Efficient production of saffron crocins and picrocrocin in · 2019. 12. 18. · 117. flower tissue...

1

Efficient production of saffron crocins and picrocrocin in 1

Nicotiana benthamiana using a virus-driven system 2

3

Maricarmen Martía,1, Gianfranco Direttob,1, Verónica Aragonésa, Sarah 4

Fruscianteb, Oussama Ahrazemc, Lourdes Gómez-Gómezc,*, José-Antonio Daròsa,* 5

6

aInstituto de Biología Molecular y Celular de Plantas (Consejo Superior de 7

Investigaciones Científicas-Universitat Politècnica de València), 46022 Valencia, Spain 8

bItalian National Agency for New Technologies, Energy, and Sustainable Development, 9

Casaccia Research Centre, 00123, Rome, Italy 10

cInstituto Botánico, Departamento de Ciencia y Tecnología Agroforestal y Genética, 11

Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 12

02071 Albacete, Spain 13

14

1M.M and G.D. contributed equally to this work. 15

16

*Corresponding authors e-mail addresses: [email protected] (L.Gómez-17

Gómez), [email protected] (J.A. Daròs). 18

19

20

ABSTRACT 21

22

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, 23

for the color and the unique taste of the saffron spice, known as red gold due to its 24

high price. Several studies have also shown the health-promoting properties of these 25

compounds. However, their high costs hamper the wide use of these metabolites in 26

the pharmaceutical sector. We have developed a virus-driven system to produce 27

remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana 28

plants in only two weeks. The system consists of viral clones derived from tobacco 29

etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes 30

from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues 31

demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. 32

davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. 33

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.18.880765doi: bioRxiv preprint

https://doi.org/10.1101/2019.12.18.880765

2

Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of 34

crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. 35

In an attempt to improve apocarotenoid content in N. benthamiana, co-expression 36

of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene 37

synthase (PaCrtB) and saffron -carotene hydroxylase 2 (BCH2), was performed 38

using the same viral system. This combinatorial approach led to an additional crocin 39

increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. 40

Considering that saffron apocarotenoids are costly harvested from flower stigma 41

once a year, and that Buddleja spp. flowers accumulate lower amounts, this system 42

may be an attractive alternative for the sustainable production of these appreciated 43

metabolites. 44

45

Keywords: Apocarotenoids; crocins; picrocrocin; carotenoid cleavage dioxygenase; viral 46

vector; tobacco etch virus; potyvirus 47

48

1. Introduction 49

50

Plants possess an extremely rich secondary metabolism and, in addition to food, 51

feed, fibers and fuel, also provide highly valuable metabolites for food, pharma and 52

chemical industries. However, some of these metabolites are sometime produced in very 53

limiting amounts. Plant metabolic engineering can address some of these natural 54

limitations for nutritional improvement of foods or to create green factories that produce 55

valuable compounds (Martin and Li, 2017; Yuan and Grotewold, 2015). Yet, the complex 56

regulation of plant metabolic pathways and the particularly time-consuming approaches 57

for stable genetic transformation of plant tissues highly limit quick progress in plant 58

metabolic engineering. In this context, virus-derived vectors that fast and efficiently 59

deliver biosynthetic enzymes and regulatory factors into adult plants may significantly 60

contribute to solve some challenges (Sainsbury et al., 2012). 61

Carotenoids constitute an important group of natural products that humans cannot 62

biosynthesize and must uptake from the diet. These compounds are synthesized by higher 63

plants, algae, fungi and bacteria (Rodriguez-Concepcion et al., 2018). Yet, they play 64

multiple roles in human physiology (Fiedor and Burda, 2014). Carotenoids are widely 65

used as food colorants, nutraceuticals, animal feed, cosmetic additives and health 66

supplements (Fraser and Bramley, 2004). In all living organisms, carotenoids act as 67


https://doi.org/10.1101/2019.12.18.880765

3

substrates for the production of apocarotenoids (Ahrazem et al., 2016a). These are not 68

only the simple breakdown products of carotenoids, as they act as hormones and are 69

involved in signaling (Eroglu and Harrison, 2013; Jia et al., 2018; Walter et al., 2010). 70

Apocarotenoids are present as volatiles, water soluble and insoluble compounds, and 71

among those soluble, crocins are the most valuable pigments used in the food, and in less 72

extent, in the pharmaceutical industries (Ahrazem et al., 2015). Crocins are glycosylated 73

derivatives of the apocarotenoid crocetin. They are highly soluble in water and exhibit a 74

strong coloring capacity. In addition, crocins are powerful free radical quenchers, a 75

property associated to the broad range of health benefits they exhibit (Bukhari et al., 2018; 76

Christodoulou et al., 2015; Georgiadou et al., 2012; Nam et al., 2010). The interest in the 77

therapeutic properties of these compounds is increasing due to their analgesic and 78

sedative properties (Amin and Hosseinzadeh, 2012), and neurological protection and 79

anticancer activities (Finley and Gao, 2017; Skladnev and Johnstone, 2017). Further, 80

clinical trials indicate that crocins have a positive effect in the treatment of depression 81

and dementia (Lopresti and Drummond, 2014; Mazidi et al., 2016). Other apocarotenoids, 82

such as safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) and its precursor 83

picrocrocin (β-D-glucopyranoside of hydroxyl-β-cyclocitral), have also been shown to 84

reduce the proliferation of different human carcinoma cells (Cheriyamundath et al., 2018; 85

Jabini et al., 2017; Kyriakoudi et al., 2015) and to exert anti-inflammatory effects (Zhang 86

et al., 2015). 87

Crocus sativus L. is the main natural source of crocins and picrocrocin, 88

respectively responsible for the color and flavor of the highly appreciated spice, saffron 89

(Tarantilis et al., 1995). Crocin pigments accumulate at huge levels in the flower stigma 90

of these plants conferring this organ with a distinctive dark red coloration (Moraga et al., 91

2009). Yet, these metabolites reach huge prices in the market, due to the labor intensive 92

activities associated to harvesting and processing the stigma collected from C. sativus 93

flowers (Ahrazem et al., 2015). Besides C. sativus, gardenia (Gardenia spp.) fruits are 94

also a commercial source of crocins, but at much lower scale. In addition, gardenia fruits 95

do not accumulate picrocrocin (Moras et al., 2018; Pfister et al., 1996). Some other plants, 96

such as Buddleja spp., also produce crocins, although they are not commercially exploited 97

due to low accumulation (Liao et al., 1999). All these plants share the common feature of 98

expressing carotenoid cleavage dioxygenase (CDD) activities, like the saffron CCD2L or 99

the CCD4 subfamily recently identified in Buddleja spp. (Ahrazem et al., 2017). 100


https://doi.org/10.1101/2019.12.18.880765

4

In C. sativus and Buddleja spp., zeaxanthin is the precursor of crocetin (Fig. 1). 101

Cleavage of the zeaxanthin molecule at the 7,8 and 7′,8′ double bonds render one 102

molecule of crocetin dialdehyde and two molecules of 4-hydroxy-2,6,6-trimethyl-1-103

cyclohexene-1-carboxaldehyde (HTCC) (Ahrazem et al., 2017, 2016c; Frusciante et al., 104

2014). Crocetin dialdehyde is further transformed to crocetin by the action of aldehyde 105

dehydrogenase (ALDH) enzymes (Demurtas et al., 2018; Gómez-Gómez et al., 2018, 106

2017). Next, crocetin serves as substrate of glucosyltransferase (UGT) activities that 107

catalyze the production of crocins through transfer of glucose to both ends of the molecule 108

(Côté et al., 2001; Demurtas et al., 2018; Moraga et al., 2004; Nagatoshi et al., 2012). The 109

HTCC molecule is also recognized by UGTs, resulting in production of picrocrocin (Fig. 110

1) (Diretto et al., 2019a). In C. sativus, the plastidic CsCCD2L enzyme catalyzes the 111

cleavage of zeaxanthin at 7,8;7′,8′ double bonds (Ahrazem et al., 2016c; Frusciante et al., 112

2014), which is closely related to the CCD1 subfamily (Ahrazem et al., 2016b, 2016a). 113

In Buddleja davidii, the CCD enzymes that catalyze the same reaction belong to a novel 114

group within the CCD4 subfamily of CCDs (Ahrazem et al., 2016a). These B. davidii 115

enzymes, BdCCD4.1 and BdCCD4.3, also localize in plastids and are expressed in the 116

flower tissue (Ahrazem et al., 2017). 117

Due to its high scientific and commercial interest, crocetin and crocins 118

biosynthesis has arisen great attention, and attempts to metabolically engineer their 119

pathway in microbial systems have been reported, although with limited success (Chai et 120

al., 2017; Diretto et al., 2019a; Tan et al., 2019; Wang et al., 2019). More in detail, the 121

use of the saffron CCD2L enzyme resulted in a maximum accumulation of 1.22 mg/l, 122

15.70 mg/l, and 4.42 mg/l of crocetin in, respectively, Saccharomyces cerevisiae (Chai et 123

al., 2017; Tan et al., 2019) and Escherichia coli (Wang et al., 2019). In a subsequent study 124

aimed to confirm, in planta, the role of a novel UGT in picrocrocin biosynthesis (Diretto 125

et al., 2019a), Nicotiana benthamiana leaves were transiently transformed, via 126

Agrobacterium tumefaciens, with CCD2L, alone or in combination with UGT709G1, 127

which led to the production of 30.5 g/g DW of crocins, with a glycosylation degree 128

ranging from 1 to 4 (crocin 1-4) (unpublished data). Although promising, these results 129

cannot be considered efficient and sustainable in the context of the development of an 130

industrial system for the production of the saffron high-value apocarotenoids. 131

Here we used a viral vector derived from Tobacco etch virus (TEV, genus 132

Potyvirus; family Potyviridae) (Bedoya et al., 2010) to transiently express a series of CCD 133

enzymes, alone or in combination with other carotenoid and non-carotenoid biosynthetic 134


https://doi.org/10.1101/2019.12.18.880765

5

enzymes, in N. benthamiana plants. Interestingly, tissues infected with viruses that 135

expressed CsCCD2L or BdCCD4.1 acquired yellow pigmentation visible to the naked 136

eye. Metabolite analyses of these tissues demonstrated the accumulation of crocins and 137

picrocrocin, reaching levels attractive enough for their commercial production, up to 138

0.35% and 0.8% of DW, respectively, in leaves in which CsCCD2L and Pantoea ananatis 139

phytoene synthase (PaCrtB) were simultaneously expressed. 140

141

2. Materials and Methods 142

143

2.1. Plasmid construction 144

145

Plasmids were constructed by standard molecular biology techniques, including 146

PCR amplifications of cDNAs with the high-fidelity Phusion DNA polymerase (Thermo 147

Scientific) and Gibson DNA assembly (Gibson et al., 2009), using the NEBuilder HiFi 148

DNA Assembly Master Mix (New England Biolabs). To generate a transformed N. 149

benthamiana line that stably expresses TEV NIb, we built the plasmid p235KNIbd. This 150

plasmid is a derivative of pCLEAN-G181 (Thole et al., 2007) and between the left and 151

right borders of A. tumefaciens transfer DNA (T-DNA) contains two in tandem cassettes 152

in which the neomycin phosphotransferase (NPTII; kanamycin resistance) and the TEV 153

NIb are expressed under the control of CaMV 35S promoter and terminator. The exact 154

nucleotide sequence of the p235KNIbd T-DNA is in Supplementary Fig. S1. Plasmids 155

pGTEVΔNIb-CsCCD2L, -BdCCD4.1, -BdCCD4.3, -CsCCD2L/PaCrtB, -156

CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1 were built on the basis of pGTEVa (Bedoya 157

et al., 2012) that contains a wild-type TEV cDNA (GenBank accession number 158

DQ986288 with the two silent and neutral mutations G273A and A1119G) flanked by the 159

CaMV 35S promoter and terminator in a binary vector that also derives from pCLEAN-160

G181. These plasmids were built, as explained above, by PCR amplification of CsCCD2L 161

(Ahrazem et al., 2016c), BdCCD4.1 and BdCCD4.3 (Ahrazem et al., 2017), PaCrtB 162

(Majer et al., 2017), CsBCH2 (Castillo et al., 2005) and CsLPT1 (Gómez-Gómez et al., 163

2010) cDNAs and assembly into a pGTEVa version in which the NIb cistron was deleted 164

(pGTEVaΔNIb) (Majer et al., 2015). The exact sequences of the TEV recombinant clones 165

(TEVΔNIb-CsCCD2L, -BdCCD4.1, -BdCCD4.3, -CsCCD2L/PaCrtB, -166

CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1) contained in the resulting plasmids are in 167


https://doi.org/10.1101/2019.12.18.880765

6

Supplementary Fig. S2. The plasmid expressing the recombinant TEVΔNIb-aGFP clone 168

(Supplementary Fig. S2) was previously described (Majer et al., 2015). 169

170

2.2. Plant transformation 171

172

The LBA4404 strain of A. tumefaciens was sequentially transformed with the 173

helper plasmid pSoup (Hellens et al., 2000) and p235KNIbd. Clones containing both 174

plasmids were selected in plates containing 50 µg/ml rifampicin, 50 µg/ml kanamycin 175

and 7.5 µg/ml tetracycline. A liquid culture grown from a selected colony was used to 176

transform N. benthamiana (Clemente, 2006). Briefly, N. benthamiana leaf explants were 177

incubated with the A. tumefaciens culture at an optical density (600 nm) of 0.5 for 30 min 178

and then incubated on solid media. Four days later, explants were transferred to plates 179

with organogenesis media containing 100 µg/ml of kanamycin. Explants were repeatedly 180

transferred to fresh organogenesis plates every three weeks until shoots emerged. These 181

shoots were first transferred to rooting media and, when roots appeared, cultivated in a 182

greenhouse with regular soil until seeds were harvested. Transformation with the NIb 183

cDNA was confirmed by PCR. Batches of seedlings from four independent transformed 184

lines were screened for efficient complementation of TEV clones lacking NIb by 185

mechanical inoculation of TEVΔNIb-Ros1 (Bedoya et al., 2010). 186

187

2.3. Plant inoculation 188

189

A. tumefaciens C58C1 competent cells, previously transformed with the helper 190

plasmid pCLEAN-S48 (Thole et al., 2007), were electroporated with different plasmids 191

that contained the TEV recombinant clones and selected in plates with 50 µg/ml 192

rifampicin, 50 µg/ml kanamycin and 7.5 µg/ml tetracycline. Liquid cultures of selected 193

colonies were brought to an optical density of 0.5 (600 nm) in agroinoculation solution 194

(10 mM MES-NaOH, pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone) and incubated 195

for 2 h at 28ºC. Using a needleless syringe, these cultures were used to infiltrate one leaf 196

of five-week-old plants of the selected N. benthamiana transformed line that stably 197

expresses TEV NIb. After inoculation, plants were kept in a growth chamber at 25ºC 198

under a 12 h day-night photoperiod. Leaves were collected at different dpi as indicated; 199

in the case of the time-course experiments, leaf samples were collected at 8 time-points 200

(0, 4, 8, 11, 13, 15, 18 and 20 dpi). 201


https://doi.org/10.1101/2019.12.18.880765

7

202

2.4. Analysis of virus progeny 203

204

Viral progeny was analyzed by reverse transcription (RT)-PCR using RevertAid 205

reverse transcriptase (Thermo Scientific) and Phusion DNA polymerase followed by 206

electrophoretic separation of the amplification products in 1% agarose gels that were 207

stained with ethidium bromide. RNA from N. benthamiana plants was purified from 208

upper non-inoculated leaves at 15 dpi using silica-gel columns (Zymo Research). For 209

virus infection diagnosis, a cDNA corresponding to the TEV CP cistron (804 bp) was 210

amplified. Aliquots of the RNA preparations were subjected to RT using primer PI (5’-211

CTCGCACTACATAGGAGAATTAGAC-3’), followed by PCR amplification with 212

primers PII (5’-AGTGGCACTGTGGGTGCTGGTGTTG-3’) and PIII (5’-213

CTGGCGGACCCCTAATAG-3’). To analyze the potential deletion of the inserted CDD 214

cDNAs, RNA aliquots were reverse transcribed using primer PIV (5’- 215

GCTGTTTGTCACTCAATGACACATTAT-3’) and amplified by PCR with primers PV 216

(5’-AAAATAACAAATCTCAACACAACATATAC-3’) and PVI (5’- 217

CCGCGGTCTCCCCATTATGCACAAGTTGAGTGGTAGC-3’). 218

219

2.5. Metabolite extraction and analysis 220

221

Polar and apolar metabolites were extracted from 50 mg of lyophilized leaf tissue. 222

For polar metabolites analyses (crocins and picrocrocin), the tissues were extracted in 223

cold 50% methanol. The soluble fraction was analyzed by HPLC-DAD-HRMS and 224

HPLC-DAD (Ahrazem et al., 2018; Moraga et al., 2009). The liposoluble fractions 225

(crocetin, HTCC, -cyclocitral, carotenoids and chlorophylls) were extracted with 0.5:1 ml 226

cold extraction solvents (50:50 methanol and CHCl3), and analyzed by HPLC-DAD-227

