Bioenergetics of Monoterpenoid Essential Oil Biosynthesis ... · 41 biochemical and physiological knowledge. ... Using simulated reaction deletions, this 43 model predicted that two

Bioenergetics of Monoterpenoid Essential Oil Biosynthesis in 1

Non-Photosynthetic Glandular Trichomes1 2

Sean R Johnson2 Iris Lange Narayanan Srividya B Markus Lange 4

Institute of Biological Chemistry and MJ Murdock Metabolomics Laboratory 6

Washington State University Pullman WA 99164-6340 USA 7

Short title Bioenergetics of glandular trichomes 10

13 1 This research was supported by grants from the Division of Chemical Sciences 14

Geosciences and Biosciences Office of Basic Energy Sciences US Department of 15

Energy (grant no DEndashSC0001553 to BML) and the National Institutes of Health training 16

grant program (grant no T32GM083864 to SRJ) 17

18 2 Current address Department of Biochemistry and Molecular Biology Michigan State 19

University 20

Correspondence 23

Bernd Markus Lange PhD 24 Professor Institute of Biological Chemistry 25 Co-Director MJ Murdock Metabolomics Laboratory 26 Washington State University Pullman WA 99164-6340 USA 27 E-mail lange-mwsuedu 28 29

The author responsible for the distribution of materials integral to the findings presented 31 in this article in accordance with the policy described in the Instructions for Authors 32 (wwwplantcellorg) is BM Lange (lange-mwsuedu) 33

Plant Physiology Preview Published on August 24 2017 as DOI101104pp1700551

ABSTRACT 35

The commercially important essential oils of peppermint (Mentha x piperita L) and its 37

relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical 38

structures called glandular trichomes (GTs) A genome-scale stoichiometric model of 39

secretory phase metabolism in peppermint GTs was constructed based on current 40

biochemical and physiological knowledge Fluxes through the network were predicted 41

based on metabolomic and transcriptomic data Using simulated reaction deletions this 42

model predicted that two processes the regeneration of ATP and ferredoxin (in its 43

reduced form) exert substantial control over flux toward monoterpenes Follow-up 44

biochemical assays with isolated GTs indicated that oxidative phosphorylation and 45

ethanolic fermentation were active and that cooperation to provide ATP depended on the 46

concentration of the carbon source We also report that GTs with high flux toward 47

monoterpenes express at very high levels genes coding for a unique pair of ferredoxin 48

and ferredoxin-NADP+ reductase isoforms This study provides the first evidence how 49

bioenergetic processes determine flux through monoterpene biosynthesis in GTs 50

Keywords ATP regeneration flux glandular trichome mathematical modeling 53

non-photosynthetic metabolism 54

Abbreviations and acronyms ADH alcohol dehydrogenase FBA flux balance 56

analysis Fd ferredoxin FNR ferredoxn-NADP+ reductase GT glandular trichome 57

HDR 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase HDS 58

1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase MEP methylerythritol 59

4-phosphate TPM transcripts per kilobase million 60

One Sentence Summary In non-photosynthetic glandular trichomes terpenoid 62

metabolism is powered by oxidative phosphorylation fermentation and a unique 63

ferredoxinferredoxin-NADP+ reductase pair 64

INTRODUCTION 65

Plant secondary (or specialized) metabolites are frequently synthesized and accumulated 67

in specialized tissues or cell types Anatomical structures that harbor specialized 68

metabolic pathways can take many forms and vary widely across the plant lineages 69

Some examples are resin ducts secretory cavities glandular trichomes (GTs) laticifers 70

and gum ducts (Fahn 1988) GTs are particularly widespread across the angiosperm 71

lineages and accumulate among others the specialized metabolites responsible for the 72

psychoactive effects of cannabis the bitter flavor of hops the insect-repellant principles 73

of tomato leaves the anti-malarial activity of sweet wormwood and the essential oils of 74

members of the Lamiaceae (Lange and Turner 2013) 75

Peppermint (Mentha x piperita L) and its close relatives have a long history as model 76

systems for furthering our understanding of GT structure and function Pioneering studies 77

in the late 1980s and early 1990s reported on methods for purifying secretory cells of GTs 78

away from other leaf cell types and established that the entire biosynthetic pathway from 79

imported sugars to the monoterpene end products is confined to these specialized cell 80

types (Gershenzon et al 1992 McCaskill et al 1992 McCaskill and Croteau 1995) In 81

the mid to late 1990s these protocols were modified to allow the development of an 82

expressed sequence tag (EST) library the sequencing of which revealed the stunning 83

metabolic specialization of mint GTs (Lange et al 2000) These data sets also enabled 84

the cloning and characterization of genes encoding the entire complement of enzymes 85

responsible for p-menthane monoterpenoid biosynthesis in peppermint (Lange et al 86

1998 Burke et al 1999 Lange and Croteau 1999a 1999b Ringer et al 2003 2005 87

Davis et al 2005) Kinetic mathematical models facilitated the discovery of feedback 88

control GT developmental patterns and epigenetic processes as critical determinants of 89

metabolic outcomes (Rios-Estepa 2008 2010 Lange and Rios-Estepa 2014 Ahkami et 90

al 2015) Protocols for the Agrobacterium-mediated transformation of mint species had 91

been developed in the late 1990s (Berry et al 1996 Caissard et al 1996 Diemer et al 92

1998 Niu et al 1998) and were later employed supported in part by mathematical 93

modeling to successfully modulate monoterpenoid essential composition and yield in 94

transgenic peppermint plants (Mahmoud and Croteau 2001 Mahmoud et al 2004 95

Lange et al 2011 Lange et al 2013) The draft genome sequence and genetic map of 96

Mentha longifolia L a diploid mint related to the ancestors of present-day peppermint 97

and spearmint (which are both polyploid) are resources that now also enable advanced 98

breeding approaches for cultivar improvement (Vining et al 2017) 99

The non-photosynthetic GTs of peppermint (Werker et al 1985) which fill up with 100

monoterpenoid essential oil within only one day (Turner et al 2000a 2000b) sustain an 101

exceptionally high flux through central carbon metabolism and the monoterpene 102

biosynthetic pathway A better understanding of how flux is regulated in these cells will 103

therefore have a tremendous impact on the development of novel metabolic engineering 104

and molecular breeding strategies toward mint cultivar improvement One promising 105

approach involves modeling metabolism more globally Genome-scale stoichiometric 106

models have been produced for various model plant systems including leaf cells of 107

Arabidopsis thaliana (Poolman et al 2009 de Oliveira DalrsquoMolin et al 2010a 108

Mintz-Oron et al 2012 Cheung et al 2013 Arnold and Nikoloski 2014) maize (de 109

Oliveira DalrsquoMolin et al 2010b Saha et al 2011) rice (Poolman et al 2013) and 110

embryos of rapeseed (Hay and Schwender 2011) Building on these pre-existing models 111

we developed a stoichiometric model reflecting metabolism in mint GTs by integrating 112

newer transcriptome data sets with the wealth of knowledge on this subject in the 113

published literature We then employed a series of simulations based on flux balance 114

analysis (FBA) (Fell and Small 1986 Savinell and Palsson 1992) to predict reactions 115

with critical functions in monoterpene formation under extraordinarily high flux Follow-up 116

experimental testing revealed shared carbon source-dependent roles for oxidative 117

phosphorylation and ethanolic fermentation in ATP regeneration and the occurrence of a 118

unique pair of ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) in heterotrophic 119

peppermint GTs 120

RESULTS 124

Reconstruction of Metabolism in Peppermint GTs 126

To automate various processes relevant to building metabolic models we developed a 128

software package called YASMEnv Because of space constraints the capabilities 129

cannot be outlined in the main body of this manuscript and extensive details are provided 130

in Supplemental Methods and Data File 1 Further instructions and the entire code are 131

available freely at httpsbitbucketorgseanrjohnsonyasmenv 132

To assemble the reactions required for central carbon metabolism in peppermint GTs 133

we surveyed the literature for a particularly well curated metabolic reconstruction and 134

selected the ldquoArabidopsis Core Modelrdquo (Arnold and Nikoloski 2014) to serve as the 135

foundation for our efforts Additional metabolic reactions were extracted from the AraCyc 136

and MetaCyc databases ( Mueller et al 2003 Caspi et al 2016) A process termed 137

ldquoinstantiationrdquo (Latendresse et al 2012) was employed using an implementation of the 138

relevant algorithm in YASMEnv to ensure that only mass-balanced reactions were 139

included in the model Each reaction was associated with a subcellular compartment 140

based on the records in the Arabidopsis Core Model AraCyc and MetaCyc and these 141

associations were confirmed by searches against the TargetP and SUBAcon databases 142

