Specific decorations of 17-hydroxygeranyllinalool ... · 8/26/2020 · 95 Diterpene glycosides...

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the 1

autotoxicity problem of chemical defense in Nicotiana attenuata 2

3

Sven Heiling1, Lucas Cortes Llorca1, Jiancai Li1, Klaus Gase1, Axel Schmidt2, Martin 4

Schäfer1, Bernd Schneider3, Rayko Halitschke1, Emmanuel Gaquerel4,5 and Ian Thomas 5

Baldwin1 6

7 1Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, Hans 8

Knöll Straße 8, 07745 Jena, Germany 9

2Max Planck Institute for Chemical Ecology, Department of Biochemistry, Hans Knöll 10

Straße 8, 07745 Jena, Germany 11

3Max Planck Institute for Chemical Ecology, Research Group Biosynthesis/NMR, Hans 12

Knöll Straße 8, 07745 Jena, Germany 13

4Centre for Organismal Studies Heidelberg, Im Neuenheimer Feld 360, 69120 14

Heidelberg, Germany 15

5Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357 Université de 16

Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France 17

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CORRESPONDING AUTHORS: 19

Ian T. Baldwin1 and Emmanuel Gaquerel2 20

1Max Planck Institute for Chemical Ecology, Molecular Ecology Department, Hans Knöll 21

Straße 8, 07745 Jena, Germany, E-mail: [email protected]; Fax: +49 (0)3641 22

571102; 2Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357 Université de 23

Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France, E-mail: 24

[email protected] 25

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Short Title: Glycosylation and Phytotoxicity of HGL-DTGs 27

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 27, 2020. ; https://doi.org/10.1101/2020.08.26.267690doi: bioRxiv preprint

https://doi.org/10.1101/2020.08.26.267690

The author responsible for distribution of materials integral to the findings presented in 29

this article in accordance with the policy described in the Instructions for Authors 30

(www.plantcell.org) is Ian T. Baldwin ([email protected]). 31

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Abstract 33

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17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) are abundant and potent 35

anti-herbivore defense metabolites in Nicotiana attenuata whose glycosylation and 36

malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize 37

the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of 38

uridine diphosphate glycosyltransferases (UGTs) and identified 107 members of family-1 39

UGTs. Tissue-specific time-course transcriptional profiling revealed that the transcripts 40

of three UGTs were highly correlated with two HGL-DTG key biosynthetic genes: 41

geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase 42

(NaGLS). NaGLS’s role in HGL-DTG biosynthesis was confirmed by virus-induced gene-43

silencing. Silencing the UDP-rhamnosyltransferase, UGT91T1, indicated its role in the 44

rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and 45

UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) 46

to lyciumoside I. UGT74P3 and UGT74P5 stably silenced plants were severely 47

developmentally deformed, suggesting a phytotoxic effect of 17-HGL. Applications of 48

synthetic 17-HGL and silencing of these UGTs in HGL-DTG-free plants confirmed the 49

phytotoxic effect of 17-HGL. Feeding assays with Manduca sexta larvae revealed the 50

defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG 51

biosynthesis. Glucosylation is a critical step that contributes to the metabolites’ 52

defensive function and solves the autotoxicity problem of this potent chemical defense. 53

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Introduction 60


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61

In the course of evolution, plants developed versatile defense strategies against 62

biotic stresses, such as herbivores and pathogens. Among these defensive strategies 63

are physical and chemical barriers such as trichomes (Fordyce and Agrawal, 2001), 64

volatiles that attract predators (Kessler and Baldwin, 2001; Halitschke et al., 2008) and a 65

wide array of defensive specialized metabolites. Many small molecules produced as part 66

of a plant's specialized metabolism function as direct defenses by being toxic, repellent, 67

or anti-nutritive for herbivores of different feeding guilds and degrees of specialization; 68

compounds with a broad spectrum of toxicity are likely to be of greater defensive value 69

against a greater diversity of attackers. Noteworthy examples are glucosinolates, 70

alkaloids, and terpenoids. However, these broad-spectrum toxins force the producers to 71

solve the "toxic waste dump problem" of chemical defense, as many direct defense 72

compounds are generally cytotoxic and can damage the tissues of non-adapted 73

producers. 74

Plants have evolved numerous ways of solving this “toxic waste dump problem” 75

for broad-spectrum chemical defenses. One frequently used solution is glycosylation, 76

which is one of the most prevalent and widespread biochemical modifications 77

contributing to the structural and functional diversity of specialized metabolites in plants. 78

Incorporation of sugar molecules into small lipophilic metabolites can regulate 79

storage/localization of defensive metabolites (Gachon et al., 2005; Yadav et al., 2014) 80

and change their bioactivity by detoxifying phytotoxic intermediates. For example, for the 81

defensive deployment of hydrogen cyanide (Gleadow and Moller, 2014) or steroidal 82

saponins (Mylona et al., 2008), plants store these toxins as glycosides, sometimes in 83

particular compartments, away from lytic enzymes that liberate the active toxins in 84

response to the tissue damage that frequently accompanies herbivore and pathogen 85

attack. Similarly, glucosinolates are compartmentalized away from the myrosinases that 86

rapidly hydrolyze them to toxic isothiocyanates and other biologically active products 87

(Matile, 1980; Halkier and Gershenzon, 2006). Steroidal alkaloids are also safely stored 88

as glycosides and silencing GAME-1, a UDP-galactosyltransferase responsible for the 89

glycosylation of these defense compounds in tomato, results in the accumulation of the 90

aglycone, α-tomatidine with severe developmental consequences (Itkin et al., 2011). 91

Glycosylation of the saponin, hederagenin in Medicago truncatula (Naoumkina et al., 92


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2010), and of avenacin A-1 in Avena sativa (Mylona et al., 2008) are other examples 93

which point to a similar chemical sequestration role exerted by glycosylation. 94

Diterpene glycosides (DTGs) are a diverse compound class whose members are 95

often associated with phytotoxic activities (Macias et al., 2008) and have potent anti-96

herbivore resistance/deterrence effects. For example, the abundance of monomers and 97

dimers of capsianosides is correlated with thrips resistance in pepper (Macel et al., 98

2019) and 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) in Nicotiana 99

species have been shown to function in resistance against larvae of the specialist 100

herbivore Manduca sexta (Lou and Baldwin, 2003; Jassbi et al., 2008; Heiling et al., 101

2010) and the generalist herbivore, Heliothis virescens (Snook et al., 1997). 102

HGL-DTGs constitute an abundant and structurally diverse group of specialized 103

metabolites whose function in plants is poorly understood (Heiling et al., 2010). HGL-104

DTGs occur in the aboveground tissues of the native diploid tobacco, Nicotiana 105

attenuata (Heiling et al., 2010) as well as other Nicotiana species (Shinozaki et al., 1996; 106

Snook et al., 1997; Lou and Baldwin, 2003; Jassbi et al., 2006; Heiling et al., 2010; 107

Jassbi et al., 2010; Poreddy et al., 2015; Heiling et al., 2016), several other solanaceous 108

genera including Capsicum (Izumitani et al., 1990; Hashimoto et al., 1997; Iorizzi et al., 109

2001; Lee et al., 2006, 2007, 2008; Lee et al., 2009) and Lycium (Terauchi et al., 1995; 110

Terauchi et al., 1997b, a; Terauchi et al., 1998b; Terauchi et al., 1998a; Roda et al., 111

2003), and the Asteraceae, Blumera lacera (Akter et al., 2016). HGL-DTGs consist of an 112

acyclic 17-hydroxygeranyllinalool aglycone, which is conjugated at its C-3 and C-17 113

hydroxyl groups to different sugar moieties, such as glucose and rhamnose. These 114

sugars can be further glycosylated at the C'-2, C'-4 or C'-6 hydroxyl groups and 115

acetylated or malonylated at the C'-6 hydroxyl group of the glucose(s) (Taguchi et al., 116

2005; Yu et al., 2008, Heiling et al., 2010; Jassbi et al., 2010; Heiling et al., 2016). To 117

date, 45 HGL-DTGs, which differ in their sugar or malonyl decorations, have been 118

putatively annotated or identified in N. attenuata (Figure 1) (Heiling et al., 2016). 119

However, it is unclear which of the many different HGL-DTGs or which structural 120

components of HGL-DTGs are responsible for the observed deterrent (Jassbi et al., 121

2006) and resistance effects against different herbivores (Snook et al., 1997). For 122

example, the geranyllinalool precursor is known to be insecticidal in pine wood and the 123

same compound can be found in the defensive secretions of the termite Reticulitermes 124


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lucifugus (Baker et al., 1982; Lemaire et al., 1990). Jassbi and colleagues (Jassbi et al., 125

2010) suggested that the 17-HGL aglycone is responsible for the feeding-deterrent 126

characteristics of HGL-DTGs, but this hypothesis has not been tested. 127

While the chemical identity and anti-herbivore effects of HGL-DTGs have been 128

the focus of major investigations, less is known about the possible phytotoxic effects of 129

HGL-DTGs and how producing plants cope with these phytotoxic effects. This is, in part, 130

because we know very little about the enzymes required for the biosynthesis of these 131

metabolites. The 17-HGL aglycone is derived from the condensation of three five-carbon 132

units of isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP) to 133

produce the diterpenoid precursor, geranylgeranyl-pyrophosphate (GGPP; (Ohnuma et 134

al., 1998; Dewick, 2002)). This reaction is catalyzed by a plastidial geranylgeranyl 135

pyrophosphate synthase (GGPPS; (Jassbi et al., 2008; Heiling et al., 2010)). Formation 136

of GGPP is followed by its allylic rearrangement by a geranyllinalool synthase (GLS; 137

(Falara et al., 2014)) that produces the tertiary alcohol geranyllinalool. However, the 138

enzymes necessary for further hydroxylation and glycosylation steps, which determine 139

most of the structural diversity of HGL-DTGs, remain to be characterized. Li and 140

colleagues (Li et al., 2018) identified and silenced a malonyltransferase (NaMAT1), 141

which is responsible for the malonylation of HGL-DTGs. However, all malonyl moieties 142

of HGL-DTGs are rapidly lost when leaves are ingested by M. sexta larvae, suggesting 143

that the malonylation of HGL-DTGs does not play a central role in anti-herbivore defense 144

(Poreddy et al., 2015). Interestingly, disruption of the uniform malonylation patterns of 145

HGL-DTGs leads to a specific reduction in the floral style lengths of N. attenuata flowers 146

(Li et al., 2018). This shows that specific decorations of a plant's specialized metabolites 147

can play a crucial, but poorly understood, role in plant development. Malonylation of 148

specialized metabolites, such as flavonoids or phenolic glucosides, is a common 149

phenomenon and it has been shown that malonyl moieties alter the molecular properties 150

of these compounds can enhance their solubility in water (Heller and Forkman, 1994) or 151

sequester compounds to molecular compartments, such as vacuoles (Taguchi et al., 152

2010). Whether other intermediates in the HGL-DTG pathway are similarly influential for 153

plant development remains to be explored. 154

To evaluate the defensive value of glycosylation and the potential (auto)toxicity of the 155

17-HGL aglycone for both plant cells and insect herbivores, we investigated the UDP-156


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glycosyltransferases (UDP-GTs) responsible for the glycosylation of HGL-DTGs. To this 157

end, we analyzed the tissue- and herbivory-specific co-regulation of the 107 predicted 158

UDP-GTs in N. attenuata UDP-GTs with two bait genes previously characterized for 159

their involvement in the biosynthesis of 17-HGL, which pinpointed on three novel UGT 160

candidates. RNAi silencing of these UGTs by RNAi approach in N. attenuata and the 161

related HGL-DTG-producing sympatric diploid species N. obtusifolia (syn. 162

N. trigonophylla Dunal) (Heiling et al., 2016) as well as enzyme assays with recombinant 163

proteins identified that UGT74P3 and UGT74P4 are responsible glucosylation of the C-3 164

and C-17 hydroxyl-groups to form lyciumoside I, while UGT91T1 in N. attenuata and 165

NoUGT91T1-like in N. obtusifolia are rhamnosyltransferases in the HGL-DTG pathway. 166

As summarized in one of our previous studies (Heiling et al., 2017), all rhamnosylated 167

