Short title: Roles of SCD in MEP and isoprenoid pathways E · 3 61. ABSTRACT. 62. Plastid isoprenoids, a diverse group of compounds that includes. carotenoids, 63. chlorophyll. s,

Short title Roles of SCD in MEP and isoprenoid pathways 1

Corresponding author details Xiaohong Yang (Email 3

yxiaohongcaueducn) 4

Tel +86-10-62732400 5

Fax +86-10-62732400 6

State Key Laboratory of Plant Physiology and Biochemistry National Maize 7

Improvement Center of China MOA Key Lab of Maize Biology Beijing Key 8

Laboratory of Crop Genetic Improvement Center for Crop Functional 9

Genomics and Molecular Breeding 10

China Agricultural University 11

No 2 Yuanmingyuan West Road Haidian District 12

Beijing 100193 China 13

Article title SEED CAROTENOID DEFICIENT Functions in Isoprenoid 15

Biosynthesis via the Plastid MEP Pathway 16

Lili Zhang12 Xuan Zhang12 Xiaoji Wang1 Jing Xu12 Min Wang12 Lin Li3 18

Guanghong Bai4 Hui Fang12 Shuting Hu12 Jigang Li1 Jianbing Yan3 19

Jiansheng Li12 Xiaohong Yang12 20

1State Key Laboratory of Plant Physiology and Biochemistry China Agricultural 21

University Beijing 100193 China 22

2National Maize Improvement Center of China MOA Key Lab of Maize Biology 23

Beijing Key Laboratory of Crop Genetic Improvement Center for Crop 24

Functional Genomics and Molecular Breeding China Agricultural University 25

Beijing 100193 China 26

3National Key Laboratory of Crop Genetic Improvement Huazhong 27

Agricultural University Wuhan 430070 China 28

4Agronomy College Xinjiang Agricultural University Urumqi 830052 China 29

One-sentence summary 31

SEED CAROTENOID DEFICIENT functions in the methylerythritol phosphate 32

pathway in maize and affects the accumulation of downstream isoprenoids 33

Plant Physiology Preview Published on February 4 2019 as DOI101104pp1801148

AUTHOR CONTRIBUTIONS 35

XY designed the research LZ performed most of the research and data 36

analysis XZ MW and LL analyzed the RNA-seq data XW and J-GL 37

performed immunoblot analysis GB discovered the mutant JX HF and 38

SH performed chemical analysis of leaves and seeds JY and J-SL 39

assisted with the experiments LZ and XY wrote the manuscript 40

Funding information 42

This study was supported by the National Natural Foundation of China 43

(31671697) and the National Key Research and Development Program of 44

China (2016YFD0100503) 45

Email addresses of all authors 47

Lili Zhang lilizhang0946163com 48

Xuan Zhang zhangxuan6644sinacom 49

Xiaoji Wang xiaojiwangcaueducn 50

Jing Xu xu_jing_coolyeahnet 51

Min Wang kumine163com 52

Lin Li hzaulilinmailhzaueducn 53

Guanghong Bai bgh601126com 54

Hui Fang fanghui8912126com 55

Shuting Hu hushutingcau163com 56

Jigang Li jiganglicaueducn 57

Jianbing Yan yjianbingmailhzaueducn 58

Jiansheng Li lijianshengcaueducn 59

Xiaohong Yang yxiaohongcaueducn 60

ABSTRACT 61

Plastid isoprenoids a diverse group of compounds that includes carotenoids 62

chlorophylls tocopherols and multiple hormones are essential for plant 63

growth and development Here we identified and characterized SEED 64

CAROTENOID DEFICIENT (SCD) which encodes an enzyme that functions 65

in the biosynthesis of plastid isoprenoids in maize (Zea mays) SCD converts 66

2C-methyl-D-erytrithol 24-cyclodiphosphate (MEcPP) to 67

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) in the penultimate 68

step of the methylerythritol phosphate (MEP) pathway In scd mutants plant 69

growth and development are impaired and the levels of MEP-derived 70

isoprenoids such as carotenoids chlorophylls and tocopherols as well as 71

abscisic and gibberellic acids are reduced in leaves and seeds This scd 72

metabolic alteration varies among plant tissues and under different light 73

conditions RNA-sequencing of the scd mutant and wild type identified a 74

limited number of differentially expressed genes in the MEP pathway although 75

isoprenoid levels were significantly reduced in scd seeds and dark-grown 76

leaves Furthermore SCD-overexpressing transgenic lines showed little or no 77

differences in isoprenoid levels indicating that SCD may be subject to 78

post-translational regulation or not represent a rate-limiting step in the MEP 79

pathway These results enhance our understanding of the transcriptomic and 80

metabolic regulatory roles of enzymes in the MEP pathway and of their effects 81

on downstream isoprenoid pathways in various plant tissues and under 82

different light conditions 83

INTRODUCTION 85

Isoprenoids (or terpenoids) are the most functionally and structurally diverse 86

group of metabolites in living organisms with over 55000 natural molecules 87

discovered to date (Thulasiram et al 2007) Many isoprenoids play critical 88

roles in plant growth and development and have important commercial 89

applications For example carotenoids act as light-harvesting pigments that 90

function in photoprotection against excess light during photosynthesis with 91

major impacts on plant growth and development (Demmig-Adams et al 1996 92

Ruiz-Sola and Rodriguez-Concepcion 2012) In addition carotenoids such as 93

provitamin A are valuable for human health due to their antioxidant and 94

nutritional qualities (Johnson 2002 Goff and Klee 2006) 95

All types of isoprenoids are derived from the common five-carbon 96

precursors isopentenyl diphosphate (IPP) and its isomer dimethylallyl 97

diphosphate (DMAPP) (Bouvier et al 2005) Condensation of IPP and 98

DMAPP units results in the synthesis of short-chain prenyl diphosphates 99

which serve as branch points for the subsequent synthesis of all isoprenoid 100

end products Plants synthesize IPP and DMAPP via two independent 101

pathways the mevalonate (MVA) pathway for isoprenoid synthesis in the 102

cytoplasm and the methylerythritol phosphate (MEP) pathway for isoprenoid 103

synthesis in plastids (Kuzuyama and Seto 2003 Hemmerlin et al 2012 104

Vranova et al 2013) 105

The MEP pathway has been well elucidated in the model plant 106

Arabidopsis thaliana This pathway starts with the production of 107

1-deoxy-D-xylulose-5-phosphate (DXP) from glyceraldehyde 3-P and pyruvate 108

a process catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 109

ends with the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate 110

(HMBPP) into a mixture of IPP and DMAPP by HMBPP reductase (HDR) 111

(Supplemental Fig S1) (Vranova et al 2012 2013) Almost all enzymes 112

involved in the MEP pathway have been identified and characterized in 113

Arabidopsis and are highly conserved with those in Escherichia coli (Estevez 114

et al 2000 Bauer et al 2001 Carretero-Paulet et al 2002 Querol et al 115

2002 Hsieh and Goodman 2005 2006) Most mutants related to these genes 116

have an albino seedling phenotype and impaired chloroplast development By 117

contrast little is known about the MEP pathway in maize (Zea mays) beyond 118

the isolation of DXS DXR and HDR which catalyze the first second and last 119

steps of the MEP pathway respectively (Hans et al 2004 Cordoba et al 120

2011 Lu et al 2012) 121

HDS also known as GcpE IspG CSB3 and CEH1 encodes 122

1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase which catalyzes 123

the conversion of 2C-methyl-D-erytrithol 24-cyclodiphosphate (MEcPP) to 124

HMBPP the penultimate step in the MEP pathway (Ostrovsky et al 1998 125

Altincicek et al 2001 Querol et al 2002 Gutierrez-Nava et al 2004 Xiao et 126

al 2012) Consistent with other enzymes in the MEP pathway HDS is 127

encoded by a nuclear gene and is imported into plastids to synthesize the 128

precursor of plastid isoprenoids Knockout of HDS blocks the formation of 129

plastid isoprenoids (Gutierrez-Nava et al 2004) For example the complete 130

loss of function of HDS in Arabidopsis and Nicotiana benthamiana inhibits 131

chlorophyll and carotenoid biogenesis resulting in a seedling-lethal albino 132

phenotype (Gutierrez-Nava et al 2004 Page et al 2004) By contrast 133

enhancing the level of active HDS protein in E coli and Arabidopsis does not 134

result in the increased accumulation of carotenoids suggesting that enhanced 135

flux through the MEP pathway during peak demand periods in bacteria and 136

plastids does not require increased HDS activity (Flores-Perez et al 2008) 137

Additional work is needed to understand the mechanism by which HDS 138

regulates the metabolic flux from the MEP pathway to the downstream 139

biosynthesis of isoprenoids 140

In this study we isolated the maize mutant seed carotenoid deficient (scd) 141

which is characterized by albino plants with pale-yellow seeds and determined 142

that it is a spontaneous recessive mutant We established that the causal 143

mutation is located in SCD which results in the loss of its function due to 144

alternative transcript splicing Phenotypic characterization of scd suggested 145

that SCD plays an important role in controlling metabolic flux in the MEP 146

pathway and indirectly affects the biosynthesis of a number of plastid 147

isoprenoids such as carotenoids chlorophylls tocopherols abscisic acid 148

(ABA) and gibberellic acids (GAs) This study provides molecular insights into 149

the regulation of SCD in leaves and seeds and enhances our understanding of 150

the mechanisms that control the MEP pathway during plant development 151

RESULTS 153

The scd Mutant Has Carotenoid-Deficient Seeds and an Albino 154

Seedling-Lethal Phenotype 155

We discovered the scd mutant which has a visually distinctive phenotype 156

consisting of pale-yellow seeds and white seedlings (Fig 1AndashC) as a 157

spontaneously occurring mutant in a recombinant inbred line population 158

derived from a single cross between two elite inbred lines Zong3 and Yu87-1 159

(Tang et al 2007) The white seedlings generally die at the end of the V1 160

stage 161

To obtain further insight into the nature of the scd mutant phenotype we 162

analyzed the carotenoid profiles of scd seeds and leaves and the chlorophyll 163

profiles of scd leaves The ability of the scd mutant to synthesize carotenoids 164

and chlorophylls was greatly reduced in its leaves and carotenoid levels were 165

significantly reduced in scd seeds (Fig 1D and E Supplemental Fig S2) 166

These results suggest that SCD likely functions upstream of carotenoid and 167

chlorophyll biosynthesis and that its effects vary between seeds and leaves 168

Carotenoids and chlorophylls play key roles in photoprotection and 169

photosynthesis To determine whether the effects of the scd mutation on 170

carotenoid and chlorophyll levels in leaves are due to the pleiotropic effects of 171

light damage we grew scd and wild-type (WT) plants in the dark and analyzed 172

the subsequent leaf carotenoid and chlorophyll profiles (Supplemental Fig 173

S3) The albino phenotype of scd did not revert to that of WT and little 174

difference in carotenoid and chlorophyll levels was detected between scd 175

mutants grown in either the light or the dark (Supplemental Fig S2 and S3) 176

These findings suggest that the albino phenotype of scd mutants is not due to 177

the secondary effects of photo-oxidative stress 178

The scd Mutation Affects Aleurone Cell Development and Results in 179

Impaired Chloroplasts 180

To investigate the effect of the scd mutation on seed development we 181

analyzed WT and scd seeds at 15 days after pollination (DAP) using 182

transmission electron microscopy (TEM) (Fig 2A and B) The aleurone and 183

subaleurone cells showed substantial changes especially with respect to the 184

number of aleurone grains and vacuole shape Compared to that in WT 185

aleurone cells in scd contained a greater number of smaller aleurone grains 186

and fewer larger vacuoles whereas subaleurone cells contained fewer smaller 187

aleurone grains and larger vacuoles (Fig 2C and D) It appears that the 188

development of some organelles in the aleurone and subaleurone layers is 189

blocked in the absence of functional SCD 190

We analyzed WT and scd leaf sections from V1-stage seedlings by 191

conventional optical microscopy and TEM to explore the cellular basis for the 192

albino phenotype of the mutant (Fig 2EndashJ) We detected an extreme reduction 193

in chloroplast numbers in mesophyll and vascular bundle sheath cells of scd 194

compared to the WT (Fig 2E and F) Moreover plastids in scd lacked 195

appressed internal membranes and contained short linear noncompact 196

membranes (Fig 2GndashJ) When the plants were grown in the dark chloroplasts 197

were still present in both the mesophyll and vascular bundle sheath cells of 198

V1-stage seedling leaves in scd but their structures were disrupted (Fig 2Kndash199

P) Based on plastid morphology it appears that chloroplast development in 200

scd is arrested at an early stage of development These results further 201

demonstrate that the presence of impaired chloroplasts is caused by the scd 202

mutation rather than the effect of photo-oxidative stress 203

Map-Based Cloning and Functional Verification of SCD 204

Before isolating the SCD gene we first asked whether the scd mutant is the 205

result of a single gene disruption We scored the number of seeds of each 206

color (yellow and pale-yellow) in SCDscd ears and found that the results fit a 207