HRMS and HPLC-DAD as previously described (Ahrazem et al., 2018; Castillo et al., 228

2005; D’Esposito et al., 2017; Fasano et al., 2016). Metabolites were identified on the 229

basis of absorption spectra and retention times relative to standard compounds. Pigments 230

were quantified by integrating peak areas that were converted to concentrations in 231

comparison with authentic standards. Chlorophyll a and b were determined by measuring 232

absorbance at 645 nm and 663 nm as previously described (Richardson et al., 2002). Mass 233

spectrometry carotenoid, chlorophyll derivative and apocarotenoid identification and 234

quantification was carried out as previously described (Ahrazem et al., 2018; Diretto et 235


https://doi.org/10.1101/2019.12.18.880765

8

al., 2019a; Rambla et al., 2016). Mass spectrometry analysis of other isoprenoids 236

(tocochromanols and quinones) were performed as reported before (Sulli et al., 2017). 237

Picrocrocin derivatives, as reported by (Moras et al., 2018) and (D’Archivio et al., 2016), 238

were tentatively identified according to their m/z accurate masses as included in the 239

PubChem database for monoisotopic masses or by using the Mass Spectrometry Adduct 240

Calculator from the Metabolomics Fiehn Lab for adduct ions; and were further validated 241

by isotopic pattern ratio and the comparison between theoretical and experimental m/z 242

fragmentation, by using the MassFrontier 7.0 software (Thermo Fisher Scientific). 243

244

2.6. Subcellular localization of crocins 245

246

To determine the subcellular localization of crocins in N. benthamiana tissues, LSCM 247

was performed with a Zeiss 7080 Axio Observer equipped with a water immersion 248

objective lens (C-Apochromat 40X/1.20 W). An argon ion laser at 458 nm was used for 249

excitation. Crocins and chloroplast autofluorescence were detected with 520 to 540 nm 250

and 660 to 750 nm band-pass emission filters, respectively. Images were processed using 251

the FIJI software (http://fiji.sc/Fiji). 252

253

2.7. Statistics and bioinformatics 254

255

Statistical validation of the data (ANOVA+ Tukey’s t-test) and heatmap 256

visualization were performed as previously described (Cappelli et al., 2018; Grosso et al., 257

2018). 258

259

3. Results 260

261

3.1. TEV recombinant clones that express CCD enzymes in N. benthamiana 262

263

The goal of this work was to develop a heterologous system to efficiently produce 264

highly appreciated and scarce apocarotenoids in plant tissues. For this purpose, we 265

planned to use an expression vector derived from TEV, more specifically TEVΔNIb 266

(Bedoya et al., 2010), recently employed to rewire the lycopene biosynthetic pathway 267

from the plastid to the cytosol of plant cells (Majer et al., 2017). Since CCD is the first 268

enzyme in the apocarotenoid biosynthetic pathway (Fig. 1), we started exploring the 269


http://fiji.sc/Fiji

https://doi.org/10.1101/2019.12.18.880765

9

virus-based expression of a series of CCD enzymes from well-known crocins producing 270

plants: namely C. sativus CsCCD2L (Ahrazem et al., 2016c), and B. davidii BdCCD4.1 271

and BdCCD4.3 (Ahrazem et al., 2017). 272

In TEVΔNIb, the approximately 1.6 kb corresponding to nuclear inclusion b 273

(NIb) cistron, coding for the viral RNA-dependent RNA polymerase, is deleted to 274

increase the space to insert foreign genetic information. Then, the viral recombinant 275

clones only infect plants in which NIb is supplied in trans (Bedoya et al., 2010). We chose 276

N. benthamiana as the biofactory plant for apocarotenoid production because of the 277

amenable genetic transformation, the high amounts of biomass achievable and the fast 278

growth. For this aim, as the first step in our work, we transformed N. benthamiana leaf 279

tissue using Agrobacterium tumefaciens and regenerated adult plants that constitutively 280

expressed TEV NIb under the control of Cauliflower mosaic virus (CaMV) 35S promoter 281

and terminator. To screen for transformed lines that efficiently complemented TEVΔNIb 282

deletion mutants, we inoculated the plants with TEVΔNIb-Ros1, a viral clone in which 283

the NIb cistron was replaced by a cDNA encoding for the MYB-type transcription factor 284

Rosea1 from Antirrhinum majus L., whose expression induces accumulation of colored 285

anthocyanins in infected tissues and facilitates visual tracking of infection (Bedoya et al., 286

2010, 2012). On the basis of anthocyanin accumulation, we selected a N. benthamiana 287

transformed line that efficiently complemented the replication and systemic movement 288

of TEVΔNIb-Ros1 (Supplementary Fig. S3). Then, by self-pollination and TEVΔNIb-289

Ros1 inoculation of the progeny, we selected a transformed homozygous line that was 290

used in all subsequent experiments. 291

Next, on the context of a TEV lacking NIb, we constructed three recombinant 292

clones (Fig. 2A) to express C. sativus CsCCD2L (TEVΔNIb-CsCCD2L), B. davidii 293

BdCCD4.1 and BdCCD4.3 (TEVΔNIb-BdCCD4.1 and TEVΔNIb-BdCCD4.3). These 294

CCDs are plastidic enzymes that are encoded in the nucleus and contain native amino-295

terminal transit peptides. To target these enzymes to plastids, we inserted their cDNAs in 296

a position corresponding to the amino terminus of the viral polyprotein in the virus 297

genome (Majer et al., 2015). CCD cDNAs also contained a sequence corresponding to an 298

artificial NIaPro cleavage site at the 3’ end to mediate the release of the heterologous 299

protein from the viral polyprotein. Based on previous observations, we used the -8/+3 site 300

that splits NIb and CP in TEV. The exact sequences of these clones are in Supplementary 301

Fig. S2. 302


https://doi.org/10.1101/2019.12.18.880765

10

Finally, transformed N. benthamiana plants that express NIb were 303

agroinoculated with the three TEV recombinant clones. As a control in this experiment, 304

some plants were agroinoculated with TEVΔNIb-aGFP (Fig. 2A and Supplementary Fig. 305

S2), which is a recombinant clone that expresses GFP from the amino-terminal position 306

in the viral polyprotein. Interestingly, 7 days post-inoculation (dpi), even before 307

symptoms were observed, we noticed a distinctive yellow pigmentation in systemic 308

tissues of the plants agroinoculated with TEVΔNIb-CsCCD2L. Symptoms of infection 309

were soon observed (approximately 8 dpi) in plants agroinoculated with TEVΔNIb-aGFP, 310

-CsCCD2L and -BdCCD4.1. Distinctive yellow pigmentation was also observed in 311

symptomatic tissues of plants agroinoculated with TEVΔNIb-BdCCD4.1, although with 312

some delay comparing to TEVΔNIb-CsCCD2L. Yellow pigmentation of symptomatic 313

tissues in plants inoculated with TEVΔNIb-CsCCD2L was more intense than that in 314

tissues of plants inoculated with TEVΔNIb-BdCCD4.1. Plants inoculated with 315

TEVΔNIb-BdCCD4.3 showed mild symptoms of infection at approximately 13 dpi and 316

yellow pigmentation was never observed. Fig. 2B shows pictures of representative leaves 317

of all these plants at 13 dpi. Reverse transcription (RT)-PCR analysis confirmed TEV 318

infection in all inoculated plants (Fig. 2C). Observation of symptomatic tissues with a 319

fluorescence stereomicroscope confirmed expression of GFP only in plants inoculated 320

with the TEVΔNIb-aGFP control (Supplementary Fig. S4). Analysis of viral progeny at 321

15 dpi by RT-PCR indicated that, while the inserts of TEVΔNIb-aGFP, -CsCCD2L are 322

perfectly stable in the recombinant virus, those of TEVΔNIb-BdCCD4.1 and TEVΔNIb-323

BdCCD4.3 are partially and completely lost, respectively, at this time post-inoculation 324

(Fig. 2D). Based on distinctive yellow color, these results suggested that the virus-driven 325

expression of CsCCD2L or BdCCD4.1 in N. benthamiana tissues may induce 326

apocarotenoid accumulation. 327

328

3.2. Analysis of apocarotenoids in infected tissues 329

330

Symptomatic leaf tissues from plants infected with TEVΔNIb-aGFP, TEVΔNIb-331

CsCCD2L and TEVΔNIb-BdCCD4.1 were subjected to extraction and analysis by high 332

performance liquid chromatography-diode array detector-high resolution mass 333

spectrometry (HPLC-DAD-HRMS) to determine their apocarotenoid and carotenoid 334

profiles. Tissues from mock-inoculated controls were also included in the analysis. The 335

tissues from the plants infected with TEVΔNIb-BdCCD4.3 were discarded due to the 336


https://doi.org/10.1101/2019.12.18.880765

11

absence of pigmentation and rapid deletion of BdCCD4.3 cDNA in the viral progeny. 337

Analysis of the polar fraction of tissues infected with TEVΔNIb-CsCCD2L and 338

TEVΔNIb-BdCCD4.1 showed a series of peaks with maximum absorbance from 433 to 339

439 nm. These peaks were not observed in extracts from tissues from mock-inoculated 340

plants or plants infected with the TEVΔNIb-aGFP control (Fig. 3). Mass spectrometry 341

chromatograms evidenced the presence, for all the fore mentioned peaks, of the crocetin 342

aglycon (m/z 329.1747 (M+H)), thus supporting the presence of crocins (Supplementary 343

Fig. S5). Further analyses of the sugar moieties conjugated with crocetin lead to the 344

identification of crocins with different degrees of glycosylation from one glucose 345

molecule to five in the infected tissues in which CCDs were expressed (Fig. 4A and 346

Supplementary Table S1). However, predominant crocins were those conjugated with 347

three and four glucose molecules. Interestingly, CCD-infected N. benthamiana leaves, 348

displayed a different pattern of crocin accumulation compared to that typical from saffron 349

stigma (Fig. 3A), with trans-crocin 4, followed by trans-crocin 3 and 2, being the most 350

abundant crocin species. Minor changes were also observed between TEVΔNIb-351

CsCCD2L and TEVΔNIb-BdCCD4.1 samples (Supplementary Table S2): in the former, 352

trans-crocin 3 was the most abundant (30.84%), followed by cis-crocin 4 (17.04%) and 353

trans-crocin 2 (16.84%); whereas similar amounts in cis-crocin 5, cis-crocin 4 and trans-354

crocin 3 were found in the latter (20.92, 20.62 and 18.31%, respectively). Picrocrocin, 355

safranal and several derivatives were also identified in the polar fraction of tissues 356

infected with TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1 (Fig. 4B and 357

Supplementary Table S1). All these polar apocarotenoids were absent in the control 358

tissues non-infected and infected with TEVΔNIb-aGFP (Fig. 4B). Overall, and in 359

agreement with the most intense visual phenotype, TEVΔNIb-CsCCD2L-infected tissues 360

displayed a higher accumulation in all the linear and cyclic apocarotenoids than 361

TEVΔNIb-BdCCD4.1-infected tissues (Fig. 4, Table 1 and Supplementary Table S1). 362

In the polar and apolar fractions of TEVΔNIb-CsCCD2L and TEVΔNIb-363

BdCCD4.1-infected leaves, we also detected the presence of crocetin dialdehyde and 364

HTCC, which are the products of the enzymatic cleavage step, and crocetin and safranal, 365

which are produced by the activity of ALDH enzymes and the spontaneous 366

deglycosilation of picrocrocin, respectively (Fig. 4 and Supplementary Table S1). None 367

of these metabolites were found in the TEVΔNIb-aGFP-infected and mock-inoculated 368

control tissues. Subsequently, the levels of different carotenoids and chlorophylls were 369

also investigated in the apolar fractions (Fig. 5 and Supplementary Table S3). As 370


https://doi.org/10.1101/2019.12.18.880765

12

expected, virus infection negatively affected the isoprenoid pools, with most of 371

carotenoids and chlorophylls reduced in all infected tissues compared to those from 372

mock-inoculated plants. However, the comparison between tissues infected with 373

TEVΔNIb-aGFP and both CCD viruses highlighted a series of alterations at metabolite 374

level. The most striking differences were in the contents of phytoene and phytofluene, as 375

well as zeaxanthin and lutein. While the formers increased in the TEVΔNIb-CsCCD2L 376

and TEVΔNIb-BdCCD4.1-infected tissues, the levels of zeaxanthin and lutein were 377

strongly reduced (Fig. 5A and Table S3A). On the contrary, no significant alterations in 378

chlorophyll levels were observed in tissues infected with the two CCD viruses when 379

compared to the TEVΔNIb-aGFP-infected control (Fig. 5B and Supplementary Table 380

S3B). Other typical leaf isoprenoids, such as tocochromanols and quinones, displayed 381

only few changes in CCD versus control tissues: for instance, -/-tocopherol, α-382

tocopherol quinone and menaquinone-8 increased, whereas plastoquinone was reduced 383

in CsCCD2L and BdCCD4.1-infected tissues compared to mock-inoculated and GFP-384

infected leaves (Fig. 5C and Supplementary Table S3C). 385

386

3.3. Subcellular accumulation of apocarotenoids in tissues infected with CCD-expressing 387

viruses 388

389

On the basis of the differential autofluorescence of chlorophylls and carotenoids, 390

we analyzed the subcellular localization of crocins that accumulate in symptomatic 391

tissues of plants infected with TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. Laser 392

scanning confocal microscopy (LSCM) images showed an intense fluorescence signal 393

from crocins in the vacuoles, while tissues infected with TEVΔNIb only showed 394

chlorophyll fluorescence (Fig. 6). 395

396

3.4. Time course accumulation of crocins and picrocrocin in inoculated plants 397

398

Next, we researched the point with the maximum apocarotenoid accumulation in 399

inoculated plants. For this aim, we carried out a time-course analysis focusing only on the 400

virus recombinant clone TEVΔNIb-CsCCD2L that induces the highest apocarotenoid 401

accumulation in N. benthamiana tissues. After plant agroinoculation, upper systemic 402

tissues were collected at 0, 4, 8, 11, 13, 15, 18 and 20 dpi from three independent replicate 403

plants per time point. Pigments were extracted and analyzed by HPLC-DAD. In these 404


https://doi.org/10.1101/2019.12.18.880765

13

tissues, accumulation of total crocins increased from approximately 8 to 13 dpi, reaching 405

a plateau up to the end of the analysis (20 dpi) (Fig. 7A). Picrocrocin levels showed 406

identical behavior (Fig. 7B). Accumulation of lutein, -carotene and chlorophylls was 407

also evaluated in all these samples. In contrast to apocarotenoids, accumulation of lutein 408

and -carotene (Fig. 7C) as well as chlorophylls a and b (Fig. 7D) dropped as infection 409

progressed. These results indicate that the virus-driven system to produce apocarotenoids 410

in N. benthamiana developed here reaches the highest yield in only 13 days after 411

inoculating the plants and this yield is maintained during at least one week with no 412

apparent loss. This experiment also revealed the remarkable accumulation of 2.18±0.23 413

mg of crocins and 8.24±2.93 mg of picrocrocin per gram of N. benthamiana dry weight 414

leaf tissue with the sole virus-driven expression of C. sativus CsCCD2L. 415

416

3.5. Virus-based combined expression of CsCCD2L and other carotenogenic and non-417

carotenogenic enzymes 418

419

In order to further improve apocarotenoid accumulation in inoculated N. 420

benthamiana plants, we combined expression of CsCCD2L with two carotenoid enzymes 421

(Fig. 8A), acting early and late in the biosynthetic pathway, namely Pantoea ananatis 422

phytoene synthase (PaCrtB), and saffron -carotene hydroxylase2 (CsBCH2); or with a 423

non-carotenoid transporter, the saffron lipid transfer protein 1 (CsLTP1), involved in the 424

transport of secondary metabolites (Wang et al., 2016). Interestingly, virus-based co-425

expression of CsCCD2L and PaCrtB induced a stronger yellow phenotype (Fig. 8B), and 426

a higher content in crocins (3.493±0.325 mg/g DW; Fig. 8C and Table S4). Improved 427

crocins levels were also observed when CsCCD2L was co-expressed with CsBCH2, 428

although at a lesser extent in this case (2.198±0.338 mg/g DW). On the contrary, co-429

expression of LPT1 did not result in an increment of total apocarotenoid content (Fig. 8C 430

and Table S4). Interestingly, when compared to tissues in which CsCCD2L alone was 431

expressed, a preferential over-accumulation for higher glycosylated crocins was 432

observed, while lower glycosylated species were down-represented (Fig. 8D). 433

434

4. Discussion 435

436


https://doi.org/10.1101/2019.12.18.880765

14

The carotenoid biosynthetic pathway in higher plants has been manipulated by 437

genetic engineering, both at the nuclear and transplastomic levels, to increase the general 438

amounts of carotenoids or to produce specific carotenoids of interest, such as 439

ketocarotenoids (Apel and Bock, 2009; Diretto et al., 2019b, 2007; Farré et al., 2014; 440