(Emanuelsson et al 2000 Hooper et al 2014) Sequences of transcripts represented in 143

recently acquired transcriptome data sets for peppermint GTs (Ahkami et al 2015) were 144

compared based on global identity (Myers and Miller 1988 Tian and Skolnick 2003) to 145

those associated with reactions in the Arabidopsis Core Model AraCyc and MetaCyc 146

The first iteration of the menpiGT metabolic reconstruction consisted of all reactions of the 147

Arabidopsis Core Model and reactions from AraCyc and MetaCyc with at least one 148

associated peppermint transcript (details in Supplemental Methods and Data File 1) 149

The model was refined in five additional stages (1) completion of pathways to known 150

glandular trichome metabolites (2) removal of thermodynamically infeasible loops (3) 151

removal of reactions retained from the Arabidopsis Core Model that are not supported by 152

peppermint transcripts (4) removal of reactions that cannot carry flux because they are 153

not connected to the remainder of the network and (5) modifications to the model based 154

on the experimental data presented in this manuscript (details in Supplemental Methods 155

and Data File 1) The final model (termed menpiGT_2015) contains 624 reactions 156

accounting for 757 expressed transcripts The menpiGT_2015 model is a reflection of the 157

astounding specialization of secretory cells within peppermint GTs during secretory 158

phase (Fig 1A) An imported oligosaccharide (raffinose or sucrose) is converted through 159

central carbon metabolism and then dedicated pathways primarily into monoterpenes 160

(94-95 (vv) of essential oil) sesquiterpenes (4 ) and polymethoxylated flavones (1-2 161

) (Fig 1B) while genes involved in other pathways that commonly carry significant flux 162

such as amino acid conversion into proteins sugar nucleotide interconversions into cell 163

wall macromolecules and fatty acidlipid biosynthesis are only expressed at fairly low 164

levels (see Supplemental Table 1) 165

Knockout Simulations Suggest Essential Roles for ATP Regeneration and 167

Non-Photosynthetic Ferredoxin Reduction 168

To test the utility of menpiGT_2015 for identifying processes required for maintaining high 170

flux toward monoterpenes we employed a process called simulated reaction deletions 171

(Edwards and Palsson 2000) Reactions were removed from menpi_2015 one at a time 172

and the impact on monoterpene formation was evaluated (see Supplemental Table 2) 173

Thirty-five reactions were found to be essential for producing (-)-menthol from an 174

imported carbon source (raffinose) (Table 1) The requirements for catabolism of 175

raffinose (primary carbon source considered in the model) the exchange of small 176

molecules across membranes (balancing C and O budgets) and the reactions involved 177

directly in the biosynthesis of precursors and end products of the monoterpene pathway 178

were expected outcomes of these in silico predictions Interestingly the regeneration of 179

nucleotide triphosphates (in particular CTP and ATP) and the recycling of reduced 180

ferredoxin were also returned as essential reactions in the simulated knockout study 181

(Table 1) These reactions are linked to the bioenergetics of GTs which were poorly 182

understood at the outset of our study and we therefore designed experiments to further 183

test how these processes may affect monoterpene formation 184

Sensitivity of Monoterpene Production to Inhibition of Oxidative Phosphorylation 185

Depends on Concentration of Carbon Source 186

Peppermint GTs are non-photosynthetic structures (Werker et al 1985) and one 188

important question pertains to the regeneration of ATP to power their highly active 189

metabolism In heterotrophic animal cells oxidative phosphorylation in mitochondria has 190

a critical role in supplying ATP and one would thus hypothesize that this process might 191

also provide energy equivalents in non-photosynthetic GTs Indeed the genes coding for 192

the enzyme complexes of the oxidative phosphorylation pathway were expressed at fairly 193

high levels in peppermint GTs (Fig 2A) indicating that this pathway of ATP regeneration 194

is likely operational Isolated GTs are capable of synthesizing monoterpenes from 195

externally supplied sugar precursors (Gershenzon et al 1992 McCaskill et al 1992 196

McCaskill and Croteau 1995) and would thus seem to be an excellent model system to 197

study this process However previous feeding experiments employed complex media 198

that contained various enzyme cofactors including ATP and it was thus important to test 199

if ATP could be omitted from the incubation buffer In experiments that evaluated various 200

combinations of cofactors we determined that the presence of NADP+ and ADP is 201

sufficient to support monoterpene production in isolated peppermint GTs (see 202

Supplemental Methods and Data File 2) 203

A few additional experimental considerations need to be mentioned (1) an alternative 204

pathway for regenerating ATP independent of oxidative phosphorylation would be 205

through the activity of adenylate kinase (direct phosphorylation of proffered ADP) and to 206

disable this potentially cooperative reaction for ATP formation the incubation buffer was 207

supplemented with AMP a known inhibitor of the enzyme (Atkinson 1968) (2) although 208

raffinose is the primary transport oligosaccharide in many Lamiaceae (and likely imported 209

into GTs) (Buumlchi et al 1998 Olennikov and Tankhaeva 2007) there is no commercial 210

source for the radiolabeled tracer and we therefore employed [U-14C]sucrose in our 211

labeling experiments (sucrose is formed during raffinose catabolism) 212

Isolated GTs were incubated in media containing three different sucrose 213

concentrations and subjected to oligomycin (omitted in controls) which inhibits the ATP 214

synthase component of the enzyme complexes performing oxidative phosphorylation 215

(Huijing and Slater 1961 Penefsky 1985) At the end of the experimental period 216

radiolabeled monoterpenes were extracted into hexaneether (11 vv) and quantified by 217

liquid scintillation counting Based on data obtained in control experiments (no 218

oligomycin) the maximum incorporation of label from [U-14C]sucrose into monoterpenes 219

occurs at 1 mM sucrose (58 nmol per million gland cells) with a slight decrease at 10 mM 220

(Fig 2B) Oligomycin had no effect on monoterpene accumulation at 002 mM sucrose 221

when the monoterpene yield (and likely the ATP demand) was very low At 1 mM sucrose 222

the addition of oligomycin resulted in a significant two-fold decrease in the quantity of 223

monoterpenes produced (from 58 to 32 nmol per million gland cells) Surprisingly at 10 224

mM sucrose (highest concentration tested) oligomycin had no impact on monoterpene 225

production (45 nmol per million gland cells) These results could not be explained with 226

the reactions included in version 5 of menpiGT_2015 and indicated that alternative 227

mechanisms for ATP regeneration particularly at high sugar concentrations needed to 228

be considered 229

One such possibility would be the coupling of ATP recycling to fermentative steps 230

such as those occurring in microbes and plants under oxygen-limiting conditions To the 231

best of our knowledge fermentative reactions have not been included in any metabolic 232

reconstruction for plants and we therefore added the relevant reactions manually to 233

menpiGT_2015 (version 6) In addition transcriptome data obtained with isolated 234

peppermint GTs at secretory stage (Ahkami et al 2015) were incorporated using a 235

modified SPOT algorithm (Kim et al 2016) (details outlined in Supplemental Methods 236

and Data 1) Allowed carbon sources were oligosaccharides ammonia oxygen 237

hydrogen sulfide sulfate inorganic phosphate water and carbon dioxide The 238

experimentally derived biomass export reactions as presented in Fig 1B were disabled 239

and therefore became values to be predicted by the model based on gene expression 240

patterns Allowed outputs were monoterpenes sesquiterpenes polymethoxylated 241

flavones ethanol lactate cellulose amino acids water and carbon dioxide (see 242

Supplemental Table S3) Interestingly our simulations predicted a significant formation of 243

fermentative reaction products (lactate and ethanol) (Fig 3B) and we thus embarked on 244

experiments described in the following paragraph to test the hypothesis that 245

fermentation plays a role in energizing peppermint GTs 246

Fermentation is Active in Peppermint GTs 247

In fermentative processes NADH and pyruvate formed in glycolysis are converted into 249

