Expression of Tyrosine Hydroxylase Gene from Rat Leads to Oxidative Stress in Potato Plants

bioRxiv preprint doi: https://doi.org/10.1101/2020.05.15.098210; this version posted May 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Short title: Oxidative stress upon HT expression in potato.

3 Expression of tyrosine hydroxylase gene from rat leads to oxidative stress in potato plants.

5 Kamil Kostyn1*, Aleksandra Boba2, Anna Kostyn2,3, Michał Starzycki4, Jan Szopa1,2, Anna Kulma2

7 1 Department of Genetics, Plant Breeding and Seed Production, Faculty of Life Sciences and Technology, Wroclaw University 8 of Environmental and Plant Sciences, pl. Grunwaldzki 24A, 50-363 Wroclaw, Poland

9 2 Department of Genetic Biochemistry, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63, 51-148 10 Wroclaw, Poland

11 3 Institute of Genetics and Microbiology, Faculty of Biological Sciences, University of Wroclaw, Przybyszewskiego 63, 51-148 12 Wroclaw, Poland

13 4 The Plant Breeding and Acclimatization Inst. (IHAR)—National Research Inst., Research Div, Poznan, ul. Strzeszyńska 36, 14 60-479 Poznan, Poland 15 * corresponding author ([email protected]; [email protected]) 16

17 One sentence summary: Introduction of rat tyrosine hydroxylase gene to potato disturbs 18 catecholamine synthesis, causes oxidative stress and activates antioxidant response.

20 Author Contributions

21 K.K. generated the transgenic lines, analyzed the GC-MS data, performed phenolics assessment with 22 UPLC, performed statistical analysis, wrote the manuscript, agrees to serve as the author responsible 23 for contact and ensures communication; A.B. performed gene transcript level assessment with RT-

24 qPCR; A.K. performed antioxidant potential assays and measured H2O2 levels; M.S. performed 25 infection tests; J.S. conceived the project; A.K. supervised the project.

27 Funding information: This study was supported by grant No. 2017/24/C/NZ1/00393 from National 28 Science Centre (NCN, Poland).

30 Abstract

31 Catecholamines are biogenic aromatic amines common among both animals and plants. In animals 32 they are synthesized via tyrosine hydroxylation, while in plants, both hydroxylation or 33 decarboxylation of tyrosine are possible, depending on the species, though no tyrosine hydroxylase – 34 a counterpart of animal enzyme has been identified yet. It is known that in potato plants it is the 35 decarboxylation of tyrosine that leads to catecholamine production. In this paper we present the 36 effects of induction of an alternative route of catecholamine production by introducing tyrosine 37 hydroxylase gene from rat. We demonstrate that an animal system can be used by the plant, 38 however, it does not function to synthesize catecholamines. Instead it leads to elevated reactive

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39 oxygen species content and constant stress condition to the plant which responds with elevated 40 antioxidant level and further with improved resistance to infection.

42 Keywords

43 catecholamine, potato, tyrosine hydroxylase, ROS, oxidative stress, antioxidants

45 Introduction

46 Catecholamines are natural biogenic amines found both in animals and plants. Their biosynthesis 47 route in animals has been well recognized. Shortly, it starts with tyrosine hydroxylation to L-DOPA, 48 catalyzed by tyrosine hydroxylase (TH, EC: 1.14.16.2). L-DOPA is further decarboxylated to dopamine 49 by L-DOPA decarboxylase (DD, EC: 4.1.1.28). This neurotransmitter may undergo hydroxylation by 50 dopamine β-monooxygenase (DH, EC: 1.14.17.1) to norepinephrine, and further to epinephrine by 51 phenylethanolamine N-methyltransferase (PNMT, EC: 2.1.1.28). This main route of catecholamine 52 biosynthesis may be complemented by that employing tyramine as a substrate for dopamine 53 biosynthesis in a reaction driven by a cytochrome P450 isoform (CYP2D6 in human brain or CYP2D in 54 rats) (Wang et al., 2014). Functions of catecholamines in animals (hormones, neurotransmitters) are 55 well recognized, as they have been studied extensively for decades (Goldstein, 2010).

56 Interestingly, the presence of these compounds in plants was shown as early as in the fifties of the 57 previous century (Udenfriend et al., 1959) and confirmed in a number of plant species since (Kulma 58 and Szopa, 2007). The role of catecholamines in plants is not fully recognized. Numerous publications 59 report either on their structural or regulatory functions. They may be substrates for other relevant 60 compounds, like betalains (Gandia-Herrero and Garcia-Carmona, 2013), alkaloids, like mescaline 61 (Aniszewski, 2015), melanins or hydroxycinnamic acid amides. Catecholamine conjugates with 62 hydroxycinnamic acids are important molecules produced upon pathogen infection that may be 63 incorporated into the cell wall reinforcing its structure and providing a tighter barrier for the invading 64 microorganisms (Newman et al., 2001). Their role may be also explained by their antioxidant 65 properties, since catecholamine molecules are potent free radical scavenging agents (Kostyn et al., 66 2012). This activity is even more pronounced in their amide conjugates. Due to neighboring hydroxyl 67 groups in the catechol ring, catecholamines possess remarkable ability to quench free radicals 68 (Kanazawa and Sakakibara, 2000). The DPPH and superoxide scavenging activities of catecholamines 69 were shown to be much higher than those of ascorbic acid and comparable to those of catechin and 70 flavan (Rahman et al., 2006). Moreover, catecholamines reacted with single oxygen distinctively 71 stronger than catechin or sodium azide (Shimizu et al., 2010). Dopamine was shown to be a stronger 72 antioxidant than tocopherol and glutathione (Kostyn et al., 2012), and flavonoids – luteolin and 73 quercetin in vitro (Huang et al., 2019). However, it must be noted that oxidized forms of 74 catecholamines might be toxic and can pose threat to cellular components. Analogously to their 75 function in animals, catecholamines were suggested to control carbohydrate metabolism. In 76 transgenic potatoes they influenced simple sugar content with simultaneous decrease in starch 77 (Swiedrych et al., 2004, Skirycz et al., 2005). Moreover, catecholamine interaction with 78 phytohormones was reported. Catecholamines were shown to stimulate ethylene biosynthesis (Dai 79 et al., 1993) or induce flowering in Lemna paucicostata (Yokoyama et al., 2000). Dopamine was 80 indicated as a mediator of gibberellic acid activity in lettuce hypocotyl (Kamisaka, 1979) and inhibited 81 auxin oxidase in tobacco and Acmella oppositifolia leading to growth stimulation (Protacio et al., 82 1992).

