carotenoids) stress-59

responses (redox ascorbate and glutathione) and development and (3) maintenance of the 60

proteins for signalling and DNAampRNA Interestingly strong increase in abundance of several 61

plastoglobule-localized proteins coincided with the formation of plastoglobules in the 62

chromoplast The proteomic data also showed that stable functioning of protein import 63

suppression of ribosome assembly and accumulation of chromoplast proteases are correlated 64

with amylo-to-chromoplast transition and thus these processes may play a collective role in 65

chromoplast biogenesis and differentiation By contrast chromoplast senescence process was 66

inferred to be associated with a significant increase in stress response and energy supply In 67

conclusion this comprehensive proteomic study identified many potentially new plastid-68

localized proteins and provides insights into the potential developmental and molecular 69

mechanisms underlying chromoplast biogenesis differentiation and senescence in orange 70

flesh 71

Introduction 75

Chromoplasts are special organelles with superior ability to synthesize and store massive 76

amounts of carotenoids bringing vivid red orange and yellow colours to many flowers fruits 77

and vegetables (Li and Yuan 2013) Chromoplasts exhibit various morphologies such as 78

crystalline globular tubular and membranous structures (Egea et al 2010) The relationship 79

between the architecture and carotenoid composition has been well stated in diverse 80

Capsicum annuum and Solanum lycopersicum fruits (Kilcrease et al 2013 Nogueira et al 81

2013) Crystalline bodies have been observed in carrots (Frey-Wyssling and Schwegler 1965) 82

and tomatoes (Harris and Spurr 1969) which predominantly consist of β-carotene and 83

lycopene respectively Globular andor tubular-globular chromoplasts in which numerous 84

lipid droplets (also named plastoglobules) that act as passive storage compartments for 85

triglycerides sterol ester and some pigments are accumulated were described for yellow 86

fruits from kiwi papaya and mango which contain lutein β-cryptoxanthin and β-carotene as 87

the major pigments respectively (Vasquez-Caicedo et al 2006 Montefiori et al 2009 88

Schweiggert et al 2011) Carotenoid composition has been reported to be regulated by the 89

expression of carotenogenic genes in the flesh of various citrus fruits differing in their internal 90

colors (Fanciullino et al 2006 2008) Chromoplasts are frequently derived from fully 91

developed chloroplasts as seen during fruit ripening from green to red or yellow fruits in 92

tomato and pepper (Egea et al 2010) In some cases chromoplasts also arise from non-93

photosynthetic plastids such as colorless proplastids leucoplasts or amyloplasts (Knoth et al 94

1986 Schweiggert et al 2011) To date most studies on chromoplast differentiation have 95

been focused on the synthesis of carotenoids by combining biochemical and molecular 96

analyses (Egea et al 2010 Bian et al 2011 Li and Yuan 2013 Cazzonelli and Pogson 97

2010) and little is known about the molecular mechanisms underlying chromoplast 98

biogenesis (Li and Yuan 2013) 99

Recently proteomics has become an efficient tool to study protein composition of 100

subcellular organelles such as chromoplasts and their dynamic changes during the 101

development of a particular plant organtissue The majority of chromoplast-related studies 102

are concerned about the functions of these organelles in various crops such as pepper tomato 103

watermelon carrot cauliflower and papaya (Siddique et al 2006 Wang et al 2013) 104

However only a few of such studies addressed the mechanisms underlying plastid 105

differentiation such as transition from proplastid to chloroplast in maize (Majeran et al 106

2010) from etioplast to chloroplast in pea and rice (Kleffmann et al 2007 Kanervo et al 107

2008) and from chloroplast to chromoplast in tomato (Barsan et al 2012) In tomato 108

chromoplastogenesis appears to be associated with major metabolic shifts including a strong 109

decrease in abundance of the proteins involved in light reaction and an increase in terpenoid 110

biosyntheisis and stress-response proteins (Barsan et al 2012) These changes in proteins are 111

in agreement with the structural changes occurring in tomato during fruit ripening which is 112

characterized by the loss of chlorophyll and the synthesis of colored compounds Chromoplast 113

differentiation from non-photosynthetic plastids occurs frequently in a number of plant tissues 114

such as watermelon flesh and carrot root (Kim et al 2010 Wang et al 2013) However to 115

the best of our knowledge no large-scale proteomic study for understanding this 116

developmental process has been reported 117

Citrus is one of the most economically important fruit crops in the world Different from 118

the model fruit tomato which represents climacteric fruits citrus shows non-climacteric 119

characteristics during fruit maturation Additionally citrus fruits exhibit a unique anatomical 120

fruit structure consisting of two major sections the pericarp and the edible flesh Considerable 121

progresses have been made in the understanding of chromoplast differentiation in the pericarp 122

of citrus fruits (Eilati et al 1969 Iglesias et al 2007) which is a process similar to that of 123

tomato and pepper (Egea et al 2010) However little is known about the molecular basis of 124

chromoplast differentiation in the edible flesh even though there are increasing evidences 125

suggesting an essential role of carotenoid synthesis in inducing chromoplast differentiation 126

(Egea et al 2010 Bian et al 2011 Li and Yuan 2013) Recently we successfully isolated 127

and purified intact chromoplasts containing a large number of plastoglobules from the flesh of 128

orange fruits at the maturation stage (Zeng et al 2011) The same method has also been 129

successfully used for isolating plastids from orange flesh in earlier maturation stages (Zeng et 130

al 2014) thus making comparative and quantitative proteomic analysis of plastid 131

differentiation possible In the present study we investigated how ultrastructural changes of 132

plastidschromoplasts during orange fruit maturation might be associated with the changes in 133

the composition of carotenoids and the expression of carotenogenic genes in red and yellow 134

flesh of the fruits Furthermore we employed the iTRAQ-based (isobaric tag for relative and 135

absolute quantitation) technology to investigate how protein compositional changes might be 136

correlated with metabolic and structural changes in the plastids of orange flesh during their 137

transformation from amyloplasts to chromoplasts 138

Results 139

Structural diversity of chromoplasts in sweet orange flesh 140

Citrus fruits are rich in carotenoids and most of them have yellow flesh but occasionally red-141

fleshed fruits are produced due to spontaneous mutations In the present study mutant lsquoHong 142

Anliursquo (Fig 1A) and its wild-type fruit lsquoAnliursquo (Fig 1B) as well as mutant lsquoCara Cararsquo (Fig 143

1E) and its wild-type fruit lsquoNewhallrsquo Navel at maturation (Fig 1F) were used as materials for 144

observing chromoplast types Based on the citrus anatomy the edible flesh mainly consists of 145

segment membrane and sac (Fig 1 C and D G and H) In this study we further separated the 146

sac into two independent parts one is sac membrane and the other is juice (Fig 1G) 147

Crystalloid and globular chromoplast types were observed in the flesh of sweet orange 148

Light microscopy and transmission electron microscopy (TEM) were used to examine the 149

formation of carotenoid sequestering structures in sweet orange Visible red carotenoid 150

sequestering structures were found in the membrane of sac and segment from lsquoHong Anliursquo 151

(Fig 2 A and B) and lsquoCara Cararsquo (Supplemental Fig S1 A and B) and polarization 152

microscopy confirmed their crystal nature (Fig 2 G and H Supplemental Fig S1 G and H) 153

According to a protocol for isolating plastids (Zeng et al 2011) abundant carotenoid crystals 154

with the size from 2 μm to 10 μm were detected in the isolation buffer (Fig 3A) Their TEM 155

images showed structural diversities in lsquoHong Anliursquo (Fig 3 B-E) and lsquoCara Cararsquo 156

(Supplemental Fig S2 A-D) 157

Globular-shaped chromoplasts with yellow-orange color were frequently observed in the 158

juice of lsquoHong Anliursquo (Fig 2C) and lsquoCara Cararsquo (Supplemental Fig S1C) as well in the 159

edible flesh of lsquoAnliursquo (Fig 2 D-F) and lsquoNewhallrsquo Navel (Supplemental Fig S1 D-F) TEM 160

analysis showed that the globular chromoplasts isolated from different sweet orange tissues 161

including the juice of lsquoHong Anliursquo (Fig 3 F and G) and lsquoCara Cararsquo (Supplemental Fig 162

S2E) and the flesh of lsquoAnliursquo (Fig 3 H and I) and lsquoNewhallrsquo Navel (Supplemental Fig S2F) 163

were found to contain plastoglobules varying in number size and electron density Taken 164

together the above results suggest that the red color of the mutants is attributable to the 165

membranes of sacs and segments rather than to the juice 166

Relationship between the architecture of globular and crystalloid chromoplasts and 167

their carotenoid content and composition 168

To determine whether the differences in chromoplast ultrastructure were associated with the 169

alteration of pigment storage we measured the content and composition of carotenoids in the 170

membranes of sac and segment as well as in the juice fractions of lsquoHong Anliursquo and lsquoAnliursquo 171

With regard to the sac membranes we found that the total amount of carotenoids of lsquoHong 172

Anliursquo was about 15-fold higher than that of lsquoAnliursquo and the levels of most carotenoids in 173

both cultivars were similar except for lycopene and beta-carotene which were relatively 174

higher in lsquoHong Anliursquo and lsquoAnliursquo respectively (Fig 4A) Interestingly we observed 175

remarkable differences of carotenoid content and composition in the segment membranes 176

The total amount of carotenoids in lsquoHong Anliursquo was 4-fold higher than that of lsquoAnliursquo In 177

addition the composition of carotenoids was also very different between these two cultivars 178

with lycopene accounting for 918 and violaxanthin 36 of the total carotenoids in lsquoHong 179

Anliursquo (Fig 4B right) and with violaxanthin accounting for 734 (356 μgg of FW Fig 180

4D) and lycopene being almost absent in the total carotenoids in lsquoAnliursquo (Fig 4B left) 181

However both cultivars had a similar carotenoid profile in their juice fractions in which 182

violaxanthin and lutein were dominant and lycopene was absent (Fig 4C) suggesting that the 183

chromoplasts from the juice may be structurally similar between these two cultivars 184

Ultrastructural differentiation of crystalloid and globular chromoplasts during fruit 185

maturation 186

For understanding the origin of chromoplasts the sac membrane and juice of lsquoHong Anliursquo 187

fruit were examined throughout the fruit-maturation period till typical crystalloid and globular 188

chromoplasts were finally formed (Fig 3 A and F) Amyloplasts with large starch granules 189

were frequently observed in the sac membrane at the immature green (IMG) stage (see 190

definition in lsquoMaterials and methodsrsquo) but plastoglobules were invisible (Fig 5A) 191

Subsequently these starch granules gradually disappeared at the mature green (MG) stage 192

which coincided with the formation of plastids containing a small number of plastoglobules 193

(Fig 5B) These observations suggest that the chromoplasts might be transformed from 194

amyloplasts At the colour-breaker (Br) stage there was a dramatic increase in the number of 195

plastoglobules with the appearance of a few slightly undulated tuber bodies in the plastids 196

(Fig 5C) As fruit maturation proceeded elongated (8 to 10 μm in length) or irregular 197

crystalloid bodies were formed which were often surrounded by a graywhitish membrane 198

structure (Fig 5D) that disappeared before the formation of a crystalloid chromoplast filled 199

with a few plastoglobules and some undulated membranes The undulated membranes 200

represented the remnants of membrane-coated carotenoid crystals which were extracted 201

during the fixation process of samples for TEM (Fig 5E) It seems that the large 202

accumulation of the crystalloid bodies might contribute to the change of the plastid shape 203

from round to oval (Fig 5 C-E) 204

To investigate the pattern of plastid differentiation in the juice of sweet orange we 205

conducted cytological analyses using purified plastids from the flesh of lsquoHong Anliursquo as 206

previously described (Zeng et al 2014) Based on our observations plastids in the juice of 207

lsquoHong Anliursquo also appeared to be differentiated from amyloplasts (Fig 5F) and the typical 208

globular chromoplasts were strongly accumulated in the juice of lsquoHong Anliursquo from MG stage 209

to full maturation (FMa) stage (Fig 5 G-I) Once the plastids appeared the pigment-210

accumulation areas within plastids gradually increased from IMG to Ma stages (Fig 5J) 211

which was largely correlated with a gradual increase of the average number of plastoglobules 212

per plastid from 321 at the MG stage to ~1370 at the Br stage and the FMa stage (Fig 5K) 213

However the number of plastoglobules showed a slight decline to 113 at the over-maturation 214

(OMa) stage Meanwhile the size of the plastids increased from 229 μm in diameter at the 215

MG stage to 259 μm at the Br stage and then stayed unchanged till the OMa stage (Fig 4L) 216

It is worth noting that the outer monolayer of plastoglobules appeared to be separate from the 217

internal main body (Supplemental Fig S3) at the OMa stage We also noted that the 218

chromoplasts from the juice maintained a round shape throughout the fruit maturation period 219

and that typical globular chromoplasts often contained concentrically arranged tubular 220

lamellae in the shape of a lsquohollowrsquo structure (Fig 4 H and I) More interestingly crystal 221

remnants frequently found in the chromoplasts of the sac membrane were not observed in the 222

chromoplasts of the juice suggesting that plastid differentiation in these two types of tissues 223

may follow different patterns To test if chloroplasts were present in the flesh of lsquoHong Anliursquo 224

during fruit maturation we performed an immunoblotting assay for two thylakoid-targeted 225

proteins involved in photosynthesis (Supplemental Fig S4) No signal for either protein was 226

detected from the total proteins of flesh throughout the entire fruit maturation process 227

including the earlier maturation stage (30 days after flowering) As positive control both 228

proteins were detected in the total protein extracts of the lsquoHong Anliursquo leaves These results 229

suggest that the chromoplasts in the flesh were probably not directly derived from 230

chloroplasts Thus it can be speculated that citrus flesh chromoplasts may be differentiated 231

from amyloplasts which is similar to the results of studies on the origin of chromoplasts in 232