NAD+ and an organic molecule (most often ethanol or lactic acid) which is coupled to 250

reactions of the bottom half of glycolysis that regenerate ATP (Fig 3A) Based on 251

transcriptome data (Ahkami et al 2015) genes involved in both glycolysis 252

(glyceraldehyde 3-phosphate dehydrogenase phosphoglycerate kinase enolase and 253

pyruvate kinase) and fermentation (pyruvate decarboxylase alcohol dehydrogenase 254

(ADH)) are expressed at high (in some cases very high) levels in peppermint GTs (Fig 255

3A) When isolated GTs were supplied with appropriate reactants significant alcohol 256

dehydrogenase (ADH) activity (assayed with NAD+ nitroblue tetrazolium and ethanol) 257

was detected in about 12 of gland cell clusters (observed as dark staining) (Fig 3C-E) 258

which corresponds well with the proportion of GTs in secretory phase on young leaves 259

(Turner et al 2000a 2000b) Enzyme assays performed with crude protein extracts 260

indicated a fairly high specific ADH activity in GTs isolated from young leaves (08 nmol 261

(min bull mg protein)-1) but no measurable activity in GTs obtained from mature leaves 262

(where no monoterpene biosynthesis occurs) (Fig 3F) These data support the 263

hypothesis that fermentation plays an important role in energizing non-photosynthetic 264

peppermint GTs and justify the inclusion of these reactions in version 6 of the 265

menpiGT_2015 model 266

Given that monoterpenol dehydrogenases are among the most highly expressed 267

genes in secretory-phase GTs (Ahkami et al 2015 Lange et al 2000) one may 268

postulate that the ADH activity we observed is a non-specific side activity of these 269

enzymes To test this hypotheses we first performed a Blastx search with the 270

well-characterized ADH1 sequence from potato (Matton et al 1990) against the 271

peppermint GT transcriptome data set This search indicated the presence of two putative 272

orthologs (at roughly 88 identity for the translated peptide sequence) that were 273

expressed at high levels in peppermint GTs (accession numbers MF579445 and 274

MF579446) (Supplemental Fig S1) The cDNAs corresponding to the peppermint ADH 275

candidates were cloned by PCR with gene-specific primers and ligated into an 276

appropriate expression vector Each vector carrying a peppermint candidate cDNA was 277

transformed into E coli the expression of the transgene induced and the recombinant 278

protein purified The sizes of the proteins encoded by MF579445 and MF579446 were 279

both roughly 41 kDa (Fig 3G) One ADH-like candidate (recombinant protein produced 280

from MF579445) turned out to be inactive in our assays while the other (produced from 281

MF579446 henceforth referred to as ADH1) had the properties of a typical ADH (km 282

(ethanol) = 539 mM kcat = 6035 s-1) (Fig 3H I) These results confirm the presence of a 283

specific ADH activity which is independent of monoterpenol dehydrogenases in 284

non-photosynthetic peppermint GTs 285

Mint GTs Contain a Unique FdFNR Complex 287

The precursors for mono- and sesquiterpene biosynthesis in peppermint GTs are 289

provided exclusively by the methylerythritol phosphate (MEP) pathway (McCaskill and 290

Croteau 1995 Eisenreich et al 1997 Lange et al 2001) The genes involved in the 291

MEP pathway and downstream monoterpene-specific reactions are expressed at very 292

high levels in peppermint GTs (Fig 4) The electron transfer reactions of reductive steps 293

of the MEP pathway catalyzed by 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate 294

synthase (HDS) and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) 295

employ Fd and FNR (Seemann et al 2006 Rekittke et al 2013) This cofactor 296

requirement is in accordance with our modeling prediction that ferredoxin recycling is 297

essential for monoterpene biosynthesis (Table 1) The non-photosynthetic isoforms of Fd 298

and FNR have redox potentials that are significantly different from those in photosynthetic 299

cells (Aliverti et al 2001) and electrons can thus flow in a different direction from NADPH 300

to FNR then from FNR to Fd and finally from Fd to the iron-sulfur clusters of HDS and 301

HDR (Fig 4) Thus far non-photosynthetic Fd and FNR isoforms have only been 302

characterized from roots (Hanke et al 2005 Aliverti et al 2008 Onda et al 2014) and 303

it was therefore of interest to evaluate which isoforms are present in non-photosynthetic 304

GTs 305

Public databases were surveyed for data sets covering leaf root and GT 306

transcriptomes of angiosperms Phylogenetic trees were then generated based on 307

alignments of nucleotide sequences of Fd and FNR isoforms (see Supplemental Table 4) 308

FNRs expressed in GTs (in some cases more than one isoform) separated into two 309

different clades those expressed in non-photosynthetic GTs (Cannabis sativa L Mentha 310

x piperita L Ocimum basilicum L) were identical to the root isoform or clustered with root 311

isoforms whereas those expressed in photosynthetic GTs (Solanum lycopersicum L 312

Artemisia annua L) clustered with leaf isoforms (Fig 5A) Generally speaking similar 313

patterns were observed with GT-expressed isoforms of Fd (separation of leaf from 314

rootGT isoforms) However there was one significant difference the major isoforms 315

expressed in non-photosynthetic GTs (particularly in the mint family) formed a sub-clade 316

that was distinguishable from that of root isoforms (Fig 5B) 317

Did GT-specific Fd isoforms evolve in mint The hybrid hexaploid nature of 318

peppermint makes it difficult to answer this question due to the potential for the presence 319

of many Fd isoforms in genomes retained from three different ancestral species 320

However the recently completed draft genome assembly for M longifolia L a diploid 321

species related to one of the ancestors of present-day peppermint (Vining et al 2017) 322

allowed us to examine all Fd isoforms Organ- and tissue-specific transcriptome data for 323

M longifolia were recently deposited in public archives (root and GT transcriptomes with 324

NCBI short read archive (SRA) accession numbers SRP083887 and SRR3623199 325

respectively) The sequence of the minor Fd isoform of M longifolia GTs (average 326

expression of 289 transcripts per kilobase million (TPM)) was identical to that of the root 327

isoform whereas the major isoform (average expression of 2586 TPM) was distinct from 328

the root isoform (Fig 5B) The major and minor isoforms from Mentha GTs differed in the 329

mature peptide sequence at 12 positions (Fig 6A) To ensure that these findings were not 330

due to assembly errors from short reads we PCR-amplified and sequenced cDNAs 331

corresponding to both Fd isoforms (KY748231 (GT major) and KY748232 (GT 332

minorroot)) and thereby confirmed the unique sequence of the major GT isoform The 333

major peppermint GT-specific isoform of Fd was essentially identical to the major isoform 334

of other mint species and the same sequence conservation across species was observed 335

for the minor GTroot isoform (Fig 5B) Mapping of the Fd isoforms to the M longifolia 336

genome revealed that they map to different contigs the major Fd isoform of GTs was 337

mapped to contig 19491 (refers to genome assembly with NCBI BioProject accession 338

number PRLNA310613) whereas the minor GT isoform mapped to contig 5531 339

indicating that these genes likely have separate origins 340

We also obtained transcriptome data for GTs isolated from Perilla frutescens L (short 341

read archive accession number SRR4106089) and included previously published 342

GT-specific cDNA sequences from basil (Ocimum basilicum L) (Gang et al 2001) in our 343

phylogenetic analysis which expands the scope of sequence comparisons to other 344

Lamiaceae with high monoterpenoid essential oil content The GT-specific Fd isoforms of 345

these species clustered with the major isoform of mint species indicating that 346

non-photosynthetic GTs with high flux toward monoterpenes may harbor unique Fd 347

isoforms across the mint family 348

To assess the potential implications of a GT-specific Fd isoform in peppermint we 349

modeled the structures of Fd (GT major isoform) FNR (GTroot isoform) and HDR based 350

on published data for closely related isoforms from plants (Fig 6) Because of the more 351

speculative nature of this analysis we are providing the details in the Discussion section 352