HGL-DTG identified so far possess rhamosyl moieties attach to glucose ones, pointing 168

to the conclusion that rhamnosylation requires prio glucosylation. Finally, genetic 169

manipulations revealed that glycosylation of the 17-HGL aglycone by UGT74P3 is 170

crucial to prevent toxic effects to the plant as well as contributes to the metabolites` 171

defensive function during the attack by M. sexta larvae 172

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Results 175

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Putative identification of UGTs responsible for HGL-DTG biosynthesis 177

To identify UGTs, a genome-wide survey of N. attenuata was performed and a 178

total of 107 putative UGT sequences containing the PSPG motif at the C-terminus were 179

detected (Supplemental Figure 1). These were phylogenetically characterized 180

(Supplemental Figure 2, Supplemental Table 2), their amino acid sequence examined 181

(Supplemental Figure 3, Supplemental Table 1, 3, 4) and their expression analyzed in a 182

full-transcriptome microarray dataset obtained from leaf and root tissues collected at 183

several time-points following simulated leaf herbivory (wounding and application with 184

oral secretions [OS]) as described in supplemental method file 1. 185

HGL-DTGs are secondary metabolites of N. attenuata, whose biosynthetic steps 186

of glycosylation are regulated by the defense phytohormone, jasmonic acid (Heiling et 187

al., 2010). Genes involved in a shared biological process tend to be co-expressed in 188


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large-scale expression datasets due to the fact that they are frequently under the control 189

of a common regulatory network (Saito et al., 2008). Following this rationale, we 190

identified UGT candidates putatively responsible for the biosynthesis of HGL-DTGs by 191

exploring gene co-expression in tissue-specific (local and systemic leaves as well as 192

root tissue) transcriptomes analyzed at 1, 5, 9, 13, 17 and 21 h after the simulated 193

herbivory treatment. For the resulting compendium consisting of 150 microarray 194

expression profiles, we calculated the Pearson Correlation Coefficients (PCC) for 150 195

microarrays of the expressed UGTs with previously identified genes of the HGL-DTG 196

pathway (NaGGPPS and NaGLS, Supplemental Table 5a/b). Transcript accumulation of 197

three UGTs was increased in response to wounding and simulated herbivory and 198

significantly correlated with GGPPS (UGT91T1 – PCC = 0.823, UGT74P3 – PCC = 199

0.608 and UGT74P5 – PCC = 0.684) and GLS (UGT91T1 – PCC = 0.899, UGT74P3 – 200

PCC = 0.872 and UGT74P5 – PCC = 0.868). The PCC value between GLS and GGPPS 201

transcript levels was 0.799 (Supplemental Figure 4). Furthermore, we compared the 202

expression levels between shoot and root tissues of the above genes. Consistent with 203

the absence of HGL-DTG accumulation in N. attenuata roots, transcript accumulation 204

was 50-fold and 3225-fold lower in roots compared to leaves for GGPPS and GLS, 205

respectively. The three candidate UGTs showed a similar profile with 2190-, 127-, and 206

20-fold lower root transcript levels of UGT91T1, UGT74P3, and UGT74P5, respectively. 207

Focusing on the 5 h time point collected from systemic leaves, we detected that GGPPS 208

(increased 6-fold), GLS (increased 8-fold), UGT91T1 (increased 7-fold), UGT74P3 209

(increased 8-fold) and UGT74P5 (increased 8-fold) were all highly up-regulated, relative 210

to untreated control and mechanical wounding conditions, in response to the simulated 211

herbivory treatment (Supplemental Figure 4 and 5, Supplemental Data 1). 212

A phylogenetic alignment with functionally characterized UGTs (Supplemental 213

Figure 6) showed a close relationship of UGT74P3 and UGT74P5 protein sequences 214

with two previously characterized diterpene glucosyltransferases (SrUGT74G1 – steviol 215

glycoside glucosyltransferase from Stevia rebaudiana and CsGIT2 - crocetin 216

glucosyltransferase from Crocus sativus). UGT91T1 showed a close phylogenetic 217

relationship to three of the very few functionally characterized flavonoid 218

rhamnosyltransferases (CmF7G12RT – flavonoid-1, 2-rhamnosyltransferase from Citrus 219

maxima, GmF3G6R – flavonol-3-O-glucoside-α-1, 6-L-rhamnosyltransferase from 220


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Glycine max and PhA3ART – anthocyanidin-3-O-glucoside-α-1, 6-L-221

rhamnosyltransferase from Petunia x hybrida). 222

Based on the co-expression with known genes of the HGL-DTG pathway, the 223

localization of high expression levels in leaf tissues and their phylogenetic relationships, 224

we selected these three candidate UGTs for further characterization. 225

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Virus-induced gene-silencing reveals a role of three UGTs in HGL-DTG production 227

Using a well-established transient virus-induced gene-silencing (VIGS) approach 228

(Saedler and Baldwin, 2004), we first examined the consequences of independently 229

silencing the three candidate UGTs for HGL-DTG production. Seventeen days after 230

inoculation with Agrobacterium tumefaciens harboring the appropriate constructs, leaf 231

transcript abundance was reduced by 98.5% for UGT91T1, 85% for UGT74P3 and 232

94.3% for UGT74P5 relative to the empty vector controls (EV, pTV00) (Supplemental 233

Figure 7). Co-silencing resulted in reduced transcript abundance of UGT74P3 and 234

UGT74P5 in both, pTVUGT74P3 and pTVUGT74P5 plants (Supplemental Figure 8). 235

To test the hypothesis that UGT91T1, UGT74P3 and UGT74P5 control the 236

glycosylation steps in the HGL-DTG pathway, we analyzed the leaf metabolome by 237

UPLC/TOF-MS of the different VIGS plants under control conditions (lanolin, Lan) or 238

after treatment with a lanolin paste containing methyl jasmonate (Lan + MeJA), known to 239

strongly induce the de novo production of HGL-DTGs (Heiling et al., 2010). We 240

putatively identified and annotated HGL-DTGs using a previously established rapid de-241

replication and identification workflow, which is based on high resolution MS data 242

analysis and a library of putative and identified HGL-DTGs (Heiling et al., 2016). 243

Levels of rhamnosylated HGL-DTGs, most particularly of lyciumoside IV and 244

attenoside, were reduced compared to pTV00 controls, while the non-rhamnosylated 245

HGL-DTGs lyciumoside I and lyciumoside II increased in the HGL-DTG chemotype of 246

pTVUGT91T1 VIGS plants (Figure 2A, Supplemental Data 2). This shift was more 247

pronounced after the Lan + MeJA treatment: in transiently transformed pTVUGT91T1 248

plants, most rhamnosylated HGL-DTGs were barely detectable and non-rhamnosylated 249

HGL-DTGs were dramatically increased compared to pTV00 controls (Figure 2A and B). 250

Furthermore, we detected the 17-HGL aglycone as well as several novel compounds in 251

pTVUGT74P3 and pTVUGT74P5 VIGS plant profiles (Figure 2A). We defined this class 252


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of compounds as intermediate HGL-DTGs and our MS annotation workflow suggested 253

compounds with only one or two sugar moieties, malonylated or not at either the C-3 or 254

C-17 hydroxy-group of the aglycone. 255

Furthermore, we searched for orthologues to these three UGTs in N. obtusifolia, a 256

sympatric tobacco species to N. attenuata. We identified NoUGT74P4 (95% homology 257

to UGT74P3), NoUGT74P6 (93% homology to UGT74P5) and NoUGT91T1-like (92% 258

homology to UGT91T1). Using VIGS, we inoculated A. tumefaciens harboring the 259

appropriate constructs into 25-day-old N. obtusifolia plants and detected transcript 260

reductions of 91.2% in NoUGT91T1-like, 98.5% in NoUGT74P3 and 93.1% in 261

pTVNoUGT74P6, 14 days after inoculation (Supplemental Figure 9). 262

pTVNoUGT91T1-like VIGS plants showed an overall decrease in rhamnosylated 263

HGL-DTGs compared to pTV00 controls and an increase in non-rhamnosylated 264

compounds (Figure 3A and 3B, Supplemental Data 3). In pTVNoUGT74P4 and 265

pTVNoUGT74P6 VIGS plants compared to pTV00 controls, the abundance of 266

intermediate HGL-DTGs as well as the 17-HGL aglycone was increased and non-267

rhamnosylated HGL-DTGs were decreased. Silencing both glucosyltransferases in N. 268

obtusifolia by means of a double construct resulted in the same phenotypic alterations, 269

namely the appearance of the intermediate HGL-DTGs and of the 17-HGL aglycone. 270

Ten novel intermediate HGL-DTGs were putatively identified based on the de-replication 271

workflow for HGL-DTGs. A detailed MS analysis and putative structure description of N. 272

attenuata and N. obtusifolia HGL-DTGs can be found in the Supplemental Table 6 and 7 273

and Supplemental Figure 10a and b and 11a-c. 274

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17-HGL glucosylation activities of UGT74P3/P4 and UGT74P5 proteins 276

The co-silencing in both UGT74P3 and UGT74P5 resulted in strong overlap in the 277

metabolic alterations of the HGL-DTG profiles produced by pTVUGT74P3 and 278

pTVUGT74P5 plants and suggested that at least one of these enzymes might play a 279

concerted role in the formation of lyciumoside I and lyciumoside II through glucosylation 280

of the C-3 or C-17 hydroxyl-group of 17-HGL. HGL-DTGs typically contain one D-Glc 281

moiety at the C-3 and one at the C-17 hydroxyl group. Lyciumoside II derives from 282

lyciumoside I by the attachment of a second D-Glc at the C’-2 position of the C-17 D-283

Glc. To test this hypothesis, we analyzed the in vitro activity of the UDP-GT enzymes 284


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when recombinantly produced in E. coli. UDP-glucose was used as sugar donor with a 285

mixture of all six isomers of synthesized 17-HGL (Supplemental Figure 12) as 286

substrates. NaUGT74P3 and its N. obtusifolia homologue, NoUGT74P4, readily used 287

UDP-glucose as a donor producing several novel products with a mass-to-charge ratio 288

of m/z 491.3003 [M+Na]+ corresponding to a mono-glucosylated 17-HGL and two 289

additional products with m/z 653.3509 [M+Na]+ corresponding to lyciumoside I and 290

another di-glucosylated 17-HGL (Figure 4). Lyciumoside I was identified by retention 291

time and MS/MS fragmentation to a purified authentic standard and the novel 292

compounds were annotated based comparisons on their MS/MS fragmentation to an in-293

house database of HGL-DTG spectra. Thus, NaUGT74P3 and NoUGT74P4 function as 294

UDP-glucosyltransferases for HGL-DTGs synthesis via their ability to catalyze 295

glucosylation at C-3 and C-17 of the 17-HGL aglycone. When using UDP-glucose as 296

donor and 17-HGL as substrate, NaUGT74P5 and NoUGT74P6 did not show any 297

detectable product in the enzymatic assays. Furthermore, the combination of 298

NaUGT74P5 with NaUGT74P3 or NoUGT74P4 did not result in any further detectable 299

products. 300

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Virus-induced gene-silencing of UGTs causes morphological defects and necrosis 304

In addition to the mild TRV infection symptoms such as curly leaves and local 305

chlorosis that were also seen in EV (pTV00) and pTVUGT91T1 plants (Supplemental 306

Figure 13a), we noticed that VIGS plants expressing the pTVUGT74P3 and 307

pTVUGT74P5 constructs displayed severe morphological alterations that ranged from 308

the presence of necrotic spots to necrotic apical meristems and a high percentage of 309

stalled flower buds (Figure 2C, Supplemental Figures 13b and c). Furthermore, we 310

isolated organs of mature flowers collected at anthesis (Figure 2D) and observed 311

significant reductions, compared to observations for pTV00 controls, in the lengths of 312

styles and corolla tubes, and in the width of the corolla limb of pTVUGT74P3 and 313

pTVUGT74P5 VIGS plants, as well as an increased combined lengths of ovary + 314

nectary (Figure 2E). 315


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N. obtusifolia plants showed the same symptoms. Plants transiently silenced with 316

pTVNoUGT91T1-like did not display any additional phenotypic alterations compared 317

with the pTV00 control plants. However, N. obtusifolia plants silenced in UGT74P4 and 318