Saffron flowers tobacco floral nectaries and peach palm fruit (Grilli Caiola and Canini 2004 233

Horner et al 2007 Hempel et al 2014) 234

their carotenogenic transcript levels 236

Carotenoid accumulation is a net result of different activities related to carotenoid metabolism 237

such as biosynthesis turnover and sequestration of carotenoids (Cazzonelli and Pogson 2010) 238

Hence it is conceivable that carotenoid accumulation during the development of globular- 239

and crystalloid- chromoplasts is an end result of coordinated expression of genes involved in 240

carotenoid biosynthesis degradation and storage To test this assumption the transcript levels 241

of the 16 carotenoid genes were selected to be compared between the segment membranes of 242

lsquoHong Anliursquo and lsquoAnliursquo where typical crystalloid and globular chromoplasts were formed 243

during fruit maturation (Fig 6) These genes included DXS and DXR involved in the plastidic 244

2-C-methyl-d-erythritol 4-phosphate (MEP) pathway eight genes in carotenoid biosynthesis 245

(PSY PDS ZDS CRTISO LCYb2 LCYe HYD and ZEP) two in carotenoid storage (FBN1 246

and FBN4) and four in carotenoid degradation (NCED2 NCED3 CCD4a and CCD4b) 247

As shown in Figure 6 most of the 16 genes showed a gradual increase in their transcript 248

levels during the formation of globular chromopalsts in lsquoAnliursquo except for CRTISO LCYe 249

CCD4a and CCD4b whose expression was relatively stable (lt2-fold change) The increased 250

expression of most carotenoid biosynthesis genes and the steady expression level of 251

carotenoid degradation gene (CCD4a and CCD4b) well explained the enhanced carotenoid 252

accumulation In contrast nearly all the tested genes showed enhanced expression during the 253

formation of crystalloid chromopalsts in lsquoHong Anliursquo except for CCD4a and CCD4b 254

Expression levels of CCD4a and CCD4b showed a ~3-fold decrease and a strong increase 255

(112-fold change) respectively (Fig 6) indicating that they might have different functions in 256

the formation of two distinct chromoplast types 257

Experimental design iTRAQ analysis and identification of plastid-localized proteins 258

Levels of gene transcription may not always be correlated with the expression of the proteins 259

of interest To understand global protein changes during chromoplast differentiation we 260

conducted an iTRAQ-based quantitative proteomic analysis of plastids purified from the flesh 261

of lsquoHong Anliursquo at four key colour stages (MG Br Ma and OMa) iTRAQ tag labelling of all 262

samples in each stage is summarized in Figure 7 To minimize system errors arising from 263

variations of the iTRAQ-based quantification system a protein mixture obtained by pooling 264

an equal amount of all tested samples was labelled with 113 tags as internal standard (IS) 265

Based on the chosen criteria for peptide and protein identification (see lsquoMaterials and 266

methodsrsquo) a total of 9236 unique peptides were identified (Supplemental Table S2) These 267

peptides were matched to 1905 unique protein groups in three independent biological 268

samples and 1596 of the 1905 unique protein groups were found to be mapped with at least 269

two unique peptides (Supplemental Table S3) with the corresponding false discovery rate of 270

each individual analysis lower than 1 (Sheng et al 2012) The proteins detected are well 271

distributed (ie approximately 10-20 of the total proteins were identified in each molecular 272

weight group) within a molecular range of 10 to 60 kDa (Supplemental Fig S6A) In addition 273

a good peptide coverage was achieved for most of the identified proteins (Supplemental Fig 274

S6B) When the cut-off value was set as 50 variation the coverage levels for the three 275

biological replicates at four chromoplast differentiation stages varied from 9899 to 9938 276

(Supplemental Fig S7A) and the coverage levels for the technological replicates at MG and 277

Br stages were 9929 and 9930 respectively (Supplemental Fig S7B) indicating that 278

there is a good biological and technological reproducibility of this iTRAQ-analysis 279

The raw data obtained from the analysis of the replicates of the citrus plastid proteins 280

were refined by comparing the set of proteins with five plastidial databases (AT-CHLORO 281

Plprot PPDB SUBA and Uniprot) and three predictors for subcellular localization (TargetP 282

Predotar and WoLF PSORT) Only proteins present in at least two plastidial databases or 283

predicted to be plastid-localized by one of the predictors were retained for further analysis 284

Additionally manual curation was performed when sequence information is available in the 285

literature An inventory of 1386 proteins (including 20 proteins encoded by the plastid 286

genome Supplemental Table S4) was constructed and 1016 of them were annotated with 287

iTRAQ values (Supplemental Table S5) The majority of these annotated proteins 288

(992=727) were predicted to be plastid-localized by at least one predictor Specifically 289

WoLF PSORT TargetP and Predotar respectively predicted 637 (870 proteins) 493 290

(673 proteins) and 395 (540 proteins) of the 1386 annotated proteins as being plastid-291

localized with a considerable degree of overlapping prediction (Supplemental Fig S8) For 292

example the percentage of proteins predicted to be plastid-localized by all the three programs 293

was as high as 332 (453 proteins) suggesting that these proteins are likely plastid-targeted 294

proteins BLAST searches against the plastid protein databases suggest that 187 of the 1386 295

proteins may be novel plastid-localized proteins (Supplemental Table S4) 296

Validation of differentially expressed proteins by Western blot 297

To test if the iTRAQ values of the predicted plastid-targeted proteins generally reflect the 298

actual protein abundance we selected three proteins that are known to have distinct plastid-299

localizations and show slight but clear development stage-fluctuations for the iTRAQ values 300

to determine protein abundance by immunoblotting analysis As shown in Figure 8 the 301

abundance of the protein binding to the chloroplast membrane (TIC40) showed a slight 302

decreasing tendency during chromoplast differentiation while plastoglobule-localized FBN1 303

and chloroplast-stroma-localized large subunit of Rubisco (RbcL) displayed a slight increase 304

from the MG stage to the Br stage followed by a decrease at the Br-to-OMa stage These 305

results were in good agreement with the corresponding iTRAQ values suggesting that the 306

data from our proteomic analysis are of high quality and may be useful for exploring how 307

changes in abundance of plastid-localized proteins are associated with chromoplast 308

differentiation on a whole proteome scale 309

Patterns of dynamic changes in protein abundance during amylo-to-chromoplast 310

transition 311

To test the idea mentioned above we subjected the iTRAQ data of the samples prepared from 312

fruit flesh of lsquoHong Anliursquo at three key colour stages (ie MG Br and Ma) to statistical 313

analyses for inferring if there is a significant change (p le005) of protein abundance for any 314

target protein The dynamic changes in protein abundance from the MG to the Ma stage could 315

be classified into seven patterns stable continuous increase early increase late increase 316

continuous decrease early decrease and late decrease (Fig 9A) Based on this classification 317

it was found that the proteins whose abundance remained constant (359) outnumbered those 318

whose abundance underwent an increase (53 continuous + 189 early + 41 late=283) or a 319

decrease (41 continuous + 92 early + 53 late =186) Notably among those proteins with a 320

change in abundance 189 (~228 of all 828 proteins examined) showed an early increase 321

and 92 (~111) showed an early decrease indicating that important changes in protein 322

abundance occurred in the early MG to Br stage (Fig 9A) 323

To get a clue as to what specific cellular processes are stable or changing during 324

chromoplast differentiation we further studied the stability or changes of proteins in different 325

functional classes Interestingly we found that 15 of 19 (789) proteins presumably 326

involved in lsquosignallingrsquo functional class remained stable in abundance In contrast only 3 of 327

18 (167) proteins predicted to be involved in metal handling remained constant The 328

percentage of proteins that showed no significant changes in abundance in the remaining 29 329

functional classes ranged from 704 to 30 (Fig 9B) Conceivably these proteins with 330

constant abundance may be important for maintaining basal metabolic functions of the 331

organelle during differentiation 332

Proteins falling into the lsquoprotein synthesisrsquo functional class showed most remarkable 333

decrease in abundance with 410 of proteins having an early decrease 131 having a 334

continuous decrease and 66 having a late decrease (Fig 9B) The majority of the proteins 335

in the lsquoelectron transportATP synthesisrsquo functional class also showed decrease in abundance 336

with 308 having an early decrease and 231 having a continuous decrease A lsquodecreasingrsquo 337

pattern of proteins involved in protein and ATP synthesis during chromplast differentiation 338

can be anticipated considering the lower metabolic activity of maturing organelles in general 339

Compared with the lsquodecreasingrsquo pattern the lsquoincreasingrsquo pattern is more popular among more 340

functional classes during chromoplast differentiation For example 442 of proteins 341

implicated in protein degradation showed an abundance increase during the chromoplast 342

differentiation with 288 having an early increase 96 having a continuous increase and 343

58 having a late increase More strikingly 833 of the proteins involved in metal 344

handling showed either early increase (778) or late increase (56) suggesting that metal 345

handling is implicated in chromoplast differentiation Also noticeable is that 50 of the 346

proteins involved in secondary metabolism and S-metabolism showed increase in abundance 347

mostly in the early stage which is consistent with the anticipated increase in the synthesis of 348

some important secondary metabolites such as carotenoids vitamin E and terpene in 349

chromoplasts at the Br stage Additionally 439 of redox amp stress-related proteins showed 350

increase in abundance possibly reflecting that more stresses were imposed to fruits which in 351

turn strengthened the stress responses during chromoplast differentiation 352

To further infer possible regulatory mechanisms underlying the changes of protein 353

abundance we constructed a heatmap that represents the hierarchical clustering of the 354

proteins into each functional class according to their abundance patterns The results indicate 355

that the four main clusters were distinguishable (Fig 10) Among them Cluster III is enriched 356

for stable proteins including those involved in lipid metabolism TCAorg hormone 357

metabolism cell protein targeting DNA amp RNA miscellaneous protein others minor CHO 358

metabolism co-factor amp vitamin metabolism and signalling Cluster II is the only cluster 359

consisting of proteins with a strong decrease in abundance including protein synthesis and 360

electron transportATP synthesis while Cluster I is enriched for proteins with increased 361

abundance that are involved in fermentation metal handling glycolysis and development 362

Two subclusters of Cluster IV (a c) contain stable proteins or proteins with increased 363

abundance and a few proteins with decreased abundance The sub-cluster IV (b) contains 364

proteins without a major bias in abundance patterns and these proteins are involved in 365

transport nucleotide metabolism redox amp stress and amino acid metabolism 366

Functional categories of differentially expressed proteins between OMa and Ma stages 367

On-tree storage is an effective method to delay the senescence process and has been widely 368

used in the citrus industry During this process a variety of physiological parameters are 369

changed such as the decrease of firmness and titratable acidity and the increase of color 370

index and maturity index (Saacutenchez et al 2013) However the mechanisms underlying the 371

regulation of chromoplast fate remains unclear To screen for differentially expressed proteins 372

during chromoplast senescence the corresponding iTRAQ-values of the samples from OMa 373

and Ma were averaged and compared Since iTRAQ quantification might underestimate the 374

amount of real fold-change between the labelled samples (Karp et al 2010) a protein with a 375

differencege14 fold and a P-valuele005 after false discovery rate control was regarded as 376

being differentially expressed between OMa and Ma (Benjamini and Hochberg 1995) 377

(Supplemental Table S6) Based on these criteria the expression of 76 proteins was 378

significantly higher in the OMa stage than in the Ma stage while the expression of 28 proteins 379

was the opposite To gain insight into the biological significance of such differential 380

expression the 104 proteins were categorized into functional classes by Mapman The 76 up-381

regulated proteins mainly fell into 5 categories stress amp redox TCAorg transport cell and 382

electron transportATP synthesis With regard to lsquostress and redoxrsquo class eleven stress-related 383

proteins had higher expression in OMa than in Ma stage for example glutathione 384

peroxidases6 (Cs5g316401) early responsive dehydration proteins (Cs2g083201) and 385

membrane-associated progesterone binding protein 3 (Cs9g171301) The significant increase 386

of these proteins involved in stress response in the present study is indicative of a high level 387

of redox activity in citrus chromoplasts and suggestive of an important role of these proteins 388

in sustaining plant defense reaction during chromoplast senescence Additionally almost all 389

the proteins categorized in lsquotransportrsquo and lsquoelectron transportATP synthesisrsquo classes showed 390

a dramatic increase in protein abundance at OMa stage Among them there were up to 14 391

ATP synthases and ATPADP carriers which were involved in energy supply (Supplemental 392

Table S6) These proteins underwent a dramatic and wide-ranging increase in protein 393

abundance suggesting that the ATP synthesis machinery was maintained at a good level 394

during chromoplast senescence The active supply of ATP might reflect the demand for 395

energy to ensure normal biological processes in the course of chromoplast senescence It is 396

well known that the ATP import or synthesis in non-green plastids is required for the 397

synthesis of several metabolites such as amino acids and carbohydrates (Neuhaus and Emes 398

2000) Consistently several proteins for amino acid metabolism were significantly increased 399

in abundance and proteins involved in the TCAorg for the supply of energy to the plastids 400

were also identified with a significant up-regulation By contrast some of the 28 down-401

regulated proteins are involved in major CHO metabolism hormone metabolism protein 402

degradation and protein synthesis Conceivably differential expression of so many plastid-403

localized proteins may directly or indirectly contribute to chromoplast senescence 404