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83 Studies on catecholamine biosynthesis pathway in plants showed their analogy to that found in 84 animals. However, while in animals the route through tyrosine hydroxylation is preferred, in plants 85 both hydroxylation and decarboxylation of tyrosine was confirmed, though not simultaneously, and 86 the active route is probably species-specific (Kulma and Szopa, 2007). Tyrosine decarboxylase (TD) 87 presence in plants has been established and its structure and functions well recognized (Zhu et al., 88 2016). Plant TDs show narrow substrate specificity and can catalyze decarboxylation of tyrosine only 89 or tyrosine and L-DOPA, depending on plant species and/or tissue and developmental stage 90 (Lehmann and Pollmann, 2009, Świędrych et al., 2004). However, despite the fact that the presence 91 of L-DOPA was evidenced in numerous plants (Soares et al., 2014), tyrosine hydroxylase was not 92 found in these organisms. Hydroxylation of tyrosine may be ascribed to other enzymes. For instance, 93 tyrosinase catalyzes the reactions leading to melanin formation, namely the ortho-hydroxylation of 94 monophenols to o-diphenols (monophenolase activity, EC 1.14.18.1) and the subsequent oxidation of 95 o-diphenols to the corresponding o-quinones (diphenolase activity, EC 1.10.3.1). The L-DOPA 96 produced in this pathway is utilized in the synthesis of DOPA-quinone rather than catecholamines 97 (Chung et al., 2015). However, L-DOPA may be decarboxylated to tyramine with the aid of tyrosine 98 decarboxylase, which further is processed to yield catecholamines (Kulma and Szopa, 2007). 99 Nevertheless, tyrosine hydroxylase activity was found in callus cultures of Portulaca grandiflora and 100 it was shown to be different from tyrosinase and polyphenol oxidase activities (Yamamoto et al., 101 2001). In another study an enzyme of tyrosine hydroxylase activity was purified and characterized in 102 Mucuna pruriens (Luthra and Singh, 2010). It was highly specific to tyrosine, but showed also a slight 103 activity to tyramine converting it to dopamine, although catalysis of such reaction is usually ascribed 104 to tyrosinases (Kahn et al., 1998).

105 L-DOPA, being the precursor for catecholamines can also serve as a substrate for melanins, producing 106 leucodopachrome and dopachrome either by auto-oxidation or due to polyphenol oxidase (PPO, EC 107 1.10.3.1) activity. This reaction produces reactive oxygen species (ROS), such like hydrogen peroxide, 108 superoxide anion and hydroxyl radical (Soares et al., 2014). L-DOPA, like other non-protein amino 109 acids are accumulated in plants to serve as deterrents to herbivores (Fürstenberg-Hägg et al., 2013). 110 Moreover, it can act as allelochemical and after exudation from the roots leads to reduction of 111 growth of neighboring plants (Fujii et al., 1991). The toxicity of L-DOPA is clearly multi-source. The 112 already mentioned production of quinones is connected with ROS generation on one hand, but it also 113 leads to quinoprotein formation on the other. These proteins can be produced by reaction between 114 dopaquinone and the sulfhydryl groups of proteins and lead to such negative effects like enzyme 115 deactivation, mitochondrial dysfunction, DNA fragmentation, and apoptosis (Mushtaq et al., 2013, 116 Jana et al., 2011). In addition, it has been shown that L-DOPA may be incorporated to proteins via 117 mimicking tyrosine or phenylalanine in respective tRNA synthesis (Rodgers and Shiozawa, 2008). 118 Nevertheless, the ROS generated due to L-DOPA oxidation turn out to be the most serious problem 119 for a plant as they cause oxidative stress with a number of diverse repercussions. Therefore, a 120 specialized machinery of ROS neutralization must be employed by the plants to cope with oxidative 121 stress. This machinery is based on two main groups of agents, one employing enzymatic apparatus to 122 neutralize reactive radicals and another using small antioxidant molecules. The enzymatic ∙- 123 mechanisms include antioxidant enzymes, such as SOD (which converts O2 to H2O2), catalases and

124 peroxidases (which remove H2O2). The non-enzymatic mechanisms of ROS removal include 125 antioxidant molecules, such as ascorbic acid, glutathione, phenylpropanoids, carotenoids, and α- 126 tocopherol. Reactive oxygen species are intrinsic element of numerous stress events and, acting as 127 signaling molecules, trigger signal transduction pathways in plant response (Huang et al., 2019). It is 128 well known that ROS is the factor that precedes activation of SA signaling in plants responding to 129 biotic and abiotic stresses (Herrera-Vásquez et al., 2015). Members of the TGA and WRKY

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130 transcription factor families involved in SA-mediated transcriptional regulation play significant role in 131 controlling biosynthesis of antioxidants, for instance phenylpropanoids, in response to stress.