31 ratio (yellow pale-yellow seeds 1205942 lt 1205940052 = 384 P gt 005 208

Supplemental Table S1) Thus scd is controlled by a single recessive gene 209

Subsequently we carried out map-based cloning using an F2 population 210

derived from the cross between B73 and SCDscd heterozygotes The SCD 211

locus was initially mapped between the insertion-deletion (InDel) markers 212

IDP7677 and IDP411 on chromosome 5 at a genetic distance of 16 cM and a 213

physical distance of 186 Mb it was subsequently fine-mapped within a 14-kb 214

region between markers MP2 and MP4 based on five recombinants (Fig 3A) 215

This region contains two predicted genes and genomic sequence analysis of 216

the refined interval in scd and WT revealed only a 76-kb insertion in the eighth 217

intron of GRMZM2G137409 (Fig 3B) BLAST analysis revealed that this 218

inserted fragment shares 99 identity with the CRM centromeric 219

retrotransposon in maize (AY1290081) which has the typical structure of a 220

long terminal repeat retrotransposon (Supplemental Fig S4) (Xu and Wang 221

2007) The long terminal repeat retrotransposon insertion in 222

GRMZM2G137409 in the scd mutant generates at least six alternatively 223

spliced transcripts in V1-stage seedling leaves and 15-DAP endosperms 224

which leads to a sharp reduction in transcript levels and consequently a lack 225

of protein from this transcript (Fig 3CndashF) Therefore we considered 226

GRMZM2G137409 as a candidate gene for the SCD locus 227

We confirmed this result by identifying additional alleles with mutations in 228

SCD The classic maize mutant lemon white2 (lw2) which was also named 229

vp12 and is referred to here as lw2-vp12 exhibits lemon-white endosperms 230

and completely albino seedlings (Fig 4AndashB Maluf et al 1997 Stinard 2013) 231

Genome sequence analysis suggested that GRMZM2G137409 is a prime 232

candidate for the lw2-vp12 locus (Stinard 2013) To investigate the 233

genotype-phenotype relationship in this mutant we sequenced 234

GRMZM2G137409 in lw2-vp12 and the WT parental line and found an 8-bp 235

insertion in the first exon resulting in a frameshift mutation (Fig 4C) 236

Expression analysis revealed severe reductions in the levels of 237

GRMZM2G137409 mRNA and protein in 15-DAP endosperms and V1-stage 238

seedling leaves of lw2-vp12 versus that in WT (Fig 4DndashE) Consistent with the 239

phenotype of scd lw2-vp12 had lower carotenoid and chlorophyll levels in 240

seeds and seedling leaves under both light and dark conditions than the WT 241

(Fig 4FndashJ) These results clearly indicate that lw2-vp12 is another 242

independent allele of scd 243

We also identified two additional mutants in GRMZM2G137409 namely 244

scd-1 and scd-2 These alleles have similar seed and seedling mutant 245

phenotypes as scd (Supplemental Fig S5A and B Supplemental Table S1) 246

Genomic sequence analysis of GRMZM2G137409 revealed that scd-1 carries 247

a Mu transposon insertion in the first exon and scd-2 has a G-to-A substitution 248

(Glu to stop codon TAG) in the twelfth exon introducing a premature stop 249

codon (Supplemental Fig S5C) These mutations also lead to decreased 250

SCD protein levels (Supplemental Fig S5D) 251

To confirm that the mutations in GRMZM2G137409 are responsible for the 252

scd phenotype we performed allelism tests using scd lw2-vp12 scd-1 and 253

scd-2 (Supplemental Fig S5) The seed phenotypes displayed a 31 254

segregation ratio of normal color to pale-yellow color for all three F1 hybrids 255

from crosses between SCDscd plants and LW2-VP12lw2-vp12 SCD-1scd-1 256

or SCD-2scd-2 plants (Supplemental Fig S5E Supplemental Table S1) 257

Therefore GRMZM2G137409 is the causal gene for the scd and lw2-vp12 258

mutants 259

We engineered mutations in SCD in the inbred line B73-329 using 260

CRISPRCas9 gene editing and obtained three null mutants referred to as 261

CR-scd-1 CR-scd-2 and CR-scd-3 (Supplemental Fig S6) Genome 262

sequencing identified multiple mutations in all mutants and reverse 263

transcription quantitative PCR (RT-qPCR) analysis revealed a drastic 264

reduction in GRMZM137409 mRNA expression in the leaves of all three 265

CR-scd plants (Supplemental Fig S6BndashD) Like the original scd mutant 266

these three CR-scd plants had albino phenotypes (Supplemental Fig S6E) 267

These findings suggest that the albino phenotype is caused by the loss of 268

function of SCD which is consistent with the original scd mutant and the 269

results of allelism tests of the three independent scd mutants 270

Characterization of SCD 271

We isolated the full-length cDNA of SCD from the leaves of WT seedlings 272

using 5prime and 3prime rapid amplification of cDNA ends (RACE) (Supplemental Fig 273

S7) In addition to the PCR product covering the whole open reading frame 274

region the full-length SCD cDNA consists of a 203-bp 5prime untranslated region 275

(UTR) a 2241-bp open reading frame (19 exons) and a 165-bp 3primeUTR (Fig 276

3B) Sequence analysis using databases such as Ensemble Plants 277

(httpplantsensemblorg) and NCBI (httpswwwncbinlmnihgov) indicated 278

that SCD is a single-copy gene in maize 279

Sequence alignment with the predicted protein indicated that SCD 280

encodes a putative HMBPP synthase (HDS) of 746 amino acids with an 281

estimated molecular mass of 82 kDa sharing high protein sequence similarity 282

with Arabidopsis HDS (Supplemental Fig S8) The HDS enzyme catalyzes 283

the conversion of MEcPP into HMBPP in the penultimate step of the MEP 284

pathway (Ostrovsky et al 1998 Altincicek et al 2001 Querol et al 2002 285

Gutierrez-Nava et al 2004 Page et al 2004 Flores-Perez et al 2008) 286

Arabidopsis plants with a knockout of HDS showed similar seed and leaf 287

phenotypes to those of the maize scd mutants (Gutierrez-Nava et al 2004) 288

confirming that SCD has conserved biological functions similar to those of its 289

Arabidopsis putative ortholog 290

The SCD protein was predicted to contain a chloroplast-targeting signal 291

peptide followed by a triose phosphate isomerase (TIM) barrel domain (A) for 292

dihydropteroate synthase an inserted TIM barrel domain (A) and a 4Fe4S 293

cluster-containing domain (B) for sulfitenitrite reductase (Supplemental Fig 294

S8) (Liu et al 2012) The A domain primarily plays a structural role and 295

interacts with the A domain to form a structure that enables the A and B 296

domains to interact in a ldquocup and ballrdquo manner during catalysis (Liu et al 2012) 297

Among these domains the A and B domains are highly conserved across 298

species (Supplemental Fig S8) 299

RT-qPCR analysis revealed that SCD was widely expressed in all maize 300

tissues examined (Supplemental Fig S9) SCD transcript levels were highest 301

in leaves followed by stems roots and other tissues In developing seeds 302

SCD was expressed at high levels at 15 and 35 DAP but at relatively low levels 303

at 20 25 and 30 DAP a similar pattern was observed in both endosperm and 304

embryo tissues at the corresponding stages When SCD was knocked out in 305

scd SCD expression was sharply downregulated especially in tissues where 306

SCD was normally expressed at high levels such as in leaves roots stems 307

and 15-DAP seeds 308

In addition to examine the subcellular localization of SCD we transiently 309

expressed SCD-enhanced green fluorescent protein (EGFP) fusions driven by 310

the 35S promoter (35SSCD-EGFP) in maize protoplasts (Supplemental Fig 311

S10) When SCD-EGFP was transiently expressed in protoplasts EGFP 312

fluorescence was observed in chloroplasts in addition to red autofluorescence 313

signals emitted from chlorophyll (Supplemental Fig S10EndashG) This result 314

indicates that SCD is imported into the plastid to carry out its function which is 315

in agreement with the movement of its Arabidopsis putative ortholog (Querol et 316

al 2002) 317

Given that SCD catalyzes the formation of HMBPP from MEcPP in the 318

MEP pathway we investigated whether the levels of metabolites in the MEP 319

pathway were altered in scd (Fig 5) The levels of the substrate metabolite 320

MEcPP were uniformly higher in scd versus WT seeds (~20-fold increase) 321

light-grown leaves (~27-fold increase) and dark-grown leaves (~230-fold 322

increase) as was also the case for the upstream metabolite MEP (~8- 68- 323

and 280-fold increase in seeds light-grown leaves and dark-grown leaves 324

respectively) By contrast the levels of the product HMBPP and the common 325

isoprenoid precursors DMAPP and IPP were significantly reduced in both light- 326

and dark-grown scd leaves specifically 607 to 879 of those in WT 327

whereas the levels of HMBPP DMAPP and IPP appeared to be reduced in 328

scd seeds but this decrease was not statistically significant These findings 329

suggest that the enzymatic activity of SCD is reduced in scd and that SCD 330

encodes an HDS protein 331

Metabolic Effects of the Loss of SCD Function 332

Due to the fundamental role of the MEP pathway in isoprenoid biosynthesis 333

we reasoned that the loss of function of SCD in this pathway might result in 334

altered levels of numerous downstream isoprenoids Indeed analysis of 335

lw2-vp12 indicated that vp12 embryos are deficient in ABA accumulation 336

during kernel development (Maluf et al 1997) Hence in addition to 337

carotenoids and chlorophylls we analyzed 24 other metabolites including 338

three tocopherols and members of six phytohormone families in seeds and 339

light- and dark-grown leaves of scd seedlings The ability of the scd and 340

lw2-vp12 mutants to synthesize tocopherols was nearly lost in leaves but still 341

remained in seeds although at a significantly reduced level (Table 1 342

Supplemental Fig S11) Light treatment had little effect on the tocopherol 343

levels in scd 344

Among the 21 metabolites of the six phytohormone families identified 16 345

17 and 18 metabolites were identified in mature seeds light-grown leaves 346

and dark-grown leaves in WT respectively (Table 1) Except for salicylic acid 347

no significant changes in the levels of these examined metabolites were 348

detected in mature scd seeds By contrast the levels of ABA and six GAs 349

including GA3 GA9 GA15 GA19 GA20 and GA53 were significantly 350

reduced in the seedling leaves of scd in both the light and dark with the effects 351

on GA19 levels highly sensitive to dark conditions In addition the levels of 352

indole-3-acetic acid 3-indolebutyric acid and jasmonic acid were significantly 353

reduced only in light-grown scd leaves By contrast the level of methyl 354

indole-3-acetate (ME-IAA) consistently was higher in both light- and 355

dark-grown scd leaves compared to the WT Collectively these results indicate 356

that the metabolic effects of SCD vary among plant tissues and growth 357

conditions 358

Identification of SCD-Associated Molecular Perturbation 359

To extend our knowledge of the changes in gene expression mediated by SCD 360

we generated transcriptome profiles for 15-DAP endosperms and embryos 361

and light- and dark-grown V1-stage seedling leaves from scd and WT The 362

abundance of reads for SCD exons downstream of the mutation site in scd 363

was strongly reduced in endosperms embryos and light-grown leaves but not 364

in dark-grown leaves (Fig 6A) which is highly consistent with the results of 365

multiple transcript analysis and RT-qPCR (Fig 3D and E Supplemental 366

Figure S12) Global transcriptome analysis identified 6000 genes with 367

significantly altered abundance in scd (gt20-fold P lt 001) including 116 in 368

endosperms 175 in embryos 735 in dark-grown leaves and 4974 in 369

light-grown leaves (Fig 6B Supplemental Dataset S1) Of these no 370

differentially expressed genes (DEGs) were common among the four tissues in 371

the up- or down-regulated groups The great differences in the altered gene 372

expression between light- and dark-grown scd leaves demonstrate that 373

transcriptional perturbation in scd leaves is greatly affected by light The large 374

differences among endosperms embryos and dark-grown leaves suggest that 375

the downstream effects of SCD at the transcriptional level might be tissue 376

specific to some extent 377

We then performed Gene Ontology (GO) enrichment analysis of the DEGs 378

to better understand the biological processes affected by SCD (Fig 6C) No 379

biological process terms were commonly enriched in the four tissues with 1 5 380

11 and 20 biological process terms enriched in endosperms embryos 381

dark-grown leaves and light-grown leaves respectively Among these 382

biological process terms photosynthesis was the most significantly enriched 383

among downregulated genes in light-grown scd leaves most likely due to 384

pleiotropic effects of light damage Of the 1026 DEGs detected in endosperms 385

embryos and dark-grown leaves ~409 (421026) of the genes were 386

considerably enriched for the biological process categories involved in 387

isoprenoid biosynthesis photosynthesis and the regulation of phytohormone 388

levels These results further confirm that SCD encodes a major enzyme in the 389

MEP pathway which produces the precursors of these isoprenoids 390

We were particularly interested in biological processes associated with 391

plastid isoprenoid biosynthesis including carotenoid chlorophyll and 392

tocopherol biosynthesis and with the biosynthesis of their precursors in the 393

MEP pathway in the plastid and isoprenoid precursor biosynthesis in the MVA 394

pathway in the cytoplasm (Fig 6D Supplemental Table S2) Among the 60 395

genes identified in these metabolic pathways 88 (5360) were sharply 396

downregulated in light-grown scd leaves especially genes in the MEP pathway 397

and its downstream pathways By contrast no gene showed a significant 398

change in expression in the endosperms and 10 (660) and 21 (1360) of 399

genes were down- or up-regulated in embryos and dark-grown scd leaves 400

respectively 401

Only two DEGs were commonly identified in embryos and dark-grown 402

leaves One of these genes is DXS2 encoding 1-deoxy-D-xylulose 5-phospate 403

(DXP) synthase which catalyzes the first step in the MEP pathway (Walter et 404

al 2002 Cordoba et al 2011) and the other is CH1 encoding chlorophyllide 405

a oxygenase which functions in chlorophyll biosynthesis (Oster et al 2000 406

Hoober and Eggink 2001 Hirashima et al 2006 Tanaka and Tanaka 2007) 407

Only DXS2 was downregulated in both tissues of scd 408

These results were confirmed by RT-qPCR analysis of the expression of 409

10 genes (Supplemental Fig S12 Supplemental Table S3) These 410

tissue-specific differences in gene expression are not consistent with the 411

similar phenotypes of both scd leaves and seeds with respect to reduced 412

carotenoid and tocopherol levels suggesting that the nature of the 413

transcriptional regulation of enzymes in the MEP pathway might vary among 414

tissues These results also indicate that this post-transcriptional regulation 415

might be involved in the metabolic flux between the MEP pathway and the 416

downstream isoprenoid biosynthesis pathway 417

Overexpression of SCD Does Not Affect the Levels of Downstream 418

Metabolites of the MEP Pathway 419

In contrast to the loss of SCD function we reasoned that an increase in SCD 420

expression might result in reduced substrate levels and enhanced metabolite 421

production consequently increasing the levels of downstream isoprenoids 422

such as carotenoids tocopherols and chlorophylls Thus we generated 423

transgenic lines expressing SCD cDNA under the control of the maize 424

Ubiquitin1 promoter (pUbi) in maize inbred line B73-329 Two positive 425

transgenic lines OE1 and OE2 were obtained and their enhanced expression 426

of SCD was confirmed by both RT-qPCR and immunoblot analysis of T2 plants 427

(Fig 7AndashD) The overexpressing line OE1 was then backcrossed to SCDscd 428

plants and self-pollinated for two generations The ears produced by selfing of 429

