Running title: Glandular trichomes development and ... · 4 84. Plant glandular trichomes: an interesting paradigm to study plant cell differentiation 85. To modulate the density

1

Running title: Glandular trichomes development and bioengineering 1

2

Corresponding author: 3

Charles Hachez 4

Institute of Life Sciences, Université catholique de Louvain, 5

Croix du sud 4-5, bte L7.07.14, 6

1348 Louvain-la-Neuve 7

Belgium 8

Email : [email protected] | Tel. + 32 (0)10 47 37 96 9

10

Update review 11

12

Plant glandular trichomes: natural cell factories of high biotechnological interest 13

Alexandre Huchelmann, Marc Boutry & Charles Hachez* Life Sciences Institute, Université 14

catholique de Louvain, Croix du Sud, 4-15 Box L7.07.14, 15

1348 Louvain-la-Neuve, Belgium 16

* Corresponding author 17

18

19

One-sentence summary: 20

Glandular trichomes: from developmental aspects to metabolic engineering approaches. 21

22

Authors’ contributions: 23

A.H., M.B. and C.H. drafted and wrote the article. 24

25

Funding information: 26

This work was partly supported by the ERA-CAP HIP project, the Belgian National Fund for 27

Scientific Research (MIS F.4522.17) and the Interuniversity Poles of Attraction Program 28

(Belgian State, Scientific, Technical and Cultural Services).29

Plant Physiology Preview. Published on July 19, 2017, as DOI:10.1104/pp.17.00727

Copyright 2017 by the American Society of Plant Biologists

www.plantphysiol.orgon January 31, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]://www.plantphysiol.org

2

Abstract: 30

31

Multicellular glandular trichomes are epidermal outgrowth characterized by the 32

presence of a head made of cells that have the ability to secrete or store large quantities of 33

specialized metabolites. Our understanding of the transcriptional control of glandular 34

trichome initiation and development is still in its infancy. This review points to some central 35

questions that need to be addressed to better understand how such specialized cell structures 36

arise from the plant protodermis. A key and unique feature of glandular trichomes is their 37

ability to synthesize and secrete large amounts, relative to their size, of a limited number of 38

metabolites. As such, they qualify as true cell factories, making them interesting targets for 39

metabolic engineering. In this review, recent advances regarding terpene metabolic 40

engineering are highlighted with a special focus on Nicotiana tabacum. In particular, the 41

choice of transcriptional promoters to drive transgene expression and the best ways to sink 42

existing pools of terpene precursors are discussed. Bioavailability of existing pools of natural 43

precursor molecules is a key parameter and is controlled by so-called “crosstalks” between 44

different biosynthetic pathways. As highlighted in this review, the exact nature and extent of 45

such crosstalks are only partially understood nowadays. In the future, awareness of, and 46

detailed knowledge on, the biology of plant glandular trichome development and metabolism 47

will generate new leads to tap the largely unexploited potential of glandular trichomes in plant 48

resistance to pests, and lead to the improved production of specialized metabolites with high 49

industrial or pharmacological value. 50

51


http://www.plantphysiol.org

3

Introduction 52

Trichomes, the epidermal outgrowths covering most of aerial plant tissues, are found 53

in a very large number of plant species, and are composed of single-cell or multicellular 54

structures. These structures are divided into two general categories: they can be glandular or 55

non-glandular depending on their morphology and secretion ability. Glandular trichomes can 56

be found on approximately 30% of all vascular plant species (Fahn 2000) and in a single plant 57

species several types of trichomes (both glandular and non-glandular) can be observed. 58

Glandular trichomes are characterized by the presence of cells that have the ability to secrete 59

or store large quantities of secondary (also called “specialized”) metabolites, which contribute 60

to increasing the plant fitness to the environment (see Box 1 for details). 61

Two main types of glandular trichomes stand out: peltate or capitate, which differ 62

according to their head size and stalk length. Capitate trichomes typically possess a stalk 63

whose length is more than half the head height, whereas peltate trichomes are defined as short 64

stalked (uni- or bicellular stalk) trichomes with a large secretory head made of four to 65

eighteen cells arranged in one or two concentric circles. Capitate trichomes are very variable 66

in their stalk cell number and length, glandular head morphology as well as secretion pattern, 67

and can be classified into various types (Glas et al. 2012). 68

A key and unique feature of glandular trichomes is their ability to synthesize and 69

secrete large amounts, relative to their size, of a limited number of specialized metabolites; 70

mainly terpenoids (Gershenzon et al. 1992; Gershenzon and Dudareva 2007), but also 71

phenylpropanoids (Gang et al. 2001; Deschamps et al. 2006; Xie et al. 2008), flavonoids 72

(Voirin et al. 1993; Tattini et al. 2000), methylketones (Fridman et al. 2005), and acyl sugars 73

(Kroumova and Wagner 2003; Schilmiller et al. 2010; Weinhold and Baldwin 2011). 74

Over the long term, the ability to modulate the density and productivity of such 75

secreting structures in plants would be of great biotechnological interest. This requires the 76

identification and characterization of the genes initiating, regulating and driving the 77

development of such glandular structures. Awareness of, and detailed knowledge on, the 78

biology of plant glandular trichome development and metabolism will generate new leads to 79

turn trichomes into biochemical factories using metabolic engineering approaches (Tissier 80

2012a), tap their largely unexploited potential in plant resistance to pests, and lead to the 81

improved production of important specialized metabolites (Lange and Turner 2013; Lange et 82

al. 2011). 83



4

Plant glandular trichomes: an interesting paradigm to study plant cell differentiation 84

To modulate the density of glandular trichomes in the epidermis or their productivity 85

for biotechnological purposes, a detailed understanding of the molecular genetic framework 86

governing their development and patterning in the plant epidermis would be beneficial. 87

Glandular trichomes have mostly been studied to decipher the biochemical pathways of the 88

compounds they produce and secrete (Champagne and Boutry 2013; Lange and Turner 2013) 89

and have thereby contributed to advancing our understanding of the secondary metabolism in 90

plants. 91

A detailed understanding of glandular trichome initiation and development is currently 92

missing 93

All multicellular organisms face the challenge of coordinating cell proliferation with 94

cell differentiation and patterning. Defects in this coordination can lead to incorrect tissue 95

formation, malformed organs, and cancerous growth. Glandular trichomes exemplify this 96

coordination challenge: they are elaborate, highly organized, and polarized cell structures 97

whose morphogenesis is modulated by an intricate array of molecular processes controlling 98

the different steps of their patterning on the leaf epidermis and subsequent differentiation. 99

Therefore, glandular trichomes can be used as a paradigm to tackle basic questions about the 100

development and differentiation of specialized multicellular secretory structures in plants. 101

A complete circuit for glandular trichome formation and patterning in the leaf 102

epidermis will require a sound understanding of their initiation process in the plant 103

protodermis and of the subsequent developmental steps leading to the formation of a polarized 104

and specialized multicellular structure, of the genes that regulate cell division, participate in 105

cell signaling, and promote specialized cell fate (Figure 1). It also requires an understanding 106

of how this developmental regulatory network affects cellular biological targets such as the 107

core cell cycle machinery. A particularly large gap in our current knowledge is the 108

identification of regulators of entry into the glandular trichome cell fate and of progression 109

through the pathway. 110

Research on non-glandular trichomes has been very fruitful in Arabidopsis thaliana. It 111

has generated a developmental framework for unicellular trichome formation and identified 112

over 30 genes involved in the initiation and development of non-glandular trichomes. We 113

chose not to review the development of trichomes in A. thaliana as this has been thoroughly 114

reviewed (Pattanaik et al. 2014; An et al. 2011; Tominaga-Wada et al. 2011; Balkunde et al. 115



5

2010; Matías-Hernández et al. 2016). Unlike the situation of A. thaliana, which contains a 116

single type of unicellular, non-glandular trichome, our understanding of the molecular genetic 117

aspects of glandular trichome development is still in its infancy but currently improving due 118

to recent progresses in -Omics and genome editing technologies as well as to a growing focus 119

from several research teams to characterize this process in different plant species (Liu et al. 120

2016; Bergau et al. 2015; Bosch et al. 2014; Li et al. 2004; Dai et al. 2010). The (draft) 121

genome sequences of a number of plant species with glandular trichomes are now available. A 122

non-exhaustive list includes different tomato species (Solanum lycopersicum and Solanum 123

pimpinellifolium (the tomato genome consortium, 2012), Solanum pennellii (Bolger et al. 124

2014)), potato (Solanum tuberosum (Xu et al. 2011)), cucumber (Cucumis sativus (Huang et 125

al. 2009)), tobacco (Nicotiana tabacum, (Sierro et al. 2014; Edwards et al., 2017)) and its 126

related species (Nicotiana sylvestris and Nicotiana tomentosiformis (Sierro et al., 2013)), hot 127

pepper (Capsicum annuum, (Kim et al., 2014)), mint (Mentha longifolia, (Vining et al., 128

2017)), Cannabis (Cannabis sativa, (van Bakel et al. 2011)) and hop (Humulus lupulus, 129

(Natsume et al. 2015)). 130

Since most of them can be genetically transformed, studying the molecular genetics of 131

trichome development in these species has become much easier (among others by using 132

genome editing or RNAi technologies). Particularly for species where genetics is poorly 133

developed (e.g. mint), the combination of transcriptomics at different stages of trichome 134

development (see below) and of genome editing or RNA interference technology should be a 135

powerful approach to address the function of candidate genes. 136

Trichome-specific data exist but cell stage-specific data are crucially needed to advance our 137

understanding of glandular trichome development 138

Extensive glandular trichome-specific EST resources were generated for a variety of 139

(non-model) plant species (Tissier 2012b; Dai et al. 2010; Akhtar et al. 2017; Chen et al. 140