132 In this study we show the effects of rat tyrosine hydroxylase gene expression in potato. We show 133 that an induction of the alternative route of tyrosine metabolism toward catecholamines results in 134 the seizure of the catecholamine biosynthesis. L-DOPA synthesized due to TH activity undergoes 135 oxidation with simultaneous ROS production, which generates a state of constant oxidative stress 136 that forces the plant to counteract with activation of antioxidant machinery.

137

138 Results

139 Construction of transgenic potato plants

140 For transgenic potato plants generation agrotransformation method was employed and resulted in 141 232 regenerants, which were further pre-selected for TH cDNA presence in genomic DNA by means 142 of PCR (Supplementary Fig. S1A). In 118 individuals a positive signal was detected. Plants positively 143 screened for the presence of the transgene in genomic DNA were submitted to a subsequent stage of 144 selection – the assessment of the level of the introduced gene’s expression with use of Northern 145 Blotting technique. Total RNA isolated from 3-week-old potato plants from in vitro culture was 146 separated in gel electrophoresis and then transferred onto nitrocellulose membrane. Among 118 147 samples hybridized with the radioactive probe in 28 of them a strong signal was detected 148 (Supplementary Fig. S1B). The 28 plants selected by means of Northern blotting analysis were 149 analyzed for the presence of tyrosine hydroxylase protein. Total proteins were separated in 150 acrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane, followed 151 by incubation with antibodies specific to R. norvegicus tyrosine hydroxylase. Out of 28 plants 18 were 152 positively selected (Supplementary Fig. S1C) for the presence of 56 kDa protein specifically 153 recognized by anti-HT antibodies and based on the results from transcript level analysis and Western 154 blotting 5 transgenic lines were selected for further analyses (HT8, HT12, HT28, HT43 and HT53).

155

156 Metabolic profile of the transgenic potato plants

157 To determine the influence of the rat tyrosine hydroxylase gene expression on metabolites their 158 contents were measured in methanolic extracts of selected transgenic lines (HTZ lines) grown in in 159 vitro culture by means of GC-MS technique. A number of primary and secondary metabolites were 160 identified. The obtained data were submitted to Independent Component Analysis (ICA). The HTZ 161 transgenic lines clearly separated from the control, and considering both, the IC01 and IC02 162 components line HTZ28 was the most different from the control (Fig. 1).

163

164 Catecholamine level in the transgenic HTZ lines

165 Levels of the metabolites of catecholamine group were measured in the HTZ lines relatively to the 166 non-transgenic control (Fig. 2). In all of the transgenic HTZ lines the level of tyrosine, a substrate for 167 tyrosine hydroxylase was considerably lower compared to the control. The level of L-DOPA, a direct 168 product of tyrosine hydroxylase was higher in all the transgenic lines reaching 157% of the control. 169 The level of tyramine did not change significantly in the HTZ lines (only in line HTZ43 it was by 30% 170 lower than in the control). Noticeable decrease in dopamine level was measured in the transgenic 171 lines to 10%-48% of the control. Hydroxylation of dopamine at C-2 yields norepinephrine which is

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172 further methylated to epinephrine. The level of the latter was lower in all transgenic lines but HTZ8 173 and the changes reached 62%-82% of the control. The level of metanephrine, a catabolite of 174 epinephrine was lower in all HTZ lines and reached 36%-69% of the control. Homovanillic acid is a 175 product of dopamine degradation. Its level was decreased in the transgenic lines to 36%-74% of the 176 control.

177

178

179

180 181 Fig. 1. Independent Component Analysis on data obtained from GC-MS analysis of transgenic HTZ 182 lines and the control. Colored areas cover agglomerations of the same sample’s datapoints.

183

184 Primary metabolite level in the transgenic HTZ lines

185 GC-MS technique allowed to determine the levels of soluble primary metabolites with sugars, organic 186 acids and amino acids among them. In all transgenic lines a decrease of glucose, fructose and sucrose 187 level was noted. The highest changes were observed for the line HTZ12 with decrease of 94%, 89,5% 188 and 59,5%, respectively. In general, decreased levels of soluble sugar derivatives (sugar acids, sugar 189 alcohols) were observed in all of the transgenic lines, except erythritol (elevated level in virtually all 190 HTZ lines) (Supplementary File S2).

191 Among the primary metabolites organic acids play an important role as they participate in vital 192 metabolic pathways, like glycolysis and Krebs cycle. The levels of these metabolites were found to be 193 changed in the THZ transgenic lines. The most profound decreases were noted in all the transgenic

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194 lines for α-ketoglutarate (90% dropdown on average). The levels of citric acid and malic acid were 195 also dropped down in all the transgenic lines but HTZ8 (Supplementary File S3).

196 Changes in free amino acids were also observed (Supplementary File S4). The highest decrease in the 197 HTZ lines was found for proline and isoleucine. Leucine, valine, serine, asparagine and tryptophan 198 levels dropped down to a lesser extent. HTZ12 line showed the most diversity from the control with 199 arginine level increase of 3-fold and ornithine level of 8-fold.