SCDOE1scd plants exhibited a 151 segregation ratio of yellow to pale-yellow 430

seeds indicating that scd was successfully complemented (Supplemental Fig 431

S13) In addition no significant differences in the levels of downstream 432

metabolites were detected between the complemented individuals and WT 433

(Supplemental Fig S14) These results suggest that the scd mutant 434

phenotype was fully rescued by overexpressing SCD 435

We subsequently measured five metabolites in the MEP pathway and the 436

downstream isoprenoids in seeds and seedling leaves of the transgenic lines 437

(Fig 7 Supplemental Table S4) Only the levels of the substrate MEcPP 438

were significantly reduced in the leaves of both transgenic lines Unexpectedly 439

the levels of the isoprenoid precursors did not increase in the seeds and 440

leaves of both transgenic lines and only a few downstream isoprenoids 441

increased in the seeds or leaves of one transgenic line 442

We also examined the expression patterns of 24 genes in 15-DAP 443

endosperms and embryos and V1-stage seedling leaves from the 444

overexpressing transgenic lines OE1 and OE2 and from WT B73-329 by 445

RT-qPCR (Supplemental Fig S15) These 24 genes included seven genes in 446

the MEP pathway two genes in the intermediate pathway between the MEP 447

pathway and its downstream pathways seven genes involved in carotenoid 448

biosynthesis four genes involved in tocopherol biosynthesis and four genes 449

involved in chlorophyll biosynthesis Only a few genes were upregulated in 450

certain tissues whereas SCD was upregulated in all three tissues These 451

findings indicate that SCD overexpression does not induce the expression of 452

genes in the MEP pathway or the downstream plastid isoprenoid pathway 453

DISCUSSION 455

The MEP pathway plays a central role in plant growth and development and 456

several isoprenoids derived from this pathway are beneficial for human health 457

and nutrition Thus identifying the major regulatory steps of the MEP pathway 458

is important for potentially modulating the production of key isoprenoids Here 459

through fine mapping allelism tests CRISPRCas9-mediated gene editing 460

and metabolite analysis we determined that SCD functions as an HDS protein 461

in the MEP pathway consequently affecting the accumulation of a variety of 462

isoprenoids such as carotenoids chlorophylls tocopherols ABA and GAs 463

These findings provide important insights into the impact of the MEP pathway 464

and its downstream metabolic and functional roles in maize 465

SCD Is an Enzyme in the MEP Pathway 466

The MEP pathway produces the common metabolic precursors IPP and 467

DMAPP for plastid isoprenoid biosynthesis which involves the consecutive 468

participation of eight enzymes including an HDS enzyme that catalyzes the 469

production of HMBPP from MEcPP in the plastid (Supplemental Fig S1) 470

Previous genome sequence analysis suggested that the lw2-vp12 locus 471

harbors a candidate gene encoding an HDS protein in maize which shares the 472

highest sequence identity with AtHDSAtGcpE in the maize genome (Stinard 473

2013) Phenotypically the lw2-vp12 mutant is a classical carotenoid mutant 474

with lemon-white endosperms and an albino seedling-lethal phenotype (Maluf 475

et al 1997) similar to that of the scd mutant identified in this study Allelism 476

tests performed with the lw2-vp12 and scd mutants demonstrated that the 477

mutation in the lw2-vp12 mutation is allelic to the scd mutation with both the 478

lw2-vp12 and scd loci resolved to be a gene encoding an HDS protein The 479

enzymatic activity and function of AtGcpEAtHDS as an HDS protein have 480

been demonstrated in Arabidopsis (Seemann et al 2005) Knockout 481

mutations in CLB4AtHDS result in white seeds and an albino seedling-lethal 482

phenotype with severely affected chloroplasts that are unable to synthesize 483

photosynthetic pigments (Gutierrez-Nava et al 2004) The similar phenotypes 484

observed in the scdlw2-vp12 mutant point to the possibility that SCD functions 485

as an HDS protein This notion was further confirmed by the observation that 486

this protein is localized to the plastid and that its substrate levels are elevated 487

in scd whereas the levels of its product are reduced 488

Visibly there is a slight difference between scdlw2-vp12 and clb4 in terms 489

of seed color in that seeds are pale yellow in scd but white in clb4 490

(Gutierrez-Nava et al 2004) These phenotypes are indicators of carotenoid 491

deficiency as observed in other carotenoid-deficient mutants such as y1 vp5 492

y9 vp9 ps1 and vp2 (Buckner et al 1990 Li et al 1996 Matthews et al 493

2003 Singh et al 2003 Li et al 2007) We found that scd seeds accumulated 494

136 of the total carotenoid levels of WT seeds (Supplemental Figure S2) 495

suggesting that carotenoid biosynthesis still occurs in scd Perhaps this is due 496

to the remaining regulatory effects of low levels of SCD since the multiple 497

alternative transcript variants in the mutant include the normal SCD transcript 498

(Figure 3CndashE Figure 6A) However we cannot exclude the possibility that an 499

active HDS enzyme other than SCD remains in the plastids of seeds since 500

immunoblot analysis revealed another visible band of the expected size that 501

was not detected in leaves (Figure 3F 4D Supplemental Figure S5D) 502

although the additional band might have been a nonspecific band detected by 503

the SCD antibody in seeds Further studies are needed to confirm the true 504

nature of the carotenoid biosynthesis ability in scd seeds 505

SCD Has Pleiotropic Effects on Metabolite Biosynthesis and Plant 506

Development 507

The MEP pathway produces metabolic precursors for plastid isoprenoids 508

suggesting that disturbing MEP pathway activity alters the accumulation of the 509

downstream isoprenoids consequently leading to pleiotropic phenotypes 510

Most MEP pathway genes identified to date are associated with carotenoid 511

accumulation (Lois et al 2000 Estevez et al 2001 Hsieh and Goodman 512

2005 Xing et al 2010 Lu et al 2012) as is SCD which was identified in this 513

study In addition to carotenoid deficiency caused by the knockout of SCD 514

SCDHDS colocalized with a QTL cluster for zeaxanthin xanthophylls and 515

total carotenoids (Venado et al 2017) pointing to the possible association 516

between SCD and carotenoids although no significant association was 517

observed in a genome-wide association study of 281 maize inbred lines 518

(Owens et al 2014) Consistent with the reduction in carotenoid levels we also 519

observed a reduction in tocopherol levels among the three tissues examined 520

Furthermore the loss of SCD function weakened the ability of the scd 521

mutant to synthesize chlorophylls ABA and several GAs in light- and 522

dark-grown leaves and indole-3-acetic acid indole-3-carboxaldehyde and 523

jasmonic acid in light-grown leaves Among the phytohormones whose levels 524

were altered in scd only ABA levels were previously shown to be reduced in 525

developing embryos of the lw2-vp12 mutant (Maluf etal 1997) However ABA 526

was not detected in scd seeds perhaps due to the fact that dried mature seeds 527

were used in our study (McCarty and Carson 1991) Nonetheless the finding 528

that ABA levels were reduced in both light- and dark-grown leaves confirms the 529

notion that SCD has a metabolic effect on the accumulation of ABA 530

In addition to the metabolic effects of SCD the scd mutant exhibited 531

severely impaired seedling development a phenotype similar to that of other 532

albino mutants in Arabidopsis and maize harboring mutations that affect the 533

enzymes involved in MEP-related biosynthetic pathways (Mandel et al 1996 534

Araki et al 2000 Estevez et al 2000 Budziszewski et al 2001 535

Gutierrez-Nava et al 2004 Guevara-Garcia et al 2005 Hsieh et al 2008 536

Cordoba et al 2011 Lu et al 2012) It is conceivable that the inability to carry 537

out photosynthesis as reflected by severely disrupted chloroplasts leads to 538

seedling lethality in the scd mutant at early stages of development As 539

chlorophylls are required for the formation of stacked thylakoids (Von Wettstein 540

et al 1995) it is reasonable to link the early arrest of chloroplast differentiation 541

in scd plants with the absence of chlorophyll However other factors could also 542

lead to the formation of albino plants such as reduced levels of some 543

phytohormones such as ABA GAs and so on (Buckner et al 1990 Li et al 544

1996 Matthews et al 2003 Singh et al 2003 Myers et al 2011) In addition 545

distinct changes were observed in the aleurone and subaleurone cells of 546

developing scd seeds including the number and size or shape of aleurone 547

grains and vacuoles How the development of the aleurone and subaleurone 548

layers in seeds is associated with SCD in the MEP pathway remains unknown 549

The metabolic and developmental effects of SCD varied among tissues 550

and light conditions A similar situation was observed in the maize w3 mutant 551

whose phenotypes result from a plastoquinone-9 deficiency that in turn limits 552

the contributions of plastoquinone-9 to the formation of downstream 553

metabolites such as carotenoids chlorophylls and tocopherols (Hunter et al 554

2018) The ability of the scd mutant to continue to accumulate isoprenoids in 555

seeds reflects the fact that these seeds are not subject to severe 556

photo-oxidative stress The light-mediated metabolic differences in leaves 557

under light and dark growth conditions can be explained primarily by the 558

different levels of damage to the photosynthesis system under different light 559

conditions Previous studies have shown that dark treatment weakens the 560

activity of the MEP pathway (Rodriguez-Concepcion et al 2004 Botella-Pavia 561

et al 2004 Hans et al 2004 Rodriguez-Villalon et al 2009) which is 562

consistent with our findings (Fig 6) Therefore the reduction in levels of the 563

isoprenoid precursor in the dark might be weakened which results in the 564

limited reduction in the levels of downstream products 565

Post-Transcriptional Regulation of SCD in the MEP Pathway 566

Although the function of SCD in maize was confirmed in this study it remains 567

unclear how SCD is regulated during the complex metabolic flux to 568

biosynthesize MEP-derived isoprenoids The MEP pathway is thought to 569

branch to produce many families of metabolites and the common core 570

reactions of this pathway are thought to be tightly regulated to assure the 571

coordination of metabolic flux Due to the pleiotropic effects of light damage 572

we only compared the DEGs identified in the developing endosperms and 573

embryos and dark-grown leaves of scd versus the WT Unexpectedly the 574

number of downregulated DEGs involved in a known biological process related 575

to isoprenoid biosynthesis was far smaller in the dark than in the light with 576

none identified in the endosperms and only two and three DEGs from the 577

carotenoid and chlorophyll pathways identified in dark-grown leaves and 578

embryos respectively (Fig 7) One possible explanation for this finding 579

involves the existence of a post-transcriptional or post-translational regulatory 580

mechanism as has been found in other species (Hemmerlin et al 2012 581

Hemmerlin 2013) This molecular regulatory mechanism operates for other 582

enzymes in the MEP pathway eg DXS in Arabidopsis (Guevara-Garcia et al 583

2005) 584

Theoretically upregulating the genes in the MEP pathway will force 585

metabolic flux to the downstream isoprenoids consequently leading to the 586

enhanced accumulation of isoprenoids For example overexpressing genes 587

for certain upstream precursor pathway enzymes (eg DXS DXR and HDR) 588

was shown to force metabolic flux to the downstream pathway (Estevez et al 589

2001 Botella-Pavia et al 2004 Carretero-Paulet et al 2006) By contrast no 590

obvious coordinated changes were detected for the levels of isoprenoids in the 591

leaves or seeds of SCD-overexpressing transgenic lines and the upregulation 592

of SCD expression was not coordinated with that of other genes controlling 593

plastid isoprenoid biosynthesis Indeed the expression levels of SCDHDS are 594

not significantly correlated with carotenoid contents in maize endosperms 595

(Valalbhaneni and Wurtzel 2009) Similar results were also obtained for SCD in 596

E coli and Arabidopsis (Flores-Perez et al 2008) and DXR another gene in 597

the MEP pathway in Arabidopsis (Xing et al 2010) Perhaps the expression 598

levels of SCD are normally higher than needed and thus metabolic changes 599

will occur only after changes in the enzyme levels reach a certain threshold 600

eg knockout of SCD In addition as suggested by the transcriptional 601

changes detected in scd SCD might undergo post-transcriptional or 602

post-translational regulation For example the mislocalization of SCD protein 603

might have a regulatory effect when the protein levels in SCD-overexpressing 604

transgenic lines are elevated However the likelihood of such an effect is 605

reduced in scd when it is rescued by SCD overexpression in transgenic plants 606

pointing to the correct localization of SCD Thus it appears that metabolic 607

balance is more sensitive to the downregulation of the MEP pathway than its 608

upregulation 609

In summary we demonstrated that SCD affects the production of plastid 610

isoprenoid precursors in the MEP pathway in maize subsequently affecting 611

the accumulation of its downstream isoprenoids thereby affecting plant 612

development The metabolic and transcriptional changes caused by the loss of 613

SCD function varied among tissues and light conditions The identification of a 614

few DEGs related to MEP-derived isoprenoid biosynthesis suggests that 615

post-transcriptional regulation might be a major regulatory mechanism of SCD 616

These findings advance our understanding of the roles of SCD in plants 617

MATERIALS AND METHODS 619

Plant Materials and Growth Conditions 620

The scd mutant in maize originated from a natural mutation that occurred in 621

recombinant inbred line RIL105 derived from a cross between two elite inbred 622

lines Zong3 and Yu87-1 (Tang et al 2007) Three additional scd mutants 623

were obtained from the Maize Genetics Cooperation Stock Center lw2-vp12 624

(stock number 512I lw2-vp12) was derived from an unknown tropical variety 625

scd-1 (stock number 523B lw2-UFMu-07766) was isolated from the UniformMu 626

line UFMu-07766 in the W22 background scd-2 (stock number 518D lw2) is a 627

spontaneous mutant in the maize variety lsquoGolden Gloryrsquo background The 628

mutation sites of these alleles were determined by sequencing with the primers 629