353 354 355

DISCUSSION 356

MenpiGT_2015 is the First Genome-Scale Reconstruction of Specialized 358

Metabolism in GTs 359

Large-scale mathematical models of plant metabolism were initially focused on species 361

for which genome sequences were available (Arabidopsis rice maize and other model 362

organisms) but with the advent of cost-competitive technologies for obtaining 363

transcriptome sequences metabolic reconstructions coupled with flux balance analysis 364

have also been performed for non-model plants (Hay and Schwender 2011) Most of 365

these models were generated to represent reactions in a major organ and only a few 366

accurately reflect metabolism specific to a certain tissue (de Oliveira DalrsquoMolin et al 367

2010b Hay and Schwender 2011 Pilalis et al 2011 Mintz-Oron et al 2012) The 368

MenpiGT_2015 model is the first attempt to gather data from various sources (literature 369

transcriptomics metabolomics and biochemical assays) and capture metabolism in 370

non-photosynthetic GTs We decided to start with a well-curated compartmentalized 371

metabolic reconstruction recently generated for a model plant (Arabidopsis Core Model) 372

(Arnold and Nikoloski 2014) and then incorporated additional information from 373

web-based databases focusing on metabolism (AraCyc and MetaCyc) to produce a 374

consensus set of reactions The YASMEnv toolbox which we developed specifically for 375

automating various steps in the generation of metabolic models was invaluable in these 376

efforts and we hope that others will take advantage of the freely available scripts 377

(httpsbitbucketorgseanrjohnsonyasmenv) Further model development steps 378

included eliminating thermodynamically infeasible loops ensuring all reactions are 379

mass-balanced and associating reactions with transcripts (adding reactions unique to 380

mint GTs and eliminating reactions not supported by mint transcripts) These adjustments 381

reduced the total number of reactions in mepiGT_2015 to 624 (from an initial 2639 382

reactions) (see Supplemental Methods and Data File 1) and are reflective of the 383

remarkable specialization of mint GTs 384

Since mint GT cells are non-photosynthetic an imported carbon source (likely 385

raffinose) (McCaskill et al 1992 Buumlchi et al 1998 Olennikov and Tankhaeva 2007) is 386

converted via central carbon metabolism to precursors for terpenoid biosynthesis (which 387

correspond to gt 95 of products accumulated in essential oil) Importantly precursors 388

for both C10-derived monoterpenes and C15-derived sesquiterpenes are provided 389

exclusively by the plastidial MEP pathway (McCaskill and Croteau 1995 Eisenreich et 390

al 1997 Lange et al 2001) which is reflected in the extremely high expression levels of 391

the relevant genes (whereas expression levels of genes of the cytosolicperoxisomal 392

mevalonate pathway are low or undetectable) (Supplemental Table S1) The pathway 393

toward methoxylated flavones is clearly active at the transcriptional level with 394

biosynthetic genes being expressed at appreciable levels (see Supplemental Table 1) 395

but the quantities of these specialized metabolites in mint GTs are generally very low (1-2 396

of the essential oil with no significant occurrence in other cell types) (Voirin and Bayet 397

1992 Sato and Tamura 2015) 398

Transcripts related to cellular maintenance reactions (eg protein synthesis and 399

degradation nucleotide biosynthesis and fatty acidlipid biosynthesis) are detectable in 400

published mint data sets (Ahkami et al 2015) but their abundance is very low compared 401

to those involved in terpenoid and polymethoxylated flavone biosynthesis Some genes 402

involved in nucleotide interconversion reactions are expressed at high levels (eg 403

adenosine kinase at 2122 TPM and adenylate kinase at 788 TPM) but these are linked to 404

the regeneration of ATP which is needed to support the highly active biosynthetic 405

pathways of GTs (the MEP pathway alone requires six ATP molecules to generate one 406

molecule of geranyl diphosphate (monoterpene precursor)) The conversions from 407

1-deoxy-D-xylulose 5-phosphate to one molecule of (-)-menthol require 10 molecules of 408

NADPH and the high expression levels of genes involved in the oxidative branch of the 409

pentose phosphate pathway (coding for glucose 6-phosphate dehydrogenase 410

6-phosphogluconolactonase and 6-phosphoglcuonate dehydrogenase) (Fig 4) which 411

support NADPH regeneration (Kruger and von Schaewen 2003) are therefore in 412

accordance with the expected demands In summary menpiGT_2015 captures the 413

specialization of mint GTs at secretory stage and allows us to test initial assumptions 414

about metabolism computationally which can then be assessed further experimentally 415

Iterative Mathematical Modeling and Experimental Testing Provides Insights into 417

the Processes that Power Metabolism in Non-Photosynthetic GTs 418

Reaction knockout simulations with menpiGT_2015 (version 5) were performed to 420

determine which reactions are essential for (-)-menthol production (Table 1 and 421

Supplemental Table S2) Surprisingly of the seven pathways or processes identified by 422

this simulation as being essential only two the MEP pathway and the monoterpene 423

biosynthetic pathway were strongly supported by prior experimental evidence (Lange 424

2015) Three other essential processes sucroseraffinose import and metabolism 425

(carbon source) CTP regeneration (required to provide CTP as reactant in MEP 426

pathway) and exchange of small molecules across membranes (trivial) did not appear to 427

warrant further exploration at this time The remaining two essential processes oxidative 428

phosphorylation and regeneration of reduced ferredoxin had not been studied at all in 429

GTs and were therefore deemed worthy of experimental testing 430

Feeding experiments with isolated GTs were employed to assess the contribution of 431

oxidative phosphorylation to the formation of organic soluble products (primarily 432

monoterpenes) from 14C-labeled sucrose One of the surprises in our experimental data 433

was the finding that monterpene production in the presence of the ATP synthase inhibitor 434

oligomycin was dependent on the concentration of sucrose We observed inhibitory 435

effects at 1 mM sucrose but not at 002 mM or 10 mM sucrose (Fig 3) The lack of 436

inhibition at 002 mM sucrose may be due to a low overall metabolic rate (measured as 437

monoterpene production per unit time) at such a low sucrose concentration At 1 mM 438

sucrose oligomycin treatment reduced monoterpene production to about half of that 439

observed in the absence of oligomycin and one might interpret this as incomplete 440

inhibition of ATP synthase However this would not explain why there was no effect of the 441

oligomycin treatment on monoterpene accumulation at 10 mM sucrose An alternative 442

explanation was the involvement of processes other than oxidative phosphorylation in 443

regenerating ATP Adenylate kinase was inhibited by addition of AMP in our assays 444

(Atkinson 1968) and was therefore unlikely to be a significant contributor to ATP 445

formation leaving fermentation as the only remaining candidate for providing energy 446

equivalents After adding fermentative reactions to our model and incorporating gene 447

expression patterns acquired previously (Ahkami et al 2015) simulations predicted that 448

fermentation products (lactate and ethanol) should indeed accumulate in peppermint 449

GTs Follow-up biochemical experiments provided evidence that peppermint GTs contain 450

highly active ADH1 the signature enzyme of fermentation (Fig 3) thereby confirming 451

model predictions It is important to note that this enzyme is clearly distinct from 452

monoterpenol dehydrogenases which were previously demonstrated to not possess an 453

ADH side activity (Kjonaas et al 1985 Gershenzon et al 1989) 454

The next question was if the consideration of fermentation could explain the data 455

obtained in the experiments in which oxidative phosphorylation was inhibited More 456

specifically why did we observe a significant reduction of monoterpene production in 457

isolated GTs treated with oligomycin at 1 mM but not at 10 mM sucrose concentration 458

In all plant ADH enzymes characterized thus far the km value is always in the millimolar 459

range (Mayne and Lea 1984 Tong and Lin 1988 Osterman et al 1993 Hadizadeh and 460