200

201 202 Fig. 2. Levels of catecholamine group metabolites in HTZ transgenic lines relatively to the control 203 (non-transgenic potato) obtained with GC-MS technique presented as means of 6 biological 204 replicates ± standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

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205

206 Phenolic level in the transgenic HTZ lines

207 As catecholamine synthetic route is connected with the large pathway of phenylalanine 208 transformation, the phenylpropanoid pathway, levels of particular phenolic derivatives (both free 209 and cell wall bound) were determined by means of LC-MS technique. Based on retention times and 210 absorption and mass spectra the presence of feruloyl-tyramine, kaempferol glycoside and 211 chlorogenic acid was determined in the methanolic extracts (free phenolic fraction). In addition, 212 based on spectral analysis, putative ferulic acid derivative and caffeic acid derivative were measured. 213 The amount of feruloyl-tyramine was higher in all HTZ lines except HTZ43 compared to the control 214 (1.66 ± μg/g DW) (Fig. 3). Similarly, the level of kaempferol glycoside was higher in all HTZ lines but 215 HTZ43 in comparison to control (2.24 ± μg/g DW). Higher level of chlorogenic acid was found in HTZ8 216 and HTZ12 relatively to control (8.78 ± μg/g DW). The levels of the ferulic acid derivative and caffeic 217 acid derivative was higher in all the transgenic lines (Supplementary File S5).

218 In the cell wall bound phenolic fraction 4-hydroxybenzoic acid (4HBA), 4-hydroxybenzaldehyde 219 (4HBAl), vanillin, p-coumaric acid, ferulic acid and feruloyl-tyramine were measured. In addition, 220 based on spectral analysis putative catechin derivative, p-coumaric acid derivative and tri-ferulic acid 221 were identified. Levels of 4HBA and 4HBAl were elevated in all THZ lines (maximally 2-fold) in 222 comparison to the control (0.71 μg/g DW and 0.32 μg/g DW, respectively). Similarly, vanillin level was 223 increased in the HTZ lines (except HTZ43 – no change) relatively to control (0.76 μg/g DW). Levels of 224 p-coumaric acid, ferulic acid and feruloyl-tyramine were most increased in HTZ8 and HTZ12, while 225 insignificant changes were measured in the remaining transgenic lines (Fig. 3) (Supplementary File 226 S5).

227 Total level of phenolic derivatives was higher in all the transgenic lines, both in methanolic extracts 228 (free phenolics) and after hydrolysis (bound phenolics). The highest increase was noted for line 229 HTZ28 and the smallest for line HTZ53 (Table 1).

230

231 232 Fig. 3. Amounts of free phenolic compounds in HTZ transgenic lines relatively to the control (CTRL – 233 non-transgenic potato) obtained with LC-MS technique presented as means of 6 biological replicates 234 ± standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

235

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236 Table. 1. Total phenolic content assayed in the transgenic HTZ lines in the methanolic extracts (free 237 phenolics) and after hydrolysis (cell wall bound phenolics) compared to the control (CTRL).

cell wall bound free phenolics [%] phenolics [%] CTRL 100 100 HTZ8 212,3 148,5 HTZ12 194,6 145,3 HTZ28 249,7 169,4 HTZ43 198,5 137,4 HTZ53 132,5 107,5 238

239 Antioxidant potential of the transgenic potato extracts

240 Phenolics are known to be strong antioxidants, thus their elevated level in the transgenic lines 241 implies a higher antioxidant potential. The antioxidant potential of free phenolic extract and bound 242 phenolic extract was measured using the DPPH method (Kedare and Singh, 2011). Methanol extracts 243 from the transgenic plants were characterized with higher antioxidant potential (up to 5-times) 244 compared to control (Fig. 4A). For cell wall hydrolysates improved antioxidant potential was noted 245 for lines HTZ8 and HTZ12, but the changes were weaker (Fig. 4B).

246

247 248 Fig. 4. Antioxidant potential expressed as percent of quenched DPPH radical measured in methanol 249 extracts (A) and extracts after hydrolysis (B) from transgenic HTZ lines compared to the non- 250 transgenic potato (CTRL), presented as means of 6 biological replicates ± standard deviation. 251 Statistically significant changes (p<0.05) are marked with asterisks.

252

253 Red-ox analysis in the transgenic HTZ lines

254 Analysis of H2O2 level in transgenic potato plants

255 Level of H2O2, a gauge of the red-ox state was measured in the transgenic lines in relation to the

256 control (Fig. 5). The level of H2O2 was elevated in all HTZ lines by 44% on average. The highest 257 increase was measured in HTZ43 and HTZ53 (by 77% and 72%, respectively).

258

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259

260 Fig. 5. The level of H2O2 in HTZ potato lines compared to the non-transgenic potato (CTRL) presented 261 as means of 6 biological replicates ± standard deviation. Statistically significant changes (p<0.05) are 262 marked with asterisks.