listed in Supplemental Table S5 The plants were cultivated in a greenhouse 630

under light or dark conditions or in an experimental field at the China 631

Agricultural University in Beijing and Hainan China Seedlings were grown in 632

growth chambers under a 16-h-light8-h-dark cycle for light conditions or a 633

24-h-dark cycle for dark conditions 634

Measurement of the Chemical Composition of Seeds and Leaves 635

To analyze the chemical composition of seeds 50 seeds were randomly 636

chosen from each well-grown ear To analyze the chemical composition of 637

leaves 01ndash02 g of tissue was collected from fresh seedling leaves The 638

chlorophyll pigments were extracted in 95 ethanol (vv) and the levels of 639

chlorophyll a and chlorophyll b were determined with a UVVis 640

spectrophotometer to measuring absorbance at 665 and 649 nm respectively 641

(Lichtenthaler 1987) To measure carotenoid and tocopherol contents in both 642

seeds and leaves high-performance liquid chromatography (HPLC) was 643

performed (Chander et al 2008) All measurements were replicated at least 644

three times 645

Microscopy 646

To visualize leaf and seed development leaves sampled from V1-stage WT 647

and scd seedlings and seeds dissected from self-pollinated WT and SCDscd 648

ears at 15 DAP were fixed for 2 h in 25 glutaraldehyde (vv) buffered with 01 649

M pH 72 phosphate buffer washed three times in 01 M pH 72 phosphate 650

buffer post-fixed for 2 h in 1 (wv) osmic acid washed in phosphate buffer 651

dehydrated in an acetone gradient up to 100 (vv) and slowly embedded in 652

Spurr epoxy resin 653

For light microscopy analysis of leaf tissue 075-μm semithin sections 654

were cut with an OM-U3 microtome (Reichert-Jung) and photographed under 655

an Olympus BX-60 microscope using a Jenoptik C5 camera system For TEM 656

of leaves and seeds samples were sectioned at 80 nm with a Leica EM UC6 657

ultramicrotome affixed to grids and stained with uranyl acetate and lead 658

citrate Digital images were acquired using JEOL 2100 200 kV scanning and 659

JEM-1230 transmission electron microscopes 660

Map-Based Cloning of SCD 661

To map the SCD locus an SCDscd plant was crossed with B73 plants 662

Primary mapping was performed with 356 simple sequence repeat (SSR) or 663

InDel markers using 509 individuals from the F2 mapping population 664

Subsequently 828 recessive individual plants with pale-yellow seeds and 665

white leaves were used for fine mapping of SCD with 11 markers 666

(Supplemental Table S5) To identify the mutation site the corresponding 667

genomic fragments in the refined interval were sequenced in scd and WT 668

using 27 primer pairs (Supplemental Table S5) 669

RNA Extraction and RT-qPCR 670

Total RNA was extracted from various organs using an RNAprep Pure Plant Kit 671

(Tiangen Dalian China) First-strand cDNA was synthesized using a 672

PrimeScript II 1st strand cDNA synthesis kit (TAKARA Dalian China) 673

RT-qPCR was carried out in triplicate for each sample using a SYBR Green I 674

Kit (TAKARA Dalian China) on a 7500 Real Time PCR System (ABI CA 675

USA) Maize TUBBLIN was used as a control for normalization between 676

samples (Supplemental Table S2) Relative transcript levels were calculated 677

using the comparative threshold cycle method (Livak and Schmittgen 2001) 678

RACE and Identification of Various Transcripts in the scd Mutant 679

Total RNA was extracted from V1-stage leaves and 15-DAP endosperms from 680

the scd mutant and WT For RACE first-strand cDNA from WT leaf tissue was 681

synthesized using a 5prime-Full RACE Kit and 3prime-Full RACE Core Set (TAKARA 682

Dalian China) SCD-specific primers were designed to amplify 5prime- and 683

3prime-RACE-ready cDNAs (Supplemental Table S5) Sequences from the 5prime- and 684

3prime-RACE products were assembled to obtain full-length SCD cDNA To isolate 685

various transcripts in scd leaves and endosperms the synthesized first-strand 686

cDNA was amplified using primer pair SCD_FL (Supplemental Table S5) The 687

PCR products from WT were directly sequenced whereas the PCR products 688

from the scd mutant were cloned into the pMDTM20 T-Vector (TAKARA 689

Dalian China) and at least 30 clones were sequenced 690

Antibody Preparation Protein Extraction and Immunoblot Analysis 691

A polypeptide containing SCD amino acid residues 733ndash745 was synthesized 692

and used to raise polyclonal antibodies in rabbits which were then affinity 693

purified by the Beijing ComWin Biotech Company (China) A 1100 dilution of 694

the resulting antibody was used for protein blotting The specificity of SCD 695

antibodies was confirmed by immunoblot analysis of WT Total proteins were 696

isolated from various organs as described (Bernard et al 1994) An equal 697

amount of protein from each sample (30 μg) was loaded onto an 8 698

SDS-polyacrylamide gel and blotted onto a 02-microm nitrocellulose membrane 699

(Schleicher and Schuell Dassel Germany) The membranes were blocked 700

with 5 (wv) nonfat dry milk in Tris-buffered saline with 01 (vv) Tween-20 701

and incubated with primary antigen-specific antiserum at a 1200 dilution and 702

secondary alkaline phosphatasendashconjugated goat anti-rabbit IgG (Bio-Rad) at 703

13000 dilution Immunoblot analysis was performed using the SCD antibody 704

at 1100 dilution with Actin antibody (ABclonal Boston MA) used as the 705

control at 11000 dilution 706

CRISPRCas9 Gene Editing and Genotyping 707

CRISPRCas9 mutagenesis and the generation of transgenic maize plants 708

were performed as described previously (Xing et al 2014) Briefly the 709

CRISPRCas9 binary vector pBUE-2gRNA-SCD was constructed to produce 710

defined deletions in the coding region of SCD using two sgRNAs alongside the 711

Cas9 endonuclease gene (Supplemental Table S5) The construct was 712

transformed into Agrobacterium tumefaciens strain EHA105 and used to 713

transform immature embryos of maize inbred line B73-329 via 714

Agrobacterium-mediated transformation at China Agricultural University 715

Transgenic Facility Center To genotype the first-generation (T0) transgenic 716

lines the white and green leaves of three regenerated plants were separately 717

collected DNA and RNA were simultaneously extracted using an RNAprep 718

Pure Plant Kit (Tiangen Dalian China) Each sample was genotyped using a 719

forward primer based on the sequence to the left of sgRNA1 and a reverse 720

primer to the right of sgRNA2 (Supplemental Table S5) PCR products were 721

cloned into the pMDTM20 T-Vector (TAKARA Dalian China) and at least six 722

clones per PCR product were sequenced Three positive transgenic plants 723

were subjected to RT-qPCR using the SCD primer pair (Supplemental Table 724

S3) 725

Subcellular Localization 726

The coding sequence of SCD was amplified using cDNA from V1-stage WT 727

seedlings as the template The resulting PCR product was inserted into the 728

pEZS-NL vector to generate the SCD-EGFP fusion protein driven by the 35S 729

promoter Maize protoplasts were isolated as described (Yoo et al 2007) 730

transformed with the 35SSCD-EGFP construct incubated for 16ndash20 h in the 731

dark at 25degC and analyzed for GFP expression under a Zeiss 710 confocal 732

microscope 733

Measurement of Metabolites in the MEP Pathway 734

The relative levels of the targeted metabolites in the MEP pathway in maize 735

seed and leaf samples were quantified by liquid chromatographyndashelectrospray 736

ionizationndashtandem mass spectrometry (LC-ESI-MSMS) (Li and Sharkey 737

2013) The metabolites were extracted from 50 mg of powdered tissue using 1 738

mL of 50 methanol (vv) and separated and quantified on a C18 5u column 739

(150 mm times 46 mm Phenomenex CA USA) fitted to a 5500 QTRAP mass 740

spectrometer (AB Sciex Ontario Canada) Standards for MEP pathway 741

metabolites including MEP MEcPP HMBPP IPP and DMAPP were acquired 742

from Echelon Biosciences (Logan UT USA) The metabolites were quantified 743

using the scheduled multiple reaction monitoring (MRM) method (Liu et al 744

2017) Among the detected metabolites DMAPP and IPP could not be 745

differentiated from each other thus the metabolite content was calculated as 746

the average of DMAPP and IPP 747

Analysis of Phytohormone Levels by Ultra-HPLC Tandem Electrospray 748

Mass Spectrometry 749

Phytohormones were extracted and purified in three biological replicates using 750

a protocol modified from Cai et al (2016) Fresh plant materials were 751

harvested immediately frozen in liquid nitrogen and stored at 80degC until use 752

The samples were ground in liquid nitrogen and 120 mg (fresh weight) of 753

material per sample was transferred into a 15-mL centrifuge tube Following 754

the addition of solution containing 80 methanol (vv 12 mL) the mixture was 755

vortexed six times every 05 h After incubation at 4degC for 12 h the supernatant 756

was collected following centrifugation at 12000 g for 15 mins at 4degC The 757

supernatant was evaporated to dryness under a mild nitrogen stream at 35degC 758

and redissolved in 01 mL solution containing 30 methanol (vv) The solution 759

was centrifuged and the supernatant was collected for phytohormone analysis 760

Twenty-one standards (Supplemental Table S6) were purchased from 761

Olchemim Ltd (Olomouc Czech Republic) and Sigma (St Louis MO USA) 762

Stock solutions of all standards were prepared at a concentration of 10 mgmL 763

in acetonitrile and stored at 18degC until use To construct the standard curve 764

the stock solutions were diluted with acetonitrile into working solutions and 18 765

gradient-mixed standard solutions ranging from 002 to 2000 ngmL (002 005 766

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

The levels of auxins GAs cytokinins jasmonic acids salicylic acid and 769

ABA in each sample and mixed standard were detected by ultra-HPLC tandem 770

electrospray mass spectrometry (UPLC-MSMS AB Sciex USA) Separation 771

was performed on an ACQUITY UPLC HSS T3 C18 column (18 microm 21 mm times 772

100 mm Waters USA) at 40degC with a mobile phase flow rate of 035 mLmin 773

The mobile phase consisted of water (A) and acetonitrile (B) both containing 774

004 acetic acid (vv) A linear gradient elution program was applied as 775

follows initial 5 B for 11 mins 95 B at 111 min for 1 min 5 B at 121 min 776

for 3 mins and 5 B at 15 min The injection volume was 50 microL The effluent 777

was alternatively connected to an ESI-triple quadrupole-linear ion trap 778

(QTRAP) mass spectrometer The API 6500 QTRAP LCMSMS system 779

equipped with an ESI Turbo Ion-Spray interface was controlled by Analyst 16 780

software (AB Sciex) The ESI source operation parameters were as follows 781

ion source turbo spray source temperature 500degC ion spray voltage (IS) 782

5500 V curtain gas (CUR) 350 psl and collision gas (CAD) set to medium 783

Detailed information about each measured metabolite is provided in 784

Supplemental Table S6 Data acquisition and processing were performed 785

using Analyst 16 software (AB Sciex USA) 786

RNA-seq Analysis 787

Eighteen samples including 15-DAP endosperms and embryos and V1-stage 788

leaves from scd and WT plants were subjected to RNA-seq Three 789

independent biological replicates were analyzed per tissue RNA-seq libraries 790

were constructed according to the standard Illumina RNA-seq protocol 791

followed by paired-end sequencing on the Illumina HiSeq 2500 platform 792

Reads were sequenced demultiplexed and filtered using the default pipeline 793

and parameters On average 1261 million 125-bp paired-end reads were 794

generated per sample (Supplemental Dataset S1) The reads were aligned to 795

the maize B73 RefGen_V3 reference genome using Tophat2 (Kim et al 2013) 796

and the number of uniquely mapped reads covering each gene model (AGPv3) 797

was determined using HTSeq software (Anders et al 2015) The counts were 798

normalized according to library size and genes with less than one count per 799

million were filtered out DEGs between scd and WT plants were identified 800

using the software package edgeR (Robinson et al 2010) allowing a false 801

discovery rate of 001 The potential functions and enriched GO terms of the 802

DEGs were analyzed using MapMan software (httpmapmangabipdorg) 803

(Thimm et al 2004) To investigate the enrichment of GO categories Fisherrsquos 804

exact test was used to analyze the number of genes per bin P-values were 805

adjusted for multiple tests based on the false discovery rate using the BH 806

method (Benjamini and Hochberg 1995) Bins for which the false discovery 807

rate was lt005 were considered to be enriched 808

Overexpression of SCD and Genotyping and Phenotyping of the 809

Resulting Lines 810

The full-length coding sequence of SCD was amplified from cDNA with primer 811

pair PBCXUN_SCD (Supplemental Table S6) and cloned into the binary 812

vector PBCXUN under the control of the maize ubiquitin-1 promoter using 813

XcmI as the restriction enzyme The recombinant plasmid PBCXUN-SCD was 814

introduced into Agrobacterium strain EHA105 which was then transformed 815

into B73-329 at the China Agricultural University Transgenic Facility Center 816

The SCD-specific primer pair ZmSCD was used to screen for 817

transgene-positive plants which were further confirmed by RT-qPCR using the 818

same primers (Supplemental Table S3) To evaluate the phenotypic effects of 819

overexpressing SCD the levels of metabolites in the MEP pathway and 820

downstream isoprenoids ie carotenoids and tocopherols were measured in 821

seedlings and seeds as described above as were chlorophyll levels in 822

seedlings 823

Statistical Analysis 824

Two-tailed Studentrsquos t-test was used to test the significance of expression and 825

metabolite differences between mutants or overexpressing transgenic lines 826

and wide types 827

Accession Numbers 829

Sequence data from this study can be found in the GenBankEMBL data 830

libraries under accession number MF784564 for SCD MG181953 for scd and 831

SRP116562 and SRP159627 for the RNA-seq data 832

Competing interests 834

The authors declare that they have no competing interests 835

Supplemental Data 837

Supplemental Figure S1 Simplified diagram of the MVA and MEP pathways 838

and the biosynthesis of their downstream products in plant cells 839

Supplemental Figure S2 Comparison of carotenoid and chlorophyll contents 840

in scd vs WT 841

Supplemental Figure S3 Phenotypes of seedlings grown in the dark 842

Supplemental Figure S4 Schematic representation of the 76-kb terminal 843

repeat retrotransposon insertion in scd 844

Supplemental Figure S5 Allelism tests using three additional scd alleles 845

Supplemental Figure S6 CRISPRCas9-induced mutations in SCD result in 846

lethal seedlings 847

Supplemental Figure S7 Full-length SCD cDNA isolated using 5rsquo and 3rsquo rapid 848