Keyhani 2004) We calculated a km of 663 mM for peppermint ADH1 which is consistent 461

with these earlier studies We therefore hypothesize that acetaldehyde does not reach an 462

intracellular concentration that is sufficiently high to drive ADH activity toward ethanolic 463

fermentation at lower sucrose concentrations Under those conditions ATP regeneration 464

therefore does not rely on fermentation and is primarily dependent on oxidative 465

phosphorylation (thus the effect of the ATP synthase inhibitor on monoterpene production 466

at 1 mM sucrose) The situation would be different at high sucrose concentrations when 467

intracellular levels of acetaldehyde can be high enough for ADH to operate in the direction 468

of ethanol production which complements or maybe even substitutes for oxidative 469

phosphorylation (thus no effect of the ATP synthase inhibitor on monoterpene production 470

at 10 mM sucrose) Based on measurements in various plants the intracellular 471

concentration of oligosaccharides can be up to 100 mM (Nadwodnik and Lohaus 2008) 472

indicating that the concentration of acetaldehyde in mint GTs could be sufficiently high to 473

support ethanolic fermentation 474

Peppermint GTs synthesize oxygenated monoterpenes at high flux (each individual 475

gland fills within about 24 h after reaching secretory stage) (Turner et al 2000a 2000b) 476

which places significant demands on the availability of molecular oxygen for biosynthetic 477

cytochrome P450-dependent monooxygenases (Lupien et al 1999 Bertea et al 2001) 478

Mint GTs are covered by a thick waxy cuticle (Turner et al 2000a 2000b) which may 479

prohibit a direct diffusion of atmospheric oxygen into the cells responsible for 480

monoterpenoid essential oil biosynthesis Together these factors may contribute to 481

oxygen-limiting conditions in GTs which would favor fermentation as a mechanism for 482

ATP regeneration (Fig 4) 483

Occurrence of GT-Specific FdFNR Isoforms in Monoterpene-Accumulating Plants 485

The regeneration of reduced Fd was identified in this study as an essential process by 487

computational knockout simulations (Table 1) This is a logical consequence of the fact 488

that Fd is a cofactor for electron transfer reactions toward two reductive enzymes of the 489

MEP pathway (Seemann et al 2006 Rekittke et al 2013) and flavone hydroxylases 490

(Berim et al 2014) which are involved in the formation of the major products of mint GTs 491

(monoterpenes and methoxylated flavones respectively) In earlier versions of 492

menpiGT_2015 Fd served as a substrate for FNR which is the direction of electron flow 493

in photosynthetic cells With the removal of photosystem I from the model due to lack of 494

support from gene expression data the direction of FNR had to be reversed in order to 495

maintain the capacity for monoterpene production which is the direction of electron flow 496

suggested to occur in non-photosynthetic root cells (Aliverti et al 2001) 497

A phylogenetic analysis indicated that the most highly expressed FNR isoform of mint 498

GTs clustered with the non-photosynthetic isoforms of others plants which was an 499

expected outcome Surprisingly however two different Fd isoforms were found to be 500

expressed in GTs of the genus Mentha The minor isoform (lower expression levels) of M 501

x piperita and M longifolia was identical to the isoform most highly expressed in roots of 502

the same species The major isoform (10-fold higher expression than minor isoform) was 503

unique to GTs The major isoform was also conserved across several members of the 504

Lamiaceae This begs the questions if there is a functional consequence to this sequence 505

diversity One possibility would be that the interaction between the major Fd isoform of 506

GTs and reductive enzymes of the MEP pathway HDS and HDR facilitates high flux 507

through these steps and the remainder of the monoterpene biosynthetic pathway 508

What are the potential implications of a novel Fd isoform specifically expressed in 509

non-photosynthetic GTs of the genus Mentha (and possibly across the Lamiaceae) 510

Based on a molecular model of ternary peppermint FNR-Fd-HDR complexes we asked if 511

there were differences among the sequences of peppermint Fd isoforms (Fig 6A) that 512

had the potential to affect the interaction(s) with FNR andor HDR (Fig 6B) Prior crystal 513

structure data of the electron transfer complex between Fd and FNR provided evidence 514

for a close proximity of the [2Fe-2S] cluster of Fd and the FAD prosthetic group of FNR 515

(Kurisu et al 2001) The authors suggested that pairs of charged residues form 516

intermolecular salt bridges which was in accordance with prior biochemical data 517

(reviewed in the context of plant Fd-FNR interactions by Aliverti et al 2008) Two 518

residues previously implicated in the interaction of Fd and FNR were different in 519

peppermint isoforms (Shinohara et al 2017) S32 and G95 in the GT major isoform 520

corresponded to T32 and S95 respectively in the minor isoform (numbering based on 521

mature polypeptide excluding plastidial targeting sequence of the major isoform of 522

peppermint) (Fig 6C) 523

Crystal structures have been resolved for both HDS and HDR but only one study 524

modeled the binding with Fd Rekittke et al (2013) employed docking calculations to 525

select the energetically most favorable complexes between HDR and the cognate Fd 526

from Plasmodium falciparum The authors concluded that ferredoxin was most likely to 527

attach to a shallow well formed by the N-terminus helix α1B and the loops near strands 528

β4A and β4B Our FNR-Fd-HDR docking simulations indicated that one of the lowest 529

energy ternary complexes had a molecular surface that was very similar to that suggested 530

previously (Rekittke et al 2013) Interestingly five residues purportedly involved in the 531

FdndashHDR interaction differed between the major and minor Fd isoforms of peppermint GTs 532

(E54 K55 T57 K69 E73 and A85 in major isoform corresponded to V54 S55 S57 N69 533

Q73 and S85 in the minor isoform respectively) (Fig 6D) 534

We hypothesize that the differences between the major and minor Fd isoforms in 535

GTs may affect the stability of the FNRndashFdndashHDR complex which in turn may impact flux 536

through the biosynthetic step catalyzed by HDR The situation would be different in 537

photosynthetic GTs characterized thus far In type VI glandular trichomes of Solanum 538

habrochaites S Knapp amp DM Spooner the accumulation of terpenoids occurs over a 539

longer period of time while in peppermint GT filling concludes within a matter of days 540

(Turner et al 2000a b) Terpenoids in S habrochaites GTs are derived from both the 541

MVA and MEP pathways (Balcke et al 2017) whereas in peppermint terpenoids of GTs 542

are produced solely via the MEP pathway and therefore the reactions catalyzed by HDS 543

and HDR (McCaskill and Croteau 1995 Eisenreich et al 1997 Lange et al 2001) It is 544

therefore tempting to speculate that the major GT-specific Fd isoform of peppermint (and 545

possibly other Lamiaceae) might have evolved to facilitate the particularly high flux 546

through the reactions catalyzed by HDS and HDR en route to monoterpenoid end 547

products 548

METHODS 552

Simulations Using the MenpiGT_2015 Model 554

The step-wise development of a metabolic reconstruction and its data-driven iterations 556

are described in the Supplemental Methods and Data File 1 To simulate gene knockouts 557

upper and lower bounds were set such that CO2 and raffinose were the only permitted 558

carbon sources in the network O2 uptake was permitted but NH4 import was disabled 559

The upper and lower flux bounds for menthol export were set to 1 and the objective 560

function was set to minimize raffinose import A reference minimum for raffinose import 561

was calculated with no internal reactions disabled Upper and lower flux bounds for each 562

reaction were then set to 0 one at a time and raffinose import was computed 563

The SPOT algorithm was implemented according to Kim et al (2016) with the 564

addition of Boolean constraints to prevent reversible reactions from being counted twice 565

in the objective function (referred to as Boolean constrained SPOT or BC-SPOT) Prior to 566

running BC-SPOT boundary fluxes were modified to disable the experimentally derived 567

biomass export reaction and to allow optional import and export of small molecules 568

Allowed carbon sources were sucrose glucose fructose or raffinose Additional allowed 569

imports were ammonia oxygen hydrogen sulfide sulfate inorganic phosphate water 570

and carbon dioxide Allowed exports were monoterpenes sesquiterpenes 571

polymethoxylated flavones ethanol lactate cellulose amino acids water and carbon 572

dioxide Optimization problems were solved with Gurobi Optimizer (Gurobi Optimization 573

Inc Houston Texas USA) accessed through YASMEnv a modeling tool we developed 574

specifically for this study (details in Supplemental Methods and Data File 1) 575