Giorio et al., 2007; Hasunuma et al., 2008; Nogueira et al., 2019; Zhu et al., 2018). 441

However, these projects also showed that the stable genetic transformation of nuclear or 442

plastid genomes from higher plants is a work-intensive and time-consuming process, 443

which frequently drives to unpredicted results, due to the complex regulation of metabolic 444

pathways. Although no exempted of important limitations, chief among them is the 445

amount of exogenous genetic information plant virus-derived expression systems can 446

carry, they represent an attractive alternative for some plant metabolic engineering goals. 447

Here, we used a virus-driven system that allows the production of noteworthy amounts 448

of appreciated apocarotenoids, such as crocins and picrocrocin, in adult N. benthamiana 449

plants in as little as 13 days (Fig. 7). For this, we manipulated the genome of a 10-kb 450

potyvirus, a quick straight-forward process that can be easily scaled-up for the high-451

throughput analysis of many enzymes and regulatory factors and engineered versions 452

thereof. 453

In this virus-driven system, the expression of an appropriate CCD in N. 454

benthamiana is sufficient to trigger the apocarotenoid pathway in this plant. Recently, it 455

was observed the same situation in a transient expression system (Diretto et al., 2019a). 456

These observations indicate that the zeaxanthin cleavage is the limiting step of 457

apocarotenoid biosynthesis in this species, while aldehyde dehydrogenation and 458

glycosylation steps can be efficiently complemented by endogenous orthologue enzymes. 459

A similar situation was previously observed with other secondary metabolites. Indeed, a 460

large portion of hydroxyl- and carboxyl-containing terpenoid compounds heterologously 461

produced in plants are glycosylated by endogenous UGTs. For example, in transgenic 462

maize expressing a geraniol synthase gene from Lippia dulcis, geranoyl-6-O-malonyl-β-463

D-glucopyranoside was the most abundant geraniol-derived compound (Yang et al., 464

2011). In another study, N. benthamiana plants transiently expressing the Artemesia 465

annua genes of the artemisin biosynthetic pathway accumulated glycosylated versions of 466

intermediate metabolites (Ting et al., 2013). Likewise, in tomato plants expressing the 467

chrysanthemyl diphosphate synthase gene from Tanacetum cinerariifolium, the 62% of 468

trans-chrysanthemic acid was converted into malonyl glycosides (Xu et al., 2018). 469


https://doi.org/10.1101/2019.12.18.880765

15

Zeaxanthin, the crocins precursor, is not a major carotenoid in plant leaves grown 470

under regular light conditions, although its level increases during high light stress 471

(Demmig-Adams et al., 2012). However, the efficient virus-driven production of all the 472

intermediate and final products of the apocarotenoid biosynthetic pathway in N. 473

benthamiana (Fig. 4) suggests that CCD is able to intercept the leaf metabolic flux in 474

order to diverge it towards the production of apocarotenoids. Similarly, the observed 475

reduction in lutein (Fig. 5A) may result from the CCD activity on the -ring of this 476

molecule, which confirms, at least for the C. sativus enzyme, the previous in vitro data 477

showing that lutein can act as substrate of this cleavage activity (Frusciante et al., 2014). 478

Although N. benthamiana leaves infected with CsCCD2L and BdCCD4.1 recombinant 479

viruses are able to accumulate crocins, it has to be mentioned that, at the qualitative level, 480

their profiles are distinct with respect to the saffron stigma: while in the formers the major 481

crocin species are the trans-crocin 3, together with cis-crocin 4, trans-crocin 2, cis-crocin 482

3 and cis-crocin 5, saffron stigmas are characterized by the presence, in decreasing order 483

of accumulation, of trans-crocin 4, trans-crocin 3 and trans-crocin 2. These results suggest 484

not only the implication of promiscuous glucosyltransferase enzymes differing form those 485

in saffron, but also the different conditions in which crocins are produce in saffron and 486

N. benthamiana, which implies respectively the absence or presence of light during this 487

process, which can effectively influence the cis- or trans- configuration of crocetin 488

(Rubio-Moraga et al., 2010). A higher bioactivity of the trans- forms has been previously 489

demonstrated, at least for crocetin (Zhang et al., 2017), thus making the crocins pool 490

accumulated in the CsCCD2L and BdCCD4.1 leaves a good source of bioactive 491

molecules. The C. sativus and B. davidii enzymes also generated a different crocins 492

pattern: this finding can be explained by a distinct specificity and kinetics, which might 493

influence the subsequent glucosylation step. Another remarkable observation is the 494

dramatic increase of phytoene and phytofluene levels in N. benthamiana tissues as a 495

consequence of the virus-driven expression of CCDs (Fig. 5A). These are upstream 496

intermediates in the carotenoid biosynthetic pathway. Previous reports indicated that the 497

overexpression of downstream enzymes or the increased storage of end-metabolites in 498

the carotenoid pathway can drain the metabolic flux towards the synthesis and 499

accumulation of the end-products (Diretto et al., 2007; Lopez et al., 2008). In our virus-500

driven system, expression of CsCCD2L and BdCCD4.1 seem to induce the same effect, 501

promoting the activities of the phytoene synthase (PSY) and phytoene desaturase (PDS), 502

which results in a higher accumulation of their metabolic intermediate products phytoene 503


https://doi.org/10.1101/2019.12.18.880765

16

and phytofluene. In this context, the observed amounts of phytoene and phytofluene 504

might be a consequence of PSY and PDS catalyzing the rate-limiting steps of the pathway 505

in N. benthamiana, as previously described in other species (Maass et al., 2009; Xu et al., 506

2006). Levels of additional plastidic metabolites in the groups of tocochromanols and 507

quinones only showed minor fluctuations, which indicates that perturbations in 508

carotenoid and apocarotenoid biosynthesis do not necessarily affect other related 509

pathways that share common precursors, such as geranylgeranyl diphosphate (GGPP) 510

(Fig. 5C). Our results also indicate that the decrease in the chlorophyll levels do not 511

depend on CCD expression, but on viral infection, since it was also observed in the control 512

tissues infected with TEVΔNIb-aGFP (Fig. 5B). This finding agrees with the effect of 513

plant virus infection on photosynthetic machinery (Li et al., 2016). 514

In our virus-driven experiments, CsCCD2L was more efficient in the production 515

of crocetin dialdehyde and crocins than BdCCD4.1. This suggests that the C. sativus CCD 516

used here (CsCCD2L) is definitively a highly active enzyme, completely compatible with 517

the virus vector and the host plant. Indeed, C. sativus stigma is the main commercial 518

source for these apocarotenoids (Castillo et al., 2005; Rubio Moraga et al., 2013). 519

Apocarotenoids are also present, although at lower levels, in the flowers of Buddleja spp., 520

where they are restricted to the calix. 521

Virus-driven expression of CsCCD2L in adult N. benthamiana plants allowed the 522

accumulation of the notable amounts of 2.18±0.23 mg of crocins and 8.24±2.93 mg of 523

picrocrocin per gram (dry weight) of infected tissues in only 13 dpi (Fig. 7). These 524

amounts are much higher than those obtained with a classic A. tumefaciens-mediated 525

transient expression of the CCD2L enzyme in N. benthamiana leaves, alone or in 526

combination with UGT709G1 (30.5 g/g DW of crocins (unpublished data) and 4.13 g/g 527

DW of picrocrocin (Diretto et al., 2019a), and highlight the opportunity to exploit the 528

viral system for the production of valuable secondary metabolites. A huge range of values 529

has been reported for crocins and picrocrocin in saffron in different studies. Crocins 530

reported values in saffron samples from Spain ranged from 0.85 to 32.40 mg/g dry weight 531

(ALONSO et al., 2001). Other reports showed 24.87 mg/g from China (Tong et al., 2015), 532

26.60 mg/g from Greece (Koulakiotis et al., 2015), 29.00 mg/g from Morocco (Gonda et 533

al., 2012), 32.60 mg/g from Iran, 49.80 mg/g from Italy (Masi et al., 2016) or 89.00 mg/g 534

from Nepal (Li et al., 2018). In Gardenia fruits, crocins levels range from 2.60 to 8.37 535

mg/g (Ouyang et al., 2011; Wu et al., 2014). The picrocrocin content ranges from 7.90 to 536

129.40 mg/g in Spanish saffron. In Greek saffron, 6.70 mg/g were reported (Koulakiotis 537


https://doi.org/10.1101/2019.12.18.880765

17

et al., 2015), and 10.70-2.16 mg/g in Indian, 21.80-6.15 mg/g in Iranian and 42.20-280.00 538

mg/g in Moroccan saffron (Lage and Cantrell, 2009). The different values obtained in 539

those samples are also a consequence of the different procedures followed to obtain the 540

saffron spice. Most methods involve a dehydration step by heating, which causes the 541

conversion of picrocrocin to safranal. Although lower than those from natural sources, 542

the crocins and picrocrocin contents of N. benthamiana tissues achieved with our virus-543

driven system may still be attractive for commercial purposes because, in contrast to C. 544

sativus stigma or Gardenia spp. fruits, N. benthamiana infected tissues can be quickly 545

and easily obtained in practically unlimited amounts. Moreover, the virus-driven 546

production of crocins and picrocrocin in N. benthamiana may still be optimized by 547

increasing, for instance, the zeaxanthin precursor. PSY over-expression in plants was 548

shown to increase the flux of the whole biosynthetic carotenoid pathway (Nisar et al., 549

2015), as well as the zeaxanthin pool (Lagarde et al., 2000; Römer et al., 2002). In 550

addition, the virus-based cytosolic expression of a bacterial PSY (Pantoea ananatis crtB) 551

also induced general carotenoid accumulation, and particularly increased phytoene levels 552

in tobacco infected tissues (Majer et al., 2017). On the other side, BCH overexpression 553

resulted in increased zeaxanthin and xanthophyll contents, both in microbes and 554

plants (Arango et al., 2014; Du et al., 2010; Lagarde et al., 2000). In this context, we 555

selected PaCrtB and CsBCH2, together with CsLTP1, involved in carotenoid transport, 556

as additional targets to improve apocarotenoid contents in N. benthamiana leaves, which 557

allowed a further increase of 1.9 and 1.6 fold in CsCCD2L/PaCrtB and CsCCD2L/ 558

CsBCH2, respectively, compared to the sole expression of CsCCD2L (Fig. 8). Other 559

targets to improve apocarotenoid accumulation could include the knocking out of 560

enzymatic steps opposing zeaxanthin accumulation, as zeaxanthin epoxydase (ZEP), 561

which uses zeaxanthin as substrate to yield violaxanthin, or lycopene -cyclase (LCY-) 562

that converts lycopene in -carotene, thus diverging the metabolic flux towards the 563

synthesis of (lutein), rather than xanthophylls (as zeaxanthin), which will be 564

object of future attempts. 565

In summary, here we describe a new technology that allows production of 566

remarkable amounts of highly appreciated crocins and picrocrocin in adult N. 567

benthamiana plants in as little as 13 dpi. This achievement is the consequence of N. 568

benthamiana, a species that accumulate negligible amounts of apocarotenoids, only 569

requiring the expression of an appropriate CCD to trigger the apocarotenoid biosynthetic 570


https://doi.org/10.1101/2019.12.18.880765

18

pathway, and can be further improved through the combination with the overexpression 571

of early or late carotenoid structural genes. 572

573

Acknowledgements 574

575

We thank K. Schreiber and C. Mares (IBMCP, CSIC-UPV, Valencia, Spain) for 576

technical assistance during plant transformation. We thank M. Gascón and M.D. Gómez-577

Jiménez (IBMCP, CSIC-UPV, Valencia, Spain) for helpful assistance with LSCM 578

analyses. We thank D. Dubbala (IBMCP, CSIC-UPV, Valencia, Spain) for English 579

revision. This work was supported by grants BIO2016-77000-R and BIO2017-83184-R 580

from the Spanish Ministerio de Ciencia, Innovación y Universidades (co-financed 581

European Union FEDER funds). M.M. was the recipient of a predoctoral fellowship from 582

the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/05294). G.D. and 583

L.G.G. are participants of the European COST action CA15136 (EUROCAROTEN). 584

L.G.G. is a participant of the CARNET network (BIO2015-71703-REDT and BIO2017‐585

90877‐RED). 586

587

Conflict of interest 588

589

The authors declare no conflict of interest. 590

591

References 592

593

Ahrazem, O., Argandoña, J., Fiore, A., Aguado, C., Luján, R., Rubio-Moraga, Á., 594

Marro, M., Araujo-Andrade, C., Loza-Alvarez, P., Diretto, G., Gómez-Gómez, L., 595

2018. Transcriptome analysis in tissue sectors with contrasting crocins 596

accumulation provides novel insights into apocarotenoid biosynthesis and 597

regulation during chromoplast biogenesis. Sci. Rep. 8. 598

https://doi.org/10.1038/s41598-018-21225-z 599

Ahrazem, O., Diretto, G., Argandoña, J., Rubio-Moraga, A., Julve, J.M., Orzáez, D., 600

Granell, A., Gómez-Gómez, L., 2017. Evolutionarily distinct carotenoid cleavage 601

dioxygenases are responsible for crocetin production in Buddleja davidii. J. Exp. 602

Bot. 68, 4663–4677. https://doi.org/10.1093/jxb/erx277 603

Ahrazem, O., Gómez-Gómez, L., Rodrigo, M.J., Avalos, J., Limón, M.C., 2016a. 604


https://doi.org/10.1101/2019.12.18.880765

19

Carotenoid cleavage oxygenases from microbes and photosynthetic organisms: 605

Features and functions. Int. J. Mol. Sci. https://doi.org/10.3390/ijms17111781 606

Ahrazem, O., Rubio-Moraga, A., Argandoña-Picazo, J., Castillo, R., Gómez-Gómez, L., 607

2016b. Intron retention and rhythmic diel pattern regulation of carotenoid cleavage 608

dioxygenase 2 during crocetin biosynthesis in saffron. Plant Mol. Biol. 91, 355–609

374. https://doi.org/10.1007/s11103-016-0473-8 610

Ahrazem, O., Rubio-Moraga, A., Berman, J., Capell, T., Christou, P., Zhu, C., Gómez-611

Gómez, L., 2016c. The carotenoid cleavage dioxygenase CCD2 catalysing the 612

synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New 613

Phytol. 209, 650–663. https://doi.org/10.1111/nph.13609 614

Ahrazem, O., Rubio-Moraga, A., Nebauer, S.G., Molina, R.V., Gómez-Gómez, L., 615

2015. Saffron: Its Phytochemistry, Developmental Processes, and Biotechnological 616

Prospects. J. Agric. Food Chem. 63, 8751–8764. 617

https://doi.org/10.1021/acs.jafc.5b03194 618

ALONSO, G.L., SALINAS, M.R., GARIJO, J., SÁNCHEZ-FERNÁNDEZ, M.A., 619

2001. COMPOSITION OF CROCINS AND PICROCROCIN FROM SPANISH 620

SAFFRON (CROCUS SATIVUS L.). J. Food Qual. 24, 219–233. 621

https://doi.org/10.1111/j.1745-4557.2001.tb00604.x 622

Amin, B., Hosseinzadeh, H., 2012. Evaluation of aqueous and ethanolic extracts of 623

saffron, Crocus sativus L., and its constituents, safranal and crocin in allodynia and 624

hyperalgesia induced by chronic constriction injury model of neuropathic pain in 625

rats. Fitoterapia 83, 888–895. https://doi.org/10.1016/j.fitote.2012.03.022 626

Apel, W., Bock, R., 2009. Enhancement of carotenoid biosynthesis in transplastomic 627

tomatoes by induced lycopene-to-provitamin A conversion. Plant Physiol. 151, 59–628

66. https://doi.org/10.1104/pp.109.140533 629

Arango, J., Jourdan, M., Geoffriau, E., Beyer, P., Welsch, R., 2014. Carotene 630

hydroxylase activity determines the levels of both α-carotene and total carotenoids 631

in orange carrots. Plant Cell 26, 2223–2233. 632

https://doi.org/10.1105/tpc.113.122127 633

Bedoya, L., Martínez, F., Rubio, L., Daròs, J.A., 2010. Simultaneous equimolar 634

expression of multiple proteins in plants from a disarmed potyvirus vector. J. 635

Biotechnol. 150, 268–275. https://doi.org/10.1016/j.jbiotec.2010.08.006 636

Bedoya, L.C., Martínez, F., Orzáez, D., Daròs, J.A., 2012. Visual tracking of plant virus 637

infection and movement using a reporter MYB transcription factor that activates 638


https://doi.org/10.1101/2019.12.18.880765

20

anthocyanin biosynthesis. Plant Physiol. 158, 1130–1138. 639

https://doi.org/10.1104/pp.111.192922 640

Bukhari, S.I., Manzoor, M., Dhar, M.K., 2018. A comprehensive review of the 641

pharmacological potential of Crocus sativus and its bioactive apocarotenoids. 642

Biomed. Pharmacother. 98, 733–745. https://doi.org/10.1016/j.biopha.2017.12.090 643

Cappelli, G., Giovannini, D., Basso, A.L., Demurtas, O.C., Diretto, G., Santi, C., 644