2014; Jin et al. 2014; Soetaert et al. 2013; Trikka et al. 2015). Dedicated trichome-related 141

open access resources exist that help researchers mine this vast and increasing amount of 142

trichome-related data. One such resource is the TrichOME database (Dai et al. 2010) which 143

hosts functional -omics data, including transcriptomics (ESTs/unigene sequences) 144

metabolomics (mass spectrometry-based trichome metabolite profiles) as well as trichome-145

related genes curated from published literature from various species. These include members 146

of Lamiaceae (Mentha piperita, Salvia fruticosa, Cistus creticus, Ocimum basilicum), 147

Solanaceae (Nicotiana benthamiana, Nicotiana tabacum, Solanum habrochaites, Solanum 148



6

lycopersicum, and Solanum pennellii), Asteraceae (Artemisia annua), Fabaceae (Medicago 149

sativa, Medicago truncatula) and Cannabaceae (Cannabis sativa, Humulus lupulus). 150

These trichome-specific expression data are particularly useful to identify trichome-151

specific genes involved in particular biosynthetic pathways as well as in other trichome-152

related processes. A key question is how to extract the most significant data out of this 153

massive and growing amount of bulk information to advance our understanding of specific 154

aspects of glandular trichome biology (Tissier 2012a)? From a developmental perspective, 155

one of the main limitations of such resources is that most of these trichome-specific 156

expression data were derived from mature glandular trichomes (which are usually easier to 157

isolate than developing structures) or from a population of trichomes at mixed developmental 158

stages. Indeed, glandular trichome initiation and differentiation occur at different times in 159

different locations within a single leaf. Therefore, even very young leaves contain a trichome 160

population of mixed developmental stages (Bergau et al. 2015). Given the way most EST 161

resources were generated up to now, expression of genes playing a role in early 162

developmental steps may be, in the best case, underestimated or, in the worst case, even not 163

detected. This is particularly the case of genes active in the initiation phase (selection of 164

trichome initials) or early on in the development. 165

In addition to trichome-specific EST resources, numerous glandular trichome-specific 166

gene promoters have been reported in the literature for a variety of plants including (but not 167

restricted to): Antirrhinum majus, A. annua, Cucumis sativus, H. lupulus, Mentha sp., N. 168

tabacum, S. lycopersicum, S. habrochaites (Ennajdaoui et al. 2010; Wang et al. 2002; Sallaud 169

et al. 2012; Choi et al. 2012; Gutiérrez-Alcalá et al. 2005; Spyropoulou et al. 2014; Okada and 170

Ito 2001; Shangguan et al. 2008; Vining et al. 2017; Liu et al. 2006; Kim et al. 2008; Wang et 171

al. 2011; Wang et al. 2013; Laterre et al. 2017; Jaffe et al. 2007; Kortbeek et al. 2016); 172

thoroughly reviewed in (Tissier 2012b). It is worth noting that most of these trichome-specific 173

promoters are active in mature glandular trichomes (mostly in glandular cells at the tip of the 174

trichome). Their activity during early trichome development has been barely analyzed. 175

Molecular data pointing to genes playing a specific role in glandular trichome 176

development already exist, especially concerning some transcription factors, cell cycle 177

regulators as well as receptors involved in phytohormones-induced signalling cascades. 178

Several transcription factors belonging to different protein families and playing a role in 179

glandular trichome development have indeed been identified: AmMIXTA, a MYB 180

transcription factor from Antirrhinum majus whose ectopic expression in tobacco induces the 181

development of additional long glandular trichomes (Glover et al. 1998); GoPGF, a basic 182



7

helix-loop-helix (bHLH) transcription factor from Gossypium sp., acting as a positive 183

regulator of glandular trichome formation, its silencing leading to a completely glandless 184

phenotype (Ma et al. 2016); AaHD1, a homeodomain-leucine zipper transcription factor 185

required for jasmonate-mediated glandular trichome initiation in Artemisia annua (Yan et al. 186

2017); AtGIS, a C2H2 zinc-finger transcription factor from A. thaliana whose ectopic 187

expression in tobacco regulates glandular trichome development through gibberellic acid 188

signaling (Liu et al. 2017); AaMYB1, a MYB transcription factor from Artemisia annua 189

whose overexpression induces the formation of a greater number of trichomes (Matías-190

Hernández et al. 2017), CsGL3, a HD-Zip transcription factor whose mutation leads to a 191

glabrous phenotype in cucumber (Cui et al. 2016). 192

In tomato, several genes required for proper development and function of different 193

types of glandular trichomes have been reported. The woolly (Wo) gene, encoding a class IV 194

homeodomain-leucine zipper protein homolog to the A. thaliana GL2, and a B-type cyclin 195

gene, SlCycB2 (possibly regulated by Wo), control the initiation and development of type I 196

trichomes. Mutant alleles of Wo triggered a hairy phenotype due to the overproduction of type 197

I trichomes, while suppression of Wo or SlCycB2 expression by RNAi decreased their density 198

in tomato (Yang et al. 2011a; Yang et al. 2011b). Another mutation (hairless) affects the 199

SRA1 (Specifically Rac1-associated protein) subunit of the WAVE regulatory complex. 200

SRA1 controls the branching of actin filaments and is required for the normal development of 201

all trichome types of tomato, which suggests that proper actin-cytoskeleton dynamics is a 202

basal requirement for normal trichome morphogenesis (Kang et al. 2016). Mutations of some 203

enzymes involved in secondary metabolism also impact trichome density and/or metabolic 204

activity of glandular trichomes. For example, in the anthocyanin-free (af) mutant, loss of 205

function of the chalcone isomerase (SlCHI1) triggers a reduction of type VI trichome density 206

and metabolic output (Kang et al. 2014) while downregulation of DXS2, a MEP enzyme, 207

increases their density (Paetzold et al. 2010). The molecular mechanisms through which these 208

genes affect trichome density are currently unknown. 209

The tomato Wov allele was shown to promote abnormal multicellular trichome 210

differentiation when ectopically overexpressed in N. tabacum (Yang et al., 2015). This hints 211

at conserved transcriptional networks among Solanaceae species. However, the trichomes in 212

this Wov overexpressor line failed to develop glandular heads and appeared as rather 213

aggregated and undifferentiated structures. Whether a WD40-bHLH-MYB regulatory 214

mechanism similar to the one in A. thaliana also controls glandular trichome development in 215

Solanaceae is still unclear (Serna and Martin 2006; Yang et al. 2015). A recent RNA-seq 216



8

analysis of N. tabacum trichomes showed that orthologs of A. thaliana genes involved in 217

trichome formation via the WD40-bHLH-MYB regulatory mechanism (such as TTG1, GL2, 218

GL3, and several MYB transcription factors) are expressed in these structures (Yang et al. 219

2015). However, their transcriptional levels were not significantly altered in response to the 220

overexpression of a Wov transgene, which induced a clear trichome proliferation phenotype. 221

On the contrary, homologs of genes (Wo and SlCycB2) involved in trichome formation in 222

Asterids (Yang et al. 2011a; Yang et al. 2011b) were significantly upregulated in Wov 223

transgenic N. tabacum plants (Yang et al. 2015). 224

Differentiation of long-stalked glandular trichomes may be initiated and controlled in 225

N. tabacum by the activity of another MYB transcription factor. Indeed, ectopic expression of 226

MIXTA from Antirrhinum majus (Glover et al. 1998) or of its Gossypium hirsutum ortholog 227

CotMYBA (Payne et al. 1999) resulted in the development of excess long-stalked trichomes. It 228

is therefore likely that another unidentified N. tabacum MYB gene, an ortholog to AmMIXTA 229

and CotMYBA, plays a role in the development of long-stalked glandular trichomes in this 230

species. Whether this MYB gene needs to be part of a regulatory complex to initiate and 231

promote glandular trichome development is not yet known. 232

Five trichome-related mutants of the genes CsGL3, TRIL, MICT, TBH, and CsGL1, all 233

of them encoding homeodomain-leucine zipper transcription factors from different 234

subfamilies, have been reported in Cucumis sativus (Chen et al. 2014; Zhao et al. 2015; Cui et 235

al. 2016; Li et al. 2015; Pan et al. 2015; Wang et al. 2016). Based on the observed 236

phenotypes, a molecular mechanism underlying the development of multicellular trichomes in 237

this species has been proposed in a recent review (Liu et al. 2016) and seems to confirm that 238

the transcriptional control of multicellular trichome in C. sativus differs from the one 239

observed in A. thaliana. 240

The development of glandular trichome is tightly regulated by the integration of 241

diverse environmental and endogenous signals. In this respect, some phytohormones, 242

especially jasmonate (JA) and possibly gibberellins (GA) elicit glandular trichome 243

development via signaling cascades and the activation of trichome-specific transcriptional 244

regulators (Tian et al. 2014; Bosch et al. 2014; Koo and Howe 2009; Li et al. 2004; Liu et al. 245

2017; Bose et al. 2013; Yan et al. 2017). In Mentha arvensis, exogenous application of GA 246

resulted in a moderate increase in trichome density and diameter of the gland, suggesting a 247

positive, although moderate, effect of GA on trichome initiation and development in mint 248

(Bose et al. 2013). For example, reduced JA levels (through silencing of OPR3, a key enzyme 249

in the biosynthesis of the precursor of JA) led to impaired glandular trichome development in 250



9

tomato: the density of type VI trichomes was drastically reduced and their metabolite content 251

was different from that of the wild-type (Bosch et al. 2014). The JA receptor JASMONIC 252

ACID INSENSITIVE 1 (JAI1), the tomato ortholog of the ubiquitin ligase CORONATINE 253

INSENSITIVE1 in A. thaliana is involved in this JA-mediated signaling cascade (Li et al. 254

2004; Bosch et al. 2014; Katsir et al. 2008; Kang et al. 2010). It remains to be investigated 255

whether a synergistic effect between GA and JA signalling, similar to that observed in A. 256

thaliana (Qi et al. 2014) also promotes glandular trichome development. 257

Time course analysis of glandular trichome development coupled to cell type- and 258 stage-specific expression data as a way to advance our understanding of glandular 259 trichome development 260