263

264 Analysis of expression of genes involved in free radical processing in transgenic potato plants

265 The red-ox state depends mainly on the enzymatic system of free radical quenching, therefore the 266 levels of transcripts of catalase (CAT), ascorbate peroxidase (APX) and three superoxide dismutase 267 (SOD) genes were measured (Fig. 6). For catalase gene expression, a dropdown in all transgenic lines 268 was noted (by 42% on average) with the highest changes for HTZ8 and HTZ12 (by 62% and 77%, 269 respectively). Ascorbate peroxidase expression did not change in the transgenic lines, except HTZ53, 270 where it reached 177% of the control. Three superoxide dismutase (iron, copper/zinc and 271 manganese) gene expression levels were measured. For one of them – Cu/Zn SOD the average 272 transcript level was considerably higher compared to the control (3.8-fold). The highest changes 273 were detected for HTZ8 and HTZ12 (467% and 627% of the control, respectively). For Fe SOD and Mn 274 SOD no significant changes were observed, except HTZ8, where a decrease (by 36% and 41%, 275 respectively) was detected. In addition, a polyphenol oxidase (PPO) gene expression level was 276 measured. The PPO is responsible for o-hydroxylation of monophenols to o-diphenols (catechols) and 277 further to o-quinones. The expression level of PPO gene was substantially elevated in all the 278 transgenic lines compared to the control (over 4 to 6 times).

279

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280 281 Fig. 6. The level of transcript of genes involved in free radical processing in the HTZ lines presented as 282 relative quantification (RQ) in relation to the non-transgenic control (horizontal line at RQ = 1). 283 Elongation factor gene was used as a reference gene. The results were obtained with RT-qPCR 284 method on cDNA matrix as mean values of 3 biological repeats ± standard deviation. Statistically 285 significant changes (p<0.05) are marked with asterisks.

286

287 Analysis of H2O2 level in potato plants after exogenous L-DOPA treatment

288 Potato plants (wild type) from in vitro culture were treated with 0.1 mg/ml L-DOPA and then H2O2 289 level in 3, 6 and 12 hours later was measured (Fig. 7). Hydrogen peroxide level has increased in all 290 three timepoints in comparison to the control, with the highest change at 3 hours after the 291 treatment.

292

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293

294 Fig. 7. The level of H2O2 in wild type potato plants treated with 0.1 mg/ml L-DOPA (brighter bars) 295 compared to the non-treated control (darker bars) presented as means of 6 biological replicates ± 296 standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

297

298 Analysis of expression of genes involved in free radical processing in potato plants after exogenous L- 299 DOPA treatment

300 In potato plants from in vitro culture treated with 0.1 mg/ml L-DOPA the levels of transcripts of 301 genes involved in free radical processing were measured (Fig. 8). Transcript levels of genes connected

302 with H2O2 neutralization was higher than in the control at 3 and 6 hours after L-DOPA treatment – 303 catalase by ca. 30% and ascorbate peroxidase by ca. 45% and decreased below the control level at 12 304 hours after the treatment. Among the superoxide dismutase genes, expression of the one encoding 305 the Cu/Zn SOD was higher at 3 and 6 hours after the treatment, by 74% and 94% compared to the 306 control, respectively. Polyphenol oxidase gene was considerably activated (ca. 3-fold) at 6 and 12 307 hours after the treatment.

308

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309 310 Fig. 8. The level of transcript of genes involved in free radical processing in wild type potato plants 311 treated with 0.1 mg/ml L-DOPA presented as relative quantification (RQ) in relation to the non- 312 treated control (horizontal line at RQ = 1). Elongation factor gene was used as a reference gene. The 313 results were obtained with RT-qPCR method on cDNA matrix as mean values of 3 biological repeats ± 314 standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

315

316 Resistance of the transgenic potatoes to Phytophtora infestans attack

317 It is suggested that catecholamines participate in plants’ response to stress, including pathogen 318 attack (Świędrych et al., 2004). Moreover, the transgenic HTZ potato lines are characterized by 319 higher antioxidant potential, which is known to positively correlate with resistance to pathogens 320 (Kasote et al., 2015). In order to investigate the effect of expression of tyrosine hydroxylase on 321 potato susceptibility to infection, the HTZ lines were submitted to infection with Phytophtora 322 infestans, a common and dangerous potato pathogen. Lines HTZ12, HTZ28 and HTZ53 were the most 323 resistant in comparison to the control (Fig. 9). No correlation was noted between the degree of 324 infection and the phenolic compound level nor antioxidant potential.

325

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326 327 Fig. 9. Degree of infection of transgenic HTZ lines with P. infestans in comparison to the non- 328 transgenic potato (CTRL) (A). Sample photos of infected HTZ lines (B-E).

329

330 Discussion

331 Although the literature on catecholamine biosynthesis in plants remains scanty, it is generally 332 believed that the main pathway proceeds through decarboxylation of tyrosine to tyramine 333 conducted by tyrosine decarboxylase (TD). The presence of the ‘alternative pathway’ involving 334 hydroxylation of tyrosine to L-DOPA by tyrosine hydroxylase (TH), which is the main route of 335 catecholamine production in animals, was not fully confirmed in plants, although L-DOPA, product of 336 tyrosine hydroxylation, was found in these organisms. Based on the available plant genome 337 sequence databases, no gene encoding tyrosine hydroxylase has been found. In this study, using 338 transgenic potato plants with rat tyrosine hydroxylase gene, we analyzed the effect of the 339 ‘alternative pathway’ of catecholamine synthesis on the plant’s metabolism. As anticipated, the 340 obtained transgenic lines were characterized by increased L-DOPA levels, but despite further 341 expectations we observed decreased levels of dopamine and subsequent compounds of this route. In 342 light of the results obtained for transgenic potatoes with overexpression of tyrosine decarboxylase, 343 where the catecholamine level was significantly increased (Swiedrych et al., 2004), the observed

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344 results suggest that the catecholamine biosynthesis route through tyrosine hydroxylation is non- 345 functional in the potato plants, while the induction of the L-DOPA synthesis by heterologous tyrosine 346 hydroxylase reorganizes catecholamine biosynthesis and leads to redirection of the substrate 347 towards the production of other types of compounds.