Girelli, G., Bacchetta, L., Mariani, F., 2018. A Corylus avellana L. extract 645

enhances human macrophage bactericidal response against Staphylococcus aureus 646

by increasing the expression of anti-inflammatory and iron metabolism genes. J. 647

Funct. Foods 45, 499–511. https://doi.org/10.1016/j.jff.2018.04.007 648

Castillo, R., Fernández, J.A., Gómez-Gómez, L., 2005. Implications of carotenoid 649

biosynthetic genes in apocarotenoid formation during the stigma development of 650

Crocus sativus and its closer relatives. Plant Physiol. 139, 674–689. 651

https://doi.org/10.1104/pp.105.067827 652

Chai, F., Wang, Y., Mei, X., Yao, M., Chen, Y., Liu, H., Xiao, W., Yuan, Y., 2017. 653

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces 654

cerevisiae. Microb. Cell Fact. 16, 54. https://doi.org/10.1186/s12934-017-0665-1 655

Cheriyamundath, S., Choudhary, S., Lopus, M., 2018. Safranal Inhibits HeLa Cell 656

Viability by Perturbing the Reassembly Potential of Microtubules. Phytother. Res. 657

32, 170–173. https://doi.org/10.1002/ptr.5938 658

Christodoulou, E., Kadoglou, N.P., Kostomitsopoulos, N., Valsami, G., 2015. Saffron: 659

A natural product with potential pharmaceutical applications. J. Pharm. Pharmacol. 660

67, 1634–1649. https://doi.org/10.1111/jphp.12456 661

Clemente, T., 2006. Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana). Methods 662

Mol. Biol. 343, 143–154. https://doi.org/10.1385/1-59745-130-4:143 663

Côté, F., Cormier, F., Dufresne, C., Willemot, C., 2001. A highly specific 664

glucosyltransferase is involved in the synthesis of crocetin glucosylesters in Crocus 665

sativus cultured cells. J. Plant Physiol. 158, 553–560. https://doi.org/10.1078/0176-666

1617-00305 667

D’Archivio, A.A., Giannitto, A., Maggi, M.A., Ruggieri, F., 2016. Geographical 668

classification of Italian saffron (Crocus sativus L.) based on chemical constituents 669

determined by high-performance liquid-chromatography and by using linear 670

discriminant analysis. Food Chem. 212, 110–116. 671

https://doi.org/10.1016/j.foodchem.2016.05.149 672


https://doi.org/10.1101/2019.12.18.880765

21

D’Esposito, D., Ferriello, F., Molin, A.D., Diretto, G., Sacco, A., Minio, A., Barone, A., 673

Di Monaco, R., Cavella, S., Tardella, L., Giuliano, G., Delledonne, M., Frusciante, 674

L., Ercolano, M.R., 2017. Unraveling the complexity of transcriptomic, 675

metabolomic and quality environmental response of tomato fruit. BMC Plant Biol. 676

17, 66. https://doi.org/10.1186/s12870-017-1008-4 677

Demmig-Adams, B., Cohu, C.M., Muller, O., Adams, W.W., 2012. Modulation of 678

photosynthetic energy conversion efficiency in nature: From seconds to seasons, 679

in: Photosynthesis Research. pp. 75–88. https://doi.org/10.1007/s11120-012-9761-680

6 681

Demurtas, O.C., Frusciante, S., Ferrante, P., Diretto, G., Azad, N.H., Pietrella, M., 682

Aprea, G., Taddei, A.R., Romano, E., Mi, J., Al-Babili, S., Frigerio, L., Giuliano, 683

G., 2018. Candidate Enzymes for Saffron Crocin Biosynthesis Are Localized in 684

Multiple Cellular Compartments. Plant Physiol. 177, 990–1006. 685

https://doi.org/10.1104/pp.17.01815 686

Diretto, G., Ahrazem, O., Rubio-Moraga, Á., Fiore, A., Sevi, F., Argandoña, J., Gómez-687

Gómez, L., 2019a. UGT709G1: a novel uridine diphosphate glycosyltransferase 688

involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron 689

(Crocus sativus). New Phytol. 224, 725–740. https://doi.org/10.1111/nph.16079 690

Diretto, G., Al-Babili, S., Tavazza, R., Papacchioli, V., Beyer, P., Giuliano, G., 2007. 691

Metabolic engineering of potato carotenoid content through tuber-specific 692

overexpression of a bacterial mini-pathway. PLoS One 2, e350. 693

https://doi.org/10.1371/journal.pone.0000350 694

Diretto, G., Frusciante, S., Fabbri, C., Schauer, N., Busta, L., Wang, Z., Matas, A.J., 695

Fiore, A., Rose, J.K.C., Fernie, A.R., Jetter, R., Mattei, B., Giovannoni, J., 696

Giuliano, G., 2019b. Manipulation of β‐carotene levels in tomato fruits results in 697

increased ABA content and extended shelf‐life. Plant Biotechnol. J. pbi.13283. 698

https://doi.org/10.1111/pbi.13283 699

Du, H., Wang, N., Cui, F., Li, X., Xiao, J., Xiong, L., 2010. Characterization of the β-700

carotene hydroxylase gene DSM2 conferring drought and oxidative stress 701

resistance by increasing xanthophylls and abscisic acid synthesis in rice. Plant 702

Physiol. 154, 1304–1318. https://doi.org/10.1104/pp.110.163741 703

Eroglu, A., Harrison, E.H., 2013. Carotenoid metabolism in mammals, including man: 704

Formation, occurrence, and function of apocarotenoids. J. Lipid Res. 705

https://doi.org/10.1194/jlr.R039537 706


https://doi.org/10.1101/2019.12.18.880765

22

Farré, G., Blancquaert, D., Capell, T., Van Der Straeten, D., Christou, P., Zhu, C., 2014. 707

Engineering complex metabolic pathways in plants. Annu. Rev. Plant Biol. 65, 708

187–223. https://doi.org/10.1146/annurev-arplant-050213-035825 709

Fasano, C., Diretto, G., Aversano, R., D’Agostino, N., Di Matteo, A., Frusciante, L., 710

Giuliano, G., Carputo, D., 2016. Transcriptome and metabolome of synthetic 711

Solanum autotetraploids reveal key genomic stress events following 712

polyploidization. New Phytol. 210, 1382–94. https://doi.org/10.1111/nph.13878 713

Fiedor, J., Burda, K., 2014. Potential role of carotenoids as antioxidants in human health 714

and disease. Nutrients 6, 466–488. https://doi.org/10.3390/nu6020466 715

Finley, J.W., Gao, S., 2017. A Perspective on Crocus sativus L. (Saffron) Constituent 716

Crocin: A Potent Water-Soluble Antioxidant and Potential Therapy for 717

Alzheimer’s Disease. J. Agric. Food Chem. 65, 1005–1020. 718

https://doi.org/10.1021/acs.jafc.6b04398 719

Fraser, P.D., Bramley, P.M., 2004. The biosynthesis and nutritional uses of carotenoids. 720

Prog. Lipid Res. 43, 228–265. https://doi.org/10.1016/j.plipres.2003.10.002 721

Frusciante, S., Diretto, G., Bruno, M., Ferrante, P., Pietrella, M., Prado-Cabrero, A., 722

Rubio-Moraga, A., Beyer, P., Gomez-Gomez, L., Al-Babili, S., Giuliano, G., 2014. 723

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron 724

crocin biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 111, 12246–12251. 725

https://doi.org/10.1073/pnas.1404629111 726

Georgiadou, G., Tarantilis, P.A., Pitsikas, N., 2012. Effects of the active constituents of 727

Crocus Sativus L., crocins, in an animal model of obsessive-compulsive disorder. 728

Neurosci. Lett. 528, 27–30. https://doi.org/10.1016/j.neulet.2012.08.081 729

Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison 3rd, C.A., Smith, 730

H.O., 2009. Enzymatic assembly of DNA molecules up to several hundred 731

kilobases. Nat. Methods 6, 343–345. https://doi.org/10.1038/nmeth.1318 732

Giorio, G., Stigliani, A.L., D’Ambrosio, C., 2007. Agronomic performance and 733

transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro 734

and control tomato lines under field conditions. Transgenic Res. 16, 15–28. 735

https://doi.org/10.1007/s11248-006-9025-3 736

Gómez-Gómez, L., Feo-Brito, F., Rubio-Moraga, A., Galindo, P.A., Prieto, A., 737

Ahrazem, O., 2010. Involvement of lipid transfer proteins in saffron 738

hypersensitivity: Molecular cloning of the potential allergens. J. Investig. Allergol. 739

Clin. Immunol. 20, 407–412. 740


https://doi.org/10.1101/2019.12.18.880765

23

Gómez-Gómez, L., Pacios, L.F., Diaz-Perales, A., Garrido-Arandia, M., Argandoña, J., 741

Rubio-Moraga, Á., Ahrazem, O., 2018. Expression and interaction analysis among 742

saffron ALDHs and crocetin dialdehyde. Int. J. Mol. Sci. 19. 743

https://doi.org/10.3390/ijms19051409 744

Gómez-Gómez, L., Parra-Vega, V., Rivas-Sendra, A., Seguí-Simarro, J.M., Molina, 745

R.V., Pallotti, C., Rubio-Moraga, Á., Diretto, G., Prieto, A., Ahrazem, O., 2017. 746

Unraveling massive crocins transport and accumulation through proteome and 747

microscopy tools during the development of saffron stigma. Int. J. Mol. Sci. 18. 748

https://doi.org/10.3390/ijms18010076 749

Gonda, S., Parizsa, P., Surányi, G., Gyémánt, G., Vasas, G., 2012. Quantification of 750

main bioactive metabolites from saffron (Crocus sativus) stigmas by a micellar 751

electrokinetic chromatographic (MEKC) method. J. Pharm. Biomed. Anal. 66, 68–752

74. https://doi.org/10.1016/j.jpba.2012.03.002 753

Grosso, V., Farina, A., Giorgi, D., Nardi, L., Diretto, G., Lucretti, S., 2018. A high-754

throughput flow cytometry system for early screening of in vitro made polyploids 755

in Dendrobium hybrids. Plant Cell. Tissue Organ Cult. 132, 57–70. 756

https://doi.org/10.1007/s11240-017-1310-8 757

Hasunuma, T., Miyazawa, S., Yoshimura, S., Shinzaki, Y., Tomizawa, K., Shindo, K., 758

Choi, S.K., Misawa, N., Miyake, C., 2008. Biosynthesis of astaxanthin in tobacco 759

leaves by transplastomic engineering. Plant J. 55, 857–868. 760

https://doi.org/10.1111/j.1365-313X.2008.03559.x 761

Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S., Mullineaux, P.M., 2000. pGreen: 762

a versatile and flexible binary Ti vector for Agrobacterium -mediated plant 763

transformation. Plant Mol.Biol. 42, 819–832. 764

Jabini, R., Ehtesham-Gharaee, M., Dalirsani, Z., Mosaffa, F., Delavarian, Z., Behravan, 765

J., 2017. Evaluation of the Cytotoxic Activity of Crocin and Safranal, Constituents 766

of Saffron, in Oral Squamous Cell Carcinoma (KB Cell Line). Nutr. Cancer 69, 767

911–919. https://doi.org/10.1080/01635581.2017.1339816 768

Jia, K.P., Baz, L., Al-Babili, S., 2018. From carotenoids to strigolactones. J. Exp. Bot. 769

https://doi.org/10.1093/jxb/erx476 770

Koulakiotis, N.S., Gikas, E., Iatrou, G., Lamari, F.N., Tsarbopoulos, A., 2015. 771

Quantitation of crocins and picrocrocin in saffron by hplc: Application to quality 772

control and phytochemical differentiation from other crocus taxa. Planta Med. 81, 773

606–612. https://doi.org/10.1055/s-0035-1545873 774


https://doi.org/10.1101/2019.12.18.880765

24

Kyriakoudi, A., O’Callaghan, Y.C., Galvin, K., Tsimidou, M.Z., O’Brien, N.M., 2015. 775

Cellular Transport and Bioactivity of a Major Saffron Apocarotenoid, Picrocrocin 776

(4-(beta-D-Glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde). 777

J. Agric. Food Chem. 63, 8662–8668. https://doi.org/10.1021/acs.jafc.5b03363 778

Lagarde, D., Beuf, L., Vermaas, W., 2000. Increased production of zeaxanthin and other 779

pigments by application of genetic engineering techniques to Synechocystis sp. 780

strain PCC 6803. Appl. Environ. Microbiol. 66, 64–72. 781

https://doi.org/10.1128/AEM.66.1.64-72.2000 782

Lage, M., Cantrell, C.L., 2009. Quantification of saffron (Crocus sativus L.) metabolites 783

crocins, picrocrocin and safranal for quality determination of the spice grown 784

under different environmental Moroccan conditions. Sci. Hortic. (Amsterdam). 785

121, 366–373. https://doi.org/10.1016/j.scienta.2009.02.017 786

Li, S., Shao, Q., Lu, Z., Duan, C., Yi, H., Su, L., 2018. Rapid determination of crocins 787

in saffron by near-infrared spectroscopy combined with chemometric techniques. 788

Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 789

https://doi.org/10.1016/j.saa.2017.09.030 790

Li, Y., Cui, H., Cui, X., Wang, A., 2016. The altered photosynthetic machinery during 791

compatible virus infection. Curr. Opin. Virol. 17, 19–24. 792

https://doi.org/10.1016/j.coviro.2015.11.002 793

Liao, Y.H., Houghton, P.J., Hoult, J.R.S., 1999. Novel and known constituents from 794

Buddleja species and their activity against leukocyte eicosanoid generation. J. Nat. 795

Prod. 62, 1241–1245. https://doi.org/10.1021/np990092+ 796

Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O’Neill, J., Li, L., 2008. Effect of 797

the cauliflower or transgene on carotenoid accumulation and chromoplast 798

formation in transgenic potato tubers. J. Exp. Bot. 59, 213–223. 799

https://doi.org/10.1093/jxb/erm299 800

Lopresti, A.L., Drummond, P.D., 2014. Saffron (Crocus sativus) for depression: A 801

systematic review of clinical studies and examination of underlying antidepressant 802

mechanisms of action. Hum. Psychopharmacol. https://doi.org/10.1002/hup.2434 803

Maass, D., Arango, J., Wüst, F., Beyer, P., Welsch, R., 2009. Carotenoid crystal 804

formation in arabidopsis and carrot roots caused by increased phytoene synthase 805

protein levels. PLoS One 4, e6373. https://doi.org/10.1371/journal.pone.0006373 806

Majer, E., Llorente, B., Rodríguez-Concepción, M., Daròs, J.A., 2017. Rewiring 807

carotenoid biosynthesis in plants using a viral vector. Sci. Rep. 7. 808


https://doi.org/10.1101/2019.12.18.880765

25

https://doi.org/10.1038/srep41645 809

Majer, E., Navarro, J.A., Daròs, J.A., 2015. A potyvirus vector efficiently targets 810

recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when 811

expressed at the amino terminus of the polyprotein. Biotechnol. J. 10, 1792–1802. 812

https://doi.org/10.1002/biot.201500042 813

Martin, C., Li, J., 2017. Medicine is not health care, food is health care: plant metabolic 814

engineering, diet and human health. New Phytol. 216, 699–719. 815

https://doi.org/10.1111/nph.14730 816

Masi, E., Taiti, C., Heimler, D., Vignolini, P., Romani, A., Mancuso, S., 2016. PTR-817

TOF-MS and HPLC analysis in the characterization of saffron (Crocus sativus L.) 818

from Italy and Iran. Food Chem. 192, 75–81. 819


Mazidi, M., Shemshian, M., Mousavi, S.H., Norouzy, A., Kermani, T., Moghiman, T., 821

Sadeghi, A., Mokhber, N., Ghayour-Mobarhan, M., Ferns, G.A.A., 2016. A 822

double-blind, randomized and placebo-controlled trial of Saffron (Crocus sativus 823

L.) in the treatment of anxiety and depression. J. Complement. Integr. Med. 13, 824

195–199. https://doi.org/10.1515/jcim-2015-0043 825

Moraga, A.R., Nohales, P.F., Pérez, J.A., Gómez-Gómez, L., 2004. Glucosylation of the 826

saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus 827

sativus stigmas. Planta 219, 955–966. https://doi.org/10.1007/s00425-004-1299-1 828

Moraga, Á.R., Rambla, J.L., Ahrazem, O., Granell, A., Gómez-Gómez, L., 2009. 829

Metabolite and target transcript analyses during Crocus sativus stigma 830

development. Phytochemistry 70, 1009–1016. 831

https://doi.org/10.1016/j.phytochem.2009.04.022 832

Moras, B., Loffredo, L., Rey, S., 2018. Quality assessment of saffron (Crocus sativus 833

L.) extracts via UHPLC-DAD-MS analysis and detection of adulteration using 834

gardenia fruit extract (Gardenia jasminoides Ellis). Food Chem. 257, 325–332. 835


Nagatoshi, M., Terasaka, K., Owaki, M., Sota, M., Inukai, T., Nagatsu, A., Mizukami, 837