UGT74P6 expression showed similar morphological modifications as observed in N. 319

attenuata, ranging from necrotic spots, misshaped and deformed leaves to stalled flower 320

buds (Figure 3C, Supplemental Figure 14) with the exception of the apical meristem 321

necrosis. Floral morphology was not examined in N. obtusifolia. 322

Finally, we generated a VIGS construct (pTVGLS) targeting the geranyllinalool 323

synthase NaGLS which was also identified from the co-expression analysis. The 324

encoded protein is predicted to be part of the HGL-DTG biosynthetic pathway by 325

hydroxylating geranyllinalool to produce 17-HGL (Falara et al., 2014). 14 days after 326

inoculation of A. tumefaciens with pTVGLS, we detected a 97.5% reduction of the GLS 327

transcript abundance compared to pTV00 controls, which was associated with a strong 328

reduction in almost all HGL-DTG types (Supplemental Figure 15 and Supplemental Data 329

4). No novel intermediate HGL-DTGs were detected, consistent with the expected 330

absence of the precursor molecule, geranyllinalool. Beyond the typical TRV infection 331

symptoms, no morphological alterations were detected, suggesting that the phenotype 332

of the glycosyltransferase-impaired lines resulted from accumulations of intermediate 333

HGL-DTGs or the aglycone 17-HGL. 334

335

Plants stably-silenced for UGT74P3 and UGT74P5 display severe developmental 336

defects 337

To further elucidate the role of UGT74P3 and UGT74P5 in controlling the flux of 338

HGL-DTG synthesis in N. attenuata as well as the mechanisms responsible for the 339

developmental defects detected during transient gene silencing, we generated 36 340

independent stably-transformed transgenic N. attenuata plants harboring UGT74P3 and 341

UGT74P5 inverted-repeat (IR) silencing constructs in their genomes. Similar to the VIGS 342

experiments, IRugt74p3 and IRugt74p5-silenced plants displayed strong effects on 343

growth and development. Almost all stable T0-transformants developed either a dwarfish 344

growth or a “broom-like” appearance (Figure 5D). Compared to wild-type (WT) plants, 345

many of the transformed plants were strongly retarded in their growth, with thicker 346

woody, or possibly suberized side branches and deformed or thick, succulent-like 347


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leaves. Most flower buds of these transformed plants were small and aborted before 348

fertilization. Few T0-transformants of both constructs exhibited a milder phenotype and 349

produced mature flowers and seed capsules. The phenotypic characteristics of 350

IRugt74p3 transformants could not be transferred to the T2 generation, as T1 plants 351

aborted most flower buds early during development and did not produce fertile flowers. 352

IRugt74p5 transformants could be propagated and two lines from the same T1 parental 353

plant were established (Supplemental Figure 16). Similar to the T0 transformants, both 354

IRugt74p5 lines displayed a milder phenotype compared to IRugt74p3, but still with 355

severe morphological alterations ranging from necrotic spots and necrotic apical 356

meristem to small deformed or thicker succulent leaves (Supplemental Figure 17a-c) 357

and numerous stalled flower buds (Figure 5C). IRugt74p5 plants were also smaller 358

compared to WT (Supplemental Figure 18). Additionally, we established a heterozygous 359

double construct UGT74P3/UGT74P5 for which ~75% of the silenced plants exhibited 360

similar morphological alterations. Similar to the IRugt74p3 plants, the homozygous 361

double construct was lethal and did not produce seeds. 362

In contrast to the IRugt74p3 and IRugt74p5-silenced plants, two independent 363

stably-silenced UGT91T1 lines did not show any morphological alteration compared to 364

WT (Supplemental Figure 19). 365

In addition to the careful examination of shoot morphological alterations (roots 366

were not examined), we analyzed transcript abundance of UGT74P3, UGT74P5 and 367

UGT91T1 in leaves of 52-day-old plants of the different transgenic lines (Figure 6). The 368

silencing efficiency for UGT91T1 transcript abundance was 97.4% for IRugt91t1a and 369

97.6% for IRugt91t1b. UGT74P5 transcript abundance was strongly repressed in all 370

IRugt74p5 lines tested (83.9% in IRugt74p5b, 96.8% in IRugt74p5a and 92.3% in 371

IRugt74p3/ugt74p5). The silencing efficiency for UGT74P3 in IRugt74p3/ugt74p5 was 372

99.3%, but we also detected a strong co-silencing of UGT74P3 expression in 373

IRugt74p5b (96.7%) and IRugt74p5a (99.4%). 374

375

Metabolic profiling confirms the unique HGL-DTG profiles of stably-silenced UGT 376

lines 377

From UPLC/qTOF-MS measurements and the application of our de-replication 378

workflow, we identified 60 HGL-DTGs in the leaf extracts of the different stably-silenced 379


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lines. HGL-DTGs were annotated and classified, based on their chemical composition, 380

into three categories as rhamnosylated, non-rhamnosylated and biosynthetic 381

intermediates only detectable upon disruption of glucosylation steps (Figure 5). Both 382

stable lines impaired in UGT74P5 expression showed the highest overall HGL-DTG 383

levels (Supplemental Figure 20). Non-rhamnosylated lyciumoside I and its malonylated 384

forms, nicotianoside IXa-d and Xa-e, were barely detectable and levels of 385

rhamnosylated lyciumoside IV was strongly reduced. While rhamnosylated nicotianoside 386

I and II remained constant compared to WT plants, both transgenic IRugt74p5 lines 387

exhibited elevated levels of lyciumoside II and attenoside and their malonylated forms, 388

nicotianoside XIa-d, XIIa-c, XIIIa-f and nicotianoside VIa-c, VIIa-c and VIIIa-b, 389

respectively. More complex reconfiguration patterns were also detected such as for 390

nicotianoside III which remained at unchanged levels in transgenic IRugt74p5 lines but 391

whose malonylated forms, nicotianoside IV and V, increased in concentrations. 392

Furthermore, we putatively identified 10 novel highly abundant intermediate HGL-DTGs 393

and the 17-HGL aglycone which were not detected in WT plants. Annotation of the 394

MS/MS spectra of these novel HGL-DTGs indicated lower molecular weight HGL-DTG 395

biosynthetic intermediates with either one (G-3-HGL, G-17-HGL) or two (RGHGL and 396

DTG 648) sugar moieties attached to the 17-HGL (Figure 5, Supplemental Figures 11a-397

c, Supplemental Table 7). Additionally, malonylated forms of these compounds were 398

also detected (DTG 572, DTG 718, DTG 734 and DTG 820) (Figure 5A and B, 399

Supplemental Figures 11a-c, Supplemental Table 7). The transgenic line IRugt74p5b 400

was used for all further experiments. The heterozygous double construct 401

IRugt74p3/ugt74p5 exhibited an almost identical pattern of accumulation of known and 402

novel intermediate HGL-DTGs. The most prominent difference compared to the 403

IRugt74p5 lines was the specific increase detected for lyciumoside II and lyciumoside IV. 404

In both stable lines impaired in UGT91T1 expression, all rhamnosylated HGL-405

DTGs were strongly decreased. Reciprocally, all non-rhamnosylated HGL-DTGs were 406

highly increased leading to a complete shift between rhamnosylated and non-407

rhamnosylated compounds within the HGL-DTG profile (Figure 5A and B). Neither 408

intermediate HGL-DTGs nor the 17-HGL aglycone could be detected in either of the 409

transgenic IRugt91t1 lines. IRugt91t1 line A was selected for all further experiments. 410

Additionally we analyzed the HGL-DTG profiles in IRggpps plants (Figure 5). 411


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Geranylgeranyl-pyrophosphate synthase (GGPPS) is responsible for the synthesis of 412

GGPP, the precursor for all HGL-DTGs (Jassbi et al., 2008) and almost all HGL-DTGs 413

were decreased and no intermediates could be detected. Only levels of nicotianoside 414

VIIIa and VIIIb were increased. Surprisingly, traces of the 17-HGL aglycone were 415

detected. A more detailed analysis of the HGL-DTG profiles in all stable silenced lines 416

can be found in Supplemental Data 5. 417

In addition, we performed a detailed metabolite profiling of central carbon 418

metabolism intermediates and specialized metabolites, which is further described in 419

Supplemental Method File 2. This included quantitative profiles of 23 amino acids and 420

biogenic amines, small organic acids, phenylpropanoids and derivatives, sugars and 421

phytohormones, such as gibberellins, cytokinins and jasmonates (Supplemental Figures 422

21 and 22, Supplemental Data 6 and 7). 423

424

Abolishing 17-HGL aglycone synthesis in IRggpps prevents the strong 425

morphological alterations that result from silencing UGT74P3 and UGT74P5 426

To determine if the morphological alterations of plants transiently- and stably-427

silenced in UGT74P3 and UGT74P5, are mediated by altered HGL-DTG profiles, we 428

performed a VIGS experiment silencing UGT74P3 and UGT74P5 in N. attenuata plants 429

impaired in GGPPS expression (IRggpps) and WT plants. Quantification of 17-HGL 430

concentrations in leaf tissues using a quantitative U(H)PLC-triple quadrupole-MS 431

method (Supplemental Figure 23) showed accumulation of 17-HGL only in WT N. 432

attenuata plants transformed with the pTVUGT74P3 or pTVUGT74P5 VIGS construct 433

(Supplemental Figure 24). 17 days after inoculation, we analyzed the morphological 434

alterations triggered by UGT74P3 and UGT74P5 silencing (Figure 7). WT plants 435

transiently transformed with pTVUGT74P3 and pTVUGT74P5 exhibited the phenotype 436

described above, ranging from necrotic spots and necrotic apical meristems to stalled 437

and aborted flower buds (Figure 7A, Supplemental Figure 25, 26 and 27). The number 438

of side branches increased and the rosette diameter decreased. IRggpps plants 439

transformed with pTVUGT74P3 and pTVUGT74P5 did not show any severe 440

morphological alterations. Specifically, the number of side branches and rosette 441

diameters of these plants were not different from those of WT plants (Figure 7B). This 442

combination of genetic manipulations indicates that the ectopic accumulation of several 443


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intermediates and of 17-HGL might be directly responsible for the observed 444

morphological alterations. 445

446

Excess of 17-HGL triggers cell necrosis in N. attenuata leaves 447

We determined the absolute concentration of 17-HGL in leaves of N. attenuata 448

plants (N=5) impaired in UGT74P5 or UGT74P3/UGT74P5 expression which exhibited 449

the severe phenotype reported above. The concentration (mean ± SD) of 17-HGL was 450

94 ± 8 nmol/g FW in IRugt74p5 and 85.4 ± 17.6 nmol/g FW in IRugt74p4/ugt74p5 451

(Figure 8A). For the determination of the phytotoxic effect of 17-HGL, we used 32-day-452

old early-elongated N. attenuata (N=3) WT plants and 48-day-old flowering N. attenuata 453

plants impaired in GGPPS expression (N=5). The average leaf mass was estimated 454

between 1.3 – 1.6 g for the rosette leaves of WT and the stem leaves of transgenic 455

IRggpps plants. Three leaves of each plant were treated with either 20 µL of DMSO or 456

DMSO with 140, 280, or 9800 nmol 17-HGL. Independent of the concentration of 17-457

HGL, DMSO application resulted in a mild dissolution of the epidermal surface. Analysis 458

of the damaged leaf area, 1 day after application revealed strong necrotic regions at all 459

three 17-HGL concentrations (Figure 8B). Significant increases in leaf damage were 460

detected starting at 140 nmol/g FW for IRggpps plants and 280 nmol/g FW in WT plants 461

(Figure 8C). 462

463

Manduca sexta larvae perform poorly on transformed lines impaired in HGL-DTG 464

glycosylation 465

HGL-DTGs are abundant and potent anti-herbivore defense metabolites in the 466

aboveground tissues of N. attenuata (Heiling et al., 2010). Although 17-HGL has been 467

suggested as a feeding deterrent (Jassbi et al., 2010), which of the many different HGL-468

DTGs or which structural component of HGL-DTGs accounts for the observed deterrent 469

(Jassbi et al., 2006) and resistance effects against herbivores (Snook et al., 1997) 470

remains unknown. To elucidate the defensive value of glucosylated and rhamnosylated 471