amplification of cDNA ends 849

Supplemental Figure S8 Amino acid sequence alignment of SCD in maize 850

and its putative orthologs in Arabidopsis thaliana Cyanidioschyzon merolae 851

and Escherichia coli 852

Supplemental Figure S9 Expression patterns of SCD in various tissues at 853

different developmental stages in WT and scd 854

Supplemental Figure S10 Subcellular localization of SCD 855

Supplemental Figure S11 Comparison of tocopherol contents between the 856

lw2-vp2 mutant and WT 857

Supplemental Figure S12 Expression patterns of 10 genes in 15-DAP 858

endosperms and embryos and leaves from light-grown and dark-grown 859

V1-stage scd and WT seedlings as estimated by RT-qPCR 860

Supplemental Figure S13 Schematic diagram of functional complementation 861

of scd via crossing with OE1 an SCD-overexpressing transgenic line 862

Supplemental Figure S14 Comparison of carotenoid chlorophyll and 863

tocopherol contents between the complemented individuals SCD 864

overexpressors and wild type 865

endosperms and embryos and V1-stage seedling leaves from overexpressing 867

transgenic lines OE1 OE2 and WT as estimated by RT-qPCR 868

Supplemental Table S1 Chi-square test of the ratio of yellow seeds to 870

pale-yellow seeds (31) derived from heterozygotes of four scd mutant alleles 871

and from the F1 hybrids between the heterozygotes for scd lw2-vp12 and two 872

additional alleles scd-1 and scd-2 873

Supplemental Table S2 List of the 60 genes related to carotenoid 874

biosynthesis providing an overview of the transcriptome profiles 875

Supplemental Table S3 Primers used for RT-qPCR in this study 876

Supplemental Table S4 Comparison of metabolite levels downstream of the 877

MEP pathway in seeds and seedlings between the two SCD-overexpressing 878

lines and wild type 879

Supplemental Table S5 Primers used for mapping sequencing and vector 880

construction in this study 881

Supplemental Table S6 ESI+-MSMS parameters used for phytohormone 882

measurements 883

Supplemental Dataset S1 List of DEGs in 15-DAP endosperms 15-DAP 884

embryos and light- and dark-grown seedling leaves 885

ACKNOWLEDGMENTS 887

We greatly appreciate Dr Philip Stinard from the Maize Genetics Cooperation 888

Stock Center for providing maize genetic stocks Dr Byung-Ho Kang at 889

University of Florida for his help in identifying plastids in maize seeds and the 890

Maize Functional Genomic Project of China Agricultural University for 891

generating the CRISPRCas9-mediated mutants and overexpressing seeds 892

Table 1 Comparison of tocopherol and phytohormone levels in scd and WT seeds and seedling leaves 894

Metabolites Mature seeds Light-grown leaves Dark-grown leaves

WT scd WT scd WT scd

Tocopherols (microgg)

DT 111 plusmn 023 053 plusmn 013 1147 plusmn 282 000 1118 plusmn 070 000

GT 1700 plusmn 100 552 plusmn 033 2543 plusmn 162 000 2721 plusmn 169 000

AT 1034 plusmn 043 564 plusmn 039 2098 plusmn 265 000 1715 plusmn 103 000

Abscisic acid (ngg)

ABA 000 000 239 plusmn 019 074 plusmn 006 916 plusmn 389 182 plusmn 017

Salicylic acid (ngg)

SA 49252 plusmn 4660 31344 plusmn 2174 27539 plusmn 5691 41513 plusmn 4106 2775 plusmn 209 2805 plusmn 173

Auxins (ngg)

IAA 26449 plusmn 5144 23227 plusmn 616 087 plusmn 002 064 plusmn 001 275 plusmn 014 317 plusmn 017

ME-IAA 081 plusmn 032 068 plusmn 003 001 plusmn 000 003 plusmn 000 003 plusmn 000 012 plusmn 001

IBA 000 000 419 plusmn 008 149 plusmn 024 700 plusmn 075 144 plusmn 328

ICA 2516 plusmn 373 3366 plusmn 353 034 plusmn 008 061 plusmn 023 029 plusmn 002 067 plusmn 016

Cytokinins (ngg)

IP 001 plusmn 000 001 plusmn 000 002 plusmn 001 003 plusmn 000 001 plusmn 000 001 plusmn 000

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

cZ 018 plusmn 005 019 plusmn 003 091 plusmn 008 120 plusmn 002 113 plusmn 001 100 plusmn 004

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

Jasmonic acids (ngg)

JA 181 plusmn 024 169 plusmn 027 235 plusmn 016 097 plusmn 010 262 plusmn 093 183 plusmn 038

H2JA 194 plusmn 137 080 plusmn 023 008 plusmn 001 011 plusmn 003 010 plusmn 002 006 plusmn 001

JA-ILE 704 plusmn 044 527 plusmn 079 082 plusmn 009 072 plusmn 005 093 plusmn 018 138 plusmn 033

Gibberellic acids (ngg)

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA3 000 000 030 plusmn 001 016 plusmn 002 037 plusmn 002 000

GA9 040 plusmn 001 037 plusmn 003 026 plusmn 003 000 091 plusmn 010 031 plusmn 004

GA15 011 plusmn 003 007 plusmn 000 010 plusmn 001 000 032 plusmn 004 000

GA19 4865 plusmn 012 4848 plusmn 004 5231 plusmn 035 4811 plusmn 006 5707 plusmn 060 000

GA20 873 plusmn 062 652 plusmn 052 558 plusmn 111 029 plusmn 002 1343 plusmn 158 083 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Values are averages of at least three biological replicates plusmn SE P lt 005 P lt 001 Studentrsquos t-test compared with the corresponding wild type DT 895

δ-tocopherol GT γ-tocopherol AT α-tocopherol ABA (+)-cis trans-Abscisic acid SA Salicylic acid IAA Indole-3-acetic acid ME-IAA Methyl 896

indole-3-acetate IBA 3-Indolebutyric acid ICA Indole-3-carboxaldehyde IP N6-Isopentenyladenine tZ trans-Zeatin cZ cis-Zeatin DZ 897

Dihydrozeatin JA (+)-Jasmonic acid H2JA (+)-Dihydrojasmonic acid JA-ILE N-[(-)-Jasmonoyl]-(L)-Isoleucine GA1 Gibberellin A1 GA3 Gibberellin 898

A3 GA9 Gibberellin A9 GA15 Gibberellin A15 GA19 Gibberellin A19 GA20 Gibberellin A20 GA24 Gibberellin A24 GA53 Gibberellin A53 899

Figure legends 901

Figure 1 Phenotypes of seeds and seedlings A A self-pollinated ear from an 902

SCDscd heterozygote Homozygous mutant seeds exhibit carotenoid 903

deficiency B and C Phenotypes of mature WT and scd seeds (B) and 904

V1-stage seedlings (C) D and E HPLC analysis of carotenoid profiles in 905

maize seeds (D) and V1-stage maize seedlings (E) 906

Figure 2 Effects of the scd mutation on the morphology of developing seeds 908

and seedling leaves of light- and dark-grown plants A and B Ultrastructures of 909

cells in the aleurone and subaleurone layers of 15-DAP endosperms from WT 910

(A) and scd (B) Bar = 5 μm C Relative number of aleurone grains and 911

vacuoles Error bars indicate the SE based on at least 10 biological replicates 912

D Relative sizes of aleurone grains and vacuoles Error bars indicate the SE 913

based on at least 10 biological replicates P lt 005 P lt 001 Studentrsquos t-test 914

E and F Transverse sections of seedling leaves from WT (E) and scd (F) Bar 915

= 10 μm G and H Bundle sheath cells in seedling leaves from WT (G) and scd 916

(H) visualized by TEM Bar = 5 μm I and J Ultrastructure of the chloroplasts in 917

bundle sheath cells in seedling leaves from WT (I) and scd (J) Bar = 1 μm K 918

and L Transverse sections of dark-grown seedling leaves from WT (K) and 919

scd (L) Bar = 10 μm M and N Bundle sheath cells in dark-grown seedling 920

leaves from WT (M) and scd (N) visualized by TEM Bar = 5 μm O and P 921

Ultrastructures of chloroplasts in bundle sheath cells of dark-grown seedling 922

leaves from WT (O) and scd (P) Bar = 1 μm BSc bundle sheath cell Mc 923

mesophyll cell Ch chloroplast M mitochondrion AL aleurone layer AG 924

aleurone grain SG starch grain PB protein body sER smooth endoplasmic 925

reticulum rER rough endoplasmic reticulum N nucleus LB lipid body V 926

vacuole VP vacuolar precipitate CW cell wall 927

Figure 3 Identification of the gene underlying the SCD locus A SCD was 929

fine-mapped to a 14-kb region on chromosome 5 The markers for primary 930

mapping their genetic distances and the genotypes and phenotypes of five 931

key recombinants are shown The black white and gray rectangles represent 932

SCD scd and heterozygous genotypes respectively B Gene structure of 933

SCD and the position of the scd mutation Introns are shown as solid lines 934

exons as black boxes and untranslated regions as white boxes The mutation 935

site is represented by an inverted triangle C PCR product of the full-length 936

SCD cDNA in 15-DAP endosperms and seedling leaves D Schematic 937

representation of multiple SCD transcripts produced in scd 15-DAP 938

endosperms and V1-stage seedling leaves A 120-bp insertion in the 5rsquo 939

untranslated region (5rsquoUTR) and a footprint fragment with an insertion length 940

ranging from 111 to 1593 bp between the eighth and ninth exons are shown as 941

gray boxes all other symbols are as in B E Relative expression of SCD in 942

15-DAP endosperms and seedling leaves The RT-qPCR primers were 943

designed based on exons 13 (forward) and 15 (reverse) which captures all 944

transcript variants Error bars indicate the SE based on at least three biological 945

replicates P lt 005 P lt 001 Studentrsquos t-test F Immunoblot analysis of 946

SCD in 15-DAP endosperms and seedling leaves Red arrow shows the target 947

bands 948

Figure 4 Molecular and genetic identification of lemon white 2 (lw2-vp12) as 950

scd A and B Seed (A) and seedling (B) phenotypes of the lw2-vp12 mutant C 951

The position of the causal mutation of SCD in the lw2-vp12 mutant Introns are 952

shown as solid lines exons as black boxes and untranslated regions as white 953

boxes The inverted triangles represent the insertions D and E Relative 954

expression (D) and immunoblot analysis (E) of SCD in 15-DAP endosperms 955

and seedling leaves of lw2-vp12 The RT-qPCR primers were designed based 956

on exons 13 (forward) and 15 (reverse) Red arrow shows the target bands Fndash957

H Carotenoid levels in mature seeds (F) and light- (G) and dark-grown (H) 958

seedling leaves I and J Chlorophyll levels in light- (I) and dark-grown (J) 959

seedling leaves Error bars indicate the SE based on at least three biological 960

replicates P lt 005 P lt 001 Studentrsquos t-test 961

Figure 5 Comparison of the levels of five metabolites of the MEP pathway in 963

scd and WT seeds and seedlings A Simplified diagram of the MEP pathway in 964

plastids The five metabolites shown in red were measured in this study BndashD 965

Content of the five metabolites in seeds (B) and light- (C) and dark-grown (D) 966

seedling leaves DMAPPIPP represents the average of DMAPP and IPP Error 967

bars indicate the SE based on at least three biological replicates P lt 005 968

P lt 001 Studentrsquos t-test 969

Figure 6 Overview of the transcriptomic changes caused by the loss of SCD 971

in 15-DAP endosperms and embryos and leaves from V1-stage seedlings 972

grown in the light and dark A Read abundance across full-length SCD The 973

range of values of each read number is shown below the tissue name B 974

Overlap of DEGs between the scd mutant and WT C Gene Ontology (GO) 975

enrichment analysis of functional categories of DEGs between scd and WT PS 976

photosynthesis CHO carbohydrate OPP oxidative phosphorylation Misc 977

miscellaneous D Changes in the expression of genes in the major isoprenoid 978

biosynthesis pathways FDR lt 001 The names of all 60 genes are listed in 979

Supplemental Table S2 980

Figure 7 Characteristics of two SCD-overexpressing maize lines A Ears from 982

OE1 OE2 and WT plants B Seedling phenotypes of OE1 OE2 and WT C 983

Relative expression levels of SCD in 15-DAP endosperms embryos and 984

seedling leaves from OE1 OE2 and WT D Immunoblot analysis of SCD in 985

15-DAP endosperms and embryos and seedling leaves from OE1 OE2 and 986

WT E and F Levels of metabolites in the MEP pathway in seeds (E) and 987

seedling leaves (F) of OE1 OE2 and WT DMAPPIPP represents the average 988

of DMAPP and IPP Error bars indicate the SE based on at least three 989

biological replicates P lt 005 P lt 001 Studentrsquos t-test 990

LITERATURE CITED 992

Altincicek B Kollas AK Sanderbrand S Wiesner J Hintz M Beck E Jomaa H (2001) 993

GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid 994

biosynthesis in Escherichia coli J Bacteriol 183 2411ndash2416 995

Anders S Pyl PT Huber W (2015) HTSeqmdasha Python framework to work with 996

high-throughput sequencing data Bioinformatics 31 166ndash169 997

Araki N Kusumi K Masamoto K Niwa Y Iba K (2000) Temperature-sensitive 998

Arabidopsis mutant defective in 1-deoxy-D-xylulose 5-phosphate synthase within the 999

plastid non-mevalonate pathway of isoprenoid biosynthesis Physiol Plantarum 108 1000

19ndash24 1001

Bauer J Hiltbrunner A Kessler F (2001) Molecular biology of chloroplast biogenesis 1002

gene expression protein import and intraorganellar sorting Cell Mol Life Sci 58 420ndash1003