H., 2012. UGT75L6 and UGT94E5 mediate sequential glucosylation of crocetin to 838

crocin in Gardenia jasminoides. FEBS Lett. 586, 1055–1061. 839

https://doi.org/10.1016/j.febslet.2012.03.003 840

Nam, K.N., Park, Y.M., Jung, H.J., Lee, J.Y., Min, B.D., Park, S.U., Jung, W.S., Cho, 841

K.H., Park, J.H., Kang, I., Hong, J.W., Lee, E.H., 2010. Anti-inflammatory effects 842


https://doi.org/10.1101/2019.12.18.880765

26

of crocin and crocetin in rat brain microglial cells. Eur. J. Pharmacol. 648, 110–843

116. https://doi.org/10.1016/j.ejphar.2010.09.003 844

Nisar, N., Li, L., Lu, S., Khin, N.C., Pogson, B.J., 2015. Carotenoid metabolism in 845

plants. Mol. Plant 8, 68–82. https://doi.org/10.1016/j.molp.2014.12.007 846

Nogueira, M., Enfissi, E.M.A., Welsch, R., Beyer, P., Zurbriggen, M.D., Fraser, P.D., 847

2019. Construction of a fusion enzyme for astaxanthin formation and its 848

characterisation in microbial and plant hosts: A new tool for engineering 849

ketocarotenoids. Metab. Eng. 52, 243–252. 850

https://doi.org/10.1016/j.ymben.2018.12.006 851

Ouyang, E., Zhang, C., Li, X., 2011. Simultaneous determination of geniposide, 852

chlorogenic acid, crocin1, and rutin in crude and processed fructus gardeniae 853

extracts by high performance liquid chromatography. Pharmacogn. Mag. 7, 267–854

270. https://doi.org/10.4103/0973-1296.90391 855

Pfister, S., Meyer, P., Steck, A., Pfander, H., 1996. Isolation and Structure Elucidation 856

of Carotenoid-Glycosyl Esters in Gardenia Fruits (Gardenia jasminoides Ellis) and 857

Saffron (Crocus sativus Linne). J. Agric. Food Chem. 44, 2612–2615. 858

https://doi.org/10.1021/jf950713e 859

Rambla, J.L., Trapero-Mozos, A., Diretto, G., Moraga, A.R., Granell, A., Gómez, L.G., 860

Ahrazem, O., 2016. Gene-metabolite networks of volatile metabolism in Airen and 861

Tempranillo grape cultivars revealed a distinct mechanism of aroma bouquet 862

production. Front. Plant Sci. 7. https://doi.org/10.3389/fpls.2016.01619 863

Richardson, A.D., Duigan, S.P., Berlyn, G.P., 2002. An evaluation of noninvasive 864

methods to estimate foliar chlorophyll content. New Phytol. 153, 185–194. 865

https://doi.org/10.1046/j.0028-646X.2001.00289.x 866

Rodriguez-Concepcion, M., Avalos, J., Bonet, M.L., Boronat, A., Gomez-Gomez, L., 867

Hornero-Mendez, D., Limon, M.C., Meléndez-Martínez, A.J., Olmedilla-Alonso, 868

B., Palou, A., Ribot, J., Rodrigo, M.J., Zacarias, L., Zhu, C., 2018. A global 869

perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition 870

and health. Prog. Lipid Res. 70, 62–93. 871

https://doi.org/10.1016/j.plipres.2018.04.004 872

Römer, S., Lübeck, J., Kauder, F., Steiger, S., Adomat, C., Sandmann, G., 2002. 873

Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and co-874

suppression of carotenoid epoxidation. Metab. Eng. 4, 263–272. 875

https://doi.org/10.1006/mben.2002.0234 876


https://doi.org/10.1101/2019.12.18.880765

27

Rubio-Moraga, A., Trapero, A., Ahrazem, O., Gómez-Gómez, L., 2010. Crocins 877

transport in Crocus sativus: The long road from a senescent stigma to a newborn 878

corm. Phytochemistry 71, 1506–1513. 879

https://doi.org/10.1016/j.phytochem.2010.05.026 880

Rubio Moraga, A., Ahrazem, O., Rambla, J.L., Granell, A., Gómez Gómez, L., 2013. 881

Crocins with High Levels of Sugar Conjugation Contribute to the Yellow Colours 882

of Early-Spring Flowering Crocus Tepals. PLoS One 8. 883


Sainsbury, F., Saxena, P., Geisler, K., Osbourn, A., Lomonossoff, G.P., 2012. Using a 885

virus-derived system to manipulate plant natural product biosynthetic pathways. 886

Methods Enzym. 517, 185–202. https://doi.org/10.1016/B978-0-12-404634-887

4.00009-7 888

Skladnev, N. V., Johnstone, D.M., 2017. Neuroprotective properties of dietary saffron: 889

More than just a chemical scavenger? Neural Regen. Res. 890

https://doi.org/10.4103/1673-5374.198976 891

Sulli, M., Mandolino, G., Sturaro, M., Onofri, C., Diretto, G., Parisi, B., Giuliano, G., 892

2017. Molecular and biochemical characterization of a potato collection with 893

contrasting tuber carotenoid content. PLoS One 12. 894


Tan, H., Chen, X., Liang, N., Chen, R., Chen, J., Hu, C., Li, Qi, Li, Qing, Pei, W., Xiao, 896

W., Yuan, Y., Chen, W., Zhang, L., 2019. Transcriptome analysis reveals novel 897

enzymes for apo-carotenoid biosynthesis in saffron and allows construction of a 898

pathway for crocetin synthesis in yeast. J. Exp. Bot. 70, 4819–4834. 899

https://doi.org/10.1093/jxb/erz211 900

Tarantilis, P.A., Tsoupras, G., Polissiou, M., 1995. Determination of saffron (Crocus 901

sativus L.) components in crude plant extract using high-performance liquid 902

chromatography-UV-visible photodiode-array detection-mass spectrometry. J. 903

Chromatogr. A 699, 107–118. https://doi.org/10.1016/0021-9673(95)00044-N 904

Thole, V., Worland, B., Snape, J.W., Vain, P., 2007. The pCLEAN dual binary vector 905

system for Agrobacterium-mediated plant transformation. Plant Physiol. 145, 906

1211–1219. 907

Ting, H.M., Wang, B., Rydén, A.M., Woittiez, L., Van Herpen, T., Verstappen, F.W.A., 908

Ruyter-Spira, C., Beekwilder, J., Bouwmeester, H.J., Van der Krol, A., 2013. The 909

metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin 910


https://doi.org/10.1101/2019.12.18.880765

28

biosynthetic pathway genes is a function of CYP71AV1 type and relative gene 911

dosage. New Phytol. 199, 352–366. https://doi.org/10.1111/nph.12274 912

Walter, M.H., Floss, D.S., Strack, D., 2010. Apocarotenoids: Hormones, mycorrhizal 913

metabolites and aroma volatiles. Planta. https://doi.org/10.1007/s00425-010-1156-914

3 915

Wang, B., Kashkooli, A.B., Sallets, A., Ting, H.M., de Ruijter, N.C.A., Olofsson, L., 916

Brodelius, P., Pottier, M., Boutry, M., Bouwmeester, H., van der Krol, A.R., 2016. 917

Transient production of artemisinin in Nicotiana benthamiana is boosted by a 918

specific lipid transfer protein from A. annua. Metab. Eng. 38, 159–169. 919


Wang, W., He, P., Zhao, D., Ye, L., Dai, L., Zhang, X., Sun, Y., Zheng, J., Bi, C., 2019. 921

Construction of Escherichia coli cell factories for crocin biosynthesis. Microb. Cell 922

Fact. 18. https://doi.org/10.1186/s12934-019-1166-1 923

Wu, X., Zhou, Y., Yin, F., Mao, C., Li, L., Cai, B., Lu, T., 2014. Quality control and 924

producing areas differentiation of Gardeniae Fructus for eight bioactive 925

constituents by HPLC-DAD-ESI/MS. Phytomedicine 21, 551–559. 926

https://doi.org/10.1016/j.phymed.2013.10.002 927

Xu, C.J., Fraser, P.D., Wang, W.J., Bramley, P.M., 2006. Differences in the carotenoid 928

content of ordinary citrus and lycopene-accumulating mutants. J. Agric. Food 929

Chem. 54, 5474–5481. https://doi.org/10.1021/jf060702t 930

Xu, H., Lybrand, D., Bennewitz, S., Tissier, A., Last, R.L., Pichersky, E., 2018. 931

Production of trans-chrysanthemic acid, the monoterpene acid moiety of natural 932

pyrethrin insecticides, in tomato fruit. Metab. Eng. 47, 271–278. 933


Yang, T., Stoopen, G., Yalpani, N., Vervoort, J., de Vos, R., Voster, A., Verstappen, 935

F.W.A., Bouwmeester, H.J., Jongsma, M.A., 2011. Metabolic engineering of 936

geranic acid in maize to achieve fungal resistance is compromised by novel 937

glycosylation patterns. Metab. Eng. 13, 414–425. 938


Yuan, L., Grotewold, E., 2015. Metabolic engineering to enhance the value of plants as 940

green factories. Metab. Eng. 27, 83–91. 941


Zhang, C., Ma, J., Fan, L., Zou, Y., Dang, X., Wang, K., Song, J., 2015. 943

Neuroprotective effects of safranal in a rat model of traumatic injury to the spinal 944


https://doi.org/10.1101/2019.12.18.880765

29

cord by anti-apoptotic, anti-inflammatory and edema-attenuating. Tissue Cell 47, 945

291–300. https://doi.org/10.1016/j.tice.2015.03.007 946

Zhang, Y., Fei, F., Zhen, L., Zhu, X., Wang, J., Li, S., Geng, J., Sun, R., Yu, X., Chen, 947

T., Feng, S., Wang, P., Yang, N., Zhu, Y., Huang, J., Zhao, Y., Aa, J., Wang, G., 948

2017. Sensitive analysis and simultaneous assessment of pharmacokinetic 949

properties of crocin and crocetin after oral administration in rats. J. Chromatogr. B. 950

Analyt. Technol. Biomed. Life Sci. 1044–1045, 1–7. 951

https://doi.org/10.1016/j.jchromb.2016.12.003 952

Zhu, Q., Zeng, D., Yu, S., Cui, C., Li, J., Li, H., Chen, J., Zhang, R., Zhao, X., Chen, L., 953

Liu, Y.G., 2018. From Golden Rice to aSTARice: Bioengineering Astaxanthin 954

Biosynthesis in Rice Endosperm. Mol. Plant 11, 1440–1448. 955

https://doi.org/10.1016/j.molp.2018.09.007 956

957


https://doi.org/10.1101/2019.12.18.880765

30

Table 1. Ratios of (A) crocins- and (B) picrocrocin-related apocarotenoid levels in tissues 958

infected with TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. 959

960

A CsCCD2L/BdCCD4.1

crocetin dialdehyde 3.206

cis-crocetin 1.139

trans-crocetin 1.113

cis-crocin 1 3.318

trans-crocin 1 1.755


trans-crocin 2' 2.420

cis-crocin 3 2.137



cis-crocin 5 2.448

trans-crocin 5 -

B CsCCD2L/BdCCD4.1

HTCC 1.573

picrocrocin isomer1 4.237

picrocrocin isomer2 1.405

picrocrocin-derivivative4 8.577

picrocrocin-derivivative7 6.474

safranal 3.805

961

962


https://doi.org/10.1101/2019.12.18.880765

31

Figures 963

964

965

Fig. 1. Schematic overview of the crocins biosynthesis pathway in C. sativus and B. 966

davidii. CrtB, phytoene synthase; PDS, phytoene desaturase; BCH2, carotene 967

hydroxylase; CsCCD2L, C. sativus carotenoid cleavage dioxygenase 2L; BdCCD4.1 and 968

4.3, B. davidii carotenoid cleavage dioxygenase 4.1 and 4.3; ALDH, aldehyde 969

dehydrogenase; UGT74AD1, UDP-glucosyltransferase 74AD1. 970


https://doi.org/10.1101/2019.12.18.880765

32

971

972

Fig. 2. Inoculation of N. benthamiana plants that stably express NIb with TEVΔNIb 973

recombinant clones that express different CCDs. (A) Schematic representation of 974

TEVΔNIb genome indicating the position where the GFP (green box) and the different 975


https://doi.org/10.1101/2019.12.18.880765

33

CCDs (CsCCD2L, orange box; BdCCD4.1, yellow box; and BdCCD4.3, gray box) were 976

inserted. The sequence of the artificial NIaPro cleavage site to mediate the release of the 977

recombinant proteins from the viral polyprotein is also indicated. The black triangle 978

indicates the exact cleavage site. Lines represent TEV 5’ and 3’ UTR and boxes represent 979

P1, HC-Pro, P3, P3N-PIPO, 6K1, CI, 6K2, VPg, NIaPro and CP cistrons, as indicated. 980

Scale bar corresponds to 1000 nt. (B) Pictures of representative leaves from plants mock-981

inoculated and agroinoculated with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L, TEVΔNIb-982

BdCCD4.1 and TEVΔNIb-BdCCD4.3, as indicated, taken at 13 dpi. Scale bars 983

correspond to 5 mm. (C) Virus diagnosis and (D) analysis of the progeny of recombinant 984

TEV in N. benthamiana plants mock-inoculated and agroinoculated with TEVΔNIb-985

aGFP, TEVΔNIb-CsCCD2L, -BdCCD4.1 and -BdCCD4.3. RNA was extracted at 15 dpi 986

and subjected to RT-PCR amplification. PCR products were separated in a 1% agarose 987

gel that was stained with ethidium bromide. Representative samples of triplicate analyses 988

are shown. (C and D) Lanes 0, DNA marker ladder with sizes (in bp) on the left; lanes 1, 989

RT-PCR controls with no RNA added; lanes 2, mock-inoculated plants; lanes 3 to 6, 990

plants inoculated with TEVΔNIb-aGFP (lanes 3), TEVΔNIb-CsCCD2L (lanes 4), 991

TEVΔNIb -BdCCD4.1 (lanes 4) and TEVΔNIb -BdCCD4.3 (lanes 5). The arrows point 992

to the bands corresponding to (C) the TEV CP, and (D) the full-length viral progenies 993

cDNAs. 994


https://doi.org/10.1101/2019.12.18.880765

34

995

996

Fig. 3. Heterologous production of crocins in N. benthamiana tissues infected with 997

recombinant viruses expressing CCD enzymes. (A) Chromatographic profile run on an 998

HPLC-PDA-HRMS and detected at 440 nm of the polar fraction of tissues infected with 999

TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. The profile of 1000


https://doi.org/10.1101/2019.12.18.880765

35

saffron stigma is also included as a control. Peaks abbreviations correspond to: c-cr, cis-1001

crocetin; t-cr, trans-crocetin; c-cr1, cis-crocin 1; t-cr1, trans-crocin 1; t-cr2, trans-crocin 1002

2; t-cr2ʹ, trans-crocin 2ʹ; c-cr3, cis-crocin 3, t-cr3, trans-crocin 3; c-cr4, cis-crocin 4; t-1003

cr4, trans-crocin 4; c-cr5, cis-crocin 5; t-cr5, trans-crocetin 5. (B) Absorbance spectra of 1004

the major crocins detected in the polar extracts of N. benthamiana tissues infected with 1005

TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1. Analyses were performed at 13 dpi. 1006


https://doi.org/10.1101/2019.12.18.880765

36

1007

Fig. 4. Apocarotenoid content and composition of N. benthamiana tissues from mock-1008

inoculated plants and plants infected with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L and 1009

TEVΔNIb-BdCCD4.1. (A) Crocetin dialdehyde, crocetin and crocins accumulation. (B) 1010

Levels of safranal, picrocrocin and its derivatives, and HTCC. Data are averages ± sd of 1011

three biological replicates and expressed as % of fold internal standard (IS) levels. 1012

Analyses were performed at 13 dpi. 1013


https://doi.org/10.1101/2019.12.18.880765

37

1014

Fig. 5. Relative quantities of (A) carotenoids (B) chlorophylls and (C) quinones and 1015

tocochromanols detected by HPLC-DAD-HRMS in tissues of mock-inoculated and 1016

infected (TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L and TEVΔNIb-BdCCD4.1) N. 1017

benthamiana plants. Data are averages ± sd of three biological replicates and expressed 1018

as fold internal standard (IS). Analyses were performed at 13 dpi. 1019


https://doi.org/10.1101/2019.12.18.880765

38

1020

1021

Fig. 6. LSCM images of crocins and chlorophyll autofluorescence in symptomatic leaves 1022

of N. benthamiana plants infected with TEVΔNIb, TEVΔNIb-CsCCD2L and TEVΔNIb-1023

BdCCD4.1, as indicated, at 13 dpi. Merged images of both fluorescent signals and images 1024

under white light are also shown. Scale bars indicate 10 μm. 1025


https://doi.org/10.1101/2019.12.18.880765

39

1026

1027

Fig. 7. Time-course accumulation of (A) crocins, (B) picrocrocin, (C) β-carotene and 1028

lutein, and (D) chlorophyll a and b, as indicated, in systemic tissues of N. benthamiana 1029

plants agroinoculated with TEVΔNIb-CsCCD2L. Data are average ± sd of at least three 1030

biological replicates. 1031


https://doi.org/10.1101/2019.12.18.880765

40

1032

Fig. 8. Apocarotenoid accumulation in N. benthamiana tissues inoculated with viral 1033

vectors carrying CsCCD2L, alone or in combination with PaCrtB, CsBCH2 and CsLTP1. 1034