The development of multicellular glandular trichomes proceeds through the 261

enlargement of single epidermal cells, followed by several cell divisions to generate a 262

structure perpendicular to the epidermal surface (see Figure 2) and specific types of glandular 263

trichomes seem to have a well-defined developmental plan (Tissier 2012a; Bergau et al. 264

2015). This highly regulated differentiation program also includes a polarized and localized 265

cell wall lysis and remodelling (Bergau et al. 2015). 266

Within a given plant species, it would be interesting to dissect the developmental 267

sequence of glandular trichome formation (including a time course analysis) and to sort the 268

cells at specific stages using marker assisted cell sorting. The difficulty resides in the specific 269

isolation of cells at early developmental stages. Recently, flow cytometry was used to 270

specifically separate young and mature type VI trichomes from the wild tomato species S. 271

habrochaites based on their distinct autofluorescence signals. This allowed the analysis of 272

their transcriptomic and metabolomic profile in a cell stage specific way (Bergau et al. 2016). 273

Such systematic dissection of the development of glandular trichomes seems very 274

promising but could be refined. Ideally, instead of autofluorescence (which may span various 275

developmental stages), a series of trichome-specific transcriptional promoters driving the 276

expression of a fluorescent marker (used as cell-stage marker) and closely associated to well 277

defined developmental stages should be used. Such genetic resources are not yet available in 278

the field of glandular trichome development. Focus should be set on identifying markers of 279

entry into the glandular trichome pathway, as well as those labelling subsequent early 280

differentiation steps. Some published gene promoters, like the one of AmMYBML3 (Jaffe et 281

al., 2007), are already known to specifically label developing trichomes and could be used to 282

drive the expression of a fluorescent reporter protein. Flow cytometry-assisted cell (or 283



10

nucleus) sorting would then permit to characterize the transcriptomic changes in a stage-284

specific way, in a similar manner as what was done to characterize stomatal development 285

(Adrian et al. 2015). In an iterative fashion, the data could be mined to identify additional 286

marker genes, so that only a few markers are necessary to initiate such transcriptomic studies. 287

As an alternative approach, ectopic overexpression of some transcription factors is known to 288

induce glandular trichome development (Yang et al., 2015, Payne et al., 1999). Inducible 289

overexpression of such transcription factors could be used in RNA-seq assays as a molecular 290

switch to identify genes acting during early developmental stages (Yang et al., 2015) that 291

could in turn be used as cell-stage markers. Further research is definitely needed to identify 292

key genes driving the post-embryonic development of glandular trichomes, and to generate a 293

molecular toolbox facilitating more applied genetic engineering approaches. 294

Glandular trichome development: a diversity of model species! 295

The field of glandular trichome development lacks a unique and robust model system. 296

This is partly due to the difficulty of finding an appropriate model system. Glandular 297

trichomes are extremely diverse in terms of shape, cell number, and type of secreted 298

compounds, and may not be the result of a single evolutionary event (Serna and Martin 2006). 299

This implies that their development may not be under similar transcriptional control in 300

different plant families, or even within a single plant species between different trichome types 301

(Serna and Martin 2006). The current view is that no single species can serve as a unique 302

model to study the biology of glandular trichomes, but certain species or families of species 303

progressively emerge as references for certain types of trichomes, such as Lamiaceae for 304

peltate trichomes or Solanaceae for capitate trichomes as suggested in Tissier (2012a). So far, 305

published data suggest that multicellular trichome formation probably occurs through 306

different transcriptional regulatory networks than those regulating trichome formation in A. 307

thaliana, so that mere orthologous relationships may not be inferred (Payne et al. 1999; Serna 308

and Martin 2006; Yang et al. 2015; Liu et al. 2016). 309

310

Turning glandular trichomes into chemical factories 311

Plant specialized metabolites have been used for centuries as a source for fragrances 312

and medicine. Since the discovery of their molecular structures and the elucidation of their 313

biosynthesis pathways, breeders and chemists have been trying to select the best compounds 314

by crossing species and varieties. For small molecules, chemical synthesis is another option 315



11

once the structure of the molecule has been determined. However, the size and complexity of 316

the stereochemistry of some plant metabolites make their chemical synthesis extremely 317

complicated, and expensive. 318

The rise of molecular genetics and a better understanding of the genomes has changed 319

the way breeders work: they now use molecular genetic screening approaches to help them 320

select the best breeding candidates and descendants. This speeds up the selection process and 321

optimizes the breeding program. 322

Among plant specialized metabolites, terpenoids are the most abundant in term of 323

quantity and diversity (reviewed in (Croteau et al. 2000; Bouvier et al. 2005; Gershenzon and 324

Dudareva 2007)). Some of them are renowned not only for their economic value but also for 325

their molecular complexity. 326

Metabolic engineering of terpenoids emerged as a new method to produce naturally 327

occurring products. Microorganisms have been heavily used for the heterologous expression 328

of plant metabolites (reviewed by (Kirby and Keasling 2009; Keasling 2010; Marienhagen 329

and Bott 2013)), and nowadays plants have also become a host of interest for the heterologous 330

or homologous production of some plant specialized metabolites (reviewed by (Aharoni et al. 331

2005; Dixon 2005; Wu and Chappell 2008)). 332

The biosynthesis of isoprenoids in plants is quite unique and many reviews have 333

already covered the different aspects of their production and regulation (Bouvier et al. 2005; 334

Hemmerlin et al. 2012; Lipko and Swiezewska 2016). Plants synthesize the common 335

precursor for isoprenoids, isopentenyl diphosphate (IPP), and its allylic isoform dimethylallyl 336

diphosphate (DMAPP), by two distinct and compartmentalized pathways, the cytosolic 337

mevalonate (MVA) pathway and the plastidial methylerythritol 4-phosphate (MEP) pathway 338

(Figure 3). Specialized terpenoids are generally synthesized by either the MEP or the MVA 339

pathway depending on their length: sesquiterpenes (C15) and triterpenes (C30) mainly derive 340

from the MVA pathway, while monoterpenes (C10) and diterpenes (C20) derive from the 341

MEP pathway. 342

Metabolic engineering in Nicotiana tabacum 343

N. tabacum is an interesting model system for metabolic engineering of terpenoid 344

compounds because it synthesizes an important pool of natural precursors (IPP/DMAPP) and, 345

besides the essential metabolites derived from the isoprenoid biosynthesis pathways, it 346

produces a very high amount of a limited range of specialized metabolites. These consist of a 347



12

few types of terpenoids, namely sesquiterpenes (Back and Chappell 1996; Starks et al. 1997, 348

Ralston et al.2001), as phytoalexins specifically produced in response to a pathogen attack 349

(Stoessl et al. 1976), and diterpenes, constitutively synthesized and secreted by the head of 350

glandular trichomes (Keene and Wagner 1985; Kandra and Wagner 1988; Guo and Wagner 351

1995; Wang and Wagner 2003) (Figure 3). Capsidiol, one of the main phytoalexin 352

sesquiterpenes produced in response to a fungal attack, and cembratriendiol, one of the main 353

diterpenes found in the cuticle, derive from the MVA and the MEP pathway, respectively 354

(Huchelmann et al. 2014). Both types of metabolites are not required for the growth and 355

development of the plants, and do not involve a complex metabolic pathway. Changing the 356

fate of the metabolic fluxes normally used for sesquiterpenes and diterpenes production looks, 357

in theory, quite simple. As the phytoalexin sesquiterpenes are only synthesized in response to 358

a pathogen attack, the main pool of terpenoid precursors available under normal conditions is 359

that involved in diterpene synthesis. 360

Terpenoids engineering with constitutive and ubiquist promoters 361

Metabolic engineering of tobacco plants to produce various terpenoids is widely 362

described, and reviews already covered the methods and the final metabolic profiles 363

(reviewed in (Verpoorte and Memelink 2002; Lange and Ahkami 2013; Moses et al. 2013)). 364

Most of the engineered metabolites were synthesized using the original precursor pools, 365

derived from the MEP pathway for mono- and diterpenes and from the MVA pathway for 366

sesqui- and triterpenes. Promoters used to drive expression of the transgenes were ubiquist 367

and constitutive. Although successful, the amount of metabolites produced in transgenic 368

tobacco lines is usually relatively low (in the range of a few ng.g-1

FW (Lücker et al. 2004; 369

Wei et al. 2004; Farhi et al. 2011)), compared to the endogenous production of capsidiol (up 370

to 100 µg.g-1

FW (Dokládal et al. 2012)), or of diterpenes (up to 75 µg.cm-2

depending on the 371

variety (Severson et al. 1984)). To be economically viable, engineering of terpenoids in plant 372

should reach yields comparable to those occurring naturally. 373

As mentioned previously, sesquiterpenes (C15) and triterpenes (C30) are considered to 374

derive from the (cytosolic) MVA pathway while monoterpenes (C10) and diterpenes (C20) are 375

derived from the (plastidial) MEP pathway. However, such a distinction is actually not so 376

strict given the existence of a crosstalk between the MVA and MEP pathways, which consists 377

of an exchange of prenyl diphosphates between the cytosol and the plastids (reviewed in 378

(Hemmerlin et al. 2012)). More and more metabolites have been shown not to derive from a 379



13

strictly cytosolic or plastidial pool of precursors but the exact way the crosstalk works is still 380

unclear (Box 2). As an example, sesquiterpenes can be synthesized using a plastidial pool of 381

IPP (Dudareva et al. 2005; Bartram et al. 2006). Monoterpenes and diterpenes can also have 382

mixed origins (Itoh et al. 2003; Wungsintaweekul and De-Eknamkul 2005; Hampel et al. 383