433 1004

Benjamini Y Hochberg Y (1995) Controlling the false discovery rate a practical and 1005

powerful approach to multiple testing J R Stat Soc B 57 289ndash300 1006

Bernard L Ciceri P Viotti A (1994) Molecular analysis of wild-type and mutant alleles at 1007

the Opaque-2 regulatory locus of maize reveals different mutations and types of O2 1008

products Plant Mol Biol 24 949ndash959 1009

Botella-Pavia P Besumbes O Phillips MA Carretero-Paulet L Boronat A 1010

Rodriguez-Concepcion M (2004) Regulation of carotenoid biosynthesis in plants 1011

evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling 1012

the supply of plastidial isoprenoid precursors Plant J 40 188-199 1013

Bouvier F Rahier A Camara B (2005) Biogenesis molecular regulation and function of 1014

plant isoprenoids Prog Lipid Res 44 357ndash429 1015

Buckner B Kelson TL Robertson DS (1990) Cloning of the y1 Locus of maize a gene 1016

involved in the biosynthesis of carotenoids Plant Cell 2 867ndash876 1017

Budziszewski GJ Lewis SP Glover LW Reineke J Jones G Ziemnik LS Lonowski 1018

J Nyfeler B Aux G Zhou Q McElver J Patton DA Martienssen R 1019

Grossniklaus U Ma H Law M Levin JZ (2001) Arabidopsis genes essential for 1020

seedling viability isolation of insertional mutants and molecular cloning Genetics 159 1021

1765ndash1778 1022

Carretero-Paulet L Ahumada I Cunillera N Rodriguez-Concepcion M Ferrer A 1023

Boronat A Campos N (2002) Expression and molecular analysis of the Arabidopsis 1024

DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase the first 1025

committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway Plant Physiol 1026

129 1581ndash1591 1027

Carretero-Paulet L Cairo A Botella-Pavia P Besumbes O Campos N Boronat A 1028

Rodriguez-Concepcion M (2006) Enhanced flux through the methylerythritol 1029

4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 1030

5-phosphate reductoisomerase Plant Mol Biol 62 683ndash695 1031

Cai WJ Ye TT Wang Q Cai BD Feng YQ (2016) A rapid approach to investigate 1032

spatiotemporal distribution of phytohormones in rice Plant Methods 12 47 1033

Chander S Guo YQ Yang XH Zhang J Lu XQ Yan JB Song TM Rocheford TR Li 1034

JS (2008) Using molecular markers to identify two major loci controlling carotenoid 1035

contents in maize grain Theor Appl Genet 116 223ndash233 1036

Cordoba E Porta H Arroyo A San Roman C Medina L Rodriguez-Concepcion M 1037

Leon P (2011) Functional characterization of the three genes encoding 1038

1-deoxy-D-xylulose 5-phosphate synthase in maize J Exp Bot 62 2023ndash2038 1039

Demmig-Adams B Gilmore AM Adams WW (1996) Carotenoids 3 in vivo functions of 1040

carotenoids in higher plants FASEB J 10 403ndash412 1041

Estevez JM Cantero A Reindl A Reichler S Leon P (2001) 1042

1-Deoxy-D-xylulose-5-phosphate synthase a limiting enzyme for plastidic isoprenoid 1043

biosynthesis in plants J Biol Chem 276 22901ndash22909 1044

Estevez JM Cantero A Romero C Kawaide H Jimenez LF Kuzuyama T Seto H 1045

Kamiya Y Leon P (2000) Analysis of the expression of CLA1 a gene that encodes 1046

the 1-deoxyxylulose 5-phosphate synthase of the 1047

2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis Plant Physiol 124 95ndash1048

104 1049

Flores-Perez U Perez-Gil J Rodriguez-Villalon A Gil M Vera P 1050

Rodriguez-Concepcion M (2008) Contribution of hydroxymethylbutenyl diphosphate 1051

synthase to carotenoid biosynthesis in bacteria and plants Biochem Biophys Res 1052

Commun 371 510ndash514 1053

Goff SA Klee HJ (2006) Plant volatile compounds sensory cues for health and 1054

nutritional value Science 311 815ndash819 1055

Guevara-Garcia A San Roman C Arroyo A Cortes ME Gutierrez-Nava MD Leon P 1056

(2005) Characterization of the Arabidopsis clb6 mutant illustrates the importance of 1057

posttranscriptional regulation of the methyl-D-erythritol 4-phosphate pathway Plant 1058

Cell 17 628ndash643 1059

Gutierrez-Nava MDL Gillmor CS Jimenez LF Guevara-Garcia A Leon P (2004) 1060

Chloroplast biogenesis genes act cell and noncell autonomously in early chloroplast 1061

development Plant Physiol 135 471ndash482 1062

Hans J Hause B Strack D Walter MH (2004) Cloning characterization and 1063

immunolocalization of a mycorrhiza-inducible 1-deoxy-d-xylulose 5-phosphate 1064

reductoisomerase in arbuscule-containing cells of maize Plant Physiol 134 614ndash624 1065

Hemmerlin A (2013) Post-translational events and modifications regulating plant 1066

enzymes involved in isoprenoid precursor biosynthesis Plant Sci 203 41ndash54 1067

Hemmerlin A Harwood JL Bach TJ (2012) A raison decirctre for two distinct pathways in 1068

the early steps of plant isoprenoid biosynthesis Prog Lipid Res 51 95ndash148 1069

Hirashima M Satoh S Tanaka R Tanaka A (2006) Pigment shuffling in antenna 1070

systems achieved by expressing prokaryotic chlorophyllide a oxygenase in 1071

Arabidopsis J Biol Chem 281 15385-15393 1072

Hoober JK Eggink LL (2001) A potential role of chlorophylls b and c in assembly of 1073

light-harvesting complexes FEBS Lett 489 1-3 1074

Hsieh MH Chang CY Hsu SJ Chen JJ (2008) Chloroplast localization of 1075

methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial 1076

genes in ispD and ispE albino mutants in Arabidopsis Plant Mol Biol 66 663ndash673 1077

Hsieh MH Goodman HM (2005) The Arabidopsis IspH homolog is involved in the plastid 1078

nonmevalonate pathway of isoprenoid biosynthesis Plant Physiol 138 641-653 1079

Hsieh MH Goodman HM (2006) Functional evidence for the involvement of Arabidopsis 1080

IspF homolog in the nonmevalonate pathway of plastid isoprenoid biosynthesis 1081

Planta 223 779ndash784 1082

Hunter CT Saunders JW Magallanes-Lundback M Christensen SA Willett D 1083

Stinard PS Li QB Lee K DellaPenna D Koch KE (2018) Maize w3 disrupts 1084

homogentisate solanesyl transferase (ZmHst) and reveals a plastoquinone-9 1085

independent path for phytoene desaturation and tocopherol accumulation in kernels 1086

Plant J 93 799ndash813 1087

Johnson EJ (2002) The role of carotenoids in human health Nutr Clin Care 5 56ndash65 1088

Kim D Pertea G Trapnell C Pimentel H Kelley R Salzberg SL (2013) TopHat2 1089

accurate alignment of transcriptomes in the presence of insertions deletions and 1090

gene fusions Genome Biol 14 R36 1091

Kuzuyama T Seto H (2003) Diversity of the biosynthesis of the isoprene units Nat Prod 1092

Rep 20 171ndash183 1093

Li F Murillo C Wurtzel ET (2007) Maize Y9 encodes a product essential for 1094

15-cis-zeta-carotene isomerization Plant Physiol 144 1181-1189 1095

Li ZH Matthews PD Burr B Wurtzel ET (1996) Cloning and characterization of a maize 1096

cDNA encoding phytoene desaturase an enzyme of the carotenoid biosynthetic 1097

pathway Plant Mol Biol 30 269ndash279 1098

Li ZR Sharkey TD (2013) Metabolic profiling of the methylerythritol phosphate pathway 1099

reveals the source of post-illumination isoprene burst from leaves Plant Cell Environ 1100

36 429ndash437 1101

Lichtenthaler HK (1987) Chlorophylls and carotenoids pigments of photosynthetic 1102

biomembranes Method Enzymol 148 350ndash382 1103

Liu RX Hong J Xu XQ Feng Q Zhang DY Gu YY Shi J Zhao SQ Liu W Wang XK 1104

Xia HH Liu ZP Cui B Liang PW Xi LQ Jin JB Ying XY Wang XL Zhao XJ Li 1105

WY Jia HJ Lan Z Li FY Wang R Sun YK Yang ML Shen YX Jie ZY Li JH 1106

Chen XM Zhong HZ Xie HL Zhang YF Gu WQ Deng XX Shen BY Xu X Yang 1107

HM Xu GW Bi YF Lai SH Wang J Qi L Madsen L Wang JQ Ning G 1108

Kristiansen K Wang WQ (2017) Gut microbiome and serum metabolome 1109

alterations in obesity and after weight-loss intervention Nat Med 23 859ndash868 1110

Liu YL Guerra F Wang K Wang WX Li JK Huang C Zhu W Houlihan K Li Z Zhang 1111

Y Nair SK Oldfield E (2012) Structure function and inhibition of the two- and 1112

three-domain 4Fe-4S IspG proteins Proc Natl Acad Sci U S A 109(22) 8558-8563 1113

Livak KJ Schmittgen TD (2001) Analysis of relative gene expression data using 1114

real-time quantitative PCR and the 2(T)(-Delta Delta C) method Methods 25 402ndash1115

408 1116

Lois LM Rodriguez-Concepcion M Gallego F Campos N Boronat A (2000) 1117

Carotenoid biosynthesis during tomato fruit development regulatory role of 1118

1-deoxy-D-xylulose 5-phosphate synthase Plant J 22 503ndash513 1119

Lu XM Hu XJ Zhao YZ Song WB Zhang M Chen ZL Chen W Dong YB Wang ZH 1120

Lai JS (2012) Map-based cloning of zb7 encoding an IPP and DMAPP synthase in 1121

the MEP pathway of maize Mol Plant 5 1100ndash1112 1122

Maluf MP Saab IN Wurtzel ET Sachs MM (1997) The viviparous12 maize mutant is 1123

deficient in abscisic acid carotenoids and chlorophyll synthesis J Exp Bot 48(311) 1124

1259-1268 1125

Mandel MA Feldmann KA HerreraEstrella L RochaSosa M Leon P (1996) CLA1 a 1126

novel gene required for chloroplast development is highly conserved in evolution 1127

Matthews PD Luo R Wurtzel ET (2003) Maize phytoene desaturase and zeta-carotene 1129

desaturase catalyse a poly-Z desaturation pathway implications for genetic 1130

engineering of carotenoid content among cereal crops J Exp Bot 54 2215ndash2230 1131

McCarty DR Carson CB (1991) The molecular genetics of seed maturation in maize 1132

Physiol Plantarum 81 267ndash272 1133

Myers AM James MG Lin QH Yi G Stinard PS Hennen-Bierwagen TA Becraft PW 1134

(2011) Maize opaque5 encodes monogalactosyldiacylglycerol synthase and 1135

specifically affects galactolipids necessary for amyloplast and chloroplast function 1136

Plant Cell 23 2331ndash2347 1137

Oster U Tanaka R Tanaka A Rudiger W (2000) Cloning and functional expression of 1138

the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from 1139

Arabidopsis thaliana Plant J 21 305-310 1140

Ostrovsky D Diomina G Lysak E Matveeva E Ogrel O Trutko S (1998) Effect of 1141

oxidative stress on the biosynthesis of 1142

2-C-methyl-D-erythritol-24-cyclopyrophosphate and isoprenoids by several bacterial 1143

strains Arch Microbiol 171 69ndash72 1144

Owens BF Lipka AE Magallanes-Lundback M Tiede T Diepenbrock CH Kandianis 1145

CB Kim E Cepela J Mateos-Hernandez M Buell CR Buckler ES DellaPenna D 1146

Gore MA Rocheford T (2014) A foundation for provitamin A biofortification of maize 1147

genome-wide association and genomic prediction models of carotenoid levels 1148

Genetics 198 1699ndash1716 1149

Page JE Hause G Raschke M Gao WY Schmidt J Zenk MH Kutchan TM (2004) 1150

Functional analysis of the final steps of the 1-Deoxy-D-xylulose 5-phosphate (DXP) 1151

pathway to isoprenoids in plants using virus-induced gene silencing Plant Physiol 1152

134 1401ndash1413 1153

Querol J Campos N Imperial S Boronat A Rodriguez-Concepcion M (2002) 1154

Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid 1155

isoprenoid biosynthesis FEBS Lett 514 343ndash346 1156

Robinson MD McCarthy DJ Smyth GK (2010) edgeR a Bioconductor package for 1157

differential expression analysis of digital gene expression data Bioinformatics 26 1158

139ndash140 1159

Rodriguez-Concepcion M Fores O Martinez-Garcia JF Gonzalez V Phillips MA 1160

Ferrer A Boronat A (2004) Distinct light-mediated pathways regulate the 1161

biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling 1162

development Plant Cell 16 144ndash156 1163

Rodriguez-Villalon A Gas E Rodriguez-Concepcion M (2009) Phytoene synthase 1164

activity controls the biosynthesis of carotenoids and the supply of their metabolic 1165

precursors in dark-grown Arabidopsis seedlings Plant J 60 424-435 1166

Ruiz-Sola MA Rodriguez-Concepcion M (2012) Carotenoid biosynthesis in Arabidopsis 1167

a colorful pathway Arabidopsis Book 10 e0158 1168

Seemann M Wegner P Schunemann V Bui BTS Wolff M Marquet A Trautwein AX 1169

Rohmer M (2005) Isoprenoid biosynthesis in chloroplasts via the methylerythritol 1170

phosphate pathway the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase 1171

(GcpE) from Arabidopsis thaliana is a [4Fe-4S] protein J Biol Inorg Chem 10 1172

131-137 1173

Singh M Lewis PE Hardeman K Bai L Rose JK Mazourek M Chomet P Brutnell 1174

TP (2003) Activator mutagenesis of the pink scutellum1viviparous7 locus of maize 1175

Stinard P (2013) Data-mining the B73 genome sequence for carotenoid biosynthesis 1177

gene candidates Maize Genet Coop Newsl 86 29-31 1178

Tanaka R Tanaka A (2007) Tetrapyrrole biosynthesis in higher plants Annu Rev Plant 1179