HGL-DTGs, we conducted performance assays with M. sexta larvae reared on leaf disks 472

of transgenic N. attenuata plants impaired in rhamnosylation (IRugt91t1) and 473

glucosylation (IRugt74p5, IRugt74p3/ugt74p5) of HGL-DTGs. Additionally, we included 474


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IRggpps, which has been shown to dramatically decrease HGL-DTG levels and enhance 475

growth of M. sexta (Heiling et al. 2010). 476

Caterpillars feeding on IRggpps plants showed enhanced growth after 6 days 477

and gained 1.6-fold higher larval mass after 12 days (P=0.000) compared to larvae 478

feeding on WT leaf disks (Figure 9A). In contrast, M. sexta feeding on leaf disks from 479

IRugt74p5 (~0.6 fold, P=0.028) and IRugt74p3/ugt74p5 (~0.6-fold, P=0.001) plants 480

showed significantly reduced growth compared to larvae feeding on WT leaf disks. 481

Caterpillars fed leaf disks of IRugt91t1 displayed only slight reductions in growth (~0.8 482

fold relative to WT). 483

Additionally, we measured the mass of leaf tissue consumed by M. sexta larvae 484

fed IRggpps, IRugt74p5, IRugt74p3/ugt74p5 and IRugt91t1 leaf disks between days 8 to 485

10 and observed a strong decrease in consumption of IRugt74p5, IRugt74p3/ugt74p5 486

and IRugt91t1 leaf disks relative to WT (Figure 9B). A detailed statistical analysis can be 487

found in Supplemental Data 8. 488

489

490

Discussion 491

492

Plants have evolved highly diversified specialized metabolism pathways to resist 493

both abiotic and biotic stresses as well as a series of mechanisms to mitigate 494

cost/benefit trade-offs of specialized metabolite production and thereby maintain 495

competitive ability. Innovations in specialized metabolism frequently result from the 496

modification or direct recruitment of pre-existing scaffolds produced by core metabolic 497

pathways. These scaffolds serve as substrates for modifying enzymes that add many 498

different types of decorations (Gachon et al., 2005). In this respect, the large numbers of 499

UGTs that populate plant genomes define a versatile glycosylation toolbox that has likely 500

facilitated the functional diversification of secondary metabolism across plant lineages. 501

Here we identified and phylogenetically characterized 107 UDP-502

glycosyltransferases of the superfamily 1 in N. attenuata. We identified three novel 503

UGTs, based on co-expression analysis, which are responsible for the synthesis of 17-504

HGL-DTGs in N. attenuata. UGT74P3 and UGT74P4 are GT-type enzymes that use 505

UDP-glucose to attach the first glucose moieties to the C-3 and C-17 hydroxyl groups of 506


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17-HGL. Additionally, we show that the glucosylation of the 17-HGL aglycone is crucial 507

to prevent an autotoxic effect of 17-HGL accumulation, which causes severe necrosis of 508

the leaves. Collectively, this functional study provides novel insights into the 509

biosynthesis of broad-spectrum anti-herbivore HGL-DTGs and provides evidence 510

consistent with the hypothesis that UGTs play a central role in a plant’s ability to manage 511

the “toxic waste dump” problem of chemical defense. 512

513

A geranyllinalool synthase (GLS) is required for HGL-DTG production 514

Geranyllinalool (GL) is an acyclic diterpene alcohol which is widely distributed 515

across the plant kingdom, occurring in several essential oils (Sandeep and Paarakh, 516

2009) and is a precursor for both HGL-DTGs in solanaceous species and for the volatile 517

C16-homoterpene 4,8,12-trimethyltrideca-1,2,7,11-tetraene (TMTT) which is emitted from 518

the foliage of a wide range of plant species including Solanum lycopersicum (Ament et 519

al., 2004), Zea mays (Hopke et al., 1994), Medicago truncatula (Leitner et al., 2010) and 520

A. thaliana (Van Poecke et al., 2001). Although geranyllinalool is present in many plant 521

species, the enzymes responsible for its biosynthesis have been discovered only 522

recently in A. thaliana (Herde et al., 2008), S. lycopersicum, and N. attenuata in which 523

GLS is constitutively expressed in leaf and flower tissues and is induced by methyl 524

jasmonate treatment (Falara et al., 2014). Based on the tissue-specific correlation of 525

GLS transcript abundance with the levels of HGL-DTGs in reproductive organs, the 526

authors predicted that NaGLS is involved in the biosynthesis of HGL-DTGs (Falara et 527

al., 2014). Here we provide additional evidence demonstrating its coordinated 528

expression with other HGL-DTG biosynthetic genes (GGPPS, UGT91T1 and UGT74P3) 529

and demonstrate that HGL-DTG levels are highly reduced after the transient silencing of 530

NaGLS. 531

532

Gene-to-gene co-expression analysis identifies three novel UGTs responsible for 533

the glycosylation of HGL-DTGs 534

To identify the UGTs responsible for the glycosylation of HGL-DTGs, we mined 535

the expression profiles of 76 UGTs in a full-transcriptome datasets created for leaf and 536

roots collected from plants after simulated herbivory. More specifically, we explored co-537

linearity patterns in the regulation of this set of UGTs and GGPPS and GLS which are 538


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involved in upstream steps of the biosynthesis of 17-HGL. A previous study by our group 539

(Gulati et al., 2013) detected, using the same transcriptome datasets, a tight co-540

regulation between genes of the non-mevalonate pathway (NaDXS, NaDXR) and 541

NaGGPPS. Our analysis pinpointed three novel glycosyltransferases strongly correlated 542

with GGPPS and GLS, which were not identified in the previous study. Gulati and 543

colleagues analyzed tissue-specific gene-to-gene and gene-to-metabolite associations 544

using fold-change data in response to herbivory and used very stringent statistical 545

parameters. Here, we used a simpler co-expression approach relying on overall gene-to-546

gene correlations across all tissues to identify significant correlations in expression 547

dynamics among UGTs and previously characterized upstream genes. Furthermore, a 548

detailed analysis of the expression pattern of each of the 76 UGTs (Supplemental Figure 549

4) revealed that herbivory has dramatic effects on the local induction of UGT expression, 550

an observation consistent with the importance of glycosylation reactions in maintaining 551

cellular homeostasis after damage. 552

Together with the three candidate UGTs identified in N. attenuata, we also 553

identified and biochemically characterized orthologues in N. obtusifolia whose genome 554

was recently published along with that of N. attenuata (Xu et al., 2017). 555

556

UDP-Rha: 17-HGL-DTG rhamnosyltransferase activity of UGT91T1 557

Very little is known about the physiological function of rhamnosylation in 558

secondary metabolism. For example, Hsu and colleagues (Hsu et al., 2017) suggested 559

that rhamnosylation is an essential step in the synthesis of lobelinin and therefore 560

responsible for the color of Lobelia flowers. Rhamnosylation of flavonols is thought to 561

modulate auxin homeostasis in rol1-2 mutants of A. thaliana (Kuhn et al., 2016). 562

However, the enzymatic characterization of UDP-rhamnosyltransferases is still thwarted 563

by the high costs of UDP-rhamnose (UDP-Rha). Despite important efforts in the 564

establishment of efficient UDP-Rha production systems (Irmisch et al., 2018), only a few 565

UDP-rhamnosyltransferases have been functionally characterized and enzymatic assays 566

remain challenging (Mo et al., 2016; Irmisch et al., 2018). 567

Here we show that UGT91T1 shares a high degree of sequence similarity with 568

functionally characterized rhamnosyltransferases for flavonoids and anthocyanins 569

(CmF7G12RT, GmF3G6R and PhA3ART – Supplemental Figure 6), is tightly and tissue-570


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specifically co-regulated with the accumulation patterns of rhamnosylated HGL-DTGs 571

and exhibits strong co-expression with GLS, GGPPS and UGT74P3. Silencing of 572

UGT91T1 expression in N. attenuata results in the almost complete loss (88.5% - 98.8% 573

in different tissues compared to WT, Supplemental Table 8) of rhamnosylated HGL-574

DTGs. These changes in HGL-DTG rhamnosylation pattern suggest that UGT91T1 is 575

responsible for the rhamnosylation at the C’-4 hydroxyl-group of glucose on both the C-3 576

and the C-17 hydroxyl-group of the aglycone. Due to the high biological variability 577

among transiently-silenced plants and the complexity of the HGL-DTG profile in N. 578

obtusifolia, which produces lower levels of rhamnosylated HGL-DTGs, the biochemical 579

function of NoUGT91T1-like, the orthologue of UGT91T1 in N. obtusifolia, could not be 580

conclusively evaluated. 581

582

UGT74P3 and UGT74P4 function as UDP-Glu: 17-HGL-DTG glucosyltransferases 583

Both UDP-glycosyltransferases clustered together in the UGT74 family, which 584

belongs to phylogenetic group L and includes glycosyltransferases that are responsible 585

for the glucosylation of indole-3-acetic acid in Zea mays (Szerszen et al., 1994) and A. 586

thaliana (Jin et al., 2013). Importantly, additional members of this family show catalytic 587

activity towards diterpene and triterpene glycosides. For example, UGT74M1 588

glucosylates the carboxylic acid moiety of the triterpene gypsogenic acid 589

(Meesapyodsuk et al., 2007), UGT74-345-2 is involved in the glucosylation of mogroside 590

(Itkin et al., 2016, 2018), and UGT74G1 catalyzes the glucosylation of cyclic diterpene 591

glycosides in Stevia rebaudiana (Richman et al., 2005). Both UGT74P3 and UGT74P4 592

are closely related to UGT74G1 and CsGLT2, a UGT responsible for crocetin 593

glucosylation in Crocus sativus (Moraga et al., 2004). The expression profile of 594

UGT74P3 in N. attenuata revealed a strong tissue-specific correlation with HGL-DTG 595

accumulation and GLS expression. 596

Recombinant enzyme activity assays demonstrated that UGT74P3 and UGT74P4 597

encode enzymes that catalyze the transfer of glucose to the C-3 and C-17 hydroxyl 598

groups of 17-HGL in order to form the putative intermediate products 3-O-599

glucopyranosyl-17-HGL (G-3-HGL) and 17-O-glucopyranosyl-17-HGL (G-17-GHL) 600

(Figure 4), as well as lyciumoside I in which both hydroxyl groups are glucosylated. 601

Neither UGT74P3 nor UGT74P4 showed activity producing lyciumoside II, which 602


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includes an additional glucose moiety linked to the C’-2 hydroxyl group of the glucose at 603

the C-17 hydroxyl group of 17-HGL, or other larger HGL-DTGs with additional sugar 604

moieties. However, the functional characterization of enzymes in the UGT family can be 605

very challenging and in vitro enzyme assays do not necessarily reflect native metabolic 606

activities in planta. 607

Metabolic profiling of transiently- and stably-silenced lines provided further 608

evidence that UGT74P3 and UGT74P4 are essential for the formation of lyciumoside I 609

through the attachment of two glucose moieties. Silencing of UGT74P3 and UGT74P4 610

expression significantly reduces the amount of lyciumoside I and its malonylated forms 611

in leaf tissue material. Interestingly, compounds further downstream in the HGL-DTG 612

biosynthetic pathway (lyciumoside II, lyciumoside IV, attenoside) are more affected in 613

the transiently than in the stably–silenced lines, which might be due to the infection with 614

the virus vector and a possible involvement of HGL-DTGs in the immune response of 615

plants against viruses (Ramegowda et al., 2014). In the stable lines impaired in 616

UGT74P3 expression, we observed a slight reduction of lyciumoside IV, but an increase 617

of attenoside and lyciumoside II, indicating that the downstream model of the HGL-DTG 618

biosynthetic pathway is incomplete and that other UGTs are involved in the glycosylation 619

of larger molecular weight HGL-DTGs. 620

In addition to the changes in known HGL-DTG patterns, we also observed an 621

accumulation of novel intermediate HGL-DTGs that lacked either glucosylation at the C-622

3 or C-17 hydroxyl group of the aglycone as well as the 17-HGL aglycone itself in both 623

stably- and transiently-silenced lines. This suggests that N. attenuata is unable to 624

reroute the excess of 17-HGL and provides us with the opportunity to study the effects of 625

glucosylation of HGL-DTGs in vivo. 626

In contrast to UGT74P3 and UGT74P4, we were not able to decipher the function 627

of UGt74P5 in N. attenuata and of its orthologue in N. obtusifolia. The high sequence 628

similarity between UGT74P3 and UGT747P5 of ~83%, indicated a related function as a 629