2007). 384

New strategies emerged for the engineering of terpenoids in plants, which consisted of 385

targeting the overexpressed enzymes to different subcellular localizations to take advantage 386

either of the MVA- or of the MEP-derived pool of precursors (Wu et al. 2006). Using such a 387

strategy, engineering of sesquiterpenes and monoterpenes was monitored in tobacco (Wu et 388

al. 2006). To produce sesquiterpenes (patchoulol or amorpha-4,11-diene), these authors 389

expressed in tobacco the corresponding terpene synthase and a farnesyl diphosphate synthase 390

(FDS) fused (or not) to a chloroplast targeting sequence to exploit either the MEP- or the 391

MVA-derived pool of precursors, respectively. In this pioneering study, expression of the 392

transgenes was driven by ubiquist promoters (a different one for each transgene). Addressing 393

the enzymes to the cytosol only led to a low yield of sesquiterpenes (a few ng.g-1

FW as in 394

previous studies), while addressing the enzyme to the plastids (to take advantage of the MEP-395

derived pool of IPP/DMAPP) led to a much higher yield (up to 25 µg.g-1

FW). Those 396

impressive results are also quite surprising as sesquiterpenes are naturally synthesized in the 397

cytosol while the engineered production is higher when the enzymes are localized in plastids. 398

Monoterpenes production in the cytosol was achieved using the same strategy to express the 399

monoterpene synthase and the geranyl diphosphate synthase. However, in this case, the 400

localisation of the enzymes seemed to be less important, as both engineering strategies (using 401

the MVA- or the MEP-derived precursors) led to roughly the same amount of R-(+)-limonene 402

(400-500 ng.g-1

FW) (Wu et al. 2006). 403

Modifying the fate of isoprenoid precursors can lead to severe phenotypes, which 404

might be expected given the importance of isoprenoids in plant growth and development, and 405

the fact that expression was ubiquist. Tobacco lines producing the highest quantity of 406

sesquiterpenes were indeed severely affected: they exhibited chlorosis and dwarfism. This 407

phenotype was also observed in metabolic engineering of the ginsenoside saponin in tobacco 408

(Gwak et al. 2017). In this case, production of the saponin led to a severe phenotype, 409

including dwarfism, change in flower and pollen morphology and impaired seed production 410

(Gwak et al. 2017). Similarly, dwarfism was observed upon metabolic engineering of tobacco 411

chloroplast to produce artemisinic acid (Saxena et al. 2014). All these approaches used 412

ubiquist promoters to drive expression of the transgenes. Because of the similarity of the 413



14

phenotypes observed (dwarfism, chlorosis, decreased seed production) between the different 414

reports, cytotoxicity of the new metabolites is probably not the only cause. The problem could 415

lay in the constitutive expression of transgenes, which might result in plant depletion of its 416

essential terpenoid precursors (IPP/DMAPP). For plant metabolic engineering to be efficient, 417

there is a necessity to better control the spatio-temporal expression of the transgenes. One 418

strategy to limit the effect of sinking essential IPP pools consists of specifically targeting the 419

expression of the genes in a cell type-specific way. In this respect, glandular trichomes are an 420

ideal expression system. 421

Terpenoids engineering specifically in trichomes 422

From a metabolic point of view, glandular trichomes are of particular interest, as they 423

are involved in the synthesis, storage, and/or excretion of specialized metabolites, making 424

these compounds easily available. The carbon metabolism of glandular trichome in 425

Solanaceae has evolved to support high metabolite production (Balcke et al. 2017). Some 426

genera, such as Nicotiana, produce up to 15% of their leaf biomass in the trichomes (Wagner 427

et al. 2004). The C20 terpenoids are exported to diffuse within the cuticle and mediate 428

resistance to insects and fungi (Chang and Grunwald 1976; Severson et al. 1984; Wang and 429

Wagner 2003; Sallaud et al. 2012). Rerouting the diterpenes production from cembrane and 430

labdane types (naturally produced in tobacco) to other diterpenes has been performed by 431

addressing the terpene synthase directly to the plastids with a trichome specific promoter. 432

433

Diterpenes engineering 434

One of the best examples is the heterologous production of taxadiene in trichomes of 435

wild tobacco, Nicotiana sylvestris (Rontein et al. 2008; Tissier et al. 2012). Taxadiene, 436

particularly taxa-4(5),11(12)-diene, is the precursor of Paclitaxel, a potent anti-cancer 437

diterpene usually extracted from the bark of Taxus species (Hezari et al. 1995; Koepp et al. 438

1995). Several groups have already tried to synthesize this precursor ubiquitously in plants 439

(reviewed in (Soliman and Tang 2015)), but the production of the diterpene triggered growth 440

defects in tomato (Kovacs et al. 2007), and a lethal phenotype in A. thaliana, most probably 441

because of an over-used GGPP pool for diterpene synthesis (Besumbes et al. 2004). As a 442

result, the MEP-derived primary metabolites were not synthesized in sufficient amounts to 443

sustain normal growth. Only few attempts led to the production of taxadiene in plants without 444



15

any defect like in ginseng (Cha et al. 2012) and Nicotiana benthamiana (Hasan et al. 2014) by 445

the ubiquitous overexpression of the taxadiene synthase, but the amount produced was quite 446

low. 447

Expression of taxadiene synthase specifically in N. tabacum trichomes, using the 448

transcriptional promoter of cembratrien-ol synthase (CBTS) led to the production in the 449

exudate of 5-10 µg/g fresh weight of taxadiene, representing only 10% of the total taxadiene 450

production, suggesting a problem of excretion (Tissier et al. 2012; further discussed in Box 3). 451

However, the amount produced is impressive compared to the 27 µg/g dry weight obtained in 452

the best stable transgenic N. benthamiana line (Hasan et al. 2014). Hasan et al. (2014) could 453

increase the production in N. benthamiana up to 48 µg/g dry weight by, in addition to 454

constitutively overexpressing the taxadiene synthase, silencing the expression of the gene 455

coding for the phytoene synthase, and thereby increasing the GGPP availability. In N. 456

tabacum, silencing the genes coding for CBTS did not increase the production of taxadiene 457

(Tissier et al. 2012). Compared to the cultivated tobacco (N. tabacum), the wild tobacco (N. 458

sylvestris) naturally synthesizes, as diterpenes, cembranoids but no labdanoids. Using the 459

CBTS promoter to drive the expression of the 8-hydroxy-copalyl diphosphate synthase and the 460

Z-abienol synthase specifically in N. sylvestris trichomes, synthesis of the labdanoid, Z-461

abienol, reached a yield of 30 µg.g-1

FW, without any major impact on the production of 462

cembranoids, nor on the plant development (Sallaud et al. 2012). Similarly, casbene, another 463

diterpene from Ricinus communis, was also produced in N. tabacum trichomes (Tissier et al. 464

2012), demonstrating that these structures can really be used as a biofactory for the production 465

of a diverse set of diterpenes. While most of the cembrane and labdane diterpenes are 466

normally found in the cuticle or in the exudate, only a small fraction (10%) of casbene or 467

taxadiene was found in the exudate, suggesting the necessity of associating a transporter when 468

engineering a metabolic pathway (Box 3). 469

Triterpenes engineering 470

Could N. tabacum trichomes be used as a production platform for terpenoids other 471

than diterpenes? In N. tabacum trichomes, the main isoprenoid-derived metabolites are 472

diterpenes from the MEP-pathway. Metabolic engineering in trichomes might take advantage 473

of this crosstalk to produce, for example, triterpenes instead of the naturally occurring 474

diterpenes by diverting the plastidic pool of IPP towards the cytosol to produce the 475

compounds of interest (Figure 4). Some plants, such as Solanum habrochaites, produce some 476



16

sesquiterpenes directly in the plastids. The corresponding enzymes, involved in the 477

biosynthesis of those C15, a sesquiterpene synthase and a farnesyl diphosphate synthase, 478

evolved to be localized to the plastids (Sallaud et al. 2009). Engineering the production of 479

MVA-derived metabolites such as sesquiterpenes (C15) or tritepernes (C30) in the plastids 480

using specifically MEP-derived IPP and DMAPP precursors may require the corresponding 481

enzymes (normally localized to the cytosol or to the ER) to be engineered to get targeted to 482

the plastids (Figure 5). 483

Triterpenes, the C30 family, are of particular interest for their substantial carbon 484

content, making them good targets for biofuel production (Gübitz et al. 1999; Khan et al. 485

2014) as well as for pharmacological purposes. The biosynthesis of triterpenes requires 486

several enzymatic steps (reviewed in (Phillips et al. 2006; Thimmappa et al. 2014)). The 487

production of squalene is the limiting regulatory step to produce triterpenes. Modifying the 488

production and fate of squalene, a key precursor of phytosterols, is risky if this modification 489

affects the whole plant as it can lead to dwarfism and loss of fertility (reviewed by (Clouse 490

2002; Schaller 2003, 2004)). Using the CBTS promoter, the production of squalene was 491

achieved in N. tabacum trichomes (Wu et al. 2012). The production was greater when the two 492

enzymes for squalene production, squalene synthase and farnesyl diphosphate synthase, were 493

targeted to the chloroplast. Even though the expression of the transgenes was expected to be 494

restricted to trichomes, those transgenic plants that expressed squalene at the highest level 495

displayed strong phenotypes such as dwarfism and chlorosis, which is similar to the 496

phenotypes observed upon ubiquist expression of transgenes impacting the pool of IPP 497

precursors (Wu et al. 2006; Saxena et al. 2014; Gwak et al. 2017). 498

Using the same approach, the production of linear triterpenes typical of the green algae 499

Botryococcus braunii was achieved in N. tabacum trichomes (Jiang et al. 2016). Once again, 500

the best production rate was when the enzymes were targeted to the plastid, which suggests 501

that the best strategy for triterpene production is to exploit the plastid pool of IPP/DMAPP. 502