(A) Schematic representation of TEVΔNIb-CsCCD2L/PaCrtB, - CsCCD2L/CsBCH2 and 1035

CsCCD2L/CsLPT1. The cDNAs corresponding to CsCCD2L, PaCrtB, CsBCH2 and 1036

CsLPT1 are indicated by orange, yellow, red and purple boxes respective. Other details 1037


https://doi.org/10.1101/2019.12.18.880765

41

are the same as in Fig. 2A. (B) Representative leaves from plants mock-inoculated and 1038

agroinoculated with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L, TEVΔNIb-1039

CsCCD2L/PaCrtB and TEVΔNIb-CsCCD2L/CsBCH2, at 13 dpi. (C) Total crocins, 1040

picrocrocin and safranal contents, expressed as mg/g of DW. Asterisks indicate 1041

significant differences according a t-test (pValue: *<0.05; **<0.01; ns, not significant) 1042

carried out in the comparisons CsCCD2L vs CsCCD2L+PaCrtB, CsCCD2L+CsBCH2 or 1043

CsCCD2L+CsLTP1. (D) Relative accumulation of single crocins, picrocrocin and 1044

safranal, normalized on the CsCCD2L-alone sample and visualized as heatmap of log2 1045

values. Data are average ± sd of three biological replicates. Analyses were performed at 1046

13 dpi. 1047


https://doi.org/10.1101/2019.12.18.880765

42

SUPPLEMENTARY FIGURES 1048

1049 Fig. S1. Nucleotide sequence of the cDNA transferred to the Nicotiana benthamiana 1050 transformed line using Agrobacterium tumefaciens to prepare the virus-drive expression 1051

system. 1052 1053 TGGCAGGATATATTGTGGTGTAAACGTTCCTGCGGCGGTCGAGATGGATCTTGGCAGGATATATTGTGGT1054 GTAAACGTTCCTGCGGCCGCAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCG1055 GATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATG1056 CCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCC1057 CCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTG1058 ATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGA1059 AGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCT1060 TTCGCAGATCTGTCGATCGACCATGGGGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGG1061 GTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGC1062 TGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAGGA1063 CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACT1064 GAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTC1065 CTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCC1066 ATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAG1067 GATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGC1068 CCGACGGCGAGGATCTCGTCGTGACACATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG1069 CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC1070 CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTC1071 CCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGA1072 TCGATCCTCTAGCTAGAGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGAT1073 AAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTG1074 TATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGAT1075 CCCCCGAATTAATTCGGCGTTAATTCAGTACATTAGCGGCCGCGATTCCATTGCCCAGCTATCTGTCACT1076 TTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCAT1077 CGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAA1078 GAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAGATTCCATTGCCCAGCTATCTGTC1079 ACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGC1080 CATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAA1081 AAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATG1082 ACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGTA1083 TTAAAATCTTAATAGGTTTTGATAAAAGCGAACGTGGGGAAACCCGAACCAAACCTTCTTCTAAACTCTC1084 TCTCATCTCTCTTAAAGCAAACTTCTCTCTTGTCTTTCTTGCGTGAGCGATCTTCAACGTTGTCAGATCG1085 TGCTTCGGCACCAGTACAACGTTTTCTTTCACTGAAGCGAAATCAAAGATCTCTTTGTGGACACGTAGTG1086 CGGCGCCATTAAATAACGTGTACTTGTCCTATTCTTGTCGGTGTGGTCTTGGGAAAAGAAAGCTTGCTGG1087 AGGCTGCTGTTCAGCCCCATACATTACTTGTTACGATTCTGCTGACTTTCGGCGGGTGCAATATCTCTAC1088 TTCTGCTTGACGAGGTATTGTTGCCTGTACTTCTTTCTTCTTCTTCTTGCTGATTGGTTCTATAAGAAAT1089 CTAGTATTTTCTTTGAAACAGAGTTTTCCCGTGGTTTTCGAACTTGGAGAAAGATTGTTAAGCTTCTGTA1090 TATTCTGCCCAAATTTGAAATGGGGGAGAAGAGGAAATGGGTCGTGGAAGCACTGTCAGGGAACTTGAGG1091 CCAGTGGCTGAGTGTCCCAGTCAGTTAGTCACAAAGCATGTGGTTAAAGGAAAGTGTCCCCTCTTTGAGC1092 TCTACTTGCAGTTGAATCCAGAAAAGGAAGCATATTTTAAACCGATGATGGGAGCATATAAGCCAAGTCG1093 ACTTAATAGAGAGGCGTTCCTCAAGGACATTCTAAAATATGCTAGTGAAATTGAGATTGGGAATGTGGAT1094 TGTGACTTGCTGGAGCTTGCAATAAGCATGCTCATCACAAAGCTCAAGGCGTTAGGATTCCCAACTGTGA1095 ACTACATCACTGACCCAGAGGAAATTTTTAGTGCATTGAATATGAAAGCAGCTATGGGAGCACTATACAA1096 AGGCAAGAAGAAAGAAGCTCTCAGCGAGCTCACACTAGATGAGCAGGAGGCAATGCTCAAAGCAAGTTGC1097 CTGCGACTGTATACGGGAAAGCTGGGAATTTGGAATGGCTCATTGAAAGCAGAGTTGCGTCCAATTGAGA1098 AGGTTGAAAACAACAAAACGCGAACTTTCACAGCAGCACCAATAGACACTCTTCTTGCTGGTAAAGTTTG1099 CGTGGATGATTTCAACAATCAATTTTATGATCTCAACATAAAGGCACCATGGACAGTTGGTATGACTAAG1100 TTTTATCAGGGGTGGAATGAATTGATGGAGGCTTTACCAAGTGGGTGGGTGTATTGTGACGCTGATGGTT1101 CGCAATTCGACAGTTCCTTGACTCCATTCCTCATTAATGCTGTATTGAAAGTGCGACTTGCCTTCATGGA1102 GGAATGGGATATTGGTGAGCAAATGCTGCGAAATTTGTACACTGAGATAGTGTATACACCAATCCTCACA1103 CCGGATGGTACTATCATTAAGAAGCATAAAGGCAACAATAGCGGGCAACCTTCAACAGTGGTGGACAACA1104 CACTCATGGTCATTATTGCAATGTTATACACATGTGAGAAGTGTGGAATCAACAAGGAAGAGATTGTGTA1105 TTACGTCAATGGCGATGACCTATTGATTGCCATTCACCCAGATAAAGCTGAGAGGTTGAGTGGATTCAAA1106 GAATCTTTCGGAGAGTTGGGCCTGAAATATGAATTTGACTGCACCACCAGGGACAAGACACAGTTGTGGT1107


https://doi.org/10.1101/2019.12.18.880765

43

TCATGTCACACAGGGCTTTGGAGAGGGATGGCATGTATATACCAAAGCTAGAAGAAGAAAGGATTGTTTC1108 TATTTTGGAATGGGACAGATCCAAAGAGCCGTCACATAGGCTTGAAGCCATCTGTGCATCAATGATCGAA1109 GCATGGGGTTATGACAAGCTGGTTGAAGAAATCCGCAATTTCTATGCATGGGTTTTGGAACAAGCGCCGT1110 ATTCACAGCTTGCAGAAGAAGGAAAGGCGCCATATCTGGCTGAGACTGCGCTTAAGTTTTTGTACACATC1111 TCAGCACGGAACAAACTCTGAGATAGAAGAGTATTTAAAAGTGTTGTATGATTACGATATTCCAACGACT1112 GAGAATCTTTATTTTCAGTAACTCTGGTTTCATTAAATTTTCTTTAGTTTGAATTTACTGTTATTCGGTG1113 TGCATTTCTATGTTTGGTGAGCGGTTTTCTGTGCTCAGAGTGTGTTTATTTTATGTAATTTAATTTCTTT1114 GTGAGCTCCTGTTTAGCAGGTCGTCCCTTCAGCAAGGACACAAAAAGATTTTAATTTTATTCGCTGAAAT1115 CACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGAT1116 AAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTT1117 GTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGGGGCCCTCGACGTT1118 CCTTGACAGGATATATTGGCGGGTAAACTAAGTCGCTGTATGTGTTTGTTTG 1119 1120

The cDNAs of the Neomycin phosphotransferase II (NPTII) and Tobacco etch virus 1121 (TEV) NIb are in blue and black, respectively. Initiation Met and stop codons are 1122

underlined. Note that an ATG has been added to the 5’ end of NIb cDNA that naturally 1123 lacks this codon because it is produced by proteolytic processing. A. tumefaciens T-DNA 1124

right border (RB) with overdrive (underlined) is in blue on yellow background and 1125

double left border (LB) is in blue on red background. Cauliflower mosaic virus 1126 (CaMV) 35S promoter and terminator are in red and fuchsia, respectively. Note that 1127 NIb is expressed from a partially duplicated version of CaMV 35S promoter. NIb open 1128 reading frame (ORF) is flanked by a modified version of the 5’ and 3’ untranslated 1129

regions (UTRs) of Cowpea mosaic virus (CPMV) RNA-2. 1130 1131

1132


https://doi.org/10.1101/2019.12.18.880765

44

Fig. S2. Nucleotide sequences of TEVΔNIb (sequence variant DQ986288 that include 1133

the two silent mutations G273A and A1119G, in red, and lacks the whole NIb cistron) 1134 and the derived recombinant viruses TEVΔNIb-aGFP, -CsCCD2L, -BdCCD4.1, -1135 BdCCD4.3, -CsCCD2L/PaCrtB, -CsCCD2L/CsBCH2 and -CsCCD2L/CsLPT1. The 1136

boundaries of viral cistrons are indicated on blue background. Initiation Met and stop 1137 codons are underlined. 1138 1139 >TEVΔNIb (8004 bp) 1140 AAAATAACAAATCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTAT1141 TGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGAT1142 AGCCATGGCACTCATCTTTGGCACAGTCAACGCTAACATCCTGAAGGAAGTGTTCGGTGGAGCTCGTATG1143 GCTTGCGTTACCAGCGCACATATGGCTGGAGCGAATGGAAGCATTTTGAAGAAGGCAGAAGAAACCTCTC1144 GTGCAATCATGCACAAACCAGTGATCTTCGGAGAAGACTACATTACCGAGGCAGACTTGCCTTACACACC1145 ACTCCATTTAGAGGTCGATGCTGAAATGGAGCGGATGTATTATCTTGGTCGTCGCGCGCTCACCCATGGC1146 AAGAGACGCAAAGTTTCTGTGAATAACAAGAGGAACAGGAGAAGGAAAGTGGCCAAAACGTACGTGGGGC1147 GTGATTCCATTGTTGAGAAGATTGTAGTGCCCCACACCGAGAGAAAGGTTGATACCACAGCAGCAGTGGA1148 AGACATTTGCAATGAAGCTACCACTCAACTTGTGCATAATAGTATGCCAAAGCGTAAGAAGCAGAAAAAC1149 TTCTTGCCCGCCACTTCACTAAGTAACGTGTATGCCCAAACTTGGAGCATAGTGCGCAAACGCCATATGC1150 AGGTGGAGATCATTAGCAAGAAGAGCGTCCGAGCGAGGGTCAAGAGATTTGAGGGCTCGGTGCAATTGTT1151 CGCAAGTGTGCGTCACATGTATGGCGAGAGGAAAAGGGTGGACTTACGTATTGACAACTGGCAGCAAGAG1152 ACACTTCTAGACCTTGCTAAAAGATTTAAGAATGAGAGAGTGGATCAATCGAAGCTCACTTTTGGTTCAA1153 GTGGCCTAGTTTTGAGGCAAGGCTCGTACGGACCTGCGCATTGGTATCGACATGGTATGTTCATTGTACG1154 CGGTCGGTCGGATGGGATGTTGGTGGATGCTCGTGCGAAGGTAACGTTCGCTGTTTGTCACTCAATGACA1155 CATTATAGCGACAAATCAATCTCTGAGGCATTCTTCATACCATACTCTAAGAAATTCTTGGAGTTGAGGC1156 CAGATGGAATCTCCCATGAGTGTACAAGAGGAGTATCAGTTGAGCGGTGCGGTGAGGTGGCTGCAATCCT1157 GACACAAGCACTTTCACCGTGTGGTAAGATCACATGCAAACGTTGCATGGTTGAAACACCTGACATTGTT1158 GAGGGTGAGTCGGGAGACAGTGTCACCAACCAAGGTAAGCTCCTAGCAATGCTGAAAGAACAGTATCCAG1159 ATTTCCCAATGGCCGAGAAACTACTCACAAGGTTTTTGCAACAGAAATCACTAGTAAATACAAATTTGAC1160 AGCCTGCGTGAGCGTCAAACAACTCATTGGTGACCGCAAACAAGCTCCATTCACACACGTACTGGCTGTC1161 AGCGAAATTCTGTTTAAAGGCAATAAACTAACAGGGGCCGATCTCGAAGAGGCAAGCACACATATGCTTG1162 AAATAGCAAGGTTCTTGAACAATCGCACTGAAAATATGCGCATTGGCCACCTTGGTTCTTTCAGAAATAA1163 AATCTCATCGAAGGCCCATGTGAATAACGCACTCATGTGTGATAATCAACTTGATCAGAATGGGAATTTT1164 ATTTGGGGACTAAGGGGTGCACACGCAAAGAGGTTTCTTAAAGGATTTTTCACTGAGATTGACCCAAATG1165 AAGGATACGATAAGTATGTTATCAGGAAACATATCAGGGGTAGCAGAAAGCTAGCAATTGGCAATTTGAT1166 AATGTCAACTGACTTCCAGACGCTCAGGCAACAAATTCAAGGCGAAACTATTGAGCGTAAAGAAATTGGG1167 AATCACTGCATTTCAATGCGGAATGGTAATTACGTGTACCCATGTTGTTGTGTTACTCTTGAAGATGGTA1168 AGGCTCAATATTCGGATCTAAAGCATCCAACGAAGAGACATCTGGTCATTGGCAACTCTGGCGATTCAAA1169 GTACCTAGACCTTCCAGTTCTCAATGAAGAGAAAATGTATATAGCTAATGAAGGTTATTGCTACATGAAC1170 ATTTTCTTTGCTCTACTAGTGAATGTCAAGGAAGAGGATGCAAAGGACTTCACCAAGTTTATAAGGGACA1171 CAATTGTTCCAAAGCTTGGAGCGTGGCCAACAATGCAAGATGTTGCAACTGCATGCTACTTACTTTCCAT1172 TCTTTACCCAGATGTCCTGAGTGCTGAATTACCCAGAATTTTGGTTGATCATGACAACAAAACAATGCAT1173 GTTTTGGATTCGTATGGGTCTAGAACGACAGGATACCACATGTTGAAAATGAACACAACATCCCAGCTAA1174 TTGAATTCGTTCATTCAGGTTTGGAATCCGAAATGAAAACTTACAATGTTGGAGGGATGAACCGAGATAT1175 GGTCACACAAGGTGCAATTGAGATGTTGATCAAGTCCATATACAAACCACATCTCATGAAGCAGTTACTT1176 GAGGAGGAGCCATACATAATTGTCCTGGCAATAGTCTCCCCTTCAATTTTAATTGCCATGTACAACTCTG1177 GAACTTTTGAGCAGGCGTTACAAATGTGGTTGCCAAATACAATGAGGTTAGCTAACCTCGCTGCCATCTT1178 GTCAGCCTTGGCGCAAAAGTTAACTTTGGCAGACTTGTTCGTCCAGCAGCGTAATTTGATTAATGAGTAT1179 GCGCAGGTAATTTTGGACAATCTGATTGACGGTGTCAGGGTTAACCATTCGCTATCCCTAGCAATGGAAA1180 TTGTTACTATTAAGCTGGCCACCCAAGAGATGGACATGGCGTTGAGGGAAGGTGGCTATGCTGTGACCTC1181 TGAAAAGGTGCATGAAATGTTGGAAAAAAACTATGTAAAGGCTTTGAAGGATGCATGGGACGAATTAACT1182 TGGTTGGAAAAATTCTCCGCAATCAGGCATTCAAGAAAGCTCTTGAAATTTGGGCGAAAGCCTTTAATCA1183 TGAAAAACACCGTAGATTGCGGCGGACATATAGACTTGTCTGTGAAATCGCTTTTCAAGTTCCACTTGGA1184 ACTCCTGAAGGGAACCATCTCAAGAGCCGTAAATGGTGGTGCAAGAAAGGTAAGAGTAGCGAAGAATGCC1185 ATGACAAAAGGGGTTTTTCTCAAAATCTACAGCATGCTTCCTGACGTCTACAAGTTTATCACAGTCTCGA1186 GTGTCCTTTCCTTGTTGTTGACATTCTTATTTCAAATTGACTGCATGATAAGGGCACACCGAGAGGCGAA1187 GGTTGCTGCACAGTTGCAGAAAGAGAGCGAGTGGGACAATATCATCAATAGAACTTTCCAGTATTCTAAG1188 CTTGAAAATCCTATTGGCTATCGCTCTACAGCGGAGGAAAGACTCCAATCAGAACACCCCGAGGCTTTCG1189 AGTACTACAAGTTTTGCATTGGAAAGGAAGACCTCGTTGAACAGGCAAAACAACCGGAGATAGCATACTT1190 TGAAAAGATTATAGCTTTCATCACACTTGTATTAATGGCTTTTGACGCTGAGCGGAGTGATGGAGTGTTC1191 AAGATACTCAATAAGTTCAAAGGAATACTGAGCTCAACGGAGAGGGAGATCATCTACACGCAGAGTTTGG1192