UGT. Furthermore, UGT74P5 expression highly correlates with that of all other genes 630

related to HGL-DTG biosynthesis (UGT74P3, UGT91T1, GLS and GGPPS), exhibits a 631

similar temporal dynamic after simulated insect herbivory and shows very low transcript 632

abundance in roots. UGT74P5 showed no activity towards the 17-HGL aglycone, 633

lyciumoside I or the putative intermediates G-3-HGL and G-17-HGL, indicating that 634


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UGT74P5 might not directly contribute to the biosynthesis of HGL-DTGs or be 635

responsible for the glucosylation of HGL-DTGs that were not tested in these enzyme 636

assays. 637

The co-silencing of UGT74P5 and UGT74P3 expression in the stable and 638

transiently silenced lines (Figure 6, Supplemental Figure 8), likely due to the high 639

sequence similarity of both UGTs, did not allow us to further characterize the specific 640

function of UGT74P5. The metabolic alterations of these lines likely reflect the silencing 641

of both genes. Based on the enzymatic assays, we inferred that the reduction of 642

lyciumoside I and the accumulation of G-3-HGL, G-17-HGL and 17-HGL directly result 643

from the silencing of UGT74P3, leaving it unclear whether UGT74P5 is involved in the 644

synthesis of HGL-DTGs, is non-functional or has a still-unknown enzymatic function 645

beyond the diterpene metabolism. 646

647

Phytotoxicity observed in UGT74P3/UGT74P5 silenced lines 648

In addition to the striking shifts in HGL-DTG metabolism detected in the stably- and 649

transiently-silenced lines impaired in UGT74P3 and UGT74P5 expression, we also 650

observed strong morphological alterations that ranged from small deformed or thicker 651

succulent leaves to numerous stalled flower buds as well as necrotic spots, stunted 652

growth and apical meristem necrosis. 653

The loss of glycosylation results in the ectopic accumulation of hydrophobic 654

aglycones, which are known to be responsible for a wide range of morphological 655

alterations and growth retardation effects. Some of the best examples, come from the 656

biosynthesis of steroidal alkaloids. Silencing GAME-1, a UDP-galactosyltransferase 657

responsible for the glycosylation of steroidal alkaloids, results in severe developmental 658

defects due to the altered sterol composition in membranes triggered by the 659

accumulation of tomatidine (Itkin et al., 2011). Moreover, alterations of the glycosylation 660

of the saponin, hederagenin, severely affected growth in M. truncatula (Naoumkina et 661

al., 2010). Loss of function mutants, sad3 and sad4, in Avena sativa accumulate the 662

intermediate monodeglucosylated diterpeneglycoside, avenacin A-1, which disrupts 663

membrane trafficking and results in epidermal degeneration and reduced root hair 664

formation (Mylona et al., 2008). Knocking out UGT74B1, a UGT responsible for 665

glucosinolate biosynthesis in A. thaliana, leads to the accumulation of toxic levels of 666


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thiohydroximates, increased auxin levels in seedlings and results in a chlorotic 667

phenotype (Grubb et al., 2004). 668

The severe growth defects, stalled flower buds and necrosis we observed in 669

UGT74P3/UGT74P5-silenced plants may have several explanations. First blocking 670

HGL-DTG glycosylation might increase the endogenous pool of UDP-glucose, resulting 671

in a preferential synthesis of other glycosides or the accumulation of other compound 672

classes (Naoumkina et al., 2010). This might influence hormone homeostasis and 673

therefore contribute to the severe alterations (Grubb et al., 2004; Kuhn et al., 2016). 674

Auxin levels, which have been suggested to mediate some of these observed 675

developmental alterations, are only changed in GGPPS-silenced plants but not in the 676

UGT-silenced lines showing the morphological defects. Alterations of the UDP-glucose 677

pool can also influence UDP-glucuronic acid (UDP-GlcA) biosynthesis. Reduction of 678

UDP-GlcA in ugd2 and ugd3 mutants of A. thaliana leads to swollen cell walls and 679

developmental defects associated with changes in the pectic network (Reboul et al., 680

2011). IRugt74p5 and IRugt74p3/ugt74p5 plants exhibit lower glucuronic acid levels 681

(Supplemental Data 6), suggesting that UDP-GlcA production is downregulated, which 682

could mediate the observed smaller leaves and the dwarfish growth phenotype. 683

However, we observed a similar reduction in glucuronic acid levels in GGPPS-silenced 684

plants, which do not show the altered growth effects. Alternatively, UGT74P3/UGT74P5-685

silenced plants also exhibited a dramatic increase in levels of intermediate HGL-DTGs 686

and the aglycone 17-HGL compared to other transgenic lines that did not show any 687

morphological phenotypes (Figure 8A). From these results we infer that the 688

accumulations of these intermediates may be toxic for the plant. 689

When UGT74P3 and UGT74P5 were silenced in the background of stably-690

silenced IRggpps lines, which do not produce the 17-HGL precursor, no developmental 691

abnormalities were detected, indicating that the developmental abnormalities are 692

associated with the altered HGL-DTG metabolism and not due to unexpected off-target 693

effects (Senthil-Kumar and Mysore, 2011). Applications of a synthetic mixture of 17-HGL 694

isomers to leaves of N. attenuata resulted in necrotic lesions that pheno-copied those 695

observed in IRugt74p5 and IRugt74p3/ugt74p5 plants. IRggpps plants, which are 696

strongly down-regulated in the expression of HGL-DTG biosynthetic genes and HGL-697

DTG accumulation, were more susceptible to the toxic effects of exogenous applications 698


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of 17-HGL than WT plants and showed necrotic lesions in response to 17-HGL 699

concentrations lower than those observed in IRugt74p3/ugt74p5 plants. The lower toxic 700

effects observed in WT leaves may be due to the chemical sequestration of 17-HGL and 701

intermediate HGL-DTGs by active glycosylation with the HGL-DTG pathway. 702

Interestingly, N. sylvestris accumulates 17-HGL without producing the glycosylated 703

forms (Heiling et al., 2016), suggesting that other species might be tolerant to or store 704

17-HGL in special compartments. In short, silencing UGT74P3 and UGT74P5 results in 705

pleiotropic morphological affects that are associated with the accumulation of the 706

phytotoxic aglycone 17-HGL. 707

708

Defensive function of HGL-DTGs 709

HGL-DTGs have been studied for more than two decades and are described to be 710

potent anti-herbivore defense compounds (Heiling et al., 2010) with deterrent (Jassbi et 711

al., 2006) and resistance (Snook et al., 1997) effects against herbivores. However, so far 712

it remains unclear which of the many HGL-DTGs, their structural components or post-713

ingestive modifications account for their mode of action. The impressive structural 714

diversity of HGL-DTGs in N. attenuata plants results largely from glycosylation and 715

malonylation reactions. While all malonyl decorations are instantaneously lost in the 716

alkaline pH environment of the midgut of M. sexta larvae and therefor may play more 717

important roles in planta (Poreddy et al., 2015; Li et al., 2018), glycosylation leads to a 718

constant and stable pool of potential defensive compounds. Larvae feeding on leaf disks 719

of transgenic IRggpps plants grow larger than larvae feeding on WT leaves, indicating 720

that the overall abundance of HGL-DTGs is a major factor of the plant’s resistance 721

against M. sexta (Figure 9), consistent with earlier studies (Jassbi et al., 2008; Heiling et 722

al., 2010; Jassbi et al., 2010). The reduced growth of larvae feeding on leaf disks of 723

transgenic IRugt74p5 and IRugt74p3/ugt74p5 plants, which have higher total levels of 724

HGL-DTGs, is also consistent with the inference that the total abundance of HGL-DTGs 725

is defensively relevant. For example, N. obtusifolia is a perennial plant, which co-occurs 726

with the annual N. attenuata in the Great Basin Desert and is much less attacked by the 727

tobacco hornworm M. sexta. This species lacks N. attenuata’s strong OS-elicited 728

signaling system and hence produces attenuated jasmonate bursts and trypsin 729

proteinase inhibitors (TPI) accumulations (Pearse et al., 2006; Anssour and Baldwin, 730


https://doi.org/10.1101/2020.08.26.267690

2010; Xu et al., 2015). Furthermore, N. obtusifolia has constitutive levels of HGL-DTGs 731

that are five times higher than those of N. attenuata and M. sexta larval performance is 732

five times lower after 22 days feeding on N. obtusifolia (Jassbi et al., 2010). However, 733

the HGL-DTG profile in N. obtusifolia is structurally different (Heiling et al., 2016), 734

suggesting that the composition or degree of glycosylation is central for the plant’s 735

defense. 736

Importantly, the specific structures responsible for the mode of action of HGL-737

DTGs remain unclear. Feeding of purified lyciumoside IV, the most abundant HGL-DTG 738

in the leaves of N. attenuata, causes mortality in M. sexta larvae silenced in β-739

glucosidase 1 (BG1) expression (Poreddy et al., 2015). However, the single-compound 740

feeding experiment does not reflect the chemical diversity of HGL-DTGs that is normally 741

consumed by larvae when they feed on plants. In contrast to the results of Poreddy and 742

colleagues, we show that, while consuming less leaf tissue, M. sexta larvae grow 743

normally when feeding on leaf disks of transgenic IRugt91t1 plants, which have reduced 744

levels of lyciumoside IV, but increased levels of the non-rhamnosylated HGL-DTGs 745

lyciumoside I and II. Furthermore, we observed increased levels of novel intermediate 746

HGL-DTGs and the aglycone, 17-HGL, in IRugt74p5 and IRugt74p3/ugt74p5 plants, 747

from which we observed a strong antifeedant/deterrent effect, which showed a strong 748

antifeedant/deterrent effect (Figure 9B) and reduced growth performance of M. sexta 749

larvae (Figure 9A). The differential consumption and growth performance responses on 750

the transgenic plants with different HGL-DTG profiles suggest that both pre- and post-751

ingestive resistance mechanisms may be at play. Given the severe metabolic and 752

pleiotropic morphological phenotypes of IRugt74p5, IRugt74p3/ugt74p5 and IRggpps 753

plants, it is challenging to draw strong inferences about the defensive function of specific 754

intermediates in the HGL-DTG biosynthetic pathway. 755

We are still in the early stages of understanding how plants solve the “toxic waste 756

dump” problem. Two common solutions are well documented: 1) producing metabolites 757

(e.g., nicotine) that are specifically toxic to tissues or organs that plants lack (e.g., 758

nervous systems and neuro-muscular junctions populated with nicotinic acetylcholine 759

receptors); and 2) sequestering pro-toxins apart from their toxin-releasing lytic enzymes 760

(e.g., compartmentalization of cyanogenic glycosides and glucosinolates and their active 761

enzymes). However, these examples are likely to be special cases and the solutions that 762


https://doi.org/10.1101/2020.08.26.267690

plants have evolved to solve the “toxic waste dump” problem for a majority of their toxic 763

defensive metabolites lie in the details of their biosynthetic pathways. Here we advance 764

our understanding of the toxicity of HGL-DTGs by showing that glucosylation plays a 765

central role in N. attenuata’s solution of maintaining an HGL-DTG-based defense. 766

Extending the metabolomics analyses to the “digestive duet” that occurs between plant 767

and insect (“frassomics”) will allow for a better understanding of the post-ingestive fate of 768

DTGs in larval guts and will help to answer a central and festering question that remains 769

unanswered from this work: are the specific molecules that are responsible for the 770

defensive function of HGL-DTGs the same as those responsible for the clear 771

autotoxicity? 772

773

Methods 774

775

Plant material and growth conditions 776

777

Seed germination and growth condition have been described previously (Krugel et al., 778

2002). Seeds of the 31th generation of an inbred line of N. attenuata Torr. Ex. Watts 779

were used as wild type plants in all experiments. N. obtusifolia plants were cultivated 780

under the same growth conditions with the exception of not applying liquid smoke to the 781

seeds. Plants for virus-induced gene-silencing were transferred after 20 days to a York 782