However, as for squalene production (Wu et al. 2012), the synthesis of triterpenes led to 503

chlorosis and dwarfism. Plastidial membranes are quite fragile and the balance of sterols is 504

important for the stability of the plastids (Babiychuk et al. 2008). Thus, the production of 505

squalene or other triterpenes in the plastids might disrupt the chloroplast integrity and lead to 506

chlorosis. Since trichomes are thought to be dispensable, the breakdown of chloroplasts is not 507

expected to have major consequences on the whole plant. A possible explanation for this 508

phenotype is that the promoter was not completely trichome specific, probably due to the 509

presence of 35S enhancers upstream of the promoter (Wu et al. 2012; Jiang et al. 2016) or to 510



17

some positional effects of the transgene affecting the specificity of the promoter. Cell type 511

specificity of the transgene(s) expression should therefore be strictly ascertained in transgenic 512

lines, and not solely based on the assessment of cell-type specificity of the promoter via the 513

analysis of transcriptional reporter lines. Another plausible explanation to such phenotype 514

could be that, although trichomes are not essential, they may transmit a stress signal to other 515

leaf tissues leading to the observed phenotypes in the transgenic lines (Wu et al. 2012). 516

Metabolic engineering in tobacco has great potential. The amount of terpenoids that 517

the plant can naturally produce is impressive. Using the plastidial pool for production of 518

terpenoids seems much more efficient than using the cytosolic one (Wu et al. 2006). 519

However, ubiquist expression alters plant development and directly impairs the yield. To be 520

economically viable, the yield of terpenoids should be increased with no or only a slight 521

impact on the plant phenotype. Lack of excretion of the potentially cytotoxic metabolites is 522

another issue (discussed in Box 3). In addition, comprehension of the exchange of prenyl 523

diphosphates between the plastids and the cytosol (Box 2) should also be investigated so as to 524

reroute the plastidic flux towards the cytosol, and avoid disrupting the stability of the 525

chloroplast with a change in sterol profile (Babiychuk et al. 2008). 526

Conclusion 527

Nowadays, plant biologists are trying to go beyond the A. thaliana model and to move 528

to non-model plant species. In this respect, the study of glandular trichome biology is greatly 529

benefitting from such change of model system. Recent advances in DNA sequencing, –omics 530

technology, and reverse genetics, including plant genome editing now offer new technical 531

resources to investigate such biological aspects in a wide variety of species. 532

Our current understanding of both the development of glandular trichomes and of the 533

biosynthetic pathways going on in these structures is improving but at this point is still quite 534

fragmentary. However, an increasing number of research groups are now focusing on various 535

aspects of glandular trichome biology including developmental aspects and bioengineering. 536

Understanding the way glandular trichomes develop to finally turn into highly 537

efficient biochemical factories in the epidermis of non-model plant species is of key 538

importance and could lead to more applied outcomes. This calls for basic research to address 539

those fascinating aspects. It is now time to consider the real biotechnological potential of 540

glandular trichomes as biochemical factories, and use up-to-date technology to fully exploit 541

the cellular machinery. Increased knowledge on these fundamental aspects will in the mid-542

term allow researchers to tap the up-to-now largely unexploited biotechnological potential of 543



18

glandular trichomes to engineer plants that would exhibit increased resistance to pests or that 544

would produce compounds of immense industrial/pharmaceutical interest (molecular 545

pharming). 546

547

Acknowledgments: 548

We wish to thank Dr. Antoine Champagne (Université catholique de Louvain) for the 549

scanning electron micrographs of N. tabacum glandular trichomes. 550

551

Figure legends: 552

Figure 1: Glandular trichome initiation and development, a process with many 553

unknowns. 554

A differentiating protodermal cell integrates both environmental and endogenous signals. 555

Such signal integration results in the selection of a pool of trichome cell precursors which will 556

initiate a specific developmental program. In these trichome initials, cell-specific 557

transcriptional control of gene expression and cell cycle regulation results in the onset of a 558

controlled cell division and trichome morphogenesis program, most of which is still not so 559

well understood in the case of glandular trichomes. It probably also involves some cell-cell 560

signaling promoting the “one cell-spacing rule” which allows a specific patterning of 561

trichomes in the epidermis. Morphogenesis of the trichome glandular head also necessitates 562

extensive remodeling of the cell wall. The extent of endoreduplication in glandular trichomes 563

is still mostly uncharacterized. The illustration shows a modified confocal picture of a long 564

glandular trichome initial from Nicotiana tabacum. Chloroplasts are shown in green, 565

propidium iodide-stained cell walls in magenta, and nuclei in cyan. 566

567

Figure 2: Glandular trichome initiation and development in Nicotiana tabacum. 568

A-F/ Confocal microscopy pictures showing the early steps of glandular trichome 569

development. The number of cells forming the developing glandular trichome is shown at the 570

bottom of each frame. A differentiating protodermal cell enlarges and forms a protuberance 571

(A), the cell nucleus migrates to the tip of the protuberance (B), cell division takes place (C) 572

forming a structure made of two cells (D). The upper cell protruding from the epidermis then 573

undergoes an asymmetric division forming one large cell (which will form the multicellular 574

stalk after several rounds of controlled cell division) and one small cell (which will give rise 575

to the multicellular glandular head) (E). A developing trichome made of five cells is shown in 576



19

(F). Magenta: cell wall (propodium iodide staining), Cyan: nuclei (DAPI staining), Green: 577

chloroplasts (Chlorophyll a autofluorescence). 578

G/ Scanning electron micrograph showing the typical cell architecture of a mature long 579

glandular trichome. 580

581

Figure 3: Isoprenoid metabolism in Nicotiana tabacum cells. 582

The red square represents the major MEP isoprenoid metabolism in plastids of developed 583

trichomes. Phytohormones are indicated in blue and specialized metabolites in violet. MEP 584

pathway, methylerythritol phosphate pathway; MVA pathway, mevalonate pathway; IPP, 585

isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; 586

FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; ABA, abscisic acid. 587

588

Figure 4: Terpenoid metabolism in engineered tobacco trichomes for triterpene 589

production in the cytosol. 590

The gray pathway represents the normal biosynthetic route for triterpenes and sterols. The 591

enzymes are targeted to the cytosol to enhance the crosstalk and sink the plastids from its 592

precursors. Overexpressed enzymes are denoted in dark blue. New products deriving from the 593

engineering metabolism are denoted in light blue. 594

MEP pathway, methylerythritol phosphate pathway; MVA pathway, mevalonate pathway; 595

IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; 596

FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; FDS, FPP synthase; SQS, 597

squalene synthase; SQE, squalene epoxidase; OSC, 2,3-oxidosqualene cyclase; CYP450, 598

cytochrome P450 599

600

Figure 5: Terpenoid metabolism in engineered tobacco trichomes for triterpene 601

production in plastids. 602

The gray pathway represents the normal biosynthetic route for triterpenes and sterols. The 603

enzymes are targeted to directly produce the triterpenes in the plastids. Overexpressed 604

enzymes are denoted in dark blue. New products deriving from the engineering metabolism 605

are denoted in light blue. Sinking plastidial isoprenoid pool might provoke undesirable 606

consequences. Potentially affected metabolites are denoted in orange. 607

MEP pathway, methylerythritol phosphate pathway; MVA pathway, mevalonate pathway; 608

IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; 609

FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; FDS, FPP synthase; SQS, 610



20

squalene synthase; SQE, squalene epoxidase; OSC, 2,3-oxidosqualene cyclase; CYP450, 611

cytochrome P450; ABA, abscisic acid. 612

613

Bibliography 614

Adam K-P, Thiel R, Zapp J (1999) Incorporation of 1-[1-13C]Deoxy-d-xylulose in 615

Chamomile Sesquiterpenes. Archives of Biochemistry and Biophysics 369 (1):127-616

132. doi:http://dx.doi.org/10.1006/abbi.1999.1346 617

Adebesin F, Widhalm JR, Boachon B, Lefevre F, Pierman B, Lynch JH, Alam I, Junqueira B, 618

Benke R, Ray S, Porter JA, Yanagisawa M, Wetzstein HY, Morgan JA, Boutry M, 619

Schuurink RC, Dudareva N (2017) Emission of volatile organic compounds from 620

petunia flowers is facilitated by an ABC transporter. Science 356 (6345):1386-1388. 621

doi:10.1126/science.aan0826 622

Adrian J., Chang J. , Ballenger C.E., Bargmann B, Alassimone J., Davies K.A., Lau OS, 623

Matos J., Hachez C., Lanctot E., Vatén A., Birnbaum K, D.C. B (2015) Transcriptome 624

dynamics of the stomatal lineage: birth, amplification and termination of a self-625

renewing population. Developmental Cell In Press 626

Akhtar MQ, Qamar N, Yadav P, Kulkarni P, Kumar A, Shasany AK (2017) Comparative 627

glandular trichome transcriptome-based gene characterization reveals reasons for 628

differential (-)-menthol biosynthesis in Mentha species. Physiologia plantarum 160 629

(2):128-141. doi:10.1111/ppl.12550 630

Aharoni A, Jongsma MA, Bouwmeester HJ (2005) Volatile science? Metabolic engineering 631

of terpenoids in plants. Trends in Plant Science 10 (12):594-602. 632

doi:http://dx.doi.org/10.1016/j.tplants.2005.10.005 633

An L, Zhou Z, Yan A, Gan Y (2011) Progress on trichome development regulated by 634

phytohormone signaling. Plant signaling & behavior 6 (12):1959-1962 635

Arigoni D, Sagner S, Latzel C, Eisenreich W, Bacher A, Zenk MH (1997) Terpenoid 636

biosynthesis from 1-deoxy-d-xylulose in higher plants by intramolecular 637

skeletal rearrangement. Proceedings of the National Academy of Sciences of the 638

United States of America 94 (20):10600-10605 639

Aschenbrenner A-K, Amrehn E, Bechtel L, Spring O (2015) Trichome differentiation on leaf 640

primordia of Helianthus annuus (Asteraceae): morphology, gene expression and 641

metabolite profile. Planta 241 (4):837-846. doi:10.1007/s00425-014-2223-y 642

Babiychuk E, Bouvier-Navé P, Compagnon V, Suzuki M, Muranaka T, Van Montagu M, 643