https://doi.org/10.1101/2019.12.18.880765

45

ATGATTACGTTACAACCTTTGATGACAATATGACAATCAACCTCGAGTTGAATATGGATGAACTCCACAA1193 GACGAGCCTTCCTGGAGTCACTTTTAAGCAATGGTGGAACAACCAAATCAGCCGAGGCAACGTGAAGCCA1194 CATTATAGAACTGAGGGGCACTTCATGGAGTTTACCAGAGATACTGCGGCATCGGTTGCCAGCGAGATAT1195 CACACTCACCCGCAAGAGATTTTCTTGTGAGAGGTGCTGTTGGATCTGGAAAATCCACAGGACTTCCATA1196 CCATTTATCAAAGAGAGGGAGAGTGTTAATGCTTGAGCCTACCAGACCACTCACAGATAACGTGCACAAG1197 CAACTGAGAAGTGAACCATTTAACTGCTTCCCAACTTTGAGGATGAGAGGGAAGTCAACTTTTGGGTCAT1198 CACCGATTACAGTCATGACTAGTGGATTCGCTTTACACCATTTTGCACGAAACATAGCTGAGGTAAAAAC1199 ATACGATTTTGTCATAATTGATGAATGTCATGTGAATGATGCTTCTGCTATAGCGTTTAGGAATCTACTG1200 TTTGAACATGAATTTGAAGGAAAAGTCCTCAAAGTGTCAGCCACACCACCAGGTAGAGAAGTTGAATTCA1201 CAACTCAGTTTCCCGTGAAACTCAAGATAGAAGAGGCTCTTAGCTTTCAGGAATTTGTAAGTTTACAAGG1202 GACAGGTGCCAACGCCGATGTGATTAGTTGTGGCGACAACATACTAGTATATGTTGCTAGCTACAATGAT1203 GTTGATAGTCTTGGCAAGCTCCTTGTGCAAAAGGGATACAAAGTGTCGAAGATTGATGGAAGAACAATGA1204 AGAGTGGAGGAACTGAAATAATCACTGAAGGTACTTCAGTGAAAAAGCATTTCATAGTCGCAACTAATAT1205 TATTGAGAATGGTGTAACCATTGACATTGATGTAGTTGTGGATTTTGGGACTAAGGTTGTACCAGTTTTG1206 GATGTGGACAATAGAGCGGTGCAGTACAACAAAACTGTGGTGAGTTATGGGGAGCGCATCCAAAGACTCG1207 GTAGAGTTGGGCGACACAAGGAAGGAGTAGCACTTCGAATTGGCCAAACAAATAAAACACTGGTTGAAAT1208 TCCAGAAATGGTTGCCACTGAAGCTGCCTTTCTATGCTTCATGTACAATTTGCCAGTGACAACACAGAGT1209 GTTTCAACCACACTGCTGGAAAATGCCACATTATTACAAGCTAGAACTATGGCACAGTTTGAGCTATCAT1210 ATTTTTACACAATTAATTTTGTGCGATTTGATGGTAGTATGCATCCAGTCATACATGACAAGCTGAAGCG1211 CTTTAAGCTACACACTTGTGAGACATTCCTCAATAAGTTGGCGATCCCAAATAAAGGCTTATCCTCTTGG1212 CTTACGAGTGGAGAGTATAAGCGACTTGGTTACATAGCAGAGGATGCTGGCATAAGAATCCCATTCGTGT1213 GCAAAGAAATTCCAGACTCCTTGCATGAGGAAATTTGGCACATTGTAGTCGCCCATAAAGGTGACTCGGG1214 TATTGGGAGGCTCACTAGCGTACAGGCAGCAAAGGTTGTTTATACTCTGCAAACGGATGTGCACTCAATT1215 GCGAGGACTCTAGCATGCATCAATAGACTCATAGCACATGAACAAATGAAGCAGAGTCATTTTGAAGCCG1216 CAACTGGGAGAGCATTTTCCTTCACAAATTACTCAATACAAAGCATATTTGACACGCTGAAAGCAAATTA1217 TGCTACAAAGCATACGAAAGAAAATATTGCAGTGCTTCAGCAGGCAAAAGATCAATTGCTAGAGTTTTCG1218 AACCTAGCAAAGGATCAAGATGTCACGGGTATCATCCAAGACTTCAATCACCTGGAAACTATCTATCTCC1219 AATCAGATAGCGAAGTGGCTAAGCATCTGAAGCTTAAAAGTCACTGGAATAAAAGCCAAATCACTAGGGA1220 CATCATAATAGCTTTGTCTGTGTTAATTGGTGGTGGATGGATGCTTGCAACGTACTTCAAGGACAAGTTC1221 AATGAACCAGTCTATTTCCAAGGGAAGAAGAATCAGAAGCACAAGCTTAAGATGAGAGAGGCGCGTGGGG1222 CTAGAGGGCAATATGAGGTTGCAGCGGAGCCAGAGGCGCTAGAACATTACTTTGGAAGCGCATATAATAA1223 CAAAGGAAAGCGCAAGGGCACCACGAGAGGAATGGGTGCAAAGTCTCGGAAATTCATAAACATGTATGGG1224 TTTGATCCAACTGATTTTTCATACATTAGGTTTGTGGATCCATTGACAGGTCACACTATTGATGAGTCCA1225 CAAACGCACCTATTGATTTAGTGCAGCATGAGTTTGGAAAGGTTAGAACACGCATGTTAATTGACGATGA1226 GATAGAGCCTCAAAGTCTTAGCACCCACACCACAATCCATGCTTATTTGGTGAATAGTGGCACGAAGAAA1227 GTTCTTAAGGTTGATTTAACACCACACTCGTCGCTACGTGCGAGTGAGAAATCAACAGCAATAATGGGAT1228 TTCCTGAAAGGGAGAATGAATTGCGTCAAACCGGCATGGCAGTGCCAGTGGCTTATGATCAATTGCCACC1229 AAAGAGTGAGGACTTGACGTTTGAAGGAGAAAGCTTGTTTAAGGGACCACGTGATTACAACCCGATATCG1230 AGCACCATTTGTCACTTGACGAATGAATCTGATGGGCACACAACATCGTTGTATGGTATTGGATTTGGTC1231 CCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGG1232 TGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATT1233 CGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCA1234 TATGTCTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATT1235 CCCTTCATCTGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTA1236 GTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATT1237 TCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTTAGTGGTTG1238 GCGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGAGCAAACCTGAAGAGCCTTTT1239 CAGCCAGTTAAGGAAGCGACTCAACTCATGAGTGAATTGGTGTACTCGCAAAGTGGCACTGTGGGTGCTG1240 GTGTTGACGCTGGTAAGAAGAAAGATCAAAAGGATGATAAAGTCGCTGAGCAGGCTTCAAAGGATAGGGA1241 TGTTAATGCTGGAACTTCAGGAACATTCTCAGTTCCACGAATAAATGCTATGGCCACAAAACTTCAATAT1242 CCAAGGATGAGGGGAGAGGTGGTTGTAAACTTGAATCACCTTTTAGGATACAAGCCACAGCAAATTGATT1243 TGTCAAATGCTCGAGCCACACATGAGCAGTTTGCCGCGTGGCATCAGGCAGTGATGACAGCCTATGGAGT1244 GAATGAAGAGCAAATGAAAATATTGCTAAATGGATTTATGGTGTGGTGCATAGAAAATGGGACTTCCCCA1245 AATTTGAACGGAACTTGGGTTATGATGGATGGTGAGGAGCAAGTTTCATACCCGCTGAAACCAATGGTTG1246 AAAACGCGCAGCCAACACTGAGGCAAATTATGACACACTTCAGTGACCTGGCTGAAGCGTATATTGAGAT1247 GAGGAATAGGGAGCGACCATACATGCCTAGGTATGGTCTACAGAGAAACATTACAGACATGAGTTTGTCA1248 CGCTATGCGTTCGACTTCTATGAGCTAACTTCAAAAACACCTGTTAGAGCGAGGGAGGCGCATATGCAAA1249 TGAAAGCTGCTGCAGTACGAAACAGTGGAACTAGGTTATTTGGTCTTGATGGCAACGTGGGTACTGCAGA1250 GGAAGACACTGAACGGCACACAGCGCACGATGTGAACCGTAACATGCACACACTATTAGGGGTCCGCCAG1251 TGATAGTTTCTGCGTGTCTTTGCTTTCCGCTTTTAAGCTTATTGTAATATATATGAATAGCTATTCACAG1252 TGGGACTTGGTCTTGTGTTGAATGGTATCTTATATGTTTTAATATGTCTTATTAGTCTCATTACTTAGGC1253


https://doi.org/10.1101/2019.12.18.880765

46

GAACGACAAAGTGAGGTCACCTCGGTCTAATTCTCCTATGTAGTGCGAGAAAAAAAAAAAAAAAAAAAAA1254 AAAAAAAAAAAAAAAAAAAAAAAA 1255 1256 >TEVΔNIb-aGFP (insert between possitions 144 and 145 of TEVΔNIb; 1257 artificial NIaPro cleavage site is on gray background) 1258 ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA1259 ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT1260 CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG1261 TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG1262 TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG1263 CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC1264 AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG1265 TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC1266 CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA1267 GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA1268 TGGACGAGCTGTACAAGACGACTGAGAATCTTTATTTTCAGGGGGAGAAG 1269 1270 >TEVΔNIb-CsCCD2L (insert between possitions 144 and 145 of TEVΔNIb; 1271 artificial NIaPro cleavage site is on gray background) 1272 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1273 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1274 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1275 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1276 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1277 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1278 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1279 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1280 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1281 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1282 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1283 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1284 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1285 CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1286 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1287 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1288 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1289 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1290 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1291 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1292 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1293 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1294 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1295 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1296 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1297 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1298 TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1299 CAGTCTGGAACA 1300 1301 >TEVΔNIb-BdCCD4.1 (insert between possitions 144 and 145 of TEVΔNIb; 1302 artificial NIaPro cleavage site is on gray background) 1303 ATGAACTCCCTTTATTCTTCCTCTTTCCTTTCCAAATTTCCAATGAATGTTTCATCACTTGGAATAATTG1304 ACAAGAACCCTCAAACAAAGATTAATCCCATTTATTCCGTACCAACGTTCCATGCCCGTCTAACCGAAAA1305 ACCCTTTCGCGGAAAGCCTTTATATGAGAGGAGAAGAAATGATGAATCTTTGTATGCAAAAATGTTGAAA1306 TCATTAGATGATTTCATATGCAAGTTCATGAGTGAAGTACCCCTGGACCCCTCCAACGATCCAAAACACA1307 TACTGGTGGGCAACTTTGCACCGGTGGACGAGCTTCCTCCGACTCCATGTGAGGTGGTGGAAGGCTCCCT1308 TCCGATATGTGTGGAGGGTGCATACATCCGCAACGGTCCCAACCCTCCGATCACCCCTCACGGTCCGTAC1309 CATCTCTTTGATGGAGATGGAATGCTTCACTCCATCACAATCTCAAAAGGCAAAGCCACCTTTTGTAGTC1310 GTTATGTTAAGACTTATAAATATTTGGTGGAACAAAACATAGGCTATCCGGTCTTCCCAAGTCCTTTCTC1311 CTCCTTTAATAATGGCCTCTCGGCTTCTATAGCGCGTTTAGTCGTATTAACGGTAGCACGAGTGCTGGTT1312 ACTAGGTGTTTGGATCCTAGACCTAATGGGTTTGGAACGGGGAATACTAGTTTAGGTTTATTTGGCGGGA1313 AACTCTTTGCGCTATGTGAGTCTGATCTTCCTTATGCTATAAAAGTGACATCGGATGGGGATATAATAAC1314


https://doi.org/10.1101/2019.12.18.880765

47

TCTTGATCGCCATGATTTTTACACCGGTGATCCGTTACTCAGAATGACTGCACACCCAAAGATTGATTTA1315 GAGACTGGACAAGTCTTCGCTTTCAGATGTGACATAACATCTCCATTCTTGACATTTTTCCGAATCGATT1316 CATCTGGAAGAAAAGGCCCCGATTTTCCAATCTTCTCCTTGCACGGACCGTCACTCATTCATGATTTTGC1317 TCTTACCAAACATTATGTCATATTCCAGGATACACAGGTTGAACTTAATCCAATGGAGATAACTAGAGGA1318 AGATCACCAATTGTAGTTGACCATACCAAAGTGCCCCGGCTAGGCATTTTACCACGCAACGCCATGAATG1319 AAACTGAACTGTCGTGGATTGATGTACCAGGATTAAACATGTCACACTTTATTAATGCTTGGGAAGAAGA1320 TGGCGGAAACAAAATAGTAATTGTTGCTTTTAATGTATTATCAGGGTTGGATTCCCTATATTTGTTCAAT1321 TCCTCAATGGAAAAAATTACAATTGACCTCAAGGCAAAGAAGATTGAAAGACATCCATTGTCCACTAAAA1322 ATCTCGAGTTAGGAGTTATTAATCCTGCTTACGCCGGAAAGAAAAACAAGTATGTATATGCGGCAATTAT1323 AAGTCAAACACCAAAGGCAGCAGGGGTGATTAAGCTTGACATATCACAGCTCCCTAATGCTGATAGTAGT1324 AATTGCATGGTGGCTAGCCGACTTTATGGTTCGAGCTGTTATGGTGGTGAACCTTACTTTGTCGCAAGGG1325 AGCCTGATAATCCAGAGGCTGAAGAGGATGATGGTTATCTAGTCACATACATGCATGATGAGAATAGCGA1326 GGAATCGTGGTTCTTGGTTATGAACGCCAAATCCCCAACTCTTGACATTGTTGCTAGTGTTAGATTACCT1327 GGTAGAGTGCCTTATGGCTTCCACGGACTTTTTGTCAGGGAAGGTGACCTCAACAAGCTGACAACCGAAA1328 ATTTATATTTCCAGTCTGGAACA 1329 1330 >TEVΔNIb-BdCCD4.3 (insert between possitions 144 and 145 of TEVΔNIb; 1331 artificial NIaPro cleavage site is on gray background) 1332 ATGGACACCCTTTCTTCCTTTTTCCTTCAAAAATATGTTCCAAGTCATTCACTCACACCATCCCCGCCAT1333 TACTCCTTAAATTCAATATTTCATCTCTTAAAATTGATAAAAAATGCGATAGATTAAATCATCAAAAATC1334 TATGATCACAACATCAATTTTGCATCAAAAAAAGCTTAAAACGTTAAACACTCCGAAAAACCCTTCAACC1335 TCAGAAATTACAAAAATAAAAAAACAATCTTTTCTTACTATTTTCTTCAACTCATTAGATAATTTCATAT1336 GCAATTTCATAGACCCCCCACTCCGCCCCTCCATCGACCCTAACCACGTCCTCTCCGGCAACTTCGCCCC1337 CGTCGGCGAGCTCCCCCCCACCGCGTGCGACGTCGTCGAGGGCTCCCTTCCGCCGTCGTTAAACGGCGCG1338 TACATTCGCAACGGCCCAAACCCTCAGTTCATCCCCCGTGGGCCCTACCACCTATTCGACGGCGATGGCA1339 TGCTCCACGCCGTCACCATCTCCGGCGGCAAAGCCACATTCTGCAGCCGCTTCGTCAAGACTTATAAGTA1340 CACGCTGGAGCGAGAGATCGGCTCGCCGGTTATTCTGAACGTGTTCTCCGCCTTCAACGGCTTGCTGGCT1341 TCGCTGGCGCGCTGCGCCGTATCATCAGGCCGCGTCCTCACCGGACAATACGACCCCCGCCGCGGCGCCG1342 GCCGCGCCAACACAAGCTTAGCGTTGATTAGCGGAAAGTTATTCGCTCACGAAGAATGTGACCTTCCCTA1343 CGGAATAAAATTAACATGTGATGGCGATATAATCACTTTAGGCCGCCATGAATTTCTCGGTGAGCAGTTT1344 ACGGCGATGACGGCGCACCCGAAAATCGACATGGAAACCGGCGAGACCTTTGCTTTCAGATACAGTTTCC1345 TGCCGCCGTTCTTAACATATTTCAGGATCAACGGGGATGGAATTATGCAACACGAAGTGCCGATTTTCTC1346 CTTGAAGCAATCGTCATTCATCCACGACTTCGCAATATCCAAAAACTACGTCGTTTTCCCAGATACGCAG1347 ATTGTGATAAAACCTAGAGATATGTTGAGAGGGAGGGTGCCATTAAGAGTTGATCTTGAAAAAGTGCCGA1348 GGCTTGGGATTATGCCGAGATACGCGGTGGATGAGAGGGAAATGTGGTGGGTTGATGTGTCGGGGTTTAA1349 TATAATACACGCGGTGAATGCGTGGGAGGAGGAAGACGACGGCGGCGACACCATTGTGATGGTGGCGCCG1350 AATGCTTTGGGGGTGGAGCATGTGTTGGAGCGTATGGATTTGGTTCACTCATCCTTGGAAAGAATTCAAA1351 TAAATTTGAAGGAGAAGAGTGTGACGAGACGCCCGGTGTCCACAGCAAATCTCGAGTTTGCGGTCATCAA1352 TCCGGCTTACGTTGGGAAGAAGAACAGGTATGTGTATGCAGCAGTAGGAGATCCAATGCCAAAGATAGTA1353 GGTGTTGTGAAGGTGGACTTATCACTTTCCACGGCCAACTCCGGCGACTGCACGGTGGCTAGACGCCTAT1354 ACGGGCCCGGCTGCTACGGCGGTGAACCATTTTTTGTGGCAAGGGAGCCGGATAATCCGGCGGCGGAGGA1355 GGATGATGGCTATCTAATAACTTATATGCATAATGAAAATACTGAAGAATCAAAGTTTTTAGTAATGGAT1356 GCAAAATCCCCTACTCTTCAAATTCTTGCTGCTGTTAAGCTACCCCAACGAGTTCCCTATGGCTTCCATG1357 GAATCTTTGTGCCGGAATCTCACTTGCGTAAACTAACAACCGAAAATTTATATTTCCAGTCTGGAACA 1358