Biol 58 321-346 1180

Tang JH Teng WT Yan JB Ma XQ Meng YJ Dai JR Li JS (2007) Genetic dissection of 1181

plant height by molecular markers using a population of recombinant inbred lines in 1182

maize Euphytica 155 117ndash124 1183

Thimm O Blasing O Gibon Y Nagel A Meyer S Kruger P Selbig J Muller LA Rhee 1184

SY Stitt M (2004) MAPMAN a user-driven tool to display genomics data sets onto 1185

diagrams of metabolic pathways and other biological processes Plant J 37 914ndash939 1186

Thulasiram HV Erickson HK Poulter CD (2007) Chimeras of two isoprenoid synthases 1187

catalyze all four coupling reactions in isoprenoid biosynthesis Science 316 73ndash76 1188

Vallabhaneni R Wurtzel ET (2009) Timing and biosynthetic potential for carotenoid 1189

accumulation in genetically diverse germplasm of maize Plant Physiol 150 562-572 1190

Venado RE Owens BF Ortiz D Lawson T Mateos-Hernandez M Ferruzzi MG 1191

Rocheford TR (2017) Genetic analysis of provitamin A carotenoid β-cryptoxanthin 1192

concentration and relationship with other carotenoids in maize grain (Zea mays L) 1193

Mol Breeding 37 127 1194

Von Wettstein D Gough S Kannangara CG (1995) Chlorophyll biosynthesis Plant Cell 1195

7 1039ndash1057 1196

Vranova E Coman D Gruissem W (2012) Structure and dynamics of the isoprenoid 1197

pathway network Mol Plant 5 318ndash333 1198

Vranova E Coman D Gruissem W (2013) Network analysis of the MVA and MEP 1199

pathways for isoprenoid synthesis Annu Rev Plant Biol 64 665ndash700 1200

Walter MH Hans J Strack D (2002) Two distantly related genes encoding 1201

1-deoxy-d-xylulose 5-phosphate synthases differential regulation in shoots and 1202

apocarotenoid-accumulating mycorrhizal roots Plant J 31 243-254 1203

Xiao YM Savchenko T Baidoo EEK Chehab WE Hayden DM Tolstikov V Corwin 1204

JA Kliebenstein DJ Keasling JD Dehesh K (2012) Retrograde signaling by the 1205

plastidial metabolite MEcPP regulates expression of nuclear stress-response genes 1206

Cell 149 1525ndash1535 1207

Xing HL Dong L Wang ZP Zhang HY Han CY Liu B Wang XC Chen QJ (2014) A 1208

CRISPRCas9 toolkit for multiplex genome editing in plants BMC Plant Biol 14 327 1209

Xing SF Miao J Li S Qin GJ Tang S Li HN Gu HY Qu LJ (2010) Disruption of the 1210

1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene results in albino 1211

dwarf and defects in trichome initiation and stomata closure in Arabidopsis Cell Res 1212

20 688ndash700 1213

Xu Z Wang H (2007) LTR_FINDER an efficient tool for the prediction of full-length LTR 1214

retrotransposons Nucleic Acids Res 35 W265ndash268 1215

Yoo SD Cho YH Sheen J (2007) Arabidopsis mesophyll protoplasts a versatile cell 1216

system for transient gene expression analysis Nat Protoc 2 1565ndash1572 1217

Figure 1 Phenotypes of seeds and seedlings A A self-pollinated ear from an

SCDscd heterozygote Homozygous mutant seeds exhibit carotenoid

deficiency B and C Phenotypes of mature WT and scd seeds (B) and

V1-stage seedlings (C) D and E HPLC analysis of carotenoid profiles in

maize seeds (D) and V1-stage maize seedlings (E)

Figure 2 Effects of the scd mutation on the morphology of developing seeds

and seedling leaves of light- and dark-grown plants A and B Ultrastructures of

cells in the aleurone and subaleurone layers of 15-DAP endosperms from WT

(A) and scd (B) Bar = 5 μm C Relative number of aleurone grains and

vacuoles Error bars indicate the SE based on at least 10 biological replicates

D Relative sizes of aleurone grains and vacuoles Error bars indicate the SE

based on at least 10 biological replicates P lt 005 P lt 001 Studentrsquos t-test

E and F Transverse sections of seedling leaves from WT (E) and scd (F) Bar

= 10 μm G and H Bundle sheath cells in seedling leaves from WT (G) and scd

(H) visualized by TEM Bar = 5 μm I and J Ultrastructure of the chloroplasts in

bundle sheath cells in seedling leaves from WT (I) and scd (J) Bar = 1 μm K

and L Transverse sections of dark-grown seedling leaves from WT (K) and

scd (L) Bar = 10 μm M and N Bundle sheath cells in dark-grown seedling

leaves from WT (M) and scd (N) visualized by TEM Bar = 5 μm O and P

Ultrastructures of chloroplasts in bundle sheath cells of dark-grown seedling

leaves from WT (O) and scd (P) Bar = 1 μm BSc bundle sheath cell Mc

mesophyll cell Ch chloroplast M mitochondrion AL aleurone layer AG

aleurone grain SG starch grain PB protein body sER smooth endoplasmic

reticulum rER rough endoplasmic reticulum N nucleus LB lipid body V

vacuole VP vacuolar precipitate CW cell wall

Figure 3 Identification of the gene underlying the SCD locus A SCD was

fine-mapped to a 14-kb region on chromosome 5 The markers for primary

mapping their genetic distances and the genotypes and phenotypes of five

key recombinants are shown The black white and gray rectangles represent

SCD scd and heterozygous genotypes respectively B Gene structure of

SCD and the position of the scd mutation Introns are shown as solid lines

exons as black boxes and untranslated regions as white boxes The mutation

site is represented by an inverted triangle C PCR product of the full-length

SCD cDNA in 15-DAP endosperms and seedling leaves D Schematic

representation of multiple SCD transcripts produced in scd 15-DAP

endosperms and V1-stage seedling leaves A 120-bp insertion in the 5rsquo

untranslated region (5rsquoUTR) and a footprint fragment with an insertion length

ranging from 111 to 1593 bp between the eighth and ninth exons are shown as

gray boxes all other symbols are as in B E Relative expression of SCD in

15-DAP endosperms and seedling leaves The RT-qPCR primers were

designed based on exons 13 (forward) and 15 (reverse) which captures all

transcript variants Error bars indicate the SE based on at least three biological

replicates P lt 005 P lt 001 Studentrsquos t-test F Immunoblot analysis of

SCD in 15-DAP endosperms and seedling leaves Red arrow shows the target

Figure 4 Molecular and genetic identification of lemon white 2 (lw2-vp12) as

scd A and B Seed (A) and seedling (B) phenotypes of the lw2-vp12 mutant C

The position of the causal mutation of SCD in the lw2-vp12 mutant Introns are

shown as solid lines exons as black boxes and untranslated regions as white

boxes The inverted triangles represent the insertions D and E Relative

expression (D) and immunoblot analysis (E) of SCD in 15-DAP endosperms

and seedling leaves of lw2-vp12 The RT-qPCR primers were designed based

on exons 13 (forward) and 15 (reverse) Red arrow shows the target bands Fndash

H Carotenoid levels in mature seeds (F) and light- (G) and dark-grown (H)

seedling leaves I and J Chlorophyll levels in light- (I) and dark-grown (J)

seedling leaves Error bars indicate the SE based on at least three biological

replicates P lt 005 P lt 001 Studentrsquos t-test

Figure 5 Comparison of the levels of five metabolites of the MEP pathway in

scd and WT seeds and seedlings A Simplified diagram of the MEP pathway in

plastids The five metabolites shown in red were measured in this study BndashD

Content of the five metabolites in seeds (B) and light- (C) and dark-grown (D)

seedling leaves DMAPPIPP represents the average of DMAPP and IPP Error

bars indicate the SE based on at least three biological replicates P lt 005

P lt 001 Studentrsquos t-test

Figure 6 Overview of the transcriptomic changes caused by the loss of SCD

in 15-DAP endosperms and embryos and leaves from V1-stage seedlings

grown in the light and dark A Read abundance across full-length SCD The

range of values of each read number is shown below the tissue name B

Overlap of DEGs between the scd mutant and WT C Gene Ontology (GO)

enrichment analysis of functional categories of DEGs between scd and WT PS

photosynthesis CHO carbohydrate OPP oxidative phosphorylation Misc

miscellaneous D Changes in the expression of genes in the major isoprenoid

biosynthesis pathways FDR lt 001 The names of all 60 genes are listed in

Supplemental Table S2

Figure 7 Characteristics of two SCD-overexpressing maize lines A Ears from

OE1 OE2 and WT plants B Seedling phenotypes of OE1 OE2 and WT C

Relative expression levels of SCD in 15-DAP endosperms and embryos and

seedling leaves from OE1 OE2 and WT D Immunoblot analysis of SCD in

15-DAP endosperms and embryos and seedling leaves from OE1 OE2 and

WT E and F Levels of metabolites in the MEP pathway in seeds (E) and

seedling leaves (F) of OE1 OE2 and WT DMAPPIPP represents the average

of DMAPP and IPP Error bars indicate the SE based on at least three biological

Parsed CitationsAltincicek B Kollas AK Sanderbrand S Wiesner J Hintz M Beck E Jomaa H (2001) GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli J Bacteriol 183 2411ndash2416

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

Anders S Pyl PT Huber W (2015) HTSeq-a Python framework to work with high-throughput sequencing data Bioinformatics 31 166ndash169

Araki N Kusumi K Masamoto K Niwa Y Iba K (2000) Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-D-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis Physiol Plantarum 108 19ndash24

Bauer J Hiltbrunner A Kessler F (2001) Molecular biology of chloroplast biogenesis gene expression protein import andintraorganellar sorting Cell Mol Life Sci 58 420ndash433

Benjamini Y Hochberg Y (1995) Controlling the false discovery rate a practical and powerful approach to multiple testing J R Stat SocB 57 289ndash300

Bernard L Ciceri P Viotti A (1994) Molecular analysis of wild-type and mutant alleles at the Opaque-2 regulatory locus of maize revealsdifferent mutations and types of O2 products Plant Mol Biol 24 949ndash959

Botella-Pavia P Besumbes O Phillips MA Carretero-Paulet L Boronat A Rodriguez-Concepcion M (2004) Regulation of carotenoidbiosynthesis in plants evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidialisoprenoid precursors Plant J 40 188-199

Bouvier F Rahier A Camara B (2005) Biogenesis molecular regulation and function of plant isoprenoids Prog Lipid Res 44 357ndash429Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Buckner B Kelson TL Robertson DS (1990) Cloning of the y1 Locus of maize a gene involved in the biosynthesis of carotenoidsPlant Cell 2 867ndash876

Budziszewski GJ Lewis SP Glover LW Reineke J Jones G Ziemnik LS Lonowski J Nyfeler B Aux G Zhou Q McElver J Patton DAMartienssen R Grossniklaus U Ma H Law M Levin JZ (2001) Arabidopsis genes essential for seedling viability isolation of insertionalmutants and molecular cloning Genetics 159 1765ndash1778

Carretero-Paulet L Ahumada I Cunillera N Rodriguez-Concepcion M Ferrer A Boronat A Campos N (2002) Expression and molecularanalysis of the Arabidopsis DXR gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase the first committed enzyme of the2-C-methyl-D-erythritol 4-phosphate pathway Plant Physiol 129 1581ndash1591

Carretero-Paulet L Cairo A Botella-Pavia P Besumbes O Campos N Boronat A Rodriguez-Concepcion M (2006) Enhanced fluxthrough the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphatereductoisomerase Plant Mol Biol 62 683ndash695

Cai WJ Ye TT Wang Q Cai BD Feng YQ (2016) A rapid approach to investigate spatiotemporal distribution of phytohormones in ricePlant Methods 12 47

Chander S Guo YQ Yang XH Zhang J Lu XQ Yan JB Song TM Rocheford TR Li JS (2008) Using molecular markers to identify twomajor loci controlling carotenoid contents in maize grain Theor Appl Genet 116 223ndash233

Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon April 19 2020 - Published by Downloaded from

Google Scholar Author Only Title Only Author and Title

Cordoba E Porta H Arroyo A San Roman C Medina L Rodriguez-Concepcion M Leon P (2011) Functional characterization of thethree genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize J Exp Bot 62 2023ndash2038

Demmig-Adams B Gilmore AM Adams WW (1996) Carotenoids 3 in vivo functions of carotenoids in higher plants FASEB J 10 403ndash412Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Estevez JM Cantero A Reindl A Reichler S Leon P (2001) 1-Deoxy-D-xylulose-5-phosphate synthase a limiting enzyme for plastidicisoprenoid biosynthesis in plants J Biol Chem 276 22901ndash22909

Estevez JM Cantero A Romero C Kawaide H Jimenez LF Kuzuyama T Seto H Kamiya Y Leon P (2000) Analysis of the expression ofCLA1 a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway inArabidopsis Plant Physiol 124 95ndash104

Flores-Perez U Perez-Gil J Rodriguez-Villalon A Gil M Vera P Rodriguez-Concepcion M (2008) Contribution of hydroxymethylbutenyldiphosphate synthase to carotenoid biosynthesis in bacteria and plants Biochem Biophys Res Commun 371 510ndash514

Goff SA Klee HJ (2006) Plant volatile compounds sensory cues for health and nutritional value Science 311 815ndash819

Guevara-Garcia A San Roman C Arroyo A Cortes ME Gutierrez-Nava MD Leon P (2005) Characterization of the Arabidopsis clb6mutant illustrates the importance of posttranscriptional regulation of the methyl-D-erythritol 4-phosphate pathway Plant Cell 17 628ndash643

Gutierrez-Nava MDL Gillmor CS Jimenez LF Guevara-Garcia A Leon P (2004) Chloroplast biogenesis genes act cell and noncellautonomously in early chloroplast development Plant Physiol 135 471ndash482

Hans J Hause B Strack D Walter MH (2004) Cloning characterization and immunolocalization of a mycorrhiza-inducible 1-deoxy-d-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize Plant Physiol 134 614ndash624

Hemmerlin A (2013) Post-translational events and modifications regulating plant enzymes involved in isoprenoid precursorbiosynthesis Plant Sci 203 41ndash54

Hemmerlin A Harwood JL Bach TJ (2012) A raison decirctre for two distinct pathways in the early steps of plant isoprenoid biosynthesisProg Lipid Res 51 95ndash148