Kushnir S, Schaller H (2008) Allelic mutant series reveal distinct functions for 644

Arabidopsis cycloartenol synthase 1 in cell viability and plastid biogenesis. 645

Proceedings of the National Academy of Sciences 105 (8):3163-3168. 646

doi:10.1073/pnas.0712190105 647

Back K, Chappell J (1996) Identifying functional domains within terpene cyclases using a 648

domain-swapping strategy. Proceedings of the National Academy of Sciences 93 649

(13):6841-6845 650

Balcke G, Bennewitz S, Bergau N, Athmer B, Henning A, Majovsky P, Jimenez-Gomez JM, 651

Hoehenwarter W, Tissier AF (2017) Multiomics of tomato glandular trichomes reveals 652

distinct features of central carbon metabolism supporting high productivity of 653

specialized metabolites. The Plant cell. doi:10.1105/tpc.17.00060 654

Balkunde R, Pesch M, Hulskamp M (2010) Trichome patterning in Arabidopsis thaliana from 655

genetic to molecular models. Curr Top Dev Biol 91:299-321. doi:10.1016/S0070-656

2153(10)91010-7 657


http://dx.doi.org/10.1006/abbi.1999.1346http://dx.doi.org/10.1016/j.tplants.2005.10.005http://www.plantphysiol.org

21

Bartram S, Jux A, Gleixner G, Boland W (2006) Dynamic pathway allocation in early 658

terpenoid biosynthesis of stress-induced lima bean leaves. Phytochemistry 67 659

(15):1661-1672 660

Bergau N, Bennewitz S, Syrowatka F, Hause G, Tissier A (2015) The development of type VI 661

glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild 662

species S. habrochaites. BMC plant biology 15:289. doi:10.1186/s12870-015-0678-z 663

Bergau N, Navarette Santos A, Henning A, Balcke GU, Tissier A (2016) Autofluorescence as 664

a Signal to Sort Developing Glandular Trichomes by Flow Cytometry. Front Plant Sci 665

7:949. doi:10.3389/fpls.2016.00949 666

Besumbes Ó, Sauret‐Güeto S, Phillips MA, Imperial S, Rodríguez‐Concepción M, Boronat A 667 (2004) Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the 668

production of taxadiene, the first committed precursor of Taxol. Biotechnology and 669

bioengineering 88 (2):168-175 670

Bick JA, Lange BM (2003) Metabolic cross talk between cytosolic and plastidial pathways of 671

isoprenoid biosynthesis: unidirectional transport of intermediates across the 672

chloroplast envelope membrane. Archives of Biochemistry and Biophysics 415 673

(2):146-154. doi:http://dx.doi.org/10.1016/S0003-9861(03)00233-9 674

Bleeker PM, Mirabella R, Diergaarde PJ, VanDoorn A, Tissier A, Kant MR, Prins M, de Vos 675

M, Haring MA, Schuurink RC (2012) Improved herbivore resistance in cultivated 676

tomato with the sesquiterpene biosynthetic pathway from a wild relative. Proceedings 677

of the National Academy of Sciences of the United States of America 109 (49):20124-678

20129. doi:10.1073/pnas.1208756109 679

Bolger A, Scossa F, Bolger ME, Lanz C, Maumus F, Tohge T, Quesneville H, Alseekh S, 680

Sorensen I, Lichtenstein G, Fich EA, Conte M, Keller H, Schneeberger K, Schwacke 681

R, Ofner I, Vrebalov J, Xu Y, Osorio S, Aflitos SA, Schijlen E, Jimenez-Gomez JM, 682

Ryngajllo M, Kimura S, Kumar R, Koenig D, Headland LR, Maloof JN, Sinha N, van 683

Ham RCHJ, Lankhorst RK, Mao L, Vogel A, Arsova B, Panstruga R, Fei Z, Rose 684

JKC, Zamir D, Carrari F, Giovannoni JJ, Weigel D, Usadel B, Fernie AR (2014) The 685

genome of the stress-tolerant wild tomato species Solanum pennellii. Nat Genet 46 686

(9):1034-1038. doi:10.1038/ng.3046 687

http://www.nature.com/ng/journal/v46/n9/abs/ng.3046.html - supplementary-information 688 Bombo AB, Appezzato-da-Glória B, Aschenbrenner AK, Spring O (2016) Capitate glandular 689

trichomes in Aldama discolor (Heliantheae – Asteraceae): morphology, metabolite 690

profile and sesquiterpene biosynthesis. Plant Biology 18 (3):455-462. 691

doi:10.1111/plb.12423 692

Bosch M, Wright LP, Gershenzon J, Wasternack C, Hause B, Schaller A, Stintzi A (2014) 693

Jasmonic Acid and Its Precursor 12-Oxophytodienoic Acid Control Different Aspects 694

of Constitutive and Induced Herbivore Defenses in Tomato. Plant Physiology 166 695

(1):396-410. doi:10.1104/pp.114.237388 696

Bose SK, Yadav RK, Mishra S, Sangwan RS, Singh AK, Mishra B, Srivastava AK, Sangwan 697

NS (2013) Effect of gibberellic acid and calliterpenone on plant growth attributes, 698

trichomes, essential oil biosynthesis and pathway gene expression in differential 699

manner in Mentha arvensis L. Plant Physiology and Biochemistry 66:150-158. 700

doi:http://doi.org/10.1016/j.plaphy.2013.02.011 701

Bouvier F, Rahier A, Camara B (2005) Biogenesis, molecular regulation and function of plant 702

isoprenoids. Progress in lipid research 44 (6):357-429 703

Cha M-J, Shim S-H, Kim S-H, Kim O-T, Lee S-W, Kwon S-Y, Baek K-H (2012) Production 704

of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene. 705

BMB reports 45 (10):589-594 706


http://dx.doi.org/10.1016/S0003-9861(03)00233-9http://www.nature.com/ng/journal/v46/n9/abs/ng.3046.html#supplementary-informationhttp://doi.org/10.1016/j.plaphy.2013.02.011http://www.plantphysiol.org

22

Champagne A, Boutry M (2013) Proteomics of nonmodel plant species. Proteomics 13 (3-707

4):663-673. doi:10.1002/pmic.201200312 708

Chang S, Grunwald C (1976) Duvatrienediols in cuticular wax of Burley tobacco leaves. 709

Journal of lipid research 17 (1):7-11 710

Chen C, Liu M, Jiang L, Liu X, Zhao J, Yan S, Yang S, Ren H, Liu R, Zhang X (2014) 711

Transcriptome profiling reveals roles of meristem regulators and polarity genes during 712

fruit trichome development in cucumber (Cucumis sativus L.). J Exp Bot 65 713

(17):4943-4958. doi:10.1093/jxb/eru258 714

Choi YE, Lim S, Kim H-J, Han JY, Lee M-H, Yang Y, Kim J-A, Kim Y-S (2012) Tobacco 715

NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from 716

glandular trichomes. The Plant Journal 70 (3):480-491. doi:10.1111/j.1365-717

313X.2011.04886.x 718

Clouse SD (2002) Arabidopsis Mutants Reveal Multiple Roles for Sterols in Plant 719

Development. The Plant cell 14 (9):1995-2000. doi:10.1105/tpc.140930 720

Croteau R, Kutchan TM, Lewis NG (2000) Natural products (secondary metabolites). 721

Biochemistry and molecular biology of plants 24:1250-1319 722

Cui J-Y, Miao H, Ding L-H, Wehner TC, Liu P-N, Wang Y, Zhang S-P, Gu X-F (2016) A 723

New Glabrous Gene (csgl3) Identified in Trichome Development in Cucumber 724

(Cucumis sativus L.). PLoS ONE 11 (2):e0148422. doi:10.1371/journal.pone.0148422 725

Dai X, Wang G, Yang DS, Tang Y, Broun P, Marks MD, Sumner LW, Dixon RA, Zhao PX 726

(2010) TrichOME: a comparative omics database for plant trichomes. Plant Physiol 727

152 (1):44-54. doi:10.1104/pp.109.145813 728

Deschamps C, Gang D, Dudareva N, Simon JE (2006) Developmental regulation of 729

phenylpropanoid biosynthesis in leaves and glandular trichomes of basil (Ocimum 730

basilicum L.). International Journal of Plant Sciences 167 (3):447-454 731

Dixon RA (2005) Engineering of plant natural product pathways. Current Opinion in Plant 732

Biology 8 (3):329-336. doi:http://dx.doi.org/10.1016/j.pbi.2005.03.008 733

Dokládal L, Obořil M, Stejskal K, Zdráhal Z, Ptáčková N, Chaloupková R, Damborský J, 734

Kašparovský T, Jeandroz S, Žd'árská M, Lochman J (2012) Physiological and 735

proteomic approaches to evaluate the role of sterol binding in elicitin-induced 736

resistance. Journal of Experimental Botany 63 (5):2203-2215. doi:10.1093/jxb/err427 737

Dudareva N, Andersson S, Orlova I, Gatto N, Reichelt M, Rhodes D, Boland W, Gershenzon 738

J (2005) The nonmevalonate pathway supports both monoterpene and sesquiterpene 739

formation in snapdragon flowers. Proceedings of the National Academy of Sciences of 740

the United States of America 102 (3):933-938 741

Edwards KD, Fernandez-Pozo N, Drake-Stowe K, Humphry M, Evans AD, Bombarely A, 742

Allen F, Hurst R, White B, Kernodle SP, Bromley JR, Sanchez-Tamburrino JP, Lewis 743