348 It is known that L-DOPA is produced by some plants and utilized as allelochemical (Fujii, 2003, 349 Nishihara et al., 2004) and its phytotoxicity is ascribed to the oxidative damage resulting from free 350 radical species including ROS, generated from the pathway of L-DOPA metabolism (either 351 autooxidation or oxidation aided by polyphenol oxidases (PPO)) to quinone and subsequently to 352 melanins. Because in our research, after transformation of potato with tyrosine hydroxylase gene, 353 we did not observe increased catecholamine synthesis, we suspected that the surplus of L-DOPA 354 might have resulted in its oxidation followed by generation of free radicals and consequently in

355 oxidative stress in the plants. Indeed, we have observed an elevated level of H2O2 in the HTZ 356 transgenic potato lines, which may translate to a constant oxidative stress present in the plants. This 357 stays in agreement with Arabidopsis response to L-DOPA treatment, which led to changes in 358 transcriptional activities of genes associated with plant’s response to stress, both biotic and abiotic, 359 with those involved in amino acid metabolism, oxidative stress, melanin synthesis and lignification 360 among them (Golisz et al., 2010). Prolonged enhanced production of ROS can pose a threat to cells 361 by causing the peroxidation of lipids, oxidation of proteins, damage to nucleic acids etc. 362 (Hasanuzzaman et al., 2012), thus it is imperative for an organism to maintain equilibrium between 363 ROS and antioxidative capacity. In order to cope with oxidative stress, plants have developed several 364 enzymatic and non-enzymatic approaches to eliminate free ROS in cells. The increased content of

365 H2O2 in the HTZ potato lines correlated positively with the activation of gene encoding the 366 cytoplasmic superoxide dismutase (SOD). At the same time, catalase (CAT) gene transcript level was 367 lowered. It may reflect the mechanism, in which transcription activity of CAT gene decreases in

368 plants during stress (e.g. pathogen infection), perhaps due to the very H2O2 molecule itself (Yi et al.,

369 2003) or accumulation of salicylic acid (Shim et al., 2003). This helps maintaining the H2O2 content at 370 higher levels, which in turn translates into activation of many antimicrobial activities (Lamb and 371 Dixon, 1997, Averyanov, 2009). The transgenic HTZ lines were also characterized with elevated levels 372 of phenolic compounds and higher antioxidant potential. The phenolics comprise of several groups of 373 compounds, like flavonoids, phenolic acids, lignin or benzoates and have a whole gamut of functions, 374 including structural, antioxidant, signaling and regulatory. Expression of gene of phenylalanine 375 ammonia lyase (PAL), which is considered a key enzyme of the phenolic synthesis, was increased in 376 the transgenic HTZ lines. Bearing in mind a constant stress triggered by L-DOPA biosynthesis this is 377 not surprising as PAL expression is known to increase upon ROS content increase and various stress 378 conditions (Anna et al., 2011, Wada et al., 2014, Darmanti et al., 2018). Our observation confirms 379 Soares et al. (2012) (Soares et al., 2012) report, where soybean which absorbed L-DOPA showed 380 increased phenolic compounds, lignin content and the activities of related enzymes such as PAL and 381 cinnamyl alcohol dehydrogenase (CAD). Although lignin content in the four-week-old plants from in 382 vitro culture is barely determinable, we observed considerably high CAD gene and shikimate/quinate 383 hydroxy-cinnamoyltransferase (HCT) gene transcript levels (Supplementary File S6). Increased 384 expression of genes connected with lignification was also observed in L-DOPA treated Arabidopsis 385 (Golisz et al., 2010). Stress conditions, especially pathogen infections coax plants to reinforce the cell 386 wall with bio-reactive compounds to hinder microbial penetration into cells. Among the phenolics 387 identified in the HTZ lines feruloyl-tyramine deserves special attention as on one hand it is a 388 catecholamine derivative and on the other, it participates in plant’s response to pathogen infections 389 (Jang et al., 2004), wounding (Pearce et al., 1998) and drought (Sprenger et al., 2016). The observed 390 increase in the hydroxycinnamic acid-tyramine amide content in the transgenic lines may be

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391 somehow incomprehensible, because the level of tyramine, a direct substrate did not differ, nor was 392 the tyramine decarboxylase gene (TD) expression higher compared to the wild-type control. 393 However, it is conceivable that tyrosine acts as the substrate for both tyrosine decarboxylase, 394 normally occurring in the plant and for tyrosine hydroxylase. Because of the oxidative stress elicited 395 by L-DOPA, the produced tyramine is immediately used in the reaction of the hydroxycinnamic acid 396 amide biosynthesis driven by tyramine n-hydroxycinnamoyl transferase (THT), while dopamine 397 synthesis is reduced. THT gene encoding this enzyme is known to be activated in the hypersensitivity 398 reaction, due to salicylic acid (SA) action (Conrath et al., 2006, Kalenahalli et al., 2015). Analysis of the 399 promoter sequence of the potato THT gene revealed the presence of a number of elements 400 interacting with WRKY and MYB transcription factors, known to be connected with oxidative stress 401 response (Supriya et al., 2013, Ma and Constabel, 2019). Such elements were also found in the 402 promoter sequences of THT from other plant organisms, like tomato (Etalo et al., 2013) or wheat 403 (Kage et al., 2017). Moreover, such promoter elements are found in a number of genes involved in 404 phenylpropanoid pathway, allowing for their control by SA in response to infection (Sarkar et al., 405 2018). WRKY among other transcription factor genes were shown to be activated in Arabidopsis upon 406 L-DOPA treatment (Golisz et al., 2010). These resemblance to pathogen attack reaction in the HTZ 407 potato lines might stand behind their better resistance to P. infestans.