Chemicals and Biochemicals 577

Chemicals were purchased from Sigma-Aldrich (St Louis MO USA) unless otherwise 579

noted Biochemical reagents were sourced from ThermoFisher Scientific (Waltham MA 580

USA) unless otherwise noted [U-14C]Sucrose (432 mCimol in 90 aq ethanol) was 581

obtained from MP Biomedicals (Santa Ana CA USA) 582

Glandular Trichome Isolation 584

GTs were isolated based on the protocol developed by Gershenzon et al (1992) with 586

modifications Roughly 20 grams of leaves were harvested into a plastic bag on ice 587

Depending on the purpose of the experiments young leaves (less than half expanded) 588

andor mature leaves (fully expanded) were harvested when the first flower buds 589

emerged Leaves were weighed and transferred into a bead beater chamber containing 590

100 g of glass beads (05 mm diameter) and 300 mL isolation buffer (250 mM sorbitol 10 591

mM KCl 2 mM MgCl2 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid 1 592

(wv) polyvinylpyrrolidone (average molecular weight of 40000 gmol) and 06 (wv) 593

methyl cellulose adjusted to pH 74) Bead beating was performed as three 1-min pulses 594

with 1 min of cooling on ice between pulses The contents of the bead beater chamber 595

were poured onto a nylon mesh (350 microm) and rinsed with wash buffer (200 mM sorbitol 596

50 mM KCl 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid 5 mM MgCl 597

1 mM ethylene glycol-bis(β-aminoethyl ether)-NNNrsquoNrsquo-tetraacetic acid 05 mM 598

Na2HPO4 and 01 mM NaP2O7 adjusted to pH 74) These wash steps were repeated 599

with nylon mesh of two sizes (first 105 microm then 20 microm) Secretory cells of GTs were 600

retained on 20 microm mesh and carefully transferred into a tube containing 1 mL wash 601

buffer To remove residual monoterpenes the mixture was centrifuged for 5 min at 2000 602

x g (at 4degC) secretory cells were resuspended in 1 mL of wash buffer and cell clusters 603

were counted using a hemacytometer 604

Conversion of Radiolabeled Sucrose to Monoterpenes by Isolated Secretory Cells 606

Feeding experiments were performed based on previously developed protocols 608

(McCaskill et al 1992 McCaskill and Croteau 1995) with modifications In a 2 mL 609

screw-cap glass vial about 80000 isolated secretory cell octets were suspended in 610

reaction buffer containing [U-14C]sucrose (05 microCi per assay) unlabeled sucrose (002 611

10 or 10 mM) 2 mM AMP (to inhibit adenylate kinase) 05 mM ADP and 01 mM NADP+ 612

which was then overlaid with 500 microL hexane ether (1 1 vv) and sealed with a 613

teflon-lined cap The mixture was incubated for 4 h at 23 degC on an orbital shaker set to 614

200 rpm Combinations of other cofactors and inhibitors were tested as described in 615

Supplemental Methods and Data File 2 At the end of the experimental period vials were 616

vortexed (30 s at highest setting) and centrifuged for 2 min at 20000 x g A 250 microL aliquot 617

of the organic phase was transferred to 3 mL EcoScint (National Diagnostics Atlanta 618

Georgia USA) and counted with a Tri-Carb 2100 TR scintillation counter (PerkinElmer 619

Waltham MA USA) 620

ADH Activity Assays 622

Intact GTs on leaves and isolated secretory cells were stained for ADH activity by a 624

method originally developed for viability staining of pollen grains (Freeling 1976 Stinson 625

and Mascarenhas 1985) For whole leaf staining leaves were harvested submerged in 626

phosphate buffer (100 mM pH 73) and maintained at -20degC for 3 h Leaves were 627

allowed to thaw at room temperature and submerged in staining solution containing 628

sodium phosphate (90 mM pH 73) nitroblue tetrazolium chloride (027 mM) NAD+ (09 629

mM) and 10 (vv) ethanol The leaves were rocked gently in the staining solution for 4 630

h at 23degC washed with water and destained with 50 aq methanol and then 90 aq 631

methanol (30 min for each wash step) Isolated secretory cells were suspended in sodium 632

phosphate (90 mM pH 73) nitroblue tetrazolium chloride (027 mM) NAD+ (027 mM) 633

phenazine methosulfate (0027 mM) and 10 (vv) ethanol Cells were rocked gently in 634

the staining solution for 4 h at 23degC and washed with 90 methanol for 45 min and then 635

sodium phosphate buffer (100 mM pH 73) for 5 min The secretory cell suspension was 636

transferred to a hemacytometer and cell disks were counted under a DMLB fluorescent 637

microscope (Leica Microsystems Buffalo Grove IL USA) Images were taken with a 638

model 320 camera (SPOT Imaging Sterling Heights MI USA) 639

For specific activity measurements isolated secretory cells (obtained as described 640

above) were suspended in lysis buffer containing tris(hydroxymethyl)aminomethane (50 641

mM pH 80) sorbitol (10 vv) E-65 proteinase inhibitor (004 mM) pepstatin A (002 642

mgmL) 110-phenanthroline (20 mM) and 4-(2-aminoethyl)benzenesulfonyl fluoride (4 643

mM) Cells were disrupted in a glass homogenizer and the lysate centrifuged at 15000 x 644

g for 10 min (4degC) The supernatant was filtered through a 022 microm filter and desalted 645

using a Bio-Gel P6 column (Bio-Rad) The concentration of total protein was determined 646

(Bradford 1976) and ADH activity measured using the method of Mayer and Arnold 647

(2002) with modifications (an aliquot of the crude protein extract was boiled for 5 min to 648

serve as negative control) The protein extract (15 microg protein per microtiter plate well for 649

young leaves 3 microg well for mature leaves) was diluted with assay buffer to finally contain 650

tris(hydroxymethyl)aminomethane (50 mM pH 80) nitroblue tetrazolium chloride (018 651

mM) NAD+ (018 mM) phenazine methosulfate (0018 mM) and gelatin (008 wv) 652

The reaction was started by addition of 02 M ethanol and the absorption at 580 nm 653

(formation of NADH) measured over a period of 4 h (micro-Quant BioTek Instruments 654

Winooski VT USA) The slope of the absorption curve was compared with that of a 655

standard curve generated by adding known quantities of NADH directly to the assay 656

buffer (no cells) The specific activity was obtained by subtracting the absorbance 657

determined in assays performed without adding substrate 658

Cloning and Characterization of ADH from Peppermint 660

The sequence of the well-characterized ADH1 from potato (P14673 Matton et al 1990) 662

was used to query our peppermint transcriptome assembly Two contigs with contained 663

genes with high identity (~ 88 at the peptide sequence level) and the corresponding 664

cDNAs were amplified using Phusion DNA polymerase (New England Biolabs Ipswich 665

MA USA) with an annealing temperature of 60degC The primer sequences were 5rsquo- 666

TGGCTGATCAAACCAAACCAG-3rsquo (ADH-like gene forward 1) 667

5rsquo-TATGGCTGATCAAACCAAACCAG-3rsquo (ADH-like gene forward 2) 668

5rsquo-GATCCTCAGAACTTGATAATAACCTTGAC-3rsquo (ADH-like gene reverse 1) 669

5rsquo-CTCAGAACTTGATAATAACCTTGAC-3rsquo (ADH-like gene reverse 2) 670

5rsquo-TGTCGACTACTCATGGTCAAG-3rsquo (ADH1 gene forward 1) 671

5rsquo-TATGTCGACTACTCATGGTCAA -3rsquo (ADH1 gene forward 2) 672

5rsquo-GATCCTTACATTTTAACAACACAACGAAG-3rsquo (ADH1 gene reverse 1) 673

5rsquo-CTTACATTTTAACAACACAACGAAG-3rsquo (ADH1 gene reverse 2) The target vector 674

(pSBET Schenk et al 1995) was digested with NdeI and BamHI and then mixed with 675