RS, Mueller LA (2017) A reference genome for Nicotiana tabacum enables map-based 744

cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC 745

genomics 18 (1):448. doi:10.1186/s12864-017-3791-6 746

Elliger CA, Benson ME, Haddon WF, Lundin RE, Waiss AC, Wong RY (1988) 747

Petuniasterones, novel ergostane-type steroids of Petunia hybridia Vilm. (Solanaceae) 748

having insect-inhibitory activity. X-Ray molecular structure of the 22,24,25-749

[(methoxycarbonyl)orthoacetate] of 7[small alpha],22,24,25-tetrahydroxy ergosta-1,4-750

dien-3-one and of 1[small alpha]-acetoxy-24,25-epoxy-7[small alpha]-hydroxy-22-751

(methylthiocarbonyl)acetoxyergost-4-en-3-one. Journal of the Chemical Society, 752

Perkin Transactions 1 (3):711-717. doi:10.1039/P19880000711 753

Elliger CA, Wong RY, Waiss AC, Benson M (1990) Petuniolides. Unusual ergostanoid 754

lactones from Petunia species that inhibit insect development. Journal of the Chemical 755

Society, Perkin Transactions 1 (3):525-531. doi:10.1039/P19900000525 756


http://dx.doi.org/10.1016/j.pbi.2005.03.008http://www.plantphysiol.org

23

Ennajdaoui H, Vachon G, Giacalone C, Besse I, Sallaud C, Herzog M, Tissier A (2010) 757

Trichome specific expression of the tobacco (Nicotiana sylvestris) cembratrien-ol 758

synthase genes is controlled by both activating and repressing cis-regions. Plant 759

molecular biology 73 (6):673-685 760

Fahn A (2000) Structure and function of secretory cells. Hallahan DL, Gray JC, Callow JA, 761

eds Advances in botanical research incorporating advances in plant pathology, volume 762

31: plant trichomes London: Academic Press 763

Farhi M, Marhevka E, Ben-Ari J, Algamas-Dimantov A, Liang Z, Zeevi V, Edelbaum O, 764

Spitzer-Rimon B, Abeliovich H, Schwartz B, Tzfira T, Vainstein A (2011) Generation 765

of the potent anti-malarial drug artemisinin in tobacco. Nat Biotechnol 29 (12):1072-766

1074. doi:10.1038/nbt.2054 767

Flügge UI, Gao W (2005) Transport of Isoprenoid Intermediates Across Chloroplast Envelope 768

Membranes. Plant Biology 7 (1):91-97. doi:10.1055/s-2004-830446 769

Fridman E, Wang J, Iijima Y, Froehlich JE, Gang DR, Ohlrogge J, Pichersky E (2005) 770

Metabolic, genomic, and biochemical analyses of glandular trichomes from the wild 771

tomato species Lycopersicon hirsutum identify a key enzyme in the biosynthesis of 772

methylketones. The Plant cell 17 (4):1252-1267 773

Fu X, Shi P, He Q, Shen Q, Tang Y, Pan Q, Ma Y, Yan T, Chen M, Hao X, Liu P, Li L, 774

Wang Y, Sun X, Tang K (2017) AaPDR3, a PDR Transporter 3, Is Involved in 775

Sesquiterpene beta-Caryophyllene Transport in Artemisia annua. Front Plant Sci 776

8:723. doi:10.3389/fpls.2017.00723 777

Gang DR, Wang J, Dudareva N, Nam KH, Simon JE, Lewinsohn E, Pichersky E (2001) An 778

investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant 779

Physiology 125 (2):539-555 780

Gerber E, Hemmerlin A, Hartmann M, Heintz D, Hartmann M-A, Mutterer J, Rodríguez-781

Concepción M, Boronat A, Van Dorsselaer A, Rohmer M, Crowell DN, Bach TJ 782

(2009) The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the 783

Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells. The Plant 784

cell 21 (1):285-300. doi:10.1105/tpc.108.063248 785

Gershenzon J, Dudareva N (2007) The function of terpene natural products in the natural 786

world. Nature chemical biology 3 (7):408-414 787

Gershenzon J, McCaskill D, Rajaonarivony JI, Mihaliak C, Karp F, Croteau R (1992) 788

Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic 789

studies of monoterpenes and other gland products. Analytical biochemistry 200 790

(1):130-138 791

Glas JJ, Schimmel BC, Alba JM, Escobar-Bravo R, Schuurink RC, Kant MR (2012) Plant 792

glandular trichomes as targets for breeding or engineering of resistance to herbivores. 793

Int J Mol Sci 13 (12):17077-17103. doi:10.3390/ijms131217077 794

Glover BJ, Perez-Rodriguez M, Martin C (1998) Development of several epidermal cell types 795

can be specified by the same MYB-related plant transcription factor. Development 796

125 (17):3497-3508 797

Gübitz GM, Mittelbach M, Trabi M (1999) Exploitation of the tropical oil seed plant Jatropha 798

curcas L. Bioresource technology 67 (1):73-82 799

Guo Z, Wagner GJ (1995) Biosynthesis of labdenediol and sclareol in cell-free extracts from 800

trichomes of Nicotiana glutinosa. Planta 197 (4):627-632 801

Gutensohn M, Nguyen TT, McMahon RD, Kaplan I, Pichersky E, Dudareva N (2014) 802

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction 803

of the non-canonical substrate neryl diphosphate. Metabolic engineering 24:107-116 804

Gutiérrez-Alcalá G, Calo L, Gros F, Caissard J-C, Gotor C, Romero LC (2005) A versatile 805

promoter for the expression of proteins in glandular and non-glandular trichomes from 806



24

a variety of plants. Journal of Experimental Botany 56 (419):2487-2494. 807

doi:10.1093/jxb/eri241 808

Gwak YS, Han JY, Adhikari PB, Ahn CH, Choi YE (2017) Heterologous production of a 809

ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs 810

the vegetative and reproductive growth. Planta:1-15. doi:10.1007/s00425-017-2668-x 811

Hampel D, Swatski A, Mosandl A, Wüst M (2007) Biosynthesis of Monoterpenes and 812

Norisoprenoids in Raspberry Fruits (Rubus idaeus L.): The Role of Cytosolic 813

Mevalonate and Plastidial Methylerythritol Phosphate Pathway. Journal of 814

Agricultural and Food Chemistry 55 (22):9296-9304. doi:10.1021/jf071311x 815

Hasan MM, Kim H-S, Jeon J-H, Kim SH, Moon B, Song J-Y, Shim SH, Baek K-H (2014) 816

Metabolic engineering of Nicotiana benthamiana for the increased production of 817

taxadiene. Plant cell reports 33 (6):895-904 818

Hemmerlin A, Harwood JL, Bach TJ (2012) A raison d’être for two distinct pathways in the 819

early steps of plant isoprenoid biosynthesis? Progress in lipid research 51 (2):95-148 820

Hemmerlin A, Hoeffler J-F, Meyer O, Tritsch D, Kagan IA, Grosdemange-Billiard C, 821

Rohmer M, Bach TJ (2003) Cross-talk between the cytosolic mevalonate and the 822

plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. 823

Journal of Biological Chemistry 278 (29):26666-26676 824

Hezari M, Lewis NG, Croteau R (1995) Purification and characterization of taxa-4 (5), 11 825

(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first 826

committed step of taxol biosynthesis. Archives of Biochemistry and Biophysics 322 827

(2):437-444 828

Huang S, Li R, Zhang Z, Li L, Gu X, Fan W, Lucas WJ, Wang X, Xie B, Ni P, Ren Y, Zhu H, 829

Li J, Lin K, Jin W, Fei Z, Li G, Staub J, Kilian A, van der Vossen EA, Wu Y, Guo J, 830

He J, Jia Z, Ren Y, Tian G, Lu Y, Ruan J, Qian W, Wang M, Huang Q, Li B, Xuan Z, 831

Cao J, Asan, Wu Z, Zhang J, Cai Q, Bai Y, Zhao B, Han Y, Li Y, Li X, Wang S, Shi 832

Q, Liu S, Cho WK, Kim JY, Xu Y, Heller-Uszynska K, Miao H, Cheng Z, Zhang S, 833

Wu J, Yang Y, Kang H, Li M, Liang H, Ren X, Shi Z, Wen M, Jian M, Yang H, 834

Zhang G, Yang Z, Chen R, Liu S, Li J, Ma L, Liu H, Zhou Y, Zhao J, Fang X, Li G, 835

Fang L, Li Y, Liu D, Zheng H, Zhang Y, Qin N, Li Z, Yang G, Yang S, Bolund L, 836

Kristiansen K, Zheng H, Li S, Zhang X, Yang H, Wang J, Sun R, Zhang B, Jiang S, 837

Wang J, Du Y, Li S (2009) The genome of the cucumber, Cucumis sativus L. Nat 838

Genet 41 (12):1275-1281. doi:10.1038/ng.475 839

Huchelmann A, Brahim MS, Gerber E, Tritsch D, Bach TJ, Hemmerlin A (2016) Farnesol-840

mediated shift in the metabolic origin of prenyl groups used for protein prenylation in 841

plants. Biochimie 127:95-102. doi:http://dx.doi.org/10.1016/j.biochi.2016.04.021 842

Huchelmann A, Gastaldo C, Veinante M, Zeng Y, Heintz D, Tritsch D, Schaller H, Rohmer 843

M, Bach TJ, Hemmerlin A (2014) S-Carvone suppresses cellulase-induced capsidiol 844

production in Nicotiana tabacum by interfering with protein isoprenylation. Plant 845

physiology 164 (2):935-950 846

Itoh D, Kawano K, Nabeta K (2003) Biosynthesis of Chloroplastidic and Extrachloroplastidic 847

Terpenoids in Liverwort Cultured Cells: 13C Serine as a Probe of Terpene 848

Biosynthesis via Mevalonate and Non-mevalonate Pathways. Journal of Natural 849

Products 66 (3):332-336. doi:10.1021/np0204141 850

Jaffe FW, Tattersall A, Glover BJ (2007) A truncated MYB transcription factor from 851

Antirrhinum majus regulates epidermal cell outgrowth. J Exp Bot 58 (6):1515-1524. 852

doi:10.1093/jxb/erm020 853

Jiang Z, Kempinski C, Bush CJ, Nybo SE, Chappell J (2016) Engineering Triterpene and 854