Chamber with 22°C for a 16h light/ 8h dark cycle. 783

784

M. sexta growth conditions 785

786

M. sexta eggs were obtained from an in-house colony in which insects are reared 787

in a growth chamber (Snijders Scientific, Tilburg, Netherlands, 788

http://www.snijderslabs.com) at 26°C:16-h light and 24°C:8-h dark, 65% relative 789

humidity, until hatching. 790

791

Performance Assays 792

793


https://doi.org/10.1101/2020.08.26.267690

Freshly hatched neonates were fed leaf disk material, taken from the between-794

vein laminal tissue of the four lowest stem leaves of 43-day-old flowering transgenic N. 795

attenuata plants (WT, IRugt91t1, IRugt74p5, IRugt74p2/ugt74p5, IRggpps), in round 796

plastic PE-packing cups. Leaf disk material was exchanged every 2 days until day 6 and 797

then exchanged every day until day 12. 798

799

Plant transformation and screening of stably-silenced N. attenuata plants 800

801

Transformation of N. attenuata was performed as described in Krugel et al. 802

(2002) using the pRESC8 vector (Gase and Baldwin, 2012) containing the hygromycin 803

phosphotransferase II gene (hptII) from pCAMBIA-1301 (GenBank AF234297) and a 804

306 bp long fragment for UGT91T1, a 310 bp long fragment for UGT74P3 or a 295 bp 805

long fragment for UGT74P5. All primers are shown in supplemental table 9. Diploid 806

plants were selected by flow cytometry of leaf material of elongated N. attenuata 807

transformants performed on a CCA-II flow cytometer (Partec, http://www.partec.com) as 808

described by Bubner et al. (Bubner et al., 2006). Afterwards, seeds were collected and 809

individuals with the T-DNA insertion were selected for hygromycin resistance by addition 810

of 35 mg/L hygromycin B to the germination medium. After 10 days, the ratio of 811

seedlings surviving the antibiotic treatment was determined. Seedlings were chosen with 812

a survival rate of 50 – 90% and 12 T1 plants per line were checked for T-DNA insertion. 813

To confirm the integrity of the T-DNA insertion, we performed a diagnostic PCR using 814

the primer pairs PROM FOR/INT REV and INT FOR/TER REV (Gase et al., 2011). 815

Genomic DNA (gDNA) was isolated from leaves of N. attenuata using a modified 816

cetyltrimethylammonium bromide method (Bubner et al., 2004). PCR was performed 817

using DreamTaqTM DNA-Polymerase (Fermentas, http://www.fermentas.com) according 818

to the instructions of the manufacturer with 1 µg of gDNA. Homozygosity of T2 plants 819

was determined by screening for resistance to hygromycin B. To confirm single 820

insertions, we performed a Southern blot as described by Jassbi et al. (Jassbi et al., 821

2008), with the exception that a 287 bp hptII probe obtained by PCR with primer pair 822

(HYG1-18/HYG2-18) was used (Gase et al., 2011). Labelling was performed with the 823

GE Healthcare (http://www.gehealthcare.com) Readyprime DNA labelling system and 824

ProbeQuant g-50 microcolumn according to the manufacturer’s protocol. 10.5 µg of 825


https://doi.org/10.1101/2020.08.26.267690

gDNA was digested with the restriction enzymes EcoRV and XbaI from New England 826

Biolabs (http://www.neb.com) and blotted onto a nylon membrane (GeneScreenPlus; 827

Perkin Elmer, http://www.perkinelmer.com) according to the manufacturer’s instructions. 828

829

Virus-induced gene-silencing (VIGS) 830

831

Vector construction, plant growth, and inoculation conditions were as described 832

by Saedler and Baldwin (Saedler and Baldwin, 2004). Briefly, 200- to 300-bp fragments 833

of N. attenuata and N. obtusifolia target genes were amplified by PCR using primer pairs 834

as listed in supplemental table 9. Amplified fragments were cloned in the vector pTV00 835

(Ratcliff et al., 2001). Agrobacterium tumefaciens strain GV3101 was transformed by 836

electroporation with the resulting plasmids. We used the empty pTV00 vector as a 837

negative control in all experiments. Four leaves of 24-27 day old N. attenuata and 25 838

day old N. obtusifolia plants were infiltrated with a 1:1 mixture of A. tumefaciens 839

transformed with pBINTRA (Ratcliff et al., 2001) and one pTV00 derivative carrying a 840

fragment of a gene of interest. pTVPDS, targeting for a Phytoene desaturase, was used 841

as a positive control to monitor silencing progress. Due to the depletion of carotenoids, 842

silencing the phytoene desaturase gene causes bleaching of tobacco leaves. VIGS-843

silenced plants were treated 14 days after inoculation, when the bleaching phenotype 844

was fully established in the pTVPDS plants. 845

846

Plant treatment 847

848

In order to analyze the regulatory function of jasmonate signaling on HGL-DTG 849

biosynthesis, petioles of five elongated plants (38-days-old) were treated with either 20 850

µL lanolin paste containing 150 µg methyl jasmonate (Lan + MeJA) or with 20 µL pure 851

lanolin (Lan). Treated leaves were harvested from elicited and unelicited plants at 72 h 852

after treatment, flash-frozen in liquid nitrogen, and stored at -80°C until use. 853

854

Determination of 17-HGL phytotoxicity 855

856


https://doi.org/10.1101/2020.08.26.267690

For the determination of the phytotoxic effect of 17-HGL, we used 32-day-old 857

early-elongated N. attenuata WT plants (N=3) and 48-day-old flowering N. attenuata 858

plants impaired in GGPPS expression (N=5). Three leaves of each plant were 859

inoculated with either 20 µL DMSO, DMSO with 140 nmol HGL, DMSO with 280 nmol 860

HGL or DMSO with 9800 nmol HGL. The damaged leaf tissue was analyzed after 24 h 861

using ImageJ (Fiji, https://fiji.sc). 862

863

RT-qPCR analysis of transcript levels 864

865

Total RNA was extracted from an aliquot of approximately 200 mg of powdered 866

leaf material of N. attenuata and N. obtusifolia ground in liquid nitrogen following the 867

protocol of Kistner and Matamoros (Kistner and Matamoros, 2005). DNase treatment 868

was performed using the TURBO DNA-free™ kit (Invitrogen). RNA quality was checked 869

on a 1% agarose gel and the concentration was determined spectrophotometrically at 870

260 nm. A total of 1 µg of DNA-free RNA was reverse transcribed using oligo(dT)18 871

primers and the SuperScript II enzyme (Invitrogen) following the manufacturer’s 872

recommendations. All RT-qPCR assays were performed using TaykonTM No ROX 873

SYBR® Master Mix dttp Blue (Eurogenetics, http://www.eurogentec.com) on a 874

Stratagene MX3005P instrument (http://www.stratagene.com) as recommended by the 875

manufacturer. To normalize transcript levels, primers specific for the Nicotiana attenuata 876

elongation factor-1α gene (EF1-α; accession no. GBGF01000210.1) were used. Specific 877

primers in the 5′ to 3′ direction used for SYBR Green-based analyses are listed in 878

supplemental table 9. 879

880

Heterologous expression of UDP-glucosyltransferases and enzymatic activity 881

assays 882

883

The four UGT cDNAs coding for NaUGT74P3, NoUGT74P4, NaUGT74P5 and 884

NoUGT74P6 were cloned into a Gateway® pDEST17 expression vector 885

(ThermoFisherScientific, http://www.thermofisher.com) using pET28a empty vector as 886

control. Integrity of the sequence was checked by Sanger-sequencing using an ABI 887


https://doi.org/10.1101/2020.08.26.267690

PRISM 3130 Genetic analyzer (AppliedBiosystems, http://www.thermofisher.com) and 888

the appropriate gene-specific primers (Supplemental Table 9). 889

The resulting plasmid was transformed into BL21 (DE3) E. coli which is optimized 890

for the expression of eukaryotic genes. 50 mL LB medium containing 50 µg/mL 891

carbenicillin were inoculated with 500 µL of an overnight culture corresponding to each 892

candidate gene. Cultures were grown at 37°C until the OD600 reached 0.6. Protein 893

expression was induced by adding 1 mM IPTG and incubation at 18 °C overnight. The 894

cells were harvested by centrifugation for 10 min at 4,500 × g. The pellet was 895

suspended in 10 mL ice cold lysis buffer (50 mM Tris HCl pH 7.5, 1% Triton 100, 200 896

mM NaCl, 1 mg/ml lysozyme, and 1 tablet of Protease inhibitor Cocktail (Roche)) and 897

sonicated 6 times for 10 s with 10 s pauses at 200-300 W, followed by centrifugation at 898

10,000 × g for 60 min. The supernatant was purified using Ni-NTA agarose (QIAGEN) in 899

accordance with the manufacturer’s instructions. The purified protein was desalted using 900

the Amicon Ultra-15 Centrifugal Filter Unit (Merck) and the desalted protein was used for 901

activity assays. Reactions were performed in 100 μL reaction volumes containing 100 902

mM Tris HCl pH 7.5, 2.5 µg protein, 5 mM UDP-α-D-glucose (Calbiochem, 903

http://www.merckmillipore.com) and 250 µg/mL 17-hydroxygeranyllinalool, for 3 h at 904

30°C. The reaction was stopped by adding 400 µL methanol, and the mixture was used 905

for HGL-DTG analysis using a high resolution time-of-flight mass spectrometer. 906

907

Quantification of primary metabolites and phytohormones in plant tissues 908

909

For the quantitative analysis of primary metabolites and phytohormones, we used 910

leaf material of 42-day-old elongated N. attenuata transgenic lines silenced in GGPPS, 911

UGT91T1, UGT74P5 and UGT74P3/UGT74P5 expression as well as WT plants. 912

Sample preparation and analysis of primary metabolites and phytohormones were 913

performed based on Schaefer et al. (Schafer et al., 2016). Peak integration was 914

performed using the operating Software MS Workstation (Bruker Daltonics). For the 915

quantitative analysis of GPP, FPP and GGPP, we followed the protocol developed by 916

Nagel et al. (Nagel et al., 2014). 917

918

Quantification of 17-hydroxygeranyllinalool in plant tissues 919


https://doi.org/10.1101/2020.08.26.267690

920

Approximately 50 mg of leaf material was flash frozen and ground in liquid 921

nitrogen and then aliquoted. Each aliquot was extracted with 500 µL 80% methanol 922

containing 0.2 ng/µL testosterone, and shaken twice at 1000 strokes for 45 sec using a 923

GenoGrinder 2000 (SPEX SamplePrep, http://www.spexsampleprep.com/). 924

Homogenized samples were then centrifuged at 16 000 × g for 20 min at 4°C. The 925

supernatant was centrifuged again at 16 000 × g for 20 min at 4°C and then diluted 1:10 926

with 80% methanol. We established a chromatographic method using a mixture of 927

solvent A: water (Milli-Q, Merck, http://www.emdmillipore.com) with 0.1% acetonitrile and 928

0.05% formic acid and solvent B: methanol. U(H)PLC for the quantification of 17-HGL 929

was performed using a Zorbax Eclipse XDB-C18 column (particle size 1.8 µm, column 930

length 3.0 × 50 mm) from Agilent Technologies (http://www.agilent.com). The 931

chromatographic separation was achieved using a U(HPLC) Advance (Bruker Daltonics) 932

with the following gradient: 0-0.5 min at 10% of B, 0.5-1 min up to 90% of B, 1-4 min up 933

to 100% of B, 4-5 min at 100% of B, 5-5.05 min down to 10% of B and from 5.05-6 min 934

at 10% of B. The injection volume was 5 µL and the flow rate 0.5mL min-1. 935

MS detection was performed on an EvoQ Elite QqQ-MS equipped with a HESI 936

(heated electrospray ionization) ion source (Bruker Daltonics) and the following HESI 937

conditions: spray voltage 4500 V, cone temperature 350°C, cone gas flow 35a (arbitrary 938

units), heated probe temperature 500°C, probe gas flow 60a and nebulizer gas 60a. 939

Compounds were detected in multiple reaction monitoring (MRM) mode using specific 940

precursor ion/product ion after positive ionization: the [M-H2O+H]+ ion was used as 941

precursor for 17-HGL, 289/81 (quantifier), 289/107 (qualifier), the [M+H]+ ion was used 942

as precursor for testosterone, 289/97 (quantifier), 289/109 (qualifier). Further details are 943

given in supplemental figure 23. The areas were analyzed using the MS Workstation 944

operating software from Bruker Daltonics (http://www.bruker.com). 945

946

Structural determination by nuclear magnetic resonance spectroscopy (NMR) 947

948

17-Hydroxygeranyllinalool was provided by HPC24 Standards (www.hpc-949

standards.com) and the general structure was verified by 1D and 2D NMR spectroscopy 950