1359 >TEVΔNIb-CsCCD2L/PaCrtB 1360 (insert between possitions 144 and 145 of TEVΔNIb; artificial NIaPro 1361 cleavage site is on gray background) 1362 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1363 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1364 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1365 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1366 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1367 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1368 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1369 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1370 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1371 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1372 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1373 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1374 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1375


https://doi.org/10.1101/2019.12.18.880765

48

CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1376 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1377 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1378 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1379 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1380 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1381 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1382 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1383 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1384 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1385 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1386 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1387 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1388 TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1389 CAGTCTGGAACA 1390 (insert between possitions 6981 and 6982 of TEVΔNIb; sequences to 1391 complete native NIaPro cleavage sites are on yellow background) 1392 GGGGAGAAGATGAATAATCCGTCGTTACTCAATCATGCGGTCGAAACGATGGCAGTTGGCTCGAAAAGTT1393 TTGCGACAGCCTCAAAGTTATTTGATGCAAAAACCCGGCGCAGCGTACTGATGCTCTACGCCTGGTGCCG1394 CCATTGTGACGATGTTATTGACGATCAGACGCTGGGCTTTCAGGCCCGGCAGCCTGCCTTACAAACGCCC1395 GAACAACGTCTGATGCAACTTGAGATGAAAACGCGCCAGGCCTATGCAGGATCGCAGATGCACGAACCGG1396 CGTTTGCGGCTTTTCAGGAAGTGGCTATGGCTCATGATATCGCCCCGGCTTACGCGTTTGATCATCTGGA1397 AGGCTTCGCCATGGATGTACGCGAAGCGCAATACAGCCAACTGGATGATACGCTGCGCTATTGCTATCAC1398 GTTGCAGGCGTTGTCGGCTTGATGATGGCGCAAATCATGGGCGTGCGGGATAACGCCACGCTGGACCGCG1399 CCTGTGACCTTGGGCTGGCATTTCAGTTGACCAATATTGCTCGCGATATTGTGGACGATGCGCATGCGGG1400 CCGCTGTTATCTGCCGGCAAGCTGGCTGGAGCATGAAGGTCTGAACAAAGAGAATTATGCGGCACCTGAA1401 AACCGTCAGGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCAGGAAGCAGAACCTTACTATTTGTCTGCCA1402 CAGCCGGCCTGGCAGGGTTGCCCCTGCGTTCCGCCTGGGCAATCGCTACGGCGAAGCAGGTTTACCGGAA1403 AATAGGTGTCAAAGTTGAACAGGCCGGTCAGCAAGCCTGGGATCAGCGGCAGTCAACGACCACGCCCGAA1404 AAATTAACGCTGCTGCTGGCCGCCTCTGGTCAGGCCCTTACTTCCCGGATGCGGGCTCATCCTCCCCGCC1405 CTGCGCATCTCTGGCAGCGCCCGCTCACGACTGAGAATCTTTATTTTCAG 1406 1407 >TEVΔNIb-CsCCD2L/CsBCH2 1408 (insert between possitions 144 and 145 of TEVΔNIb; artificial NIaPro 1409 cleavage site is on gray background) 1410 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1411 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1412 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1413 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1414 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1415 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1416 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1417 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1418 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1419 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1420 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1421 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1422 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1423 CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1424 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1425 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1426 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1427 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1428 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1429 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1430 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1431 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1432 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1433 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1434 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1435 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1436


https://doi.org/10.1101/2019.12.18.880765

49

TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1437 CAGTCTGGAACA 1438 (insert between possitions 6981 and 6982 of TEVΔNIb; sequences to 1439 complete native NIaPro cleavage sites are on yellow background) 1440 GGGGAGAAGATGTCGGCCAGAATCTCCCCCTCCGCCACCACCCTCGCCGCCTCCTTCCGCCGCCCTCCGT1441 CTGGCGCACGCATCCTCCTCCTCCCTTCGCTCCCTGTCCGCCGCCCCGTCGAACTCCGGCCGCCGTTGCT1442 TCATCGTCGGCGTCGGACGGCGACAGTGTTTTTCGTTCTCGCCGAAGAAAAAACAACTCCTTTTCTTGAC1443 GACGTGGAGGAAGAGAAGAGTATTGCGCCGTCAAATCGGGCGGCTGAGAGGTCGGCGCGGAAGCGGTCGG1444 AGCGGACCACGTACCTCATAGCCGCGGTTATGTCGAGCTTGGGCATCACATCCATGGCCGCCGCAGCCGT1445 CTACTACCGCTTCGCTTGGCAAATGGAGGGAGGGGATGTGCCAGTGACGGAGATGGCGGGAACGTTCGCT1446 CTCTCGGTCGGGGCGGCGGTGGGGATGGAGTTCTGGGCCAGGTGGGCCCACCGGGCCCTCTGGCACGCGT1447 CGCTCTGGCACATGCACGAGTCCCACCACCGGCCGAGGGAGGGCGCTTTCGAACTCAACGACGTCTTCGC1448 CATAATCAACGCGGTCCCCGCCATAGCCCTGCTCAACTTCGGCTTCTTCCACAGAGGTCTTCTCCCCGGC1449 CTCTGTTTCGGCGCCGGGCTGGGGATCACGCTGTTTGGTATTGCGTACATGTTCGTCCACGACGGGCTAG1450 TCCACCGGCGGTTCCCTGTGGGGCCCATAGCCGACGTGCCCTACTTCCAGCGCGTCGCCGCTGCTCACCA1451 GATCCACCACTCGGAGAAGTTCGAAGGGGTGCCCTATGGACTGTTCATGGGGCCCAAGGTCGGTGCGGTT1452 TCTTTTTGTTGTTTTCCTCTCTTAACGACTGAGAATCTTTATTTTCAG 1453 1454 >TEVΔNIb-CsCCD2L/CsLPT1 1455 (insert between possitions 144 and 145 of TEVΔNIb; artificial NIaPro 1456 cleavage site is on gray background) 1457 ATGGAATCTCCTGCTACTAAATTACCTGCACCTCTGCTGATGTTATCTTCTTCTCCATTCCTTCTCCCTT1458 CTCCTAATAAGAGCTCCTCCATCTTCCTTCCACGTAAATTAGGGCCGCTACCTCCAAAATATTATTATTA1459 CAATTGCTGCCATCCTAAGAGTAGATCAATCTCAGTAGTATCAATGGCAAATAAGGAGGAGGCAGAGACC1460 AGTAAGAAGAAGCCCAAACCATTAAAAGTACTAATTACCAAAGTGGATCCGAAGCCGAGGAAGGGCATGG1461 CATCCGTCGCAGTGGACTTACTCGAGAAGGCCTTTGTATACCTATTGTCCGGAAATTCTGCAGCTGATCG1462 TAGTAGTAGTAGTGGTCGTCGTCGTCGTAAAGAGCATTACTACCTCTCCGGCAATTATGCGCCCGTCGGA1463 CACGAAACCCCGCCCTCCGACCACCTCCCCATTCATGGATCCCTTCCTGAATGCTTGAATGGAGTGTTTC1464 TGAGAGTTGGTCCTAACCCCAAGTTTGCTCCCGTAGCCGGATACAATTGGGTCGATGGAGATGGAATGAT1465 TCATGGATTGCGTATTAAAGATGGAAAAGCAACTTATCTATCTCGATATATTAAAACGTCACGGTTTAAA1466 CAAGAAGAATATTTTGGAAGAGCAAAATTTATGAAGATTGGAGATCTAAGGGGATTGCTTGGGTTCTTTA1467 CGATCTTAATACTAGTACTTCGAACAACATTGAAAGTAATAGACATTTCATATGGAAGAGGGACGGGTAA1468 TACAGCTCTTGTGTATCATAATGGCTTACTATTGGCTCTATCAGAAGAAGATAAACCTTATGTTGTTAAA1469 GTTTTAGAAGATGGAGACTTGCAAACTCTTGGGATATTGGATTATGACAAGAAATTGTCACATCCATTCA1470 CCGCTCATCCAAAGATCGATCCGTTAACTGATGAGATGTTTACCTTTGGATATTCCATCTCGCCTCCGTA1471 TCTTACTTATCGAGTCATTTCCAAGGATGGAGTGATGCAAGATCCAGTGCAAATCTCAATTACATCCCCT1472 ACCATAATGCATGATTTTGCTATTACTGAAAATTATGCCATCTTCATGGACCTGCCCTTGTATTTCCAAC1473 CAGAGGAAATGGTAAAGGGGAAATTTGTCTCTTCATTTCACCCTACAAAAAGAGCTCGTATCGGTGTGCT1474 TCCACGATATGCAAAAGACGAGCATCCAATTCGATGGTTCGATCTTCCAAGTTGCTTCATGACTCATAAT1475 GCAAATGCTTGGGAAGAGAATGATGAAGTTGTGCTATTCACATGTCGCCTTGAGAGTCCTGATCTTGACA1476 TGCTTAGTGGACCTGCGGAAGAAGAGATTGGGAATTCAAAAAGTGAGCTTTACGAAATGAGGTTCAATTT1477 GAAAACTGGAATTACTTCACAAAAGCAACTATCTGTACCTAGTGTTGATTTTCCTCGGATCAACCAAAGT1478 TATACTGGCAGGAAACAGCAATATGTTTATTGTACTCTTGGCAACACCAAGATTAAGGGCATTGTGAAGT1479 TTGATCTGCAAATTGAACCAGAAGCCGGAAAGACAATGCTTGAAGTTGGAGGAAATGTACAAGGCATCTT1480 TGAGTTGGGACCTAGAAGATATGGTTCAGAGGCAATATTTGTGCCATGCCAACCTGGCATCAAATCTGAT1481 GAGGATGACGGTTACTTGATATTCTTTGTACACGACGAAAACAATGGGAAATCTGAGGTCAATGTCATCG1482 ATGCAAAGACAATGTCTGCAGAACCTGTGGCTGTTGTGGAACTTCCAAGCAGGGTTCCATATGGATTCCA1483 TGCCTTGTTTCTGAATGAGGAAGAACTTCAGAAGCACCAAGCAGAGACAACAACCGAAAATTTATATTTC1484 CAGTCTGGAACA 1485 (insert between possitions 6981 and 6982 of TEVΔNIb; sequences to 1486 complete native NIaPro cleavage sites are on yellow background) 1487 GGGGAGAAGATGGCTCGCCAAGTTGCTCTGTGCTCTGTCCTGGTGATCGCTGCCTTCCTCTTCATCTCCT1488 CCCCTCGTGCCGAGAGCGCAGTCACCTGCAGCACTGTGGCCTCTGCGGTATCACCGTGCATCGGTTATGT1489 TCGCGGCCAGGGCGCACTGACTGGCGCGTGCTGCAGCGGCGTGAAGGGCCTTGCTGCAGCGGCAACCACC1490 CCCCCTGACCGTCGGACTGCATGCAGCTGCCTCAAGTCAATGGCTGGCAAAATCAGCGGCCTGAACCCTA1491 GTCTCGCAAAAGGCCTCCCCGGCAAGTGCGGTGCCAGCGTCCCCTATGTCATCAGCACATCCACTGACTG1492 CACTAAGGTGGCAACGACTGAGAATCTTTATTTTCAG 1493

1494


https://doi.org/10.1101/2019.12.18.880765

50

1495 1496 Fig. S3. Complementation of a TEV mutant that lacks NIb in the transformed N. 1497 benthamiana line that stably expresses TEV NIb. (A) Wild-type and (B) NIb-expressing 1498

N. benthamiana plants were mechanically inoculated with TEVΔNIb-Ros1, a 1499 recombinant virus in which the NIb cistron is replaced by the Rosea1 visual marker that 1500 induces anthocyanin accumulation. Pictures were taken 11 days post-inoculation (dpi). 1501

1502


https://doi.org/10.1101/2019.12.18.880765

51

1503

1504 Fig. S4. Fluorescence stereomicroscope analysis of tissues from plants mock-inoculated 1505 and agroinoculated with TEVΔNIb-aGFP, TEVΔNIb-CsCCD2L, -BdCCD4.1 and -1506 BdCCD4.3. Pictures at 15 dpi were taken with a Leica MZ 16 F stereomicroscope under 1507 white light and under UV illumination with the GFP2 (Leica) fluorescence filter. Scale 1508 bars correspond to 5 mm. 1509

1510


https://doi.org/10.1101/2019.12.18.880765

52

1511 1512 Fig. S5. Crocetin ion extraction (m/z 329.1747 (M+H+)) in control and CsCCD2L and 1513 BdCCD4.1 polar fraction chromatograms. Metabolites were extracted from infected 1514

leaves and run on an HPLC-PDA-HRMS system. The apocarotenoids have an accurate 1515 mass and a chromatographic mobility identical to those of the authentic standards, when 1516 available. 1517

1518


https://doi.org/10.1101/2019.12.18.880765

53

SUPPLEMENTARY TABLES 1519

1520

Table S1. LC-HRMS of crocins- (A) and picrocrocin-related (B) apocarotenoid levels in 1521

infected leaves. Data are avg ± sd of, at least, three biological replicates; nd (not detected). 1522

Values are reported as fold level between the signal intensities of the apocarotenoid 1523

metabolite on the signal intensity of the internal standard (formononetin). 1524

1525

Table S2. Relative abundance, as % on the total, of apocarotenoid levels in CsCCD2L 1526

and BdCCD4.1 leaves. (A) crocins-related metabolites and (B) picrocrocin-related 1527

metabolites. 1528

1529

Table S3. LC-HRMS of carotenoid- (A), chlorophyll-related (B), and tocochromanols 1530

and quinones (C) levels in infected leaves. Data are avg ± sd of at least three biological 1531

replicates; nd (not detected). Values are reported as fold level between the signal 1532

intensities of the apocarotenoid metabolite on the signal intensity of the internal standard 1533

(a-tocopherol acetate). Asterisks indicate significant differences according a t-test 1534

(pValue: *<0.05; **<0.01) carried out in the comparisons Not infected vs GFP, CCD2L 1535

vs GFP and CCD4.1 vs GFP. 1536

1537

Table S4. LC-HRMS of crocins- (A) and picrocrocin and safranal (B) apocarotenoid 1538

levels in infected leaves. Data are avg ± sd of at least three biological replicates; nd (not 1539

detected). Values are reported as absolute amounts (in mg/g DW) by using authentical 1540

standards and external calibration curves. Asterisks indicate significant differences 1541

according a t-test (pValue: *<0.05; **<0.01) carried out in the comparisons CsCCD2L 1542

vs CsCCD2L+PaCrtB, CsCCD2L+CsBCH2 or CsCCD2L+CsLTP1. 1543


https://doi.org/10.1101/2019.12.18.880765

Efficient production of saffron crocins and picrocrocin in · 2019. 12. 18. · 117. flower tissue...

Documents

Transcript of Efficient production of saffron crocins and picrocrocin in · 2019. 12. 18. · 117. flower tissue...