Discussion 405

Two types of putative amylo-to-chromoplast transition in citrus flesh 406

Unlike flavedo of citrus fruits where chromoplasts are converted from chloroplasts (Eilati et 407

al 1975) chromoplasts in the flesh of citrus fruits seem to be differentiated from amyloplasts 408

There are two putative different amylo-to-chromoplast patterns for citrus pulp each with its 409

own unique characteristics Figure 11 depicts a tentative model for the sequential 410

development of citrus flesh plastids The starch granules were degraded rapidly and 411

transformed into plastids with a few plastoglobules (Fig 11 A and B) In Arabidopsis the 412

globules were documented to be swelled and separated from thylakoidal membranes (Ben-413

Shaul and Naftali 1969 Austin et al 2006) In our study the mechanism underlying globular 414

formation remains unknown as the association of these plastoglobules with membranes or 415

other related structural elements was not directly observed However we can not rule out the 416

possibility that the degradation of starch granules provides energy or metabolic precursors for 417

subsequent formation of globular elements as plastoglobules were generally found around the 418

degraded starch granules in the subsequent stage (Fig 11D) Since key biosynthetic enzymes 419

are targeted to plastoglobules (Ytterberg et al 2006 Schweiggert et al 2011 Davidi et al 420

2015) carotenoid biosynthesis is supposed to be initiated in these globules Additionally the 421

undulating lines in Figure 11C can also been interpreted as early carotenoid depositions in 422

tomato (Harris and Spurr 1969) In parallel with carotenoid accumulation tubular structures 423

apparently developed independently of the globular structure resulting in the formation of 424

tubular-globular chromoplasts (Fig 11 D and E) 425

The crystal remnants were accumulated in the chromoplasts of red tissues but the 426

additional formation of comparably large crystalloids was apparently induced by the 427

concomitant biosynthesis of lipid-soluble lycopene (Fig 11F Schweiggert et al 2011) There 428

are two hypotheses regarding the biogenesis of lycopene-rich structures that the structures 429

might (1) be associated with thylakoids system in the transformation of Lycopersicum 430

esculentum (Ben-Shaul and Naftali 1969) and Aglaonema commutatum (Knoth 1981) where 431

lycopene is tightly assembled into photosynthesis structures or (2) derive from plastid 432

membrane (Schweiggert et al 2011) Since such crystal remnants were frequently found to 433

be close to the plastid envelope (Fig 11F) and thylakoid structures appeared to be absent 434

within citrus flesh the lsquolycopenicrsquo membranes in red-fleshed lsquoHong Anliursquo might be derived 435

from the plastid envelop or other unknown structures specific to citrus flesh 436

Complex regulation of carotenoid metabolism during globular chromoplast 437

differentiation 438

Chromoplast development is accompanied by massive synthesis and accumulation of 439

carotenoids and is often regulated at the transcriptional level (Cazzonelli and Pogson 2010) 440

whereas the regulatory mechanism at the protein level is still poorly understood In this study 441

we found that carotenoid metabolism is co-ordinately regulated by both transcription and 442

protein levels (Fig 6 and Fig 12A) For example four members of the MEP pathway namely 443

DXR (Cs8g070201) and 2-C-methyl-D-erythritol 24-cyclodiphosphate synthase (ISPF 444

Cs5g030501) hydroxymethylbutenyl 4-diphosphate synthase (HDS Cs8g167002) and 445

hydroxymethylbutenyl 4-diphosphate reductase (HDR Cs8g070201) showed early or 446

continuous increase (plt005) in abundance during chromoplast differentiation coinciding 447

with the accumulation of isoprenoids and carotenoids Additionally the synthesis of 448

violaxanthin and lutein needs to be facilitated by HYD catalysis (Cazzonelli and Pogson 449

2010) In comparison with the significant increase of HYD transcriptional level during 450

chromoplast differentiation which may explain the large accumulation of violaxanthin and 451

lutein in globular chromoplasts we only identified a limited number of enzymes participating 452

in downstream carotenoid synthesis for instance HYD (Cs9g192702) and NCED5 453

(Cs2g032701) We therefore presume that HYD mainly plays a key role in the violaxanthin 454

biosynthesis at transcriptional level Consistently the expression of HYD promotes the 455

violaxanthin biosynthesis in Arabidopsis seeds (Yu et al 2007) and citrus callus (Cao et al 456

2012) The relatively higher abundance of upstream pathway proteins led to the biosynthesis 457

of lycopene while the downstream enzyme proteins were present at low abundance or not 458

detected which may be important for gauging the metabolic flux into carotenoid synthesis 459

pathway Consistently the carotenoid-accumulation areas were formed as maturation 460

proceeded (Fig 5J) 461

Plastoglobule composition and formation within citrus flesh chromoplasts 462

A rapid increase in the number of plastoglobules is the most representative feature during 463

chromoplastogenesis in citrus flesh We therefore performed BLASTP of the citrus plastid 464

proteome against the list of proteins of the plastoglobules established by Lundquist et al 465

(2012) Among the 30 plastoglobule-localized proteins in Arabidopsis 24 were identified to 466

be citrus homologies 16 of which were quantified (Supplemental Table S7) Among the 16 467

quantified proteins 11 had a continuouslyearly increased expression (Fig 12B) including 468

ABC1K1379 (activity of bc1complex kinases 1379) Fibrillin 24 (FBN24) Esterase 1 469

(Cs2g140901 Cs2g141101 Cs2g141201) Flavin reductase-related 2 (Cs2g036001) and 470

an unknown protein (Cs5g281301) 471

Among ABC1 kinases subfamily 6 proteins (ABC1K1 3 5 6 7 and 9) predicted to 472

localize in plastoglobules constitute the core components of the plastoglobule proteome in 473

chromoplasts of citrus flesh (Supplemental Fig S9) which is similar to the case of 474

Arabidopsis chloroplasts (Lundquist et al 2012) The AtABC1K7 mutant developed larger 475

plastoglobules and lipid composition of the chloroplast membrane when compared with wide-476

type Arabidopsis (Manara et al 2014 2015) Additionally ABC1K13 as a complex binds 477

with phosphorylated tocopherol cyclase to affect plastoquinone metabolism (Lundquist et al 478

2013 Martinis et al 2013 Manara et al 2014 Martinis et al 2014) Their double mutation 479

also leads to abnormal accumulation of plastoglobules in Arabidopsis The increase of 480

ABC1K1 3 and 7 in abundance during chromoplastogenesis suggests that they might 481

positively regulate plastoglobule development A previous study revealed that ABC1K9 482

might be co-expressed with FBNs and MEP enzymes (Lundquist et al 2012) suggesting that 483

the increase of ABC1K9 in abundance might play an essential role in plastoglobule 484

development and carotenoid metabolism In the present study we observed an early or 485

continuous increase in abundance of ABC1K1 3 7 and 9 during amylo-to-chromopalst 486

transition which confirms the speculation that these proteins may have distinct modulating 487

functions in plastogobule development in citrus 488

The FBN family is required for leaf or fruit development to regulate early generation or 489

extension of plastogobules (Lundquist et al 2012) In this study FBNs family were the most 490

abundant in chromoplast plastsoglobules accounting for 685 of total identified peptides 491

(Supplemental Fig S9) which is similar to the case in Arabidopsis chloroplasts (Lundquist et 492

al 2012) It is not surprising to find an increasing abundance of FBN24 in our study during 493

chromoplast differentiation confirming its role in the structural development of 494

plasotogobules (Singh and McNellis 2011) FBN1 has been well established in several 495

studies with a positive regulatory role in plastoglobule development (Singh and McNellis 496

2011) In our proteomic analysis it is noteworthy that FBN1 only showed a slight increase 497

(plt005) in abundance from MG to Br stages followed by a slight decrease from Br to OMa 498

stages (plt005) This change tendency was confirmed by immunoblotting analysis of FBN1 499

(Fig 8) which is also in line with the variation in the number of plastoglobules from Br to 500

OMa stages However the fold-change in protein abundance was much lower than the rapid 501

increase in the number of plastoglobules from MG to Br stages To explain this discrepancy 502

we conducted another series of immunoblotting analyses by including two earlier stages of 503

fruit development (Supplemental Fig S10) which confirmed the tendency of a slight increase 504

in abundance from MG to Br stages (Fig 8) More importantly the abundance of FBN1 was 505

enhanced rapidly from 90 DAF to 120 DAF These data indicate that FBN1 expression is 506

sensitive at earlier stages when the average number of plastoglobules per plastid increased 507

from 0 to about 32 and the subsequent slowdown of the increase tendency might be due to 508

the dull response of proteins to developmental conditions or environmental stresses on the 509

basis of the pre-existing plastoglobules or regulation via post-translational modification such 510

as protein phosphorylation (Zeng et al 2014) In this study we selected MG stage as the first 511

stage for plastid differentiation analysis for two reasons (1) the fruit at MG stage gains the 512

capacity to ripen and its plastid ultrastructure shows apparent difference from that in the 513

subsequent maturation stages (2) the accumulation of high-density starches in amyloplasts at 514

an earlier stage makes it difficult to use the same density gradient centrifugation to purify 515

plastids Taken together our data concerning these plastoglobule-localized proteins suggest 516

that they have important and exclusive functions in regulating the differentiation of 517

chromoplasts in the flesh of citrus fruits 518

Stability in protein import loss of ribosome assembly and build-up of chromoplast 519

proteases 520

Translocation of proteins into plastids is of importance for chromoplast biogenesis (Egea et 521

al 2010) Here we identified nearly all the elements of the protein import machinery and 522

classified them into lsquoprotein targetingrsquo which showed a constant abundance during amylo-to-523

chromoplast transition (Supplemental Table S5) Among these elements TOC159 which is 524

tightly bound to the transit peptides serves as receptors for chloroplast-destined preproteins 525

(Aronsson and Jarvis 2008) and is thought to interact with casein kinase 2 to affect the 526

chromoplast biogenesis (Agne et al 2010 Zeng et al 2014) TOC75 is the main pore of the 527

TOC complex deeply embedded within the outer membrane forming a translocation channel 528

(Jarvis 2008) Additionally TIC11040 major components of TIC complex play a critical 529

role in protein import (Jarvis and Lopez-Juez 2013) These four proteins remained unchanged 530

in abundance among which TIC40 was confirmed by immunoblotting analysis The constant 531

protein abundance of TOCTIC complex suggests the continuous import of nuclear-encoded 532

proteins during chromoplast differentiation 533

Consistent with a previous finding in chloro-to-chromoplast transition in tomato (Barsan 534

et al 2012) the translation machinery of the citrus chromoplast underwent a great loss of 535

efficiency with a strong decrease in abundance of ribosomal proteins of both the chloroplast-536

encoded small 30S subunit and the large 50S subunit complexes as well as those of nuclear-537

encoded small 40S subunit and the larger 60S subunit during chromoplast differentiation (Fig 538

12C) This finding is also in agreement with the gradual down-regulation of plastid translation 539

during tomato chromoplast differentiation observed by Kahlau and Bock (2008) These 540

results indicate that there probably exists a conservative property of translation machinery in 541

chromoplast biogenesis which arises not only from non-photosynthetic plastids like 542

amyloplasts but also from photosynthetic chloroplasts 543

Plastids undergo drastic changes morphologically and physiologically at different 544

developmental stages and under different environmental conditions (Egea et al 2010) 545

Keeping a balance between protein biosynthesis and degradationprocessing is crucial for 546

accomplishing structurally dynamic changes of plastids and maintaining their homeostasis 547

(Sakamoto 2006) A set of caseinolytic peptidase (Clp) proteases increased in abundance 548

during chromoplast differentiation including 5 lsquocontinuous increasersquo 15 lsquoearly increasersquo and 549

3 lsquolate increasersquo proteins while only 3 proteins showed the lsquodecreasingrsquo pattern (Fig 12D and 550

Supplemental Table S5) These Clp proteases formed a chaperone complex to drive the 551

import of proteins into the plastids cleave the transit peptide and recycle plastid components 552

In Arabidopsis knocking out most members of the Clp proteases family results in reduced 553

plant growth with pale-green or variegated leaf phenotype indicating the crucial role of Clp 554

complex in chloroplast development (Meinke et al 2008 Liu et al 2010 Olinares et al 555

2011) Previous studies have revealed that the Clp complex one of the prominent complexes 556

increases in abundance during the transition from proplastids or etioplasts into chloroplasts 557

(Kanervo et al 2008 Majeran et al 2010) Our study shows that the proteins involved in 558

protein degradation are among the proteins of the highest abundance during amylo-to-559

chromoplast transition while an apparent increasing tendency in protein abundance of Clp 560

complex suggests that the machinery of protein degradation or processing during chromoplast 561

differentiation might have distinct modulating functions that are conserved during the 562

biogenesis of both chloroplasts and chromoplasts 563

The plastid proteome of citrus flesh also consists of a number of membrane-bound ATP-564

dependent Zn-metallo proteases (FtsH1 Cs3g14380 FtsH2 Cs2g15180 FtsH5 Cs3g27030 565

FtsH7 Cs8g19900 FtsH11 Cs9g18420 FtsH12 orange11t03252 FtsHi1 Cs5g02060 566

FtsHi2 Cs1g15540 FtsHi3 Cs7g14690) (Supplemental Table S5) Several FtsH proteins 567

such as FtsH1 2 and 5 are key components of thylakoid which has been well demonstrated 568

by previous studies (Zaltsman et al 2005 Sakamoto 2006 Kato and Sakamoto 2010) Since 569

citrus flesh lacks chloroplasts and cannot perform photosynthesis throughout the entire 570

maturation stage it is reasonable to speculate that these proteins are localized in other 571

subcellular structures within plastids rather than thylakoids Therefore the identification of 572

these proteins in non-photosynthetic plastids may imply a basic role other than photosynthesis 573