(for 1H NMR, see Supplemental Figure 12). A Bruker AVANCE 400 NMR spectrometer 951


https://doi.org/10.1101/2020.08.26.267690

(Bruker, Rheinstetten, Germany), equipped with a 5 mm BBFO probe, was used to 952

record 1H NMR, DEPT 135, 1H-1H COSY, HSQC and HMBC spectra in MeOH-d4 at 300 953

K. Spectra were processed using TOPSPIN 3.1. (BrukerBiospin). 954

955

Rapid screening of HGL-DTGs via ultrahigh-pressure liquid chromatography/time 956

of flight mass spectrometry 957

958

All materials were ground in liquid nitrogen and split into aliquots of 10-100 mg 959

fresh weight (FW), dependent on the tissue. Each aliquot was extracted in 100 µL - 1 mL 960

extraction solution (80% methanol; ratio 1/10 FW/extraction solution) containing two 961

steel balls by shaking twice at 1200 strokes/min for 60 sec using a Geno/Grinder 2000. 962

Homogenized samples were then centrifuged at 16 000 × g for 20 min at 4°C. The 963

supernatant was centrifuged again at 16 000 × g for 20 min at 4°C. Two independent 964

chromatographic methods were used to resolve HGL-DTGs. Both methods used a 965

mixture of solvent A: water with 0.1% acetonitrile and 0.05% formic acid and solvent B: 966

acetonitrile and 0.05% formic acid. U(H)PLC for method A was performed using a 967

Dionex UltiMate 3000 rapid separation LC system (Thermo Fisher, 968

http://www.thermofisher.com), combined with a Thermo Acclaim RSLC 120 C18 column 969

(particle size 2.2 µm, average pore diameter 120Å, column dimension 2.1 × 150 mm). 970

Gradient elution steps were as follows: 0-0.5 min at 10% of B, 0.5–6.5 min up to 80% of 971

B and 6.5–8 min at 80% of B followed by returning to the starting conditions and column 972

equilibration. For method B sample gradient steps were as follows: 0–3 min at 10% B, 973

3–12 min up to 20% B, 12–17 min up to 35% B, 17–23 min up to 40% B, 23–25 min up 974

to 45% B, 25–30 min up to 50% B, 30–40 min up to 90% B and 40–45 min at 90% B, 975

followed by returning to the starting conditions and column equilibration. The injection 976

volume was 2 µL and the flow rate 0.4 mL/min for method A and B. 977

MS detection was performed using a micrOTOF-Q II, an Impact II and a maXis 978

UHR-Q-TOF-MS system (Bruker Daltonics) equipped with an electrospray ionization 979

(ESI) source operating in positive ion mode. ESI conditions for the micrOTOF-Q II 980

system were: end plate offset 500 V, capillary voltage 4500 V, capillary exit 130 V, dry 981

temperature 180°C and a dry gas flow of 10 L min-1. ESI conditions for the Impact II 982

UHR-Q-TOF-MS system were capillary voltage 4500 V, end plate offset 500 V, nebulizer 983


https://doi.org/10.1101/2020.08.26.267690

2 bar, dry temperature 200°C and a dry gas flow of 8 L min-1. ESI conditions for the 984

maXis UHR-Q-TOF-MS system were capillary voltage 4500 V, end plate offset 500 V, 985

nebulizer 1.8 bar, dry temperature 200°C and a dry gas flow of 8 L min-1. MS data were 986

collected over a range of m/z from 100 to 1600. Mass calibration was performed using 987

sodium formate (50 mL isopropanol, 200 µL formic acid, 1 mL 1 M NaOH in water). Data 988

files were calibrated using the Bruker high-precision calibration algorithm. Lock mass 989

calibration was performed for the profiling of the stable lines using signal m/z 622.0289 990

(molecular formula C12H19F12N3O6P3) from the ESI Tuning Mix (Agilent Technologies, 991

http://www.agilent.com). MS/MS experiments were performed using AutoMS/MS runs at 992

various CID voltages from 12.5 to 22.5 eV for ammonium adducts. Instrument control, 993

data acquisition and reprocessing were performed using HyStar 3.1 (Bruker Daltonics). 994

Molecular formulae were determined using SmartFormula 3D. SmartFormula calculates 995

the elemental compositions from accurate mass as well as the isotopic pattern 996

information using MS (SmartFormula) and MS + MS/MS information (SmartFormula 3D) 997

(Krebs and Yates, 2008; Kind and Fiehn, 2010). The mass tolerance was set to 4 mDa, 998

and the filter H/C element ratio was set between 1 and 3. Isotope peaks were assigned 999

using the Simulate Pattern Tool of the DataAnalysis software version 4.2 (Bruker 1000

Daltonics). We used QuantAnalysis (Bruker Daltonics) to integrate the peak areas. 1001

1002

Dereplication of HGL-DTGs 1003

1004

The dereplication workflow relies on a comprehensive MS and MS/MS database 1005

constructed by our group for HGL-DTGs of several solanaceous species (Heiling et al., 1006

2016) and a detailed rule-set for the annotation of fragmentation patterns of the different 1007

moieties decorating the 17-hydroxygeranyllinalool (17-HGL) aglycone. The MS and 1008

MS/MS database is based on the retention time and mass spectrometric data of purified 1009

HGL-DTGs which are used as authentic standards. Novel HGL-DTGs are annotated 1010

based on their spectral similarity to the MS and MS/MS in-house database. For the 1011

visualization and identification of HGL-DTG profiles, we computed the extracted ion 1012

chromatogram EIC m/z 271.2420. This m/z fragment corresponds to the 17-HGL 1013

aglycone lacking both hydroxyl-groups and is produced by in-source fragmentation 1014

during ionization for all HGL-DTGs independently of the type and degree of metabolic 1015


https://doi.org/10.1101/2020.08.26.267690

decorations. The trace returned for m/z 271.2420 allows for the visualization of the 1016

complete HGL-DTG chemotype and to rapidly assess variations within this chemotype 1017

that result from the single gene manipulations. Following application of the de-replication 1018

workflow, we subdivided, based on the presence of diagnostic m/z signals, the 1019

chemotype between rhamnosylated and non-rhamnosylated HGL-DTGs. Lyciumoside I 1020

and lyciumoside II and their malonylated derivatives corresponded to non-1021

rhamnosylated HGL-DTGS, while lyciumoside IV, attenoside and nicotianoside III as 1022

well as their malonylated forms, represented the major rhamnosylated compounds. We 1023

provide a detailed description of the identification of known and novel HGL-DTGs in 1024

Supplemental Tables 1a/b. The identification levels are based on community standards 1025

reported in (Sumner et al., 2007). Raw MS metabolomics data have been deposited in 1026

the open metabolomics database Metabolights, www.ebi.ac.uk/metabolights (accession 1027

no. MTBLS1819). 1028

1029

1030

Statistical analysis 1031

1032

Data were analyzed using Excel (Microsoft, http://www.microsoft.com), SPSS 1033

20.0 (SPSS Inc, http://www-01.ibm.com/software/analytics/spss/) and RStudio (RStudio 1034

Inc, https://www.r-project.org) using the package xlsx. Unless otherwise stated, 1035

parametric data were compared using ANOVA followed by Fisher LSD/Holm-Bonferroni 1036

post hoc tests or Mann-Whitney-Wilcox Pairwise Test (for heteroskedastic data). The 1037

phylogenetic tree was constructed using the maximum-likelihood method in MEGA5.0 1038

(http://www.megasoftware.net/). 1039

1040

ACCESSION NUMBERS 1041

1042

For the analysis of the HGL-DTG pathway, we used the following GenBank 1043

accessions for N. attenuata: NaGLS, KJ755868; NaGGPPS, EF382626; NaUGT74P3, 1044

KX752207; NaUGT91T1, KX752209; NaUGT74P5, KX752208), N. obtusifolia 1045

(NoUGT74P4, KX752210; NoUGT74P6, KX752211; NoUGT91T1-like, MG051326). The 1046


https://doi.org/10.1101/2020.08.26.267690

MS metabolomics data set has been deposited in the open metabolomics database 1047

Metabolights, www.ebi.ac.uk/metabolights (accession no. MTBLS1819). 1048

1049

1050

Supplemental Data: 1051

1052

Supplemental Method File 1: Phylogenetic characterization of the UDP-1053

glycosyltransferases in N. attenuata and N. obtusifolia 1054

Supplemental Method File 2: Altered general and specialized metabolite levels 1055

1056

Supplemental Figure 1: Alignment of the UGT C-terminal consensus sequence of 112 1057

family 1 glycosyltransferases from N. attenuata and N. obtusifolia. 1058

Supplemental Figure 2: Phylogenetic analysis of the N. attenuata UGT superfamily 1059

shows 16 major groups 1060

Supplemental Figure 3: Amino acid composition of all identified UGTs of the 1061

superfamily 1 in N. attenuata 1062

Supplemental Figure 4: Phylogenetic relationships and herbivory-induced tissue-1063

specific expression of 110 predicted UDP-glycosyltransferases (UGT) 1064

Supplemental Figure 5: Transcriptomic variation of UGTs after treatment with OS in N. 1065

attenuata 1066

Supplemental Figure 6: Phylogenetic tree analysis for the UDP-glycosyltransferases 1067

used for stable and transient silencing. 1068

Supplemental Figure 7: Silencing efficiency for the three transiently-silenced 17-HGL-1069

DTG biosynthetic UGTs in pTVUGT91T1, pTVUGT74P3 and pTVUGT74P5. 1070

Supplemental Figure 8: Co-silencing efficiency of UGT74P3 and UGT74P5 in 1071

pTVUGT74P3 and pTVUGT74P5. 1072

Supplemental Figure 9: Silencing efficiency for the three transiently-silenced 17-HGL-1073

DTG biosynthetic UGTs in pTVUGT91T1-like, pTVUGT74P4 and pTVUGT74P6 in N. 1074

obtusifolia. 1075

Supplemental Figure 10a/b: Mass spectrometric characterization and annotation of 1076

novel HGL-DTGs in transiently-silenced N. obtusifolia plants impaired in NoUGT74P4 1077

and NoUGT74P6 expression. 1078


https://doi.org/10.1101/2020.08.26.267690

Supplemental Figure 11a/b/c: Characterization and annotation of novel HGL-DTGs via 1079

MS/MS in stably-silenced N. attenuata plants impaired in UGT74P3 and UGT74P5 1080

expression. 1081

Supplemental Figure 12: 1H NMR spectrum of synthetic 17-hydroxygeranyllinalool (17-1082

HGL, HPC24 Standards) 1083

Supplemental Figure 13a/b/c: Morphological characterization of N. attenuata plants 1084

transiently-silenced in UGT91T1, UGT74P3 and UGT74P5 expression 1085

Supplemental Figure 14: Morphological characterization of N. obtusifolia plants 1086

transiently-silenced in NoUGT91T1-like, NoUGT74P4, NoUGT74P6 and 1087

NoUGT74P4/UGT74P6 expression 1088

Supplemental Figure 15: Metabolite profiling and morphological characterization of N. 1089

attenuata plants transiently-silenced via virus-induced gene silencing (VIGS) of 1090

geranyllinalool synthase (GLS) 1091

Supplemental Figure 16: Southern Blot 1092

Supplemental Figure 17a/b/c: Morphological characterization of the stable transformed 1093

IRugt74p5 Line A, Line B and IRugt74p3/ugt74p5. 1094

Supplemental Figure 18: Characterization of growth parameters in IRugt91t1, 1095

IRugt74p5, IRugt74p3/ugt74p5 and IRggpps 1096

Supplemental Figure 19: Morphological characterization of IRugt91t1 1097

Supplemental Figure 20: Overall abundance of HGL-DTGs 1098

Supplemental Figure 21: Disrupting HGL-DTG glycosylation reorganizes general, 1099

specialized and hormonal metabolic pathways 1100

Supplemental Figure 22: Characterization of free prenyldiphosphates in IRggpps and 1101