408 Another explanation might be rerouting of tyramine to the hydroxycinnamic acid amide production 409 due to the presence of tyrosine hydroxylase (TH). TH is a soluble enzyme occurring in cytoplasm, but 410 it has been shown to interact with plasma membrane elements, like phosphatidylserine (Kuhar et al., 411 1999). In animal neurons TH is localized in the vicinity of the synaptic vesicle and mitochondria 412 together with aromatic amino acid decarboxylase (AAD) and dopamine β-hydroxylase (DH), which, 413 thanks to the availability of substrates can swiftly convert L-DOPA to dopamine and dopamine to 414 norepinephrine, respectively (Wang et al., 2009). The co-localization of the three enzymes using 415 immunoprecipitation approach in rats was also shown by Cartier et al. (Cartier et al., 2010). As for 416 now, the presence of such or similar complexes that could involve tyrosine decarboxylase remains 417 hypothetical. However, the presence of tyrosine hydroxylase in transgenic potatoes could lead to the 418 rearrangement of the putative enzymatic complex and tyramine might be rerouted to the 419 biosynthesis of hydroxycinnamic acid amides catalyzed by THT. Nonetheless, this hypothesis requires 420 detailed research on the interactions between the enzymes of catecholamine biosynthesis in plants.

421 To summarize, the study on the effect of the alternative route of tyrosine metabolism towards 422 catecholamines led to acquisition of the new knowledge on the possibility of manipulating plant 423 metabolic pathways by introducing animal-type system. Expression of rat tyrosine hydroxylase gene 424 in potatoes resulted in elevated L-DOPA level. However, this intermediate did not serve for 425 catecholamine biosynthesis, but was oxidized with polyphenol oxidases and possibly also 426 spontaneously. These reactions produced ROS molecules, which elevated level led to the formation 427 of constant oxidative stress conditions. To compensate, antioxidant machinery was actuated, both 428 enzymatic and non-enzymatic. This in turn caused better resistance of the transgenic potatoes to P. 429 infestans infection. We suspect that introduction of an external element to the mechanism of 430 catecholamine synthesis in potato disturbed it and caused redirection of their substrates. These 431 results constitute a starting point to a new study, which will answer the question whether an 432 enzymatic complex producing catecholamines exists or not.

433

434 Materials and Methods

435 Plant material

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436 Potato plants (Solanum tuberosum L., cv. Desiree), obtained from “Saatzucht Fritz Lange KG” (Bad 437 Schwartau, FRG), were grown in tissue culture on MS medium (Murashige and Skoog, 1962) 438 supplemented with 0.8% agar, 1% sucrose and 250 mg/L claforan under 16 h light (21°C) and 8 h 439 darkness (16°C) cycle.

440

441 Construction of transgenic potato lines

442 The cDNA encoding TH from Rattus norvegicus (EMBL/GenBank database acc no. M10244.1), was 443 ligated in the sense orientation into the plant binary vector under the control of the 35S CaMV 444 promoter and OCS terminator. The vector was introduced into the Agrobacterium tumefaciens strain 445 C58C1:pGV2260 and the integrity of the plasmid was verified by restriction enzyme analysis. Young 446 leaves of wild-type potato S. tuberosum L. cv. Desiree were transformed with A. tumefaciens by 447 immersing leaf explants in bacterial suspension. A. tumefaciens inoculated leaf explants were 448 subsequently transferred to callus induction medium and shoot regeneration medium. Transgenic 449 plants were pre-selected by using PCR with the primers for the neomycin phosphotransferase 450 (kanamycin resistance) gene ((forward, CCGACCTGTCCGGTGCCC; reverse, CGCCACACCCAGCCGGCC)) 451 on genomic DNA isolated from 3-week-old tissue-cultured plants as a template and then selected by 452 northern blot analysis with a HT specific cDNA fragment as probe and subsequently by western blot 453 analysis with antibodies specific to rat HT protein (Sigma-Aldrich).

454

455 Northern blot analysis

456 Total RNA was prepared from frozen plant material using plant RNA purification kit (QIAGEN RNeasy

457 Kit). Following electrophoresis (1.5%(w/v) agarose, 15%(w/v) formaldehyde), RNA was transferred to 458 nylon membranes (Hybond N+, Amersham, UK). Membranes were hybridized overnight at 42°C in

459 250 mM sodium phosphate buffer (pH 7.2) containing 7%(w/v) SDS, 1%(w/v) bovine serum albumin 460 (BSA) and 1 mM EDTA. Radioactively labelled full-length cDNA was used as a hybridization probe.

461 Filters were washed three times in 1 × SSC containing 0.5%(w/v) SDS at 65°C (highly stringent 462 condition) or in the same buffer but at 42°C (medium stringent condition) for 30 min.