Additionally another type of ATP-independent metalloprotease present in the citrus flesh 574

plastid proteome such as stromal pre-sequence proteases (PreP2 Cs8g057201) and M24 575

peptidase (Cs4g135101) has been described as necessary for plastid development 576

(Cederholm et al 2009 Kmiec and Glaser 2012) The continuous increase of PreP2 and 577

M24 peptidase probably contributed to chromoplast differentiation by means of the removal 578

and degradation of transit peptides within the plastids during amylo-to-chromoplast transition 579

In summary our data suggest that chromoplast differentiation in citrus flesh is correlated not 580

only with the repression of protein synthesis and promotion of protein degradationprocessing 581

but also with the stable maintenance of protein import via the TOCTIC complex These 582

dynamic changes are associated with the remodelling of protein systems and may have a 583

crucial role in governing chromoplast biogenesis and differentiation 584

Conclusions 585

In this study we examined two tissue-specific chromoplast types converted from amyloplasts 586

which are associated with the composition of carotenoids in sweet orange Subsequently we 587

used iTRAQ-based high-throughput proteomics to construct an inventory of the proteins 588

present at different fruit maturation stages in sweet orange during plastid differentiation 589

which expands the knowledge of the plant plastid proteome available so far Additionally our 590

results highlight several aspects of the regulation of global reorganization during chromoplast 591

differentiation The change pattern in protein abundance reported here is partially in 592

agreement with the chromoplast differentiation in tomato (Barsan et al 2012) but shows 593

great differences with the proplastidsetioplast-to-chloroplast transition in maizerice 594

(Kleffmann et al 2007 Majeran et al 2010) The overall findings from this research provide 595

useful information about the chromoplast biogenesis in sweet orange flesh and facilitate a 596

better understanding of its chromoplast differentiation and senescence 597

Materials and methods 598

Plant materials 599

Fruits of lsquoAnliursquo sweet orange (C sinensis L Osbeck) and its red-fleshed mutant lsquoHong 600

Anliursquo lsquoNewhallrsquo Navel orange (C sinensis Osbeck) and its red-fleshed mutant lsquoCara Cararsquo 601

were harvested at the National Centre of Citrus Breeding located at Huazhong Agricultural 602

University Wuhan China Fruits of lsquoHong Anliursquo and lsquoAnliursquo were collected at six 603

maturation stages after careful selection (1) IMG (immature green stage 90 days after 604

flowering (DAF)) (2) MG (mature green stage 140 DAF) at which fruit nearly reaches full 605

size with green peel and without pigment accumulation in the flesh (3) Br (colour breaker 606

stage 170 DAF) at which fruit is characterized by a change in colour from green to a bit 607

orange in peel and a bit red in flesh (4) Ma (maturation stage 200 DAF) at which fruit has a 608

red colour in the whole flesh and is harvested 30 days after Br (5) FMa (full maturation stage 609

230 DAF) (6) OMa (over-maturation stage 260 DAF) at which fruit is at over- maturation 610

stage and stored on-tree under a low temperature in winter China Fruits at each stage were 611

harvested from three different trees and 10 representative fruits were harvested from each 612

tree thus a total of 30 fruits were obtained for each genotype The edible citrus segment was 613

then gently separated into segment membrane sac membrane and juice followed by freezing 614

in liquid nitrogen for subsequent extraction of carotenoids and RNA Fruits of lsquoHong Anliursquo 615

at MG Br Ma and OMa stage were sampled from three selected trees on each harvest day 616

with 40 representative fruits from each tree thus a total of 120 fruits per stage were harvested 617

and aliquoted into three portions for immediate chromoplast purification as previously 618

described (Zeng et al 2014) Plastids of flesh were prepared in three biological replicates 619

followed by immediate freezing in liquid nitrogen and storage at -80oC until use 620

Microscopy observation and analysis 621

The segment membrane and sac membrane were carefully cut into small slices using a frozen 622

sectioning technique (Leica CM1900 Germany) A drop of juice was gently placed onto 623

microscope slides These fresh tissues were immediately observed under a microscope (BX61 624

Olympus) equipped with a DP70 camera to compare the structural characteristics of 625

chromoplasts 626

An aliquot of the same segment membrane and sac membrane was also used for 627

transmission electron microscopy (TEM) analysis The plastids were enriched from the juice 628

using gradient centrifugation as described in Zeng et al (2014) Samples for TEM analysis 629

were fixed stained and observed as previous description (Zeng et al 2011) The diameter of 630

the plastids was measured by (major axis + minor axis)2 using Image J software 631

(httprsbwebnihgovij) 632

Carotenoid profile analysis 633

Carotenoid extraction and analysis using reversed-phase high performance liquid 634

chromatography (RP-HPLC) were conducted as previously described (Cao et al 2012) 635

Approximately 4 g of citrus fruit fresh weight (FW) was extracted separately from the 636

segment membrane sac membrane and juice from lsquoAnliursquo and lsquoHong Anliursquo at the fully 637

maturation stage The carotenoids were identified by their characteristic absorption spectra 638

and typical retention time based on the literature and standards from CaroNature Co (Bern 639

Switzerland) The output data were obtained from three experiment replicates 640

Quantitative analysis of gene expression 641

RNA was extracted from segment membranes and juice of lsquoAnliursquo and lsquoHong Anliursquo fruits as 642

described by Liu et al (2007) RNA quality and quantity measurement first-strand cDNA 643

synthesis and real-time RT-PCR were performed according to Cao et al (2012) The primer 644

pairs used in this experiment came from Cao et al (2012) or were designed using the Primer 645

Express software 3 (Applied Biosystems Foster City CA USA) both of which are shown in 646

Supplemental Table S1 Actin was used as an endogenous control to normalize the expression 647

in different samples At least three replicates were conducted in real-time RT-PCR analysis 648

Protein extraction and Western blot 649

Polyclonal antibodies at the appropriated dilution against rubisco large subunit (RbcL 650

13000) fibrillin 1 (FBN1 12000) two thylakoid membrane marker proteins (PsbA | D1 651

protein of PSII C-terminal 12000 and Lhcb2 | LHCII type II chlorophyll ab-binding 652

protein 12000) and chloroplast inner envelope membrane translocon complex protein40 653

(TIC40 12000) RbcL FBN1 PsbA and Lhcb2 were obtained from Agriserareg and TIC40 654

kindly provided by Paul Jarvisrsquos lab (University of Oxford) 30 μg proteins were separated by 655

12 SDS-PAGE and detection by antibodies were performed as described in Zeng et al 656

(2011) or Ling et al(2012) At least two independent replicates were conducted for each 657

western blot analysis 658

Protein preparation digestion and iTRAQ labelling 659

The pooled plastids were treated with acetone and the precipitated proteins were resuspended 660

by adding approximately 500 μL STD buffer (4 SDS 1 mM DTT 150 mM Tris-HCl pH 661

80 containing cocktail for proteases (EDTA-free Roche)) After 5 min incubation in boiling 662

water the homogenate was sonicated on ice The crude extract was then incubated in boiling 663

water again and clarified by centrifugation at 14000timesg for 40 min at 25 oC before the 664

supernatant was collected The protein concentration was determined using a bicinchoninic 665

acid protein assay kit (BCA Beyotime China) 666

Protein digestion was performed based on the FASP-method as previous description 667

(Wiśniewski et al 2009) and the resulting peptide mixtures were labelled with the 8-plex 668

iTRAQ (isobaric tags for relative and absolute quantification) reagent by following the 669

manufacturerrsquos instructions (Applied Biosystems) 80 μg of proteins from each sample was 670

pooled as internal standard (IS) Briefly 250 μg of proteins for each sample (including IS) 671

was incorporated into 30 μL STD (4 SDS 150 mM Tris-HCl pH 80 100 mM DTT) The 672

UA buffer (8 M Urea 150 mM Tris-HCl pH 80) was used to remove DTT detergent and 673

other low-molecular-weight components by repeated ultrafiltration (Microcon units 30 kDa) 674

Next the samples were incubated in darkness for 30 min by adding 100 μL 50 mM 675

iodoacetamide into UA buffer to block the reduced cysteine residues The filters were washed 676

with 100 μL UA buffer three times followed by two washes with 100 μL DS buffer (50 mM 677

triethylammoniumbicarbonate pH 85) Finally the protein suspension was digested 678

overnight at 37 degC with 6 μg trypsin (Promega) dissolved in 40 μL DS buffer and the 679

resulting peptides were collected as a filtrate The peptide content was estimated by UV light 680

spectral density at 280 nm using an extinction coefficient of 11 of 01 (gL) solution that 681

was calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins 682

Two iTRAQ Reagent 8-plex kits (Applied Biosystems) were used to label the peptide 683

samples (each 25 μg) of plastids isolated at each developmental stage according to 684

manufacturers specifications (Fig 7) Three biological replicates were iTRAQ-labelled for 685

MG Br Ma and OMa stages as well as one technological replicate for MG and Br stages 686

Protein samples of each stage were equally mixed as internal standard (IS) and then labelled 687

with 113 tag MG-3_E2_115 and Br-1_E1_117 were technological replicates for MG-688

4_E2_116 and Br-2_E1_121 respectively 689

Sample fractionation using SCX chromatography 690

Prior to LC-MSMS analysis peptides were purified from excess labelling reagent by strong 691

cation exchange (SCX) chromatography using AKTA Purifier 100 (GE Healthcare) Peptides 692

were dried in a vacuum concentrator and then dissolved in 2 mL buffer A (10 mM KH2PO4 in 693

25 acetonitrile (ACN) pH 30) to reconstitute and acidify the dried peptide mixtures which 694

were subsequently loaded onto a Polysμlfoethyl 46times100 mm column (5 microm 200Aring 695

PolyLCInc Maryland USA) at a flow rate of 1 mLmin A suitable gradient elution (0-696

10 for 32 min 10-20 for 10 min 20-45 for 15 min and 45-100 for 13 min) was 697

applied to separate the peptides at a flow rate of 1mLmin with buffer B (10 mM KH2PO4 698

500 mM KCl in 25 ACN pH 30) Eluted peptides were collected and desalted by an offline 699

fraction collector Finally a total of 10 sample pools including 30 collected fractions were 700

desalted on C18 Cartridges (Empore SPE Cartridges (standard density) bed ID 7 mm 701

volume 3 mL Sigma) Each final fraction was concentrated by a vacuum concentrator and 702

reconstituted in 40 microL of 01 (vv) trifluoroacetic acid All samples were stored at -80oC for 703

LC-MSMS analysis 704

LC-MSMS analysis by Q Exactive 705

A nanoflow HPLC instrument (Easy nLC ProxeonBiosystems now Thermo Fisher Scientific) 706

was coupled on-line to a Q Exactive mass spectrometer The peptide mixture (5 μg) was 707

loaded onto a C18-reversed phase column (Thermo Scientific Easy Column 10 cm long 75 708

μm inner diameter 3 μm resin) in buffer A (01 Formic acid) and separated with a linear 709

gradient of buffer B (01 Formic acid and 80 ACN) at a flow rate of 250 nLmin 710

controlled by IntelliFlow technology over 140 min 711

MS data were acquired using a data-dependent ldquotop10rdquo method dynamically choosing 712

the most abundant precursor ions from the survey scan (mass range300-1800 mz) for HCD 713

fragmentation Determination of the target value was based on predictive Automatic Gain 714

Control (pAGC) Dynamic exclusion duration was 60 s Survey scans were acquired at a 715

resolution of 70000 at mz 200 and resolution for HCD spectra was set to 17500 at mz 200 716

Normalized collision energy was 27 eV and the under fill ratio which specifies the minimum 717

percentage of the target value likely to be reached at maximum fill time was defined as 01 718

The instrument was run with peptide recognition mode enabled 719

MSMS spectra were searched using MASCOT engine (Matrix science London UK 720

version 22) embedded into Proteome Discoverer 13 (Thermo Electron San Jose CA) 721

against a non-redundant Protein Index C sinensis protein database (Huazhong Agricultural 722

University httpcitrushzaueducn) and the proteome sequences predicted from the 723

chloroplastic genome (accession no NC_008334) Proteins were identified with the following 724

parameters peptide mass tolerance of 20 ppm fragment mass tolerance of 01 Da trypsin 725

enzyme with the number of missed cleavages up to 2 carbamidomethyl cysteine residues 726

as fixed modification and oxidation of methionine residues as variable modification 727

Software Proteomics Tools (version 316) was used to further process the MASCOT search 728

results of each SCX elution The BuildSummary program was used for assembling protein 729

identifications according to a target-decoy in shotgun proteomics All reported data were 730

based on 99 confidence intervals for protein identification as determined by false discovery 731

rate (FDR) le1 FDR=N (decoy)2 (N (decoy) + N (target)) which is a widespread 732

normalization process and considered as a standard way to preprocess data in proteomics 733

(Zeng et al 2014 Zheng et al 2014) 734

Search against existing databases functional classification targeting predictions and 735

curation 736

Identified proteins were functionally classified according to Mapman 737

(httpmapmangabipdorgwebguesthome) using annotations retrieved from databases and 738

manual curation based on PPDB (httpppdbtccornelledu) Homologies of the detected 739

proteins were searched against five plastidial databases (i) two databases specific to plastids 740