PCR amplicons in a sticky-end ligation reaction The identity of constructs was confirmed 676

by sequencing Constructs were transformed into E coli BL21 cells for recombinant 677

protein production 678

A single colony was picked and grown in LB solution (5 ml) containing 50 microgml 679

kanamycin for 12 h The mixture was added to a larger Erlenmeyer flask containing 150 680

ml LB medium and cells were grown at 37ordmC to an optical density of 08 Transgene 681

expression was induced by addition of 01mM isopropyl β-D-1-thiogalactopyranoside and 682

the flask incubated at 16ordmC for 16 h The turbid broth was centrifuged in a Sorvall RC3C 683

centrifuge (ThermoFisher Waltham MA USA) at 5000 x g for 10 min Precipitated cells 684

cells were suspended in 50 mM 3-morpholino-2-hydroxypropanesulfonic acid buffer 685

(containing 10 (vv) glycerol and 1mM dithiothreitol MOPSO) and sonicated for three 686

cycles of 15 s each on ice The resulting suspension was centrifuged in a model 4515 D 687

microfuge (Eppendorf Hauppauge NY USA) at 13000 x g for 30 min The supernatant 688

was collected and loaded onto hydroxyapatite (Sigma-Aldrich St Louis MO USA) in 689

batch mode and incubated for 1 h After washing with MOPSO the sample was eluted 690

with 200 mM phosphate buffer To assess the purity of the preparation 15 ul of the eluent 691

were processed by SDS-PAGE and stained using Coomassie Brilliant Blue Following 692

destaining using a 20773 (vvv) mixture of methanolacetic acidwater protein bands 693

were detected by imaging (Alpha Innotech San Leandro CA USA) Activity assays 694

contained 10 microM purified enzyme and varying concentrations of ethanol The conversion 695

of NAD to NADH was used to monitor the progress of the reaction at 340 nm (Synergy H1 696

Hybrid Reader BioTek Winooski VT USA) The Origin 2017 software package 697

(OriginLab Northampton MA USA) was employed to fit data and deduce kinetic 698

constants 699

FNR and Fd Sequence Comparisons 701

M longifolia M arvensis and P frutescens mRNA was purified from isolated glandular 703

trichome tissue as described above For M longifolia and P frutescens strand-specific 704

paired-end libraries were prepared and sequenced using the HiSeq3000 platform 705

(Illumina San Diego CA USA) For M arvensis single-end libraries were sequenced by 706

the HiSeq2000 platform (Illumina San Diego CA USA) Sequences for other species 707

were obtained from the literature or from publicly accessible databases (details in 708

Supplemental Table 3) In some cases the literature data included information on gene 709

expression levels In other cases we assembled contigs and determined relative 710

expression levels To generate contigs reads were trimmed using Skewer (Jiang et al 711

2014) and assembled with either Trinity (Grabherr et al 2011) (in the case of Illumina 712

reads) or MIRA (Chevreux et al 2004) (in the case of 454 reads) Expression levels were 713

estimated as average read coverage in the case of MIRA assemblies or calculated using 714

Bowtie (Langmead et al 2009) and RSEM (Li et al 2010) in the case of Trinity 715

assemblies Sequences were aligned with Clustal Omega (Sievers et al 2011) (5rsquo-end 716

corresponding to plastidial targeting sequence removed) and maximum likelihood trees 717

generated using MEGA7 (Kumar et al 2016) with 1000 iterations of bootstrapping 718

Cloning of GT-Specific Isoforms of Peppermint Fd and FNR 720

GTs were isolated RNA was purified and first strand cDNA was generated as described 722

previously (Ahkami et al 2015) cDNAs were amplified using Phusion DNA polymerase 723

(New England Biolabs Ipswich MA USA) with an annealing temperature of 60degC The 724

primer sequences were 5rsquo-AGTGTACATATGATCAGAAAGTTCAGAAACTC-3rsquo (Fd 725

major GT isoform forward) 5rsquo- TCTATGGGATCCCACGATTTCGATTGTTCATCCC-3rsquo 726

(Fd major GT isoform reverse) 5rsquo- AGTGTACATATGGCAACTCCGTCTAAGAAGC-3rsquo 727

(Fd minor GT isoform forward) 5rsquo- 728

TCTATGGGATCCTTCCCTCATATCTAACAACAAGC-3rsquo (Fd minor GT isoform 729

reverse) 5rsquo- AGTGTACATAT GAGCATCGTTATTTTCCGAC-3rsquo (FNR GTroot isoform 730

forward) 5rsquo- TCTATGGGATCCGGGGTCTGTTTTAATAATTGC-3rsquo (FNR GTroot 731

isoform reverse) 5rsquo- AGTGTACATATGACTGCTGCTGTAAGTGCCGCA-3rsquo (FNR leaf 732

isoform forward) and 5rsquo-TCTATGGGATCCTCAGTACACTTCCACATTCCAC-3rsquo (FNR 733

leaf isoform reverse) Following digestion with NdeI and BamHI amplicons were ligated 734

into the pGem-T Easy vector (Promega Corp Madison WI USA) and the identity of 735

constructs was confirmed by sequencing 736

Simulating Protein-Protein Interactions in the Peppermint Glandular Trichome 738

FNR-Fd-HDR Complex 739

The sequences of peppermint Fd (minor GTroot isoform) Fd (major GT isoform) FNR 741

(GTroot isoform) and HDR were submitted to the I-TASSER server for protein structure 742

prediction (Yang et al 2015) A structural representation in Protein Data Bank (PDB) 743

format was returned and employed in structural alignments using the Chimera molecular 744

visualization program (Pettersen et al 2004) PDB files of the recently published crystal 745

structures of the root isoforms of maize Fd and FNR (Shinohara et al 2017) were 746

uploaded into Chimera A structural alignment was then performed by overlaying the 747

corresponding peppermint Fd and FNR isoforms Using the Swap function in Chimera 748

the sequences of the maize Fd and FNR isoforms were changed manually to reflect the 749

residues that are present in the appropriate peppermint Fd and FNR isoforms The 750

structures of the peppermint Fd and FNR isoforms obtained by this approach (excluding 751

iron-sulfur clusters and ligands) were energy-minimized in Chimera and the iron sulfur 752

clusters and ligands subsequently reintroduced by docking The same method was 753

employed to produce a peppermint HDR structure (following alignment to the published 754

Plasmodium HDR structure (Rekittke et al 2013)) The structure files for peppermint Fd 755

FNR and HDR were submitted to the GRAMM-X server (Tovchigrechko et al 2006) to 756

assess interactions in a three-protein complex (Fd-FNR as receptor HDR as ligand) The 757

GRAMM-X algorithm generated multiple docked confirmations The conformation that 758

most closely maintained the amino acid distances previously suggested to contribute to 759

protein-protein interaction surfaces (Rekittke et al 2013 Shinohara et al 2017) was 760

selected for structure-function studies 761

ACCESSESSION NUMBERS FOR cDNAs AND TRANSCRIPTOME DATA SETS 763

Mentha x piperita L Fd cDNA clone (major GT isoform) GenBank KY748231 764

Mentha x piperita L Fd cDNA clone (minor GTroot isoform) GenBank KY748232 765

Mentha x piperita L FNR cDNA clone (GTroot isoform) GenBank KY748233 766

Mentha x piperita L FNR cDNA clone (leaf isoform) GenBank KY748234 767

Mentha x piperita L HDR cDNA sequence GenBank KY888887 768

Mentha x piperita L ADH-like cDNA sequence GenBank MF579445 769

Mentha x piperita L ADH1 cDNA sequence GenBank MF579446 770

Perilla frutescens L GT transcriptome SRR4106089 771

Mentha arvensis L GT transcriptome SRR1271560 (young leaves) 772

Mentha arvensis L GT transcriptome SRR1271559 (mature leaves) 773

Mentha longifolia L (CMEN585 accession) GT transcriptome SRR3623199 774

ACKNOWLEDGMENTS 776

This research was supported by grants from the Division of Chemical Sciences 777

grant program (grant no T32GM083864 to SRJ) The authors would like to thank 780

Susan Vogtman for tending plants Kelly Vining (Oregon State University) for sharing data 781

prior to publication and Jeremy Jewell Amber Parrish and Glenn Turner for helpful 782

discussions and technical assistance 783

AUTHOR CONTRIBUTIONS 785

BML and SRJ designed the research SRJ IL and NS performed the 786

experiments BML SRJ IL and NS analyzed the data BML and SRJ wrote 787

the manuscript 788

789 790 791 792 793 794 795 796

LEGENDS FOR FIGURES 797

Fig 1 Reconstruction of metabolism in peppermint GTs A Leaf surfaces contain 799

abundant GTs Eight secretory cells (gray shading) arranged in a disk are responsible 800

for the biosynthesis of monoterpenoid essential oils which are then secreted into a 801

preformed cavity The major pathways operating in peppermint GTs based on 802

transcriptomic and metabolomic data are depicted B Monoterpenes sesquiterpenes 803

and polymethoxylated flavones are the most significant biomass outputs of GTs at 804

secretory stage and the values in this table were the basis for flux balance analysis using 805

the menpiGT_2015 metabolic reconstruction to determine the distribution of carbon flow 806

through the network of reactions 807

Fig 2 Relevance of oxidative phosphorylation for monoterpene formation in peppermint 809