Methylated Triterpene Production in Plants Provides Biochemical and Physiological 855


http://dx.doi.org/10.1016/j.biochi.2016.04.021http://www.plantphysiol.org

25

Insights into Terpene Metabolism. Plant Physiology 170 (2):702-716. 856

doi:10.1104/pp.15.01548 857

Jin J, Panicker D, Wang Q, Kim MJ, Liu J, Yin JL, Wong L, Jang IC, Chua NH, Sarojam R 858

(2014) Next generation sequencing unravels the biosynthetic ability of spearmint 859

(Mentha spicata) peltate glandular trichomes through comparative transcriptomics. 860

BMC plant biology 14:292. doi:10.1186/s12870-014-0292-5 861

Jozwiak A, Lipko A, Kania M, Danikiewicz W, Surmacz L, Witek A, Wojcik J, Zdanowski 862

K, Pączkowski C, Chojnacki T, Poznanski J, Swiezewska E (2017) Modelling of 863

dolichol mass spectra isotopic envelopes as a tool to monitor isoprenoid biosynthesis. 864

Plant Physiology. doi:10.1104/pp.17.00036 865

Kandra L, Wagner GJ (1988) Studies of the site and mode of biosynthesis of tobacco 866

trichome exudate components. Archives of Biochemistry and Biophysics 265 (2):425-867

432. doi:http://dx.doi.org/10.1016/0003-9861(88)90145-2 868 Kang J-H, Campos ML, Zemelis-Durfee S, Al-Haddad JM, Jones AD, Telewski FW, 869

Brandizzi F, Howe GA (2016) Molecular cloning of the tomato Hairless gene 870

implicates actin dynamics in trichome-mediated defense and mechanical properties of 871

stem tissue. Journal of Experimental Botany 67 (18):5313-5324. 872

doi:10.1093/jxb/erw292 873

Kang J-H, Liu G, Shi F, Jones AD, Beaudry RM, Howe GA (2010) The Tomato odorless-2 874

Mutant Is Defective in Trichome-Based Production of Diverse Specialized 875

Metabolites and Broad-Spectrum Resistance to Insect Herbivores. Plant Physiology 876

154 (1):262-272. doi:10.1104/pp.110.160192 877

Kang J-H, McRoberts J, Shi F, Moreno JE, Jones AD, Howe GA (2014) The Flavonoid 878

Biosynthetic Enzyme Chalcone Isomerase Modulates Terpenoid Production in 879

Glandular Trichomes of Tomato. Plant Physiology 164 (3):1161-1174. 880

doi:10.1104/pp.113.233395 881

Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008) COI1 is a critical 882

component of a receptor for jasmonate and the bacterial virulence factor coronatine. 883

Proc Natl Acad Sci U S A 105 (19):7100-7105. doi:10.1073/pnas.0802332105 884

Keasling JD (2010) Manufacturing Molecules Through Metabolic Engineering. Science 330 885

(6009):1355-1358. doi:10.1126/science.1193990 886

Keene CK, Wagner GJ (1985) Direct Demonstration of Duvatrienediol Biosynthesis in 887

Glandular Heads of Tobacco Trichomes. Plant Physiology 79 (4):1026-1032. 888

doi:10.1104/pp.79.4.1026 889

Khan NE, Myers JA, Tuerk AL, Curtis WR (2014) A process economic assessment of 890

hydrocarbon biofuels production using chemoautotrophic organisms. Bioresource 891

Technology 172:201-211. doi:http://dx.doi.org/10.1016/j.biortech.2014.08.118 892

Kim S-H, Chang Y-J, Kim S-U (2008) Tissue Specificity and Developmental Pattern of 893

Amorpha-4,11-diene Synthase (ADS) Proved by ADS Promoter-Driven GUS 894

Expression in the Heterologous Plant, Arabidopsis thaliana. Planta Med 74 (02):188-895

193. doi:10.1055/s-2008-1034276 896

Kim S, Park M, Yeom S-I, Kim Y-M, Lee JM, Lee H-A, Seo E, Choi J, Cheong K, Kim K-T, 897

Jung K, Lee G-W, Oh S-K, Bae C, Kim S-B, Lee H-Y, Kim S-Y, Kim M-S, Kang B-898

C, Jo YD, Yang H-B, Jeong H-J, Kang W-H, Kwon J-K, Shin C, Lim JY, Park JH, 899

Huh JH, Kim J-S, Kim B-D, Cohen O, Paran I, Suh MC, Lee SB, Kim Y-K, Shin Y, 900

Noh S-J, Park J, Seo YS, Kwon S-Y, Kim HA, Park JM, Kim H-J, Choi S-B, Bosland 901

PW, Reeves G, Jo S-H, Lee B-W, Cho H-T, Choi H-S, Lee M-S, Yu Y, Do Choi Y, 902

Park B-S, van Deynze A, Ashrafi H, Hill T, Kim WT, Pai H-S, Ahn HK, Yeam I, 903

Giovannoni JJ, Rose JKC, Sorensen I, Lee S-J, Kim RW, Choi I-Y, Choi B-S, Lim J-904

S, Lee Y-H, Choi D (2014) Genome sequence of the hot pepper provides insights into 905


http://dx.doi.org/10.1016/0003-9861(88)90145-2http://dx.doi.org/10.1016/j.biortech.2014.08.118http://www.plantphysiol.org

26

the evolution of pungency in Capsicum species. Nat Genet 46 (3):270-278. 906

doi:10.1038/ng.2877 907

Kirby J, Keasling JD (2009) Biosynthesis of Plant Isoprenoids: Perspectives for Microbial 908

Engineering. Annual Review of Plant Biology 60 (1):335-355. 909

doi:doi:10.1146/annurev.arplant.043008.091955 910

Koepp AE, Hezari M, Zajicek J, Vogel BS, LaFever RE, Lewis NG, Croteau R (1995) 911

Cyclization of Geranylgeranyl Diphosphate to Taxa-4 (), 11 ()-diene Is the Committed 912

Step of Taxol Biosynthesis in Pacific Yew. Journal of Biological Chemistry 270 913

(15):8686-8690 914

Koo AJK, Howe GA (2009) The wound hormone jasmonate. Phytochemistry 70 (13-915

14):1571-1580. doi:10.1016/j.phytochem.2009.07.018 916

Kortbeek RW, Xu J, Ramirez A, Spyropoulou E, Diergaarde P, Otten-Bruggeman I, de Both 917

M, Nagel R, Schmidt A, Schuurink RC, Bleeker PM (2016) Engineering of Tomato 918

Glandular Trichomes for the Production of Specialized Metabolites. Methods in 919

enzymology 576:305-331. doi:10.1016/bs.mie.2016.02.014 920

Kovacs K, Zhang L, Linforth RS, Whittaker B, Hayes CJ, Fray RG (2007) Redirection of 921

carotenoid metabolism for the efficient production of taxadiene [taxa-4 (5), 11 (12)-922

diene] in transgenic tomato fruit. Transgenic research 16 (1):121-126 923

Kroumova AB, Wagner GJ (2003) Different elongation pathways in the biosynthesis of acyl 924

groups of trichome exudate sugar esters from various solanaceous plants. Planta 216 925

(6):1013-1021. doi:10.1007/s00425-002-0954-7 926

Lange BM, Ahkami A (2013) Metabolic engineering of plant monoterpenes, sesquiterpenes 927

and diterpenes—current status and future opportunities. Plant Biotechnology Journal 928

11 (2):169-196. doi:10.1111/pbi.12022 929

Lange BM, Mahmoud SS, Wildung MR, Turner GW, Davis EM, Lange I, Baker RC, 930

Boydston RA, Croteau RB (2011) Improving peppermint essential oil yield and 931

composition by metabolic engineering. Proceedings of the National Academy of 932

Sciences 108 (41):16944-16949 933

Laterre R, Pottier M, Remacle C, Boutry M (2017) Photosynthetic Trichomes Contain a 934

Specific Rubisco with a Modified pH-Dependent Activity. Plant Physiology 173 935

(4):2110-2120. doi:10.1104/pp.17.00062 936

Li L, Zhao Y, McCaig BC, Wingerd BA, Wang J, Whalon ME, Pichersky E, Howe GA 937

(2004) The tomato homolog of CORONATINE-INSENSITIVE1 is required for the 938

maternal control of seed maturation, jasmonate-signaled defense responses, and 939

glandular trichome development. The Plant cell 16 (1):126-143 940

Li Q, Cao C, Zhang C, Zheng S, Wang Z, Wang L, Ren Z (2015) The identification of 941

Cucumis sativus Glabrous 1 (CsGL1) required for the formation of trichomes 942

uncovers a novel function for the homeodomain-leucine zipper I gene. J Exp Bot 66 943

(9):2515-2526. doi:10.1093/jxb/erv046 944

Liakopoulos G, Stavrianakou S, Karabourniotis G (2006) Trichome layers versus dehaired 945

lamina of Olea europaea leaves: differences in flavonoid distribution, UV-absorbing 946

capacity, and wax yield. Environmental and Experimental Botany 55 (3):294-304. 947

doi:http://dx.doi.org/10.1016/j.envexpbot.2004.11.008 948

Liu J, Xia K-F, Zhu J-C, Deng Y-G, Huang X-L, Hu B-L, Xu X, Xu Z-F (2006) The 949

Nightshade Proteinase Inhibitor IIb Gene is Constitutively Expressed in Glandular 950

Trichomes. Plant and Cell Physiology 47 (9):1274-1284. doi:10.1093/pcp/pcj097 951

Liu X, Bartholomew E, Cai Y, Ren H (2016) Trichome-Related Mutants Provide a New 952

Perspective on Multicellular Trichome In

Documents

Running title: Glandular trichomes development and ... · 4 84. Plant glandular trichomes: an interesting paradigm to study plant cell differentiation 85. To modulate the density