(Plprot httpwwwplprotethzch AT_CHLORO httpwwwgrenobleprabifrat_chloro) 741

and (ii) three databases comprising plastidial subsets (SUBA 742

httpsubaplantenergyuwaeduau PPDB httpppdbtccornelledu Uniprot 743

httpwwwuniprotorg) Predictions of subcellular localization were carried out using 744

TargetP (httpwwwcbsdtudkservicesTargetP) Predotar 745

(httpurgiversaillesinrafrpredotarpredotarhtml) and WoLF PSORT 746

(httpwolfpsortseqcbrcjp) Those proteins were tentatively considered to be plastid 747

proteins if they met at least one of the following two criteria (i) the protein candidates were 748

predicted to localize in plastids by at least one of the three web programs (Wolf PSORT 749

TargetP and Predotar) and (ii) the protein candidates were present in at least two of the five 750

databases mentioned above (Plprot AT_CHLORO SUBA PPDB and Uniprot) 751

Differential abundance analysis 752

The final ratios of proteins were normalized by the median average protein ratio of the equal 753

mix of different labelled samples Only proteins detected in all runs (every biological 754

replicate) were included in the data set Data were expressed as the mean plusmn SD and evaluated 755

by Studentrsquos t-test using Microsoft Excel Software (ldquoTTESTrdquo function (two tailed)) The 756

trends of changes in abundance of proteins between stages were calculated with a 5 757

significance level and referred to as 0 for no change -1 for decrease and +1 for increase 758

Acknowledgments 759

We would like to thank the Guangxi Citrus Research Institute for providing citrus materials 760

and the Shanghai Applied Protein Technology Co Ltd for technology support and helpful 761

advice We are grateful to Professor Paul Jarvis for a generous gift of antibody (TIC40) Great 762

thanks should also go to Prof Zuoxiong Liu and Hanchang Zhu for reading and editing the 763

manuscript 764

References 766 Agne B Andregraves C Montandon C Christ B Ertan A Jung F Infanger S Bischof S 767

Baginsky S Kessler F (2010) The acidic A-domain of Arabidopsis TOC159 occurs 768 as a hyperphosphorylated protein Plant Physiol 153 1016-1030 769

Aronsson H Jarvis P (2008) The chloroplast protein import apparatus its components and 770 their roles 1861-1370 771

Austin JR Frost E Vidi PA Kessler F Staehelin LA (2006) Plastoglobules are lipoprotein 772 subcompartments of the chloroplast that are permanently coupled to thylakoid 773 membranes and contain biosynthetic enzymes Plant Cell 18 1693-1703 774

Barsan C Zouine M Maza E Bian W Egea I Rossignol M Bouyssie D Pichereaux C 775 Purgatto E Bouzayen M (2012) Proteomic analysis of chloroplast-to-chromoplast 776 transition in tomato reveals metabolic shifts coupled with disrupted thylakoid 777 biogenesis machinery and elevated energy-production components Plant Physiol 778 160 708-725 779

Ben-Shaul Y Naftali Y (1969) The development and ultrastructure of lycopene bodies in 780 chromoplasts ofLycopersicum esculentum Protoplasma 67 333-344 781

Benjamini Y Hochberg Y (1995) Controlling the false discovery rate a practical and 782 powerful approach to multiple testing J Roy Stat Soc B 57 289-300 783

Bian W Barsan C Egea I Purgatto E Chervin C Zouine M Latcheacute A Bouzayen M 784 Pech J-C (2011) Metabolic and molecular events occurring during chromoplast 785 biogenesis Journal of Botany 2011 786

Cao HB Zhang JC Xu J Ye JL Yun Z Xu Q Xu J Deng XX (2012) Comprehending 787 crystalline β-carotene accumulation by comparing engineered cell models and the 788 natural carotenoid-rich system of citrus J Exp Bot 63 4403-4417 789

Cazzonelli CI Pogson BJ (2010) Source to sink regulation of carotenoid biosynthesis in 790 plants Trends Plant Sci 15 266-274 791

Cederholm SN Baumlckman HG Pesaresi P Leister D Glaser E (2009) Deletion of an 792 organellar peptidasome PreP affects early development in Arabidopsis thaliana Plant 793 Mol Biol 71 497-508 794

Davidi L Levin Y Ben-Dor S Pick U (2015) Proteome analysis of cytoplasmatic and 795 plastidic b-carotene lipid droplets in Dunaliella bardawil Plant Physiol 167 60ndash79 796

Egea I Barsan C Bian W Purgatto E Latcheacute A Chervin C Bouzayen M Pech JC 797 (2010) Chromoplast differentiation current status and perspectives Plant Cell 798 Physiol 51 1601-1611 799

Eilati S BUDOWSKJ P Monselise S (1975) Carotenoid changes in the lsquoShamoutirsquo orange 800 peel during chloroplast-chromoplast transformation on and off the tree J Exp Bot 26 801 624-632 802

Eilati S SP M Budowski P (1969) Seasonal development of external color and carotenoid 803 content in the peel of ripening Shamouti oranges J Am Soc Hortic Sci 94 346-348 804

Fanciullino AL Cercos M Dhuique-Mayer C Froelicher Y Talon M Ollitrault P 805 Morillon R (2008) Changes in carotenoid content and biosynthetic gene expression 806 in juice sacs of four orange varieties (Citrus sinensis) differing in flesh fruit color J 807 Agric Food Chem 56 3628ndash3638 808

Fanciullino AL Dhuique-Mayer C Luro F Casanova J Morillon A Ollitrault P (2006) 809 Carotenoid diversity in cultivated citrus is highly influenced by genetic factors J 810 Agric Food Chem 54 4397ndash4406 811

Frey-Wyssling A Schwegler F (1965) Ultrastructure of the chromoplasts in the carrot root J 812 Ultra Mol Struct R 13 543-559 813

Grilli Caiola M Canini A (2004) Ultrastructure of chromoplasts and other plastids in 814 Crocus sativus L(Iridaceae) Plant Biosyst 138 43-52 815

Harris WM Spurr AR (1969) Chromoplasts of tomato fruits II The red tomato Am J Bot 816 56 380-389 817

Hempel J Amrehn E Quesada S Esquivel P Jimeacutenez VM Heller A Carle R 818 Schweiggert RM (2014) Lipid-dissolved γ-carotene β-carotene and lycopene in 819 globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits Planta 240 1-820 14 821

Horner H Healy R Ren G Fritz D Klyne A Seames C Thornburg R (2007) Amyloplast 822 to chromoplast conversion in developing ornamental tobacco floral nectaries provides 823 sugar for nectar and antioxidants for protection Am J Bot 94 12-24 824

Iglesias DJ Cercoacutes M Colmenero-Flores JM Naranjo MA Riacuteos G Carrera E Ruiz-825 Rivero O Lliso I Morillon R Tadeo FR (2007) Physiology of citrus fruiting Braz 826 J Plant Physiol 19 333-362 827

Jarvis P (2008) Targeting of nucleus‐encoded proteins to chloroplasts in plants New Phytol 828 179 257-285 829

Kahlau S and Bock R (2008) Plastid transcriptomics and translatomics of tomato fruit 830 development and chloroplast-to-chromoplast differentiation chromoplast gene 831 expression largely serves the production of a single protein Plant Cell 20 856-874 832

Kanervo E Singh M Suorsa M Paakkarinen V Aro E Battchikova N Aro EM (2008) 833 Expression of protein complexes and individual proteins upon transition of etioplasts 834 to chloroplasts in pea (Pisum sativum) Plant Cell Physiol 49 396-410 835

Karp NA Huber W Sadowski PG Charles PD Hester SV Lilley KS (2010) Addressing 836 accuracy and precision issues in iTRAQ quantitation Mol Cell Proteomics 9 1885-837 1897 838

Kato Y Sakamoto W (2010) New insights into the types and function of proteases in 839 plastids Int Rev Cel Mol Bio 280 185-218 840

Kilcrease J Collins AM Richins RD Timlin JA OConnell MA (2013) Multiple 841 microscopic approaches demonstrate linkage between chromoplast architecture and 842 carotenoid composition in diverse Capsicum annuum fruit Plant J 76 1074-1083 843

Kim JE Rensing KH Douglas CJ Cheng KM (2010) Chromoplasts ultrastructure and 844 estimated carotene content in root secondary phloem of different carrot varieties 845 Planta 231 549-558 846

Kleffmann T von Zychlinski A Russenberger D Hirsch-Hoffmann M Gehrig P 847 Gruissem W Baginsky S (2007) Proteome dynamics during plastid differentiation in 848 rice Plant Physiol 143 912-923 849

Kmiec B Glaser E (2012) A novel mitochondrial and chloroplast peptidasome PreP Physiol 850 Plantarum 145 180-186 851

Knoth R (1981) Ultrastructure of lycopene containing chromoplasts in fruits of Aglaonema 852 commutatum schott (Araceae) Protoplasma 106 249-259 853

Knoth R Hansmann P Sitte P (1986) Chromoplasts of Palisota barteri and the molecular 854 structure of chromoplast tubules Planta 168 167-174 855

Li L Paolillo DJ Parthasarathy MV DiMuzio EM Garvin DF (2001) A novel gene 856 mutation that confers abnormal patterns of β‐carotene accumulation in cauliflower 857 (Brassica oleracea var botrytis) The Plant Journal 26 59-67 858

Li L Yuan H (2013) Chromoplast biogenesis and carotenoid accumulation Arch Biochem 859 Biophys 539 102-109 860

Ling Q Huang W Baldwin A Jarvis P (2012) Chloroplast biogenesis is regulated by direct 861 action of the ubiquitin-proteasome system Science 338 655-659 862

Liu Q Xu J Liu Y Zhao X Deng X Guo L Gu J (2007) A novel bud mutation that 863 confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus 864 sinensis L Osbeck) J Exp Bot 58 4161-4171 865

Liu X Yu F Rodermel S (2010) Arabidopsis chloroplast FtsH var2 and suppressors of var2 866 leaf variegation a review J Integr Plant Biol 52 750-761 867

Luft JH (1961) Improvements in epoxy resin embedding methods J Biophys Biochem Cy 9 868 409-414 869

Lundquist PK Poliakov A Bhuiyan NH Zybailov B Sun Q van Wijk KJ (2012) The 870 functional network of the Arabidopsis plastoglobule proteome based on quantitative 871 proteomics and genome-wide coexpression analysis Plant Physiol 158 1172-1192 872

Lundquist PK Poliakov A Giacomelli L Friso G Appel M McQuinn RP Krasnoff SB 873 Rowland E Ponnala L Sun Q (2013) Loss of plastoglobule kinases ABC1K1 and 874

ABC1K3 causes conditional degreening modified prenyl-lipids and recruitment of 875 the jasmonic acid pathway Plant Cell 25 1818-1839 876

Majeran W Friso G Ponnala L Connolly B Huang M Reidel E Zhang C Asakura Y 877 Bhuiyan NH Sun Q (2010) Structural and metabolic transitions of C4 leaf 878 development and differentiation defined by microscopy and quantitative proteomics 879 in maize Plant Cell 22 3509-3542 880

Manara A DalCorso G Leister D Jahns P Baldan B Furini A (2014) AtSIA1 AND 881 AtOSA1 two Abc1 proteins involved in oxidative stress responses and iron 882 distribution within chloroplasts New Phytol 201 452-465 883

Manara A DalCorso G Guzzo F Furini A (2015) Loss of the atypical kinases ABC1K7 884 and ABC1K8 changes the lipid composition of the chloroplast membrane Plant Cell 885 Physiol doi 101093pcppcv046 886

Martinis J Glauser G Valimareanu S Kessler F (2013) A chloroplast ABC1-like kinase 887 regulates vitamin E metabolism in Arabidopsis Plant Physiol 162 652-662 888

Martinis J Glauser G Valimareanu S Stettler M Zeeman SC Yamamoto H Shikanai 889 T Kessler F (2014) ABC1K1PGR6 kinase a regulatory link between 890 photosynthetic activity and chloroplast metabolism Plant J 77 269-283 891

Meinke D Muralla R Sweeney C Dickerman A (2008) Identifying essential genes in 892 Arabidopsis thaliana Trends Plant Sci 13 483-491 893

Montefiori M McGhie TK Hallett IC Costa G (2009) Changes in pigments and plastid 894 ultrastructure during ripening of green-fleshed and yellow-fleshed kiwifruit Sci 895 Hortic 119 377-387 896

Neuhaus H Emes M (2000) Nonphotosynthetic metabolism in plastids Annu Rev Plant Biol 897 51 111-140 898

Nogueira M Mora L Enfissi EM Bramley PM Fraser PD (2013) Subchromoplast 899 sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing 900 different carotenoid gene combinations Plant Cell 25 4560-4579 901

Olinares PDB Kim J van Wijk KJ (2011) The Clp protease system a central component 902 of the chloroplast protease network BBA Bioenergetics 1807 999-1011 903

Pan ZY Liu Q Yun Z Guan R Zeng WF Xu Q Deng XX (2009) Comparative 904 proteomics of a lycopene-accumulating mutant reveals the important role of oxidative 905 stress on carotenogenesis in sweet orange (Citrus sinensis L Osbeck) Proteomics 9 906 5455ndash5470 907

Saacutenchez MT La Haba D Peacuterez-Mariacuten D (2013) Internal and external quality assessment of 908 mandarins on-tree and at harvest using a portable NIR spectrophotometer Comput 909 Electron Agr 92 66-74 910

Sakamoto W (2006) Protein degradation machineries in plastids Annu Rev Plant Biol 57 911 599-621 912