WT 1102

Supplemental Figure 23: Quantitative 17-HGL method 1103

Supplemental Figure 24: 17-HGL concentration in IRggpps and WT plants transiently 1104

transformed with pTV00, pTVUGT74P3 and pTVUGT74P5 1105

Supplemental Figure 25: Characterization of the morphological phenotype of WT and 1106

IRggpps plants transiently transformed with pTV00. 1107


IRggpps plants transiently transformed with pTVUGT74P3. 1109


https://doi.org/10.1101/2020.08.26.267690


IRggpps plants transiently transformed with pTVUGt74P5. 1111

1112

Supplemental Table 1: Molecular weight of UGTs in N. attenuata 1113

Supplemental Table 2: Phylogenetic grouping of 107 UGTs in N. attenuata 1114

Supplemental Table 3: UGT Amino acid composition in N. attenuata 1115

Supplemental Table 4: SignalIP4.1 – signal peptide cleavage sites 1116

Supplemental Table 5a/b: Pearson Correlation of all UGTs to NaGLS and NaGGPPS 1117

Supplemental Table 6: MS/MS Measurements for HGL-DTGs in N. obtusifolia 1118

Supplemental Table 7: MS/MS Measurements for HGL-DTGs in N. attenuata 1119

Supplemental Table 8a-b: Tissue-specific HGL-DTG modulation 1120

Supplemental Table 9: Primers 1121

1122

Supplemental Data 1: Relative UGT expression vs. time in N. attenuata 1123

Supplemental Data 2: HGL-DTG profiles of transiently-silenced N. attenuata plants 1124

Supplemental Data 3: HGL-DTG profiles of transiently-silenced NaGLS N. attenuata 1125

plants 1126

Supplemental Data 4: HGL-DTG profiles of transiently-silenced N. obtusifolia plants 1127

Supplemental Data 5: HGL-DTG profiles of stably-silenced N. attenuata plants 1128

Supplemental Data 6: General and specialized metabolites in stably-silenced N. 1129

attenuata plants 1130

Supplemental Data 7: Phytohormone profiles in stably-silenced N. attenuata plants 1131

Supplemental Data 8: Statistical analysis of the performance assay and consumed leaf 1132

disk mass 1133

1134

ACKNOWLEDGEMENTS 1135

1136

We thank the gardening staff at the Max Planck Institute for Chemical Ecology. 1137

We thank Thomas Hahn, Nicolas Heinzel, Alexander Weinhold, Mario Kallenbach and 1138

Matthias Schoettner for the analytical and technical support as well as Dapeng Li and 1139

Felipe Yon for the fruitful discussions. We thank Raimund Nagel for his help with the 1140

terpene quantification. Additionally, we thank Eric McGale and Jyotasana Gulati for their 1141


https://doi.org/10.1101/2020.08.26.267690

statistical wisdom and Michael Court for assistance naming UGTs. We thank the Max 1142

Planck Society and the International Max Planck Research School on the Exploration of 1143

Ecological Interactions with Chemical and Molecular Techniques for financial support. 1144

We acknowledge the European Research Council advanced grant ClockworkGreen to 1145

I.T.B. (number 293926; I.T.B.) for funding. 1146

1147

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Figure 1: HGL-DTG biosynthetic pathway

Components of the diverse HGL-DTGs structures previously identified and annotated in

the leaves of N. attenuata that differ with respect to their sugar and malonyl group

composition.


https://doi.org/10.1101/2020.08.26.267690

Figure 2: Metabolite profiling and morphologies of N. attenuata plants transiently-

silenced in the expression of HGL-DTG-predicted UGTs by virus-induced gene

silencing (VIGS)

A) Extracted ion chromatograms (EIC) for identified HGL-DTGs in leaves of 37 day-old

elongated N. attenuata plants silenced in UGT91T1, UGT74P3 or UGT74P5 transcript


https://doi.org/10.1101/2020.08.26.267690

accumulation as well as in the empty vector controls (pTV00). HGL-DTGs were

categorized into rhamnosylated, non-rhamnosylated and intermediates with one or two

glucose moieties to facilitate visualization. The 17-hydroxygeranyllinallool (17-HGL)

aglycone was only detected in transiently-silenced pTVUGT74P3 and pTVUGT74P5

lines. B) Heatmap visualization of the patterns of deregulation in control plants or in

plants treated with 150 µg methyl jasmonate (MeJA) in 20 µL lanolin paste (N=5). The

color gradient visualizes fold changes in individual HGL-DTGs for each of the VIGs

constructs compared to the average in the pTV00 empty vector VIGS plants. C)

Morphological alterations observed in pTV00, pTVUGT74P3 and pTVUGT74P5

transiently-transformed plants ranged from necrotic spots and tissues to necrotic apical

meristem and flower buds frequently stalled in the opening process. Additional

phenotypic details are provided in supplemental Figures S7a-c. D) and E) Morphological

alterations of the corolla tube, corolla limb, style, ovary and nectary (N=20). Asterisks

indicate significant differences between the empty vector control and pTVUGT74P3 or

pTVUGT74P5 VIGS plants (*P ≤ 0.05, ** P < 0.01, *** P < 0.001).


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Figure 3: Metabolite profiling and morphologies of N. obtusifolia plants

transiently-silenced in the expression of HGL-DTG-predicted UGTs by VIGS

A) Extracted ion chromatograms (EIC) for the identified HGL-DTGs of 37 day-old

elongated N. obtusifolia plants silenced in NoUGT91T1-like, NoUGT74P4 and

NoUGT74P6 transcript accumulations as well as in the empty vector (pTV00) controls.

Rhamnosylated, non-rhamnosylated and intermediate HGL-DTGs with one or two


https://doi.org/10.1101/2020.08.26.267690

glucose moieties were color-categorized as in Fig. 3. The 17-HGL aglycone was only

detected in pTVNoUGT74P4, pTVNoUGT74P6 and the double construct

pTVNoUGT74P4/UGT74P6 VIGS plants. B) Heatmap visualization of deregulations in

the leaf HGL-DTG profiles of transiently-transformed pTVNoUGt91T1-like,

pTVNoUGT74P4, pTVNoUGT74P6 or pTVNoUGT74P4/UGT74P6 plants (N=5). The

color gradient visualizes fold changes in individual HGL-DTGs for each of the VIGS

constructs compared to the average in the pTV00 empty vector VIGS plants. C)

Morphological alterations in pTV00, pTVNoUGT91T1-like, pTVNoUGT74P4,

pTVNoUGT74P6 or pTVNoUGT74P4/UGT74P6 ranged from necrotic spots to a high

percentage of stalled flower buds. Additional phenotypic details are reported in

supplemental Figure 9.


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Figure 4: Recombinant UGT74P3, UGT74P4, and UGT74P5 proteins glucosylate

the 17-HGL aglycone

Enzyme activity assays of the recombinant UGT74P3, UGT74P4 and UGT74P5 proteins

expressed in E. coli BL21 DE3 cells. Chromatograms (EIC traces for aglycone m/z

329.2475, HGL-DTGs with 1 glucose moiety m/z 491.3003 and HGL-DTGs with 2

glucose moieties m/z 653.3507) of analyses of 4 UGTs recombinant protein incubated

for 3 h in 50 mM Tris HCL pH 7.0 with 5 mM 17-HGL in the presence of 5 mM UDP-

glucose. Additionally, activity assays combining UGT74P5 with UGT74P3 or UGT74P4

were performed, but did not differ from the enzyme assays using only UGT74P3 or

UGT74P4.


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Figure 5: Metabolite profiling and morphological characterization of stably

silenced N. attenuata plants

A) EICs for identified HGL-DTGs in leaves of 42 day-old elongated N. attenuata plants

silenced in GGPPS, UGT91T1, UGT74P3 and UGT74P5 transcript accumulation as well

as WT control plants. HGL-DTGs were categorized into rhamnosylated, non-

rhamnosylated HGL-DTG and intermediates with one or two glucose moieties to

facilitate visualization. The 17-HGL aglycone was only detected in IRugt74p5 and

IRugt74p3/ugt74p5 plants. B) Heatmap visualization of deregulations in the leaf HGL-

DTG profile of IRugt91t1, IRugt74p5, IRugt74p3/ugt74p5 and IRggpps (N=5). The color

gradient visualizes fold changes in individual HGL-DTGs for each of the stably-

transformed lines compared to the average in the WT plants. C) Morphological

alterations in IRugt74p5, IRugt74p3/ugt74p5 with milder phenotypes ranged from

necrotic spots and tissues, altered leaf shape and thickness, to apical meristem necrosis

and a high percentage of stalled flower buds and overall highly stunted growth.

Additional details of these phenotypes are shown in supplemental Figure S14a-c. D) 1-

year-old independent T0-transformants silenced in the expression of UGT74P3,

UGT74P5 and UGT74P3/UGT74P5. Strong morphological alterations ranging from

stunted growth, succulent leaves, stalled flower buds to a ‘broom’ like appearance were

consistently detected among T0-transformants. Viable seeds were produced by a few

transformants.


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Figure 6: Silencing efficiency for the three 17-HGL-DTG biosynthetic UGTs in

IRugt91t1, IRugt74p5, IRugt74p3/ugt74p5 plants

Relative transcript abundance of UGT91T1, UGT74P3 and UGT74P5 in leaves of stably

transformed N. attenuata plants (N=4). Asterisks indicate significant differences between

WT control and stable transformants (*P ≤ 0.05, ** P < 0.01, *** P < 0.001).


https://doi.org/10.1101/2020.08.26.267690

Figure 7: Abolishing 17-HGL aglycone synthesis by silencing NaGGPPS

abrogates morphological alterations resulting from the silencing of UGT74P3 and

UGT74P5.

A) Morphological alterations of 42-day-old N. attenuata plants stably transformed to

silence NaGGPPS expression (IRggpps) and WT after inoculation with A. tumefaciens

harboring pTVUGT74P5, pTVUGT74P3 and the empty vector control (pTV00) VIGS

constructs. B) Stem height, number of side branches and rosette diameter in WT and

IRggpps. Asterisks indicate significant differences between empty vector control and

transient silenced lines (*P ≤ 0.05, ** P < 0.01, *** P < 0.001).


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Figure 8: Application of 17-HGL aglycone results in necrotic lesions that

phenocopy those observed in IRugt74p5 and IRugt74p3/ugt74p5 plants

A) Concentrations of HGL in leaf material of WT, IRggpps, IRugt74p5,

IRugt74p3/ugt74p5 and IRugt91t1. B) Necrotic leaf tissue of 32 day-old elongated WT

and 48 day-old flowering IRggpps plants treated with DMSO, DMSO + 140nmol HGL,

DMSO + 280 nmol HGL and DMSO + 9800nmol HGL after 1 day. C) Percentage of

damaged leaf area in WT (N=3) and IRggpps (N=5). Asterisks indicate significant

differences between Control (DMSO) and treated (+HGL) leaves (*P ≤ 0.05, ** P < 0.01,

*** P < 0.001).


https://doi.org/10.1101/2020.08.26.267690

Figure 9: Performance assays show reduced growth of M. sexta fed on

transformed lines impaired in HGL-DTG glycosylation

A) Mass of M. sexta larvae feeding on leaf disk material of four stably transformed plants

impaired in glucosylation (IRugt74p5, IRugt74p3/ugt74p5) and rhamnosylation

(IRugt91t1) of HGL-DTGs as well as the formation of their precursor geranylgeranyl

diphosphate (IRggpps) (average ± SE; n=24 to 30). Larvae grow significantly larger on

IRggpps (P=0.005) and are significantly smaller on IRugt74p3/ugt74p5 (P=0.005) and

IRugt74p5 (P=0.006) by day 6 as determined by Mann-Whitney-Wilcox Pairwise Tests.

For clarity, significance is only shown for days 12: *P<0.05, **P<0.01, ***P<0.001. B)

Mass of consumed leaf disks after caterpillars fed leaf disks of transgenic plants

(average ± SE; n=26 to 30 leaf disks with one larva). Larvae fed IRugt74p5,

IRugt74p3/ugt74p5 and IRugt91t1 consumed significantly less leaf disk material as

determined by Mann-Whitney-Wilcox Pairwise Tests. *P<0.05, **P<0.01, ***P<0.001.


https://doi.org/10.1101/2020.08.26.267690

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