GTs A Mapping of gene expression patterns (values are transcript per kilobase million in 810

secretory-phase GTs) related to enzyme complexes involved in oxidative 811

phosphorylation B Monoterpene production from sucrose by isolated secretory cells 812

White and gray bars are used to indicate values obtained with controls and 813

oligomycin-treated cell clusters respectively Values are mean plusmn standard deviation 814

(n=3) The asterisk indicates a P-value lt 005 for the difference between control and 815

treated samples based on a two-tailed t-test 816

Fig 3 Relevance of fermentation for monoterpene formation in peppermint GTs A 818

Mapping of gene expression patterns (values are transcript per kilobase million in 819

secretory-phase GTs) in the context of coupling the NADH to NAD+ conversion catalyzed 820

by ADH to the ATP-generating steps of glycolysis B MenpiGT_2015 model predicts 821

fermentative reactions to carry significant flux during the secretory phase of GTs C ADH 822

activity staining darkens GTs in secretory phase on intact leaves D ADH activity staining 823

darkens isolated secretory cells E Percentage of secretory cells labeled by ADH activity 824

staining correlates with the proportion of GTs at secretory phase F ADH activity in GTs 825

isolated from young and mature leaves Values are mean plusmn standard deviation (n=3) G 826

Purification of recombinant peppermint ADH1 from E coli H Michaelis-Menton plot for 827

the dependence of reaction velocity of peppermint ADH1 on the substrate concentration 828

H Lineweaver-Burke plot to determine the km and Vmax values for peppermint ADH1 829

graphically 830

Fig 4 Relevance of Fd and FNR for reductive steps in the biosynthesis of monoterpene 832

precursors in peppermint GTs (values are transcript per kilobase million in 833

secretory-phase GTs) 834

Fig 5 A unique FdFNR pair occurs in monoterpene-accumulating GTs A Maximum 836

likelihood tree of FNR isoforms B Maximum likelihood tree of Fd isoforms Comparisons 837

employ nucleotide sequences corresponding to the mature proteins (excluding plastidial 838

targeting sequences) Scale bars represent number of substitutions per site Species 839

abbreviations Aann (Artemisia annua L) Athal (Arabidopsis thaliana (L) Heynh) Csat 840

(Cannabis sativa L) Hlup (Humulus lupulus L) Maqu (Mentha acquatica L) Marv 841

(Mentha arvensis L) Mlon (Mentha longifolia L) Mpip (Mentha x piperita L) Mspi 842

(Mentha x spicata L) Obas (Ocimum basilicum L) Pfru (Perilla frutescens) (L) Britton) 843

Slyc (Solanum lycopersicum L) Acronyms GT glandular trichome L leaf isoform maj 844

major glandular trichome isoform min minor glandular trichome isoform R root isoform 845

Colors indicate isoforms present in leaves (green) roots (gray) glandular trichomes 846

(yellow) and in both roots and glandular trichomes (yellowgray) 847

Fig 6 Protein-protein interactions in the ternary complex FNRndashFdndashHDR complex of 849

peppermint glandular trichomes A Alignment of mature peptide sequences of Fd 850

isoforms present in peppermint glandular trichomes Residues that differ between the 851

major and minor isoform are highlighted in yellow B Docking conformation of the FNRndash852

FdndashHDR ternary complex C Ribbon diagram of the FNRndashFd interaction Residues that 853

differ between the major and minor Fd isoforms of glandular trichomes are highlighted in 854

red D Ribbon diagram of the FdndashHNR interaction Residues that differ between the 855

major and minor Fd isoforms of glandular trichomes are highlighted in red 856

SUPPLEMENTAL DATA 858

Supplemental Methods and Data File S1 Reconstructing mint glandular trichome 860

metabolism (all iterations of the menpiGT_2015 model) 861

Supplemental Methods and Data File S2 Establishing experimental conditions to test 863

the effects of in vivo ATP synthase inhibition on monoterpene formation in isolated 864

peppermint GTs 865

Supplemental Table S1 Reactions included in various iterations of the menpiGT_2015 867

model 868

Supplemental Table S2 Predicted outcomes of simulated reaction deletions 870

Supplemental Table S3 Predicted reaction outcomes of menpiGT_2015 model using 872

BC-SPOT and gene expression patterns to estimate flux distribution 873

Supplemental Table S4 Sequences and relative expression levels of genes coding for 875

FNR and Fd isoforms across Angiosperms that harbor GTs 876

Supplemental Fig S1 Alignment f alcohol dehydrogenase sequences The 878

SwissProt accession numbers are provided as unique identifiers (NCBI identifiers for 879

peppermint) 880

Table 1 Reactions returned as being essential for (-)-menthol production from raffinose 881 as determined by simulated reaction deletions 882

Reaction Description Pathway Compartment

1-Deoxy-D-xylulose 5-phosphate synthase MEP pathway plastid 1-Deoxy-D-xylulose 5-phosphate reductoisomerase MEP pathway plastid 4-Diphosphocytidyl-2C-methyl-D-erythritol synthase MEP pathway plastid 4-Diphosphocytidyl-2C-methyl-D-erythritol kinase MEP pathway plastid 2C-Methyl-D-erythritol 24-cyclodiphosphate synthase MEP pathway plastid 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase MEP pathway plastid 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate reductase MEP pathway plastid Geranyl diphosphate synthase Prenyl elongation plastid (-)-Limonene 3-hydroxylase Monoterpene modifications cytosol (-)-trans-Isopiperitenol dehydrogenase Monoterpene modifications mitochondrion (-)-trans-Isopiperitenone reductase Monoterpene modifications cytosol (+)-cis-Isopulegone isomerase Monoterpene modifications cytosol (+)-Pulegone reductase Monoterpene modifications cytosol (-)-Menthone(-)-menthol reductase Monoterpene modifications cytosol (-)-Limonene transport Monoterpene modifications plastid to cytosol (ER) (-)-trans-Isopiperitenol transport Monoterpene modifications cytosol to mitochondrion(-)-trans-Isopiperitenone transport Monoterpene modifications mitochondrion to cytosol(-)-Menthol export Monoterpene modifications cytosol to oil cavityProton diffusion O2CO2H2OH diffusion mitochondrion to cytosol

CO2 diffusion O2CO2H2OH diffusion cytosol to plastid

Water diffusion O2CO2H2OH diffusion cytosol to mitochondrion

O2 diffusion O2CO2H2OH diffusion cytosol to mitochondrion

O2 exchange O2CO2H2OH diffusion external to cytosol

CO2 exchange O2CO2H2OH diffusion cytosol to external

Raffinose import Raffinose catabolism external to cytosolUTPgalactose-1-phosphate uridylyltransferase Raffinose catabolism cytosol UDPgalactose 4-epimerase Raffinose catabolism cytosol Raffinose galactosidase Raffinose catabolism cytosol Galactokinase Raffinose catabolism cytosol CDP kinase CTP regeneration plastid CMP kinase CTP regeneration plastid Inorganic phosphatase CTP regeneration plastid Respiratory complex I Oxidative phosphorylation mitochondrion ATP synthase Oxidative phosphorylation mitochondrion Ferredoxin-NADP+ reductase Ferredoxin reduction plastid

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