Schweiggert RM Steingass CB Heller A Esquivel P Carle R (2011) Characterization of 913 chromoplasts and carotenoids of red-and yellow-fleshed papaya (Carica papaya L) 914 Planta 234 1031-1044 915

Sheng Q Dai J Wu Y Tang H Zeng R (2012) BuildSummary using a group-based 916 approach to improve the sensitivity of peptideprotein identification in shotgun 917 proteomics J Proteome Res 11 1494-1502 918

Siddique MA Grossmann J Gruissem W Baginsky S (2006) Proteome analysis of bell 919 pepper (Capsicum annuum L) chromoplasts Plant Cell Physiol 47 1663-1673 920

Singh DK McNellis TW (2011) Fibrillin protein function the tip of the iceberg Trends 921 Plant Sci 16 432-441 922

Vasquez-Caicedo AL Heller A Neidhart S Carle R (2006) Chromoplast morphology and 923 β-carotene accumulation during postharvest ripening of mango cvtommy atkins J 924 Agric Food Chem 54 5769-5776 925

Wang Y-Q Yang Y Fei Z Yuan H Fish T Thannhauser TW Mazourek M Kochian 926 LV Wang X Li L (2013) Proteomic analysis of chromoplasts from six crop species 927 reveals insights into chromoplast function and development J Exp Bot 64 949-961 928

Wiśniewski JR Zougman A Nagaraj N Mann M (2009) Universal sample preparation 929 method for proteome analysis Nat Methods 6 930

Yang R Xie C Fan Q Gao C Fang L (2010) Ontogeny of the digestive tract in yellow 931 catfish Pelteobagrus fulvidraco larvae Aquaculture 302 112-123 932

Ytterberg AJ Peltier J-B Van Wijk KJ (2006) Protein profiling of plastoglobules in 933 chloroplasts and chromoplasts A surprising site for differential accumulation of 934 metabolic enzymes Plant Physiol 140 984-997 935

Yu B Lydiate DJ Schaumlfer UA Hannoufa A (2007) Characterization of a β-carotene 936 hydroxylase of Adonis aestivalis and its expression in Arabidopsis thaliana Planta 937 226 181-192 938

Zaltsman A Ori N Adam Z (2005) Two types of FtsH protease subunits are required for 939 chloroplast biogenesis and photosystem II repair in Arabidopsis Plant Cell 17 2782-940 2790 941

Zeng Y Pan Z Ding Y Zhu A Cao H Xu Q Deng X (2011) A proteomic analysis of the 942 chromoplasts isolated from sweet orange fruits [Citrus sinensis (L) Osbeck] J Exp 943 Bot 62 5297-5309 944

Zeng Y Pan Z Wang L Ding Y Xu Q Xiao S Deng X (2014) Phosphoproteomic analysis 945 of chromoplasts from sweet orange during fruit ripening Physiol Plantarum 150 946 252-270 947

Zheng B Fang Y Pan Z Sun L Deng X Grosser J Guo W (2014) iTRAQ-based 948 quantitative proteomics analysis revealed alterations of carbohydrate metabolism 949 pathways and mitochondrial proteins in a male sterile cybrid pummelo J Proteome 950 Res 13 2998-3015 951

Figure Legends 956

Figure 1 Materials used in this experiment and their diagrammatic cross-section through a 957 citrus A and C Red-fleshed lsquoHong Anliursquo sweet orange B and D Yellow-fleshed lsquoAnliursquo 958 sweet orange E and G Red-fleshed lsquoCara Cararsquo sweet orange F and H Yellow-fleshed 959 lsquoNewhallrsquo Navel orange C D G and H represent the small part of pulp (left side) and its 960 corresponding sac (upper right inset) and segment membrane (low right inset) The sac was 961 gently separated into sac membrane and juice (yellow and red arrows denote juice and sac 962 membrane respectively) 963

Figure 2 Structural diversity of chromoplasts from the flesh of lsquoHong Anliursquo and lsquoAnliursquo 965 Light microscopic inspection of the sac membrane segment membrane and juice separated 966 from lsquoHong Anliursquo (A to C) lsquoAnliursquo (D to F) under normal light field microscopy G and H 967 Carotenoid crystals within chromoplasts confirmed by polarization microscopy in sac 968 membrane and segment membrane of lsquoHong Anliursquo I and J Polarization micrographs of 969 globular chromoplasts in lsquoAnliursquo flesh and juice of lsquoHong Anliursquo Red- and yellow-arrows 970 indicate crystalloid chromoplasts and globular chromoplasts respectively The bars represent 971 10 μm 972

Figure 3 Ultrastructures of chromoplasts from the flesh of lsquoHong Anliursquo and lsquoAnliursquo A 974 Pooled crystalloid chromoplasts from segment membrane and sac membrane of lsquoHong Anliursquo 975 which showed several different ultrastructures including (B to E) Globular chromoplasts 976 containing numerous plastoglobules were observed from the juice of lsquoHong Anliursquo (F and G) and 977 the flesh of lsquoAnliursquo (H and I) lsquopgrsquo denotes plastoglobule lsquorsquo crystal remnants lsquosmsrsquo special 978 membrane structure and lsquocwrsquo denotes cell wall 979 980

Figure 4 Chromoplast formation in relation to carotenoid content and composition Pie 981 charts illustrate the carotenoid compositions (fresh weight) in the sac membrane (A) segment 982 membrane (B) and juice (C) of lsquoHong Anliursquo (HAL) and lsquoAnliursquo (AL) at maturation stage 983 (Ma) The detailed value (μgg) for each sample is shown in (D) 984

Figure 5 Ultrastructural changes during chromoplast differentiation at different maturation 986 stages of lsquoHong Anliursquo (A to E) and (F to I) were the micrographs of sac membrane and 987 juice during fruit ripening (IMG (immature green stage) MG (mature green stage) Br (colour 988 breaker stage) Ma (maturation stage) FMa (full maturation stage)) respectively A 989 Amyloplasts with starch granules at IMG B Plastids with limited number of plastoglobules 990 at MG C Chromoplasts with numerous globular and tubular structures at Br D and E 991 Crystalloid chromoplasts with crystal remnants at maturation stage and full maturation stage 992 F Amyloplasts with some starch granules at IMG G to I The formation of globular- and 993

tubular-chromoplasts with an increase in the number of plastoglobules from MG to FMa 994 Arrows denote plastoglobules (pg) sms special membrane structures s starch granules 995 asterisks crystal remnants t tubular elements J Light micrographs of purified plastids 996 which show that the pigment-enriched areas gradually increased during chromopalst 997 development The white bars are 5 μm K The number of plastoglobules significantly 998 increased from MG to Br stage then slightly decreased from Ma to OMa stage (over-999 maturation stage) L The size of plastids rapidly increased from MG to Br and then remained 1000 unchanged from Br to OMa Data are expressed as the mean plusmn SD and evaluated by Studentrsquos 1001 t-test using Microsoft Excel software (p lt 0001) n represents the number of plastids for 1002 calculation using Image J software 1003

Figure 6 Comparative transcriptional analysis of carotenoid metabolic genes among full 1005 ripening stage and immature green stage The major carotenoids accumulated in chromoplasts 1006 are shaded in gray in a square box The transcript levels are represented by segment 1007 membrane of lsquoAnliursquo (blue arrowheads) and lsquoHong Anliursquo (red arrows) and variation in the 1008 transcript levels is indicated by the different colors of the arrowheads and arrows Three 1009 experimental replicates were used to indicate up-regulation (upper arrow) or down-regulation 1010 (down arrow) The expression levels of Actin were used to normalize the expression for each 1011 sample The detailed information see Supplemental Figure S5 MEP plastidic 2-C-methyl-d-1012 erythritol 4-phosphate DXS 1-deoxy-D-xylulose 5-phosphate synthase DXR 1-deoxy-D-1013 xylulose 5-phosphate reductoisomerase PSY phytoene synthase ZISO ζ-carotene isomerase 1014 PDS phytoene desaturase ZDS ζ-carotene desaturase CRTISO carotene isomerase LCYb2 1015 lycopene β-cyclase2 LCYe lycopene ε-cyclase HYD flavedo-specific β-carotene 1016 hydroxylase HYE α-carotene hydroxylase ZEP zeaxanthin epoxidase NCED 9-cis-1017 epoxycarotenoid dioxygenase VDE violaxanthin de-epoxidase NXS neoxanthin synthsase 1018 FBN fibrillins CCDs carotenoid cleavage dioxygeneases ABA abscisic acid 1019

Figure 7 Schematic of experimental design for iTRAQ proteomic analysis Plastids were 1021 purified from 40 fruits at each ripening stage for each independent replicate (for details see 1022 lsquoMaterials and Methodsrsquo) A total of three biological replicates plus two technological 1023 replicates at MG and Br stages (MG 3+1 Br 3+1 Ma 3 and OMa 3) were performed A total 1024 of 1905 unique proteins were identified (see Supplemental Table S3A) of which 1386 1025 proteins were predicted to be plastid-localized and 1016 of which were further annotated 1026 with iTRAQ values as shown in Supplemental Table S3B 1027

1028 Figure 8 Comparison of protein abundance determined by proteomic analysis and 1029 immunoblotting The abundance of proteins determined by the iTRAQ analysis is expressed 1030 as the value of individualinternal standard (IS) 30 μg of total proteins extracted from 1031 purified plastids at the indicated stages was separated using SDS-PAGE blotted and 1032 subjected to immunoassays using specific antibodies that recognize the following three 1033 proteins TIC40 a component of the translocon complexes at the inner envelope membrane 1034

FBN1 Fibrillin 1 targeted to the plastoglobules in plastids RbcL the large subunit of 1035 Rubisco targeted to the plastid stroma 1036 1037 Figure 9 Abundance model of plastid-localized proteins during chromoplast differentiation 1038 A Abundance model of plastid-localized proteins being classified into seven categories 1039 during fruit ripening (MG-Br-Ma) These seven categories are stable early increase late 1040 increase continuous increase early decrease late decrease and continuous decrease 1041 Excluding the lsquonot assignedrsquo class and 91 proteins that lack a consistent and logical pattern of 1042 abundance we established an abundance model for 828 proteins B Patterns of protein 1043 abundance summarized according to functional classes Note that the number (right-hand 1044 side) and percentage (bottom) of proteins in the coloured-coded MapMan functional classes 1045 were shown in (A) 1046

1047 Figure 10 A heatmap showing the proportionof proteins of each functional class in each of 1048 the three abundance (stable increase and decrease) patterns during chromoplast 1049 differentiation The magnitude of the percentage is represented by a color scale (top left) 1050 going from low (white) to high (red) 1051

Figure 11 Schematic diagram of chromoplast differentiation in the flesh of citrus fruits A to 1053 E indicate the globular chromoplast formation in yellow-tissues and (A B C and F) 1054 indicate the crystalloid chromoplast development in the red-tissues A Amyloplasts B and C 1055 Starch granule degradation and the formation of plastids with globular and tubular structures 1056 D and E Typical globular and tubular chromoplasts F Chromoplasts with crystal remnants 1057

Figure 12 Abundance of proteins (y axis in ratio of iTRAQ) involved in carotenoid 1059 accumulation plastoglobule formation ribosome assembly as well as protein degradation 1060 and processing during amylo-to-chromoplast differentiation and chromoplast senescence A 1061 Proteins involved in MEP pathway and carotenoids biosynthesis B Proteins involved in 1062 plastoglobules formation C The sum of all proteins involved in protein initiation nuclear-1063 encoded 40S subunit and 60S subunit as well as plastid-encoded 30S subunit and 50S subunit 1064 D Proteins involved in protein degradation and processing Individual values are given in 1065 Supplemental Table S3B Protein abundance is represented by the average of the ratios of 1066 iTRAQ-valueISplusmnSD The full names of the proteins are indicated in the text and 1067 Supplemental Table S3B 1068

Supplemental Figure S1 Structural diversity of chromoplasts from the flesh of lsquoCara Cararsquo and 1069 lsquoNewhallrsquo Navel Light microscopic inspection of the sac membrane segment membrane and juice 1070 that were separated from lsquoCara Cararsquo (A to C) and lsquoNewhallrsquo Navel (D to F) under normal light field 1071 microscopy G and H Carotenoid crystals within crystalloid chromoplasts confirmed by polarization 1072 microscopy in sac membrane and segment membrane of lsquoCara Cararsquo I and J Polarization 1073 micrographs of globular chromoplasts in the flesh and juice of lsquoNewhallrsquo Navel Red- and yellow-1074 arrows indicate crystalloid chromoplasts and globular chromoplasts respectively The bars represent 1075 10 μm 1076

Supplemental Figure S2 Ultrastructures of chromoplasts from the flesh of lsquoCara Cararsquo and 1078 lsquoNewhallrsquo Navel A to D Ultrastructural diversity of crystalloid chromoplasts from segment 1079 membrane and sac membrane of lsquoCara Cararsquo Globular chromoplasts containing numerous 1080 plastoglobules were observed in juice of lsquoCara Cararsquo (E) and lsquoNewhallrsquo Navel flesh (F) lsquopgrsquo denotes 1081 plastoglobules lsquorsquo crystal remnants lsquotrsquo tubular structure 1082

Supplemental Figure S3 The outer monolayer of plastoglobules appeared to be separated from the 1084 internal body at OMa stage The ultrastructure of globular chromoplast of the juice in lsquoHong Anliursquo at 1085 OMa stage The bar represents 2 μm 1086