Hirashima M Satoh S Tanaka R Tanaka A (2006) Pigment shuffling in antenna systems achieved by expressing prokaryoticchlorophyllide a oxygenase in Arabidopsis J Biol Chem 281 15385-15393

Hoober JK Eggink LL (2001) A potential role of chlorophylls b and c in assembly of light-harvesting complexes FEBS Lett 489 1-3Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Hsieh MH Chang CY Hsu SJ Chen JJ (2008) Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulationof mitochondrial genes in ispD and ispE albino mutants in Arabidopsis Plant Mol Biol 66 663ndash673

Hsieh MH Goodman HM (2005) The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoidbiosynthesis Plant Physiol 138 641-653

Hsieh MH Goodman HM (2006) Functional evidence for the involvement of Arabidopsis IspF homolog in the nonmevalonate pathwayof plastid isoprenoid biosynthesis Planta 223 779ndash784

Hunter CT Saunders JW Magallanes-Lundback M Christensen SA Willett D Stinard PS Li QB Lee K DellaPenna D Koch KE (2018)Maize w3 disrupts homogentisate solanesyl transferase (ZmHst) and reveals a plastoquinone-9 independent path for phytoenedesaturation and tocopherol accumulation in kernels Plant J 93 799ndash813

Johnson EJ (2002) The role of carotenoids in human health Nutr Clin Care 5 56ndash65Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kim D Pertea G Trapnell C Pimentel H Kelley R Salzberg SL (2013) TopHat2 accurate alignment of transcriptomes in the presenceof insertions deletions and gene fusions Genome Biol 14 R36

Kuzuyama T Seto H (2003) Diversity of the biosynthesis of the isoprene units Nat Prod Rep 20 171ndash183Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Li F Murillo C Wurtzel ET (2007) Maize Y9 encodes a product essential for 15-cis-zeta-carotene isomerization Plant Physiol 144 1181-1189

Li ZH Matthews PD Burr B Wurtzel ET (1996) Cloning and characterization of a maize cDNA encoding phytoene desaturase anenzyme of the carotenoid biosynthetic pathway Plant Mol Biol 30 269ndash279

Li ZR Sharkey TD (2013) Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illuminationisoprene burst from leaves Plant Cell Environ 36 429ndash437

Lichtenthaler HK (1987) Chlorophylls and carotenoids pigments of photosynthetic biomembranes Method Enzymol 148 350ndash382Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Liu RX Hong J Xu XQ Feng Q Zhang DY Gu YY Shi J Zhao SQ Liu W Wang XK Xia HH Liu ZP Cui B Liang PW Xi LQ Jin JB YingXY Wang XL Zhao XJ Li WY Jia HJ Lan Z Li FY Wang R Sun YK Yang ML Shen YX Jie ZY Li JH Chen XM Zhong HZ Xie HLZhang YF Gu WQ Deng XX Shen BY Xu X Yang HM Xu GW Bi YF Lai SH Wang J Qi L Madsen L Wang JQ Ning G Kristiansen KWang WQ (2017) Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention Nat Med 23 859ndash868

Liu YL Guerra F Wang K Wang WX Li JK Huang C Zhu W Houlihan K Li Z Zhang Y Nair SK Oldfield E (2012) Structure functionand inhibition of the two- and three-domain 4Fe-4S IspG proteins Proc Natl Acad Sci U S A 109(22) 8558-8563

Livak KJ Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C)method Methods 25 402ndash408

Lois LM Rodriguez-Concepcion M Gallego F Campos N Boronat A (2000) Carotenoid biosynthesis during tomato fruit developmentregulatory role of 1-deoxy-D-xylulose 5-phosphate synthase Plant J 22 503ndash513

Lu XM Hu XJ Zhao YZ Song WB Zhang M Chen ZL Chen W Dong YB Wang ZH Lai JS (2012) Map-based cloning of zb7 encoding anIPP and DMAPP synthase in the MEP pathway of maize Mol Plant 5 1100ndash1112

Maluf MP Saab IN Wurtzel ET Sachs MM (1997) The viviparous12 maize mutant is deficient in abscisic acid carotenoids andchlorophyll synthesis J Exp Bot 48(311) 1259-1268

Mandel MA Feldmann KA HerreraEstrella L RochaSosa M Leon P (1996) CLA1 a novel gene required for chloroplast development ishighly conserved in evolution Plant J 9 649ndash658

Matthews PD Luo R Wurtzel ET (2003) Maize phytoene desaturase and zeta-carotene desaturase catalyse a poly-Z desaturationpathway implications for genetic engineering of carotenoid content among cereal crops J Exp Bot 54 2215ndash2230

McCarty DR Carson CB (1991) The molecular genetics of seed maturation in maize Physiol Plantarum 81 267ndash272Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Myers AM James MG Lin QH Yi G Stinard PS Hennen-Bierwagen TA Becraft PW (2011) Maize opaque5 encodesmonogalactosyldiacylglycerol synthase and specifically affects galactolipids necessary for amyloplast and chloroplast function PlantCell 23 2331ndash2347

Oster U Tanaka R Tanaka A Rudiger W (2000) Cloning and functional expression of the gene encoding the key enzyme forchlorophyll b biosynthesis (CAO) from Arabidopsis thaliana Plant J 21 305-310

Ostrovsky D Diomina G Lysak E Matveeva E Ogrel O Trutko S (1998) Effect of oxidative stress on the biosynthesis of 2-C-methyl-D-erythritol-24-cyclopyrophosphate and isoprenoids by several bacterial strains Arch Microbiol 171 69ndash72

Owens BF Lipka AE Magallanes-Lundback M Tiede T Diepenbrock CH Kandianis CB Kim E Cepela J Mateos-Hernandez M BuellCR Buckler ES DellaPenna D Gore MA Rocheford T (2014) A foundation for provitamin A biofortification of maize genome-wideassociation and genomic prediction models of carotenoid levels Genetics 198 1699ndash1716

Page JE Hause G Raschke M Gao WY Schmidt J Zenk MH Kutchan TM (2004) Functional analysis of the final steps of the 1-Deoxy-D-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing Plant Physiol 134 1401ndash1413

Querol J Campos N Imperial S Boronat A Rodriguez-Concepcion M (2002) Functional analysis of the Arabidopsis thaliana GCPEprotein involved in plastid isoprenoid biosynthesis FEBS Lett 514 343ndash346

Robinson MD McCarthy DJ Smyth GK (2010) edgeR a Bioconductor package for differential expression analysis of digital geneexpression data Bioinformatics 26 139ndash140

Rodriguez-Concepcion M Fores O Martinez-Garcia JF Gonzalez V Phillips MA Ferrer A Boronat A (2004) Distinct light-mediatedpathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development Plant Cell 16144ndash156

Rodriguez-Villalon A Gas E Rodriguez-Concepcion M (2009) Phytoene synthase activity controls the biosynthesis of carotenoids andthe supply of their metabolic precursors in dark-grown Arabidopsis seedlings Plant J 60 424-435

Ruiz-Sola MA Rodriguez-Concepcion M (2012) Carotenoid biosynthesis in Arabidopsis a colorful pathway Arabidopsis Book 10 e0158Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Seemann M Wegner P Schunemann V Bui BTS Wolff M Marquet A Trautwein AX Rohmer M (2005) Isoprenoid biosynthesis inchloroplasts via the methylerythritol phosphate pathway the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) fromArabidopsis thaliana is a [4Fe-4S] protein J Biol Inorg Chem 10 131-137

Singh M Lewis PE Hardeman K Bai L Rose JK Mazourek M Chomet P Brutnell TP (2003) Activator mutagenesis of the pinkscutellum1viviparous7 locus of maize Plant Cell 15 874ndash884

Stinard P (2013) Data-mining the B73 genome sequence for carotenoid biosynthesis gene candidates Maize Genet Coop Newsl 86 29-31 wwwplantphysiolorgon April 19 2020 - Published by Downloaded from

Tanaka R Tanaka A (2007) Tetrapyrrole biosynthesis in higher plants Annu Rev Plant Biol 58 321-346Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Tang JH Teng WT Yan JB Ma XQ Meng YJ Dai JR Li JS (2007) Genetic dissection of plant height by molecular markers using apopulation of recombinant inbred lines in maize Euphytica 155 117ndash124

Thimm O Blasing O Gibon Y Nagel A Meyer S Kruger P Selbig J Muller LA Rhee SY Stitt M (2004) MAPMAN a user-driven tool todisplay genomics data sets onto diagrams of metabolic pathways and other biological processes Plant J 37 914ndash939

Thulasiram HV Erickson HK Poulter CD (2007) Chimeras of two isoprenoid synthases catalyze all four coupling reactions inisoprenoid biosynthesis Science 316 73ndash76

Vallabhaneni R Wurtzel ET (2009) Timing and biosynthetic potential for carotenoid accumulation in genetically diverse germplasm ofmaize Plant Physiol 150 562-572

Venado RE Owens BF Ortiz D Lawson T Mateos-Hernandez M Ferruzzi MG Rocheford TR (2017) Genetic analysis of provitamin Acarotenoid β-cryptoxanthin concentration and relationship with other carotenoids in maize grain (Zea mays L) Mol Breeding 37 127

Von Wettstein D Gough S Kannangara CG (1995) Chlorophyll biosynthesis Plant Cell 7 1039ndash1057Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Vranova E Coman D Gruissem W (2012) Structure and dynamics of the isoprenoid pathway network Mol Plant 5 318ndash333Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Vranova E Coman D Gruissem W (2013) Network analysis of the MVA and MEP pathways for isoprenoid synthesis Annu Rev Plant Biol64 665ndash700

Walter MH Hans J Strack D (2002) Two distantly related genes encoding 1-deoxy-d-xylulose 5-phosphate synthases differentialregulation in shoots and apocarotenoid-accumulating mycorrhizal roots Plant J 31 243-254

Xiao YM Savchenko T Baidoo EEK Chehab WE Hayden DM Tolstikov V Corwin JA Kliebenstein DJ Keasling JD Dehesh K (2012)Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes Cell 149 1525ndash1535

Xing HL Dong L Wang ZP Zhang HY Han CY Liu B Wang XC Chen QJ (2014) A CRISPRCas9 toolkit for multiplex genome editing inplants BMC Plant Biol 14 327

Xing SF Miao J Li S Qin GJ Tang S Li HN Gu HY Qu LJ (2010) Disruption of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) gene results in albino dwarf and defects in trichome initiation and stomata closure in Arabidopsis Cell Res 20 688ndash700

Xu Z Wang H (2007) LTR_FINDER an efficient tool for the prediction of full-length LTR retrotransposons Nucleic Acids Res 35 W265ndash268

Yoo SD Cho YH Sheen J (2007) Arabidopsis mesophyll protoplasts a versatile cell system for transient gene expression analysis NatProtoc 2 1565ndash1572

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

AUTHOR CONTRIBUTIONS 35

XY designed the research LZ performed most of the research and data 36

analysis XZ MW and LL analyzed the RNA-seq data XW and J-GL 37

performed immunoblot analysis GB discovered the mutant JX HF and 38

SH performed chemical analysis of leaves and seeds JY and J-SL 39

assisted with the experiments LZ and XY wrote the manuscript 40

Funding information 42

This study was supported by the National Natural Foundation of China 43

(31671697) and the National Key Research and Development Program of 44

China (2016YFD0100503) 45

Email addresses of all authors 47

Lili Zhang lilizhang0946163com 48

Xuan Zhang zhangxuan6644sinacom 49

Xiaoji Wang xiaojiwangcaueducn 50

Jing Xu xu_jing_coolyeahnet 51

Min Wang kumine163com 52

Lin Li hzaulilinmailhzaueducn 53

Guanghong Bai bgh601126com 54

Hui Fang fanghui8912126com 55

Shuting Hu hushutingcau163com 56

Jigang Li jiganglicaueducn 57

Jianbing Yan yjianbingmailhzaueducn 58

Jiansheng Li lijianshengcaueducn 59

Xiaohong Yang yxiaohongcaueducn 60

ABSTRACT 61

INTRODUCTION 85

2011 Lu et al 2012) 121

RESULTS 153

stage 161

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

ABSTRACT 61

INTRODUCTION 85

2011 Lu et al 2012) 121

RESULTS 153

stage 161

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

INTRODUCTION 85

2011 Lu et al 2012) 121

RESULTS 153

stage 161

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

2011 Lu et al 2012) 121

RESULTS 153

stage 161

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

RESULTS 153

stage 161

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

mutants 259

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al 2002) 317

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

levels in scd 344

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

conditions 358

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

respectively 401

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

DISCUSSION 455

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Development 507

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

2005) 584

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

upregulation 609

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

three times 645

Microscopy 646

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

S3) 725

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

microscope 733

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

01 02 05 1 2 5 10 20 30 50 100 200 400 8001600 and 2000 ngmL) 767

were prepared 768

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Resulting Lines 810

seedlings 823

and wide types 827

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

in scd vs WT 841

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

measurements 883

ACKNOWLEDGMENTS 887

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Abscisic acid (ngg)

Auxins (ngg)

Cytokinins (ngg)

tZ 121 plusmn 031 165 plusmn 006 000 000 000 000

DZ 549 plusmn 108 537 plusmn 038 000 000 000 000

GA1 000 000 000 000 170 plusmn 015 155 plusmn 013

GA24 2444 plusmn 066 2429 plusmn 009 000 000 000 000

GA53 000 000 204 plusmn 010 000 217 plusmn 031 000

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Figure legends 901

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

bands 948

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

19ndash24 1001

433 1004

1765ndash1778 1022

129 1581ndash1591 1027

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

104 1049

Cell 17 628ndash643 1059

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Rep 20 171ndash183 1093

36 429ndash437 1101

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

408 1116

1259-1268 1125

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

134 1401ndash1413 1153

139ndash140 1159

131-137 1173

Biol 58 321-346 1180

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

7 1039ndash1057 1196

Cell 149 1525ndash1535 1207

20 688ndash700 1213

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

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Documents

Short title: Roles of SCD in MEP and isoprenoid pathways E · 3 61. ABSTRACT. 62. Plastid isoprenoids, a diverse group of compounds that includes. carotenoids, 63. chlorophyll. s,