Supplemental Figure S4 Immunoblotting analysis reveals the absence of chloroplasts within citrus 1088 flesh during fruit maturity Total proteins extracted from lsquoHong Anliursquo leaves (as control) or flesh at 1089 five different sampling points were subjected to gel blotting using antibodies against thylakoid 1090 membrane marker proteins (PsbA and Lhcb2) The same weight of proteins were used as loading 1091 control At least two experimental replicates were conducted for immunoblotting analysis 1092

Supplemental Figure S5 Expression of carotenoid metabolic genes during the formation of 1094 chromoplasts at different maturation stages Relative transcript levels were analysed by real-time RT-1095 PCR in the segment membrane of lsquoAnliursquo (A) and lsquoHong Anliursquo (H) Data represent mean values plusmn 1096 SD from at least three experimental replicates The expression levels of Actin were used to normalize 1097 the expression for each sample 1098

Supplemental Figure S6 Distribution of peptide molecular weight (A) and of peptide coverage (B) 1100

Supplemental Figure S7 Analysis of reproducibility of iTRAQ biological replicates and 1102 technological replicates of each development stage A Reproducibility of biological replicates (MG-1103 1_E1_114 VS MG-2_E1_119 VS MG-3_E2_115 Br-1_E1_117 VS Br-2_E1_121 VS Br-3_E2_114 1104 Ma-1_E1_116 VS Ma-2_E2_118 VS Ma-3_E2_121 OMa-1_E1_115 VS OMa-2_E1_118 VS OMa-1105 3_E2_117 as showed in Figure 7) B Reproducibility of technological replicates (MG-3_E2_115 VS 1106 MG-4_E2_116 and Br-1_E1_117 VS Br-2_E1_121) 1107 1108 Supplemental Figure S8 Venn diagram of the number of proteins of the citrus plastid proteome 1109 predicted to be plastid localized by three predictors and five plastidial databases A total of 1366 1110 proteins are predicted by at least one predictor or more than two out of five plastidial databases The 1111 20 plastid-genome encoded proteins were excluded from this analysis 1112

Supplemental Figure S9 Relative mass contributions of the 30 plastoglobule core proteins to the 1113 total core plastoglobule proteome for Arabidopsis chloroplasts (A) and orange flesh chromoplasts (B) 1114 The graph of Arabidopsis cellular ultrastructure and plastoglobule-localized proteins was taken from 1115 the study by Lundquist et al (2012) 1116

Supplemental Figure S10 Abundance of FBN1 during fruit ripening 30 μg of total proteins 1118 extracted from lsquoHong Anliursquo fruit from 60 DAF to 260 DAF were separated using SDS-PAGE 1119 blotted and subjected to immunoassays using FBN1 antibodies The equal amount of proteins was 1120 performed as a loading control 1121 1122

Supplemental Tables 1123

See excel files 1124

Supplemental Table S1 Primers used for Real-time PCR 1125

Supplemental Table S2 Identified peptides with iTRAQ-values in this experiment 1126 Supplemental Table S3 iTRAQ data of proteomics analysis during chromoplast differentiation and 1127 list of quantitative proteins 1128 Supplemental Table S4 Characteristics of the 1386 citrus pulp plastid protein candidates identified 1129 by iTRAQ 1130 Supplemental Table S5 Set of 1016 plastid proteins that have been quantified during chromoplast 1131 differentiation and senescence 1132 Supplemental Table S6 Set of 104 differentially expressed proteins between OMa and Ma stage 1133 Supplemental Table S7 The core proteome of plastoglobules in citrus chromoplasts 1134

Figure 1 Materials used in this experiment and their diagrammatic cross-section through a citrus A and C Red-fleshed lsquoHong Anliursquo sweet orange B and D Yellow-fleshed lsquoAnliursquo sweet orange E and G Red-fleshed lsquoCara Cararsquo sweet orange F and H Yellow-fleshed lsquoNewhallrsquo Navel sweet orange C and D G and H represent the small part of pulp (left side) and its corresponding sac (upper right inset) and segment membrane (low right inset) The sac was gently separated into sac membrane and juice (yellow and red arrows denote juice and sac membrane respectively)

Figure 2 Structural diversity of chromoplasts from the flesh of lsquoHong Anliursquo and lsquoAnliursquo Light microscopic inspection of the sac membrane segment membrane and juice separated from lsquoHong Anliursquo (A to C) lsquoAnliursquo (D to F) under normal light field microscopy G and H Carotenoid crystals within chromoplasts confirmed by polarization microscopy in sac membrane and segment membrane of lsquoHong Anliursquo I and J Polarization micrographs of globular chromoplasts in lsquoAnliursquo flesh and juice of lsquoHong Anliursquo Red- and yellow-arrows indicate crystalloid chromoplasts and globular chromoplasts respectively The bars represent 10 μm

Figure 3 Ultrastructures of chromoplasts from the flesh of lsquoHong Anliursquo and lsquoAnliursquo A Pooled crystalloid chromoplasts from segment membrane and sac membrane of lsquoHong Anliursquo which showed several different ultrastructures including (B to E) Globular chromoplasts containing numerous plastoglobules were observed from the juice of lsquoHong Anliursquo (F and G) and the flesh of lsquoAnliursquo (H and I) lsquopgrsquo denotes plastoglobule lsquorsquo crystal remnants lsquosmsrsquo special membrane structure and lsquocwrsquo denotes cell wall

Figure 4 Chromoplast formation in relation to carotenoid content and composition Pie charts illustrate the carotenoid compositions (fresh weight) in the sac membrane (A) segment membrane (B) and juice (C) of lsquoHong Anliursquo (HAL) and lsquoAnliursquo (AL) at maturation stage (Ma) The detailed value (μgg) for each sample is shown in (D)

Figure 5 Ultrastructural changes during chromoplast differentiation at different maturation stages of lsquoHong Anliursquo (A to E) and (F to I) were the micrographs of sac membrane and juice during fruit ripening (IMG (immature green stage) MG (mature green stage) Br (colour breaker stage) Ma (maturation stage) FMa (full maturation stage)) respectively A Amyloplasts with starch granules at IMG B Plastids with limited number of plastoglobules at MG C Chromoplasts with numerous globular and tubular structures at Br D and E Crystalloid chromoplasts with crystal remnants at maturation stage and full maturation stage F Amyloplasts with some starch granules at IMG G to I The formation of globular- and tubular-chromoplasts with an increase in the number of plastoglobules from MG to FMa Arrows denote plastoglobules (pg) sms special membrane structures s starch granules

asterisks crystal remnants t tubular elements J Light micrographs of purified plastids which show that the pigment-enriched areas gradually increased during chromopalst development The white bars are 5 μm K The number of plastoglobules significantly increased from MG to Br stage then slightly decreased from Ma to OMa stage (over-maturation stage) L The size of plastids rapidly increased from MG to Br and then remained unchanged from Br to OMa Data are expressed as the mean plusmn SD and evaluated by Studentrsquos t-test using Microsoft Excel software (p lt 0001) n represents the number of plastids for calculation using Image J software

Figure 6 Comparative transcriptional analysis of carotenoid metabolic genes among full ripening stage and immature green stage The major carotenoids accumulated in chromoplasts are shaded in gray in a square box The transcript levels are represented by segment membrane of lsquoAnliursquo (blue arrowheads) and lsquoHong Anliursquo (red arrows) and variation in the transcript levels is indicated by the different colors of the arrowheads and arrows Three experimental replicates were used to indicate up-regulation (upper arrow) or down-regulation (down arrow) The expression levels of Actin were used to normalize the expression for each sample The detailed information see Supplemental Figure S5 MEP plastidic 2-C-methyl-d-erythritol 4-phosphate DXS 1-deoxy-D-xylulose 5-phosphate synthase DXR 1-deoxy-D-xylulose 5-phosphate reductoisomerase PSY phytoene synthase ZISO ζ-carotene isomerase PDS phytoene desaturase ZDS ζ-carotene desaturase CRTISO carotene isomerase LCYb2 lycopene β-cyclase2 LCYe lycopene ε-cyclase HYD flavedo-specific β-carotene hydroxylase HYE α-carotene hydroxylase ZEP zeaxanthin epoxidase NCED 9-cis-epoxycarotenoid dioxygenase VDE violaxanthin de-epoxidase NXS neoxanthin synthsase FBN fibrillins CCDs carotenoid cleavage dioxygeneases ABA abscisic acid

lt2 fold 2~4 fold 4~6 fold 6~8 fold gt8 fold

HAL AL In De In De

Figure 7 Schematic of experimental design for iTRAQ proteomic analysis Plastids were purified from 40 fruits at each ripening stage for each independent replicate (for details see lsquoMaterials and Methodsrsquo) A total of three biological replicates plus two technological replicates at MG and Br stages (MG 3+1 Br 3+1 Ma 3 and OMa 3) were performed A total of 1905 unique proteins were identified (see Supplemental Table S3A) of which 1386 proteins were predicted to be plastid-localized and 1016 of which were further annotated with iTRAQ values as shown in Supplemental Table S3B

Figure 8 Comparison of protein abundance determined by proteomic analysis and immunoblotting The abundance of proteins determined by the iTRAQ analysis is expressed as the value of individualinternal standard (IS) 30 μg of total proteins extracted from purified plastids at the indicated stages was separated using SDS-PAGE blotted and subjected to immunoassays using specific antibodies that recognize the following three proteins TIC40 a component of the translocon complexes at the inner envelope membrane FBN1 Fibrillin 1 targeted to the plastoglobules in plastids RbcL the large subunit of Rubisco targeted to the plastid stroma

Figure 9 Abundance model of plastid-localized proteins during chromoplast differentiation A Abundance model of plastid-localized proteins being classified into seven categories during fruit ripening (MG-Br-Ma) These seven categories are stable early increase late increase continuous increase early decrease late decrease and continuous decrease Excluding the lsquonot assignedrsquo class and 91 proteins that lack a consistent and logical pattern of abundance we established an abundance model for 828 proteins B Patterns of protein abundance summarized according to functional classes Note that the number (right-hand side) and percentage (bottom) of proteins in the coloured-coded MapMan functional classes were shown in (A)

Figure 10 A heatmap showing the proportionof proteins of each functional class in each of the three abundance (stable increase and decrease) patterns during chromoplast differentiation The magnitude of the percentage is represented by a color scale (top left) going from low (white) to high (red)

Figure 11 Schematic diagram of chromoplast differentiation in the flesh of citrus fruits A to E indicate the globular chromoplast formation in yellow-tissues and (A B C and F) indicate the crystalloid chromoplast development in the red-tissues A Amyloplasts B and C Starch granule degradation and the formation of plastids with globular and tubular structures D and E Typical globular and tubular chromoplasts F Chromoplasts with crystal remnants

httpsplantphysiolorgD

ownloaded on N

ovember 22 2020 - Published by

merican Society of Plant B

Figure 12 Abundance of proteins (y axis in ratio of iTRAQ) involved in carotenoid accumulation plastoglobule formation ribosome assembly as well as protein degradation and processing during amylo-to-chromoplast differentiation and chromoplast senescence A Proteins involved in MEP pathway and carotenoids biosynthesis B Proteins involved in plastoglobules formation C The sum of all proteins involved in protein initiation nuclear-encoded 40S subunit and 60S subunit as well as plastid-encoded 30S subunit and 50S subunit D Proteins involved in protein degradation and processing Individual values are given in Supplemental Table S3B Protein abundance is represented by the average of the ratios of iTRAQ-valueISplusmnSD The full names of the proteins are indicated in the text and Supplemental Table S3B

ownloaded on N

Parsed CitationsAgne B Andregraves C Montandon C Christ B Ertan A Jung F Infanger S Bischof S Baginsky S Kessler F (2010) The acidic A-domain of Arabidopsis TOC159 occurs as a hyperphosphorylated protein Plant Physiol 153 1016-1030

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Aronsson H Jarvis P (2008) The chloroplast protein import apparatus its components and their roles 1861-1370Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Austin JR Frost E Vidi PA Kessler F Staehelin LA (2006) Plastoglobules are lipoprotein subcompartments of the chloroplast thatare permanently coupled to thylakoid membranes and contain biosynthetic enzymes Plant Cell 18 1693-1703

Barsan C Zouine M Maza E Bian W Egea I Rossignol M Bouyssie D Pichereaux C Purgatto E Bouzayen M (2012) Proteomicanalysis of chloroplast-to-chromoplast transition in tomato reveals metabolic shifts coupled with disrupted thylakoid biogenesismachinery and elevated energy-production components Plant Physiol 160 708-725

Ben-Shaul Y Naftali Y (1969) The development and ultrastructure of lycopene bodies in chromoplasts ofLycopersicumesculentum Protoplasma 67 333-344

Benjamini Y Hochberg Y (1995) Controlling the false discovery rate a practical and powerful approach to multiple testing J RoyStat Soc B 57 289-300

Bian W Barsan C Egea I Purgatto E Chervin C Zouine M Latcheacute A Bouzayen M Pech J-C (2011) Metabolic and molecularevents occurring during chromoplast biogenesis Journal of Botany 2011

Cao HB Zhang JC Xu J Ye JL Yun Z Xu Q Xu J Deng XX (2012) Comprehending crystalline szlig-carotene accumulation bycomparing engineered cell models and the natural carotenoid-rich system of citrus J Exp Bot 63 4403-4417

Cazzonelli CI Pogson BJ (2010) Source to sink regulation of carotenoid biosynthesis in plants Trends Plant Sci 15 266-274Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cederholm SN Baumlckman HG Pesaresi P Leister D Glaser E (2009) Deletion of an organellar peptidasome PreP affects earlydevelopment in Arabidopsis thaliana Plant Mol Biol 71 497-508

Davidi L Levin Y Ben-Dor S Pick U (2015) Proteome analysis of cytoplasmatic and plastidic b-carotene lipid droplets in Dunaliellabardawil Plant Physiol 167 60-79

Egea I Barsan C Bian W Purgatto E Latcheacute A Chervin C Bouzayen M Pech JC (2010) Chromoplast differentiation currentstatus and perspectives Plant Cell Physiol 51 1601-1611

Eilati S BUDOWSKJ P Monselise S (1975) Carotenoid changes in the Shamouti orange peel during chloroplast-chromoplasttransformation on and off the tree J Exp Bot 26 624-632

Eilati S SP M Budowski P (1969) Seasonal development of external color and carotenoid content in the peel of ripeninghttpsplantphysiolorgDownloaded on November 22 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

Shamouti oranges J Am Soc Hortic Sci 94 346-348Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Fanciullino AL Cercos M Dhuique-Mayer C Froelicher Y Talon M Ollitrault P Morillon R (2008) Changes in carotenoid contentand biosynthetic gene expression in juice sacs of four orange varieties (Citrus sinensis) differing in esh fruit color J Agric FoodChem 56 3628-3638

Fanciullino AL Dhuique-Mayer C Luro F Casanova J Morillon A Ollitrault P (2006) Carotenoid diversity in cultivated citrus ishighly inuenced by genetic factors J Agric Food Chem 54 4397-4406

Frey-Wyssling A Schwegler F (1965) Ultrastructure of the chromoplasts in the carrot root J Ultra Mol Struct R 13 543-559Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Grilli Caiola M Canini A (2004) Ultrastructure of chromoplasts and other plastids in Crocus sativus L(Iridaceae) Plant Biosyst 13843-52

Harris WM Spurr AR (1969) Chromoplasts of tomato fruits II The red tomato Am J Bot 56 380-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Hempel J Amrehn E Quesada S Esquivel P Jimeacutenez VM Heller A Carle R Schweiggert RM (2014) Lipid-dissolved -carotene szlig-carotene and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits Planta 240 1-14

Horner H Healy R Ren G Fritz D Klyne A Seames C Thornburg R (2007) Amyloplast to chromoplast conversion in developingornamental tobacco floral nectaries provides sugar for nectar and antioxidants for protection Am J Bot 94 12-24

Iglesias DJ Cercoacutes M Colmenero-Flores JM Naranjo MA Riacuteos G Carrera E Ruiz-Rivero O Lliso I Morillon R Tadeo FR (2007)Physiology of citrus fruiting Braz J Plant Physiol 19 333-362

Jarvis P (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants New Phytol 179 257-285Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kahlau S and Bock R (2008) Plastid transcriptomics and translatomics of tomato fruit development and chloroplast-to-chromoplastdifferentiation chromoplast gene expression largely serves the production of a single protein Plant Cell 20 856-874

Kanervo E Singh M Suorsa M Paakkarinen V Aro E Battchikova N Aro EM (2008) Expression of protein complexes andindividual proteins upon transition of etioplasts to chloroplasts in pea (Pisum sativum) Plant Cell Physiol 49 396-410

Karp NA Huber W Sadowski PG Charles PD Hester SV Lilley KS (2010) Addressing accuracy and precision issues in iTRAQquantitation Mol Cell Proteomics 9 1885-1897

Kato Y Sakamoto W (2010) New insights into the types and function of proteases in plastids Int Rev Cel Mol Bio 280 185-218Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kilcrease J Collins AM Richins RD Timlin JA OConnell MA (2013) Multiple microscopic approaches demonstrate linkagehttpsplantphysiolorgDownloaded on November 22 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit Plant J 76 1074-1083Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kim JE Rensing KH Douglas CJ Cheng KM (2010) Chromoplasts ultrastructure and estimated carotene content in root secondaryphloem of different carrot varieties Planta 231 549-558

Kleffmann T von Zychlinski A Russenberger D Hirsch-Hoffmann M Gehrig P Gruissem W Baginsky S (2007) Proteome dynamicsduring plastid differentiation in rice Plant Physiol 143 912-923

Kmiec B Glaser E (2012) A novel mitochondrial and chloroplast peptidasome PreP Physiol Plantarum 145 180-186Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Knoth R (1981) Ultrastructure of lycopene containing chromoplasts in fruits of Aglaonema commutatum schott (Araceae)Protoplasma 106 249-259

Knoth R Hansmann P Sitte P (1986) Chromoplasts of Palisota barteri and the molecular structure of chromoplast tubules Planta168 167-174

Li L Paolillo DJ Parthasarathy MV DiMuzio EM Garvin DF (2001) A novel gene mutation that confers abnormal patterns of szlig-carotene accumulation in cauliflower (Brassica oleracea var botrytis) The Plant Journal 26 59-67

Li L Yuan H (2013) Chromoplast biogenesis and carotenoid accumulation Arch Biochem Biophys 539 102-109Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Ling Q Huang W Baldwin A Jarvis P (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasomesystem Science 338 655-659

Liu Q Xu J Liu Y Zhao X Deng X Guo L Gu J (2007) A novel bud mutation that confers abnormal patterns of lycopeneaccumulation in sweet orange fruit (Citrus sinensis L Osbeck) J Exp Bot 58 4161-4171

Liu X Yu F Rodermel S (2010) Arabidopsis chloroplast FtsH var2 and suppressors of var2 leaf variegation a review J IntegrPlant Biol 52 750-761

Luft JH (1961) Improvements in epoxy resin embedding methods J Biophys Biochem Cy 9 409-414Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Lundquist PK Poliakov A Bhuiyan NH Zybailov B Sun Q van Wijk KJ (2012) The functional network of the Arabidopsisplastoglobule proteome based on quantitative proteomics and genome-wide coexpression analysis Plant Physiol 158 1172-1192

Lundquist PK Poliakov A Giacomelli L Friso G Appel M McQuinn RP Krasnoff SB Rowland E Ponnala L Sun Q (2013) Loss ofplastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening modified prenyl-lipids and recruitment of thejasmonic acid pathway Plant Cell 25 1818-1839

Majeran W Friso G Ponnala L Connolly B Huang M Reidel E Zhang C Asakura Y Bhuiyan NH Sun Q (2010) Structural andmetabolic transitions of C4 leaf development and differentiation defined by microscopy and quantitative proteomics in maize PlantCell 22 3509-3542

Manara A DalCorso G Leister D Jahns P Baldan B Furini A (2014) AtSIA1 AND AtOSA1 two Abc1 proteins involved in oxidativestress responses and iron distribution within chloroplasts New Phytol 201 452-465

Manara A DalCorso G Guzzo F Furini A (2015) Loss of the atypical kinases ABC1K7 and ABC1K8 changes the lipid composition ofthe chloroplast membrane Plant Cell Physiol doi 101093pcppcv046

Martinis J Glauser G Valimareanu S Kessler F (2013) A chloroplast ABC1-like kinase regulates vitamin E metabolism inArabidopsis Plant Physiol 162 652-662

Martinis J Glauser G Valimareanu S Stettler M Zeeman SC Yamamoto H Shikanai T Kessler F (2014) ABC1K1PGR6 kinase aregulatory link between photosynthetic activity and chloroplast metabolism Plant J 77 269-283

Meinke D Muralla R Sweeney C Dickerman A (2008) Identifying essential genes in Arabidopsis thaliana Trends Plant Sci 13 483-491

Montefiori M McGhie TK Hallett IC Costa G (2009) Changes in pigments and plastid ultrastructure during ripening of green-fleshed and yellow-fleshed kiwifruit Sci Hortic 119 377-387

Neuhaus H Emes M (2000) Nonphotosynthetic metabolism in plastids Annu Rev Plant Biol 51 111-140Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Nogueira M Mora L Enfissi EM Bramley PM Fraser PD (2013) Subchromoplast sequestration of carotenoids affects regulatorymechanisms in tomato lines expressing different carotenoid gene combinations Plant Cell 25 4560-4579

Olinares PDB Kim J van Wijk KJ (2011) The Clp protease system a central component of the chloroplast protease network BBABioenergetics 1807 999-1011

Pan ZY Liu Q Yun Z Guan R Zeng WF Xu Q Deng XX (2009) Comparative proteomics of a lycopene-accumulating mutant revealsthe important role of oxidative stress on carotenogenesis in sweet orange (Citrus sinensis L Osbeck) Proteomics 9 5455-5470

Saacutenchez MT La Haba D Peacuterez-Mariacuten D (2013) Internal and external quality assessment of mandarins on-tree and at harvest usinga portable NIR spectrophotometer Comput Electron Agr 92 66-74

Sakamoto W (2006) Protein degradation machineries in plastids Annu Rev Plant Biol 57 599-621Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Schweiggert RM Steingass CB Heller A Esquivel P Carle R (2011) Characterization of chromoplasts and carotenoids of red-andyellow-fleshed papaya (Carica papaya L) Planta 234 1031-1044

Pubmed Author and TitlehttpsplantphysiolorgDownloaded on November 22 2020 - Published by

CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Sheng Q Dai J Wu Y Tang H Zeng R (2012) BuildSummary using a group-based approach to improve the sensitivity ofpeptideprotein identification in shotgun proteomics J Proteome Res 11 1494-1502

Siddique MA Grossmann J Gruissem W Baginsky S (2006) Proteome analysis of bell pepper (Capsicum annuum L) chromoplastsPlant Cell Physiol 47 1663-1673

Singh DK McNellis TW (2011) Fibrillin protein function the tip of the iceberg Trends Plant Sci 16 432-441Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Vasquez-Caicedo AL Heller A Neidhart S Carle R (2006) Chromoplast morphology and szlig-carotene accumulation duringpostharvest ripening of mango cvtommy atkins J Agric Food Chem 54 5769-5776

Wang Y-Q Yang Y Fei Z Yuan H Fish T Thannhauser TW Mazourek M Kochian LV Wang X Li L (2013) Proteomic analysis ofchromoplasts from six crop species reveals insights into chromoplast function and development J Exp Bot 64 949-961

Wisniewski JR Zougman A Nagaraj N Mann M (2009) Universal sample preparation method for proteome analysis Nat Methods 6Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Yang R Xie C Fan Q Gao C Fang L (2010) Ontogeny of the digestive tract in yellow catfish Pelteobagrus fulvidraco larvaeAquaculture 302 112-123

Ytterberg AJ Peltier J-B Van Wijk KJ (2006) Protein profiling of plastoglobules in chloroplasts and chromoplasts A surprising sitefor differential accumulation of metabolic enzymes Plant Physiol 140 984-997

Yu B Lydiate DJ Schaumlfer UA Hannoufa A (2007) Characterization of a szlig-carotene hydroxylase of Adonis aestivalis and itsexpression in Arabidopsis thaliana Planta 226 181-192

Zaltsman A Ori N Adam Z (2005) Two types of FtsH protease subunits are required for chloroplast biogenesis and photosystem IIrepair in Arabidopsis Plant Cell 17 2782-2790

Zeng Y Pan Z Ding Y Zhu A Cao H Xu Q Deng X (2011) A proteomic analysis of the chromoplasts isolated from sweet orangefruits [Citrus sinensis (L) Osbeck] J Exp Bot 62 5297-5309

Zeng Y Pan Z Wang L Ding Y Xu Q Xiao S Deng X (2014) Phosphoproteomic analysis of chromoplasts from sweet orange duringfruit ripening Physiol Plantarum 150 252-270

Zheng B Fang Y Pan Z Sun L Deng X Grosser J Guo W (2014) iTRAQ-based quantitative proteomics analysis revealedalterations of carbohydrate metabolism pathways and mitochondrial proteins in a male sterile cybrid pummelo J Proteome Res 132998-3015

Parsed Citations

Article File

Figures

Parsed Citations

Research Paper 23

A comprehensive analysis of chromoplast differentiation reveals complex protein 24

changes associated with plastoglobule biogenesis and remodelling of protein systems in 25

orange flesh1 26

Yunliu Zeng1 Jiabin Du1 Lun Wang1 Zhiyong Pan1 Qiang Xu1 Shunyuan Xiao 12 and 27

Xiuxin Deng1 28

1Key Laboratory of Horticultural Plant Biology (Ministry of Education) College of 29

China 31

2Institute for Bioscience and Biotechnology Research University of Maryland Rockville 32

MD USA 33

Author for correspondence E-mail xxdengmailhzaueducn 34

Footnotes 36

This work was supported by the National 973 project of China (No 2011CB100601) and 37

NSFC (No 31330066 and 31221062) 38

Correspondence Author 39

Professor Xiuxin Deng email xxdengmailhzaueducn 40

Key Laboratory of Horticultural Plant Biology (Ministry of Education) College of 41

China 43

flesh 71

Introduction 75

Results 139

maturation 186

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

flesh 71

Introduction 75

Results 139

maturation 186

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Introduction 75

Results 139

maturation 186

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Results 139

maturation 186

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Results 139

maturation 186

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

maturation 186

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

transition 311

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Discussion 405

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

differentiation 438

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

proteases 520

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Conclusions 585

Plant materials 599

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

chromoplasts 626

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

curation 736

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Acknowledgments 759

manuscript 764

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Figure Legends 956

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

HAL AL In De In De

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

ownloaded on N

Parsed Citations

Article File

Figures

Parsed Citations

Article File

Figures

Parsed Citations

Article File

Figures

Parsed Citations

Article File

Figures

Parsed Citations

Article File

Figures

Parsed Citations

Article File

Figures

Parsed Citations

Documents

Plant Physiology Preview. Published on June 8, 2015, as … · 2015. 6. 8. · 23 Research Paper 24 A comprehensive analysis of chromoplast differentiation reveals complex protein