463

464 Western blot analysis

465 Proteins were extracted from frozen plant material using extraction buffer (100 mM Hepes-NaOH, pH

466 7.4, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 20%(v/v) glycerol, 0.5 mM PMSF, 70 mM beta-

467 mercaptoethanol) supplemented either with 0.1% or 1%(v/v) TritonX- 100. The assessment of the 468 expression of rat TH gene by means of western blot analysis using rabbit IgG anti HT protein was 469 conducted as described previously (Aksamit-Stachurska et al., 2008). Briefly, solubilized protein was

470 run on 12%(w/v) SDS polyacrylamide gels and blotted electrophoretically onto nitrocellulose 471 membranes (Schleicher and Schuell). Following transfer, the membrane was sequentially incubated

472 with blocking buffer (5%(w/v) defatted dry milk), and then with antibody directed against the TH 473 protein (1:5000 dilution). Formation and detection of immune complexes was performed with 474 alkaline phosphatase-conjugated goat anti-rabbit IgG at a dilution of 1:1500.

475

476 Isolation and analysis of phenolics

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477 The frozen tissue (100 mg), comminuted in liquid nitrogen was extracted to 1 ml of 0.1% HCl in 478 methanol in an ultrasonic bath for 10 minutes and centrifuged at 12000 × g, 4°C for 10 minutes. The 479 procedure was repeated twice and the collected supernatants were merged, dried under nitrogen 480 flow and re-suspended in 200 μl of methanol (free phenolic fraction). The remaining pellet was 481 hydrolyzed in 2 M NaOH at 37°C overnight. Then the pH was adjusted to 3 using concentrated HCl 482 and two volumes of ethyl acetate were added and mixed thoroughly. Following centrifugation 483 (12000 × g, 4°C, 15 minutes), the organic phase was collected and the extraction to ethyl acetate was 484 repeated two more times. All the collected volumes were merged, dried under nitrogen flow and re- 485 suspended in 200 μl of methanol (bound phenolic fraction). The obtained extracts were analyzed in 486 UPLC with diode array detector 2996 PDA and mass detector QTOF. Acquity UPLC BEH C18 (2.1 × 100

487 mm, 1.7 μm) column was used for compound separation. The mobile phase consisted of 0.1%(v/v) 488 formic acid (solvent A) and acetonitrile (solvent B) and the flow was 95A:5B for 1 minute, then 489 linearly for 11 minutes to 70A:30B, for next 4 minutes to 0A:100B and 1 minute to 95A:5B at rate of 490 0.4 ml/min. Column temperature was 25°C. Absorbance was measured at 210 nm – 500 nm range 491 and mass spectra were registered in the range of 50-1000 Da under the following conditions: 492 nitrogen flow 800 l/h, source temperature 70°C, cone temperature 400°C, capillary voltage 3.5 kV, 493 cone voltage 40 V – 60 V, scan time 0.2 s (Wojtasik et al., 2013). Identification of compounds based 494 on the comparison to the retention times, absorbance spectra and mass spectra of pure standards 495 (Sigma-Aldrich).

496

497 GC-MS analysis of metabolome

498 The amount of 150 mg of whole plant tissue ground in liquid nitrogen was extracted with methanol 499 (12 ml/g FW). Samples were incubated at 70°C for 15 min. and centrifuged at room temperature

500 (12000×g, 10 min.). Then they were supplemented with 1.5 ml H2O and extracted with 0.75 ml 501 chloroform. The polar phase was dried and resolved in piridin/metoxyamine solution (20 mg/ml) and 502 then incubated at 37°C for 2 hours. Samples were derivatized with N-methyl-N-trimethylsilyl 503 trifluoroacetamide (MSTFA) and analyzed in GC-MS. GC8000 gas chromatograph with A2000 504 autosampler and Voyager Quadrupole Mass Spectrometer (Thermoquest) were used. 505 Chromatograms and mass spectra were analyzed with TagFinder software based on mass spectra 506 database (The Max Planck Institute of Molecular Plant Physiology (MPI-MP), Golm, Germany) 507 (Roessner et al., 2000, Luedemann et al., 2008, Kopka et al., 2004). Ribitol was used as the external 508 standard.

509

510 RT-PCR

511 Total RNA prepared from frozen plant material using plant RNA purification kit (QIAGEN RNeasy Kit) 512 was used for cDNA synthesis preceded by removal of residual genomic DNA with DNase I kit 513 (Invitrogen) according to the producer’s instructions. High capacity cDNA Reverse Transcription Kit 514 (Thermo Fisher Scientific) was used for reverse transcription of the RNA as stated in the producent’s 515 protocol. Transcript levels were determined with RT-qPCR using StepOnePlus™ Real-Time OCR 516 Systems (Applied Biosystems) on the cDNA matrix. Primers used in the reactions were designed in 517 LightCycler® Probe Design v2 (Roche) software. For the reactions DyNAmo SYBR Green qPCR Kit 518 (Thermo Fisher Scientific) was used. The cDNA was dissolved 5 times prior to the experiment. The 519 annealing temperature was 57°C. Changes in the transcript levels of the examined genes were 520 presented as relative quantification in regard to control. Elongation factor gene was used as the 521 reference gene.

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522

523 Determination of antioxidant capacity

524 Methanol extracts (free phenolic fraction and bound phenolic fraction) were used for antioxidant 525 potential determination with 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Kedare and Singh, 2011). 6 μl of 526 sample extract was added to 200 μl of 0.2 mM DPPH methanolic solution and incubated at room 527 temperature in darkness for 15 min. 6 μl of methanol was used in the control and pure methanol was 528 used as blank. Subsequently, absorbance at λ=515 nm was measured. The antioxidant potential 529 corresponded to the degree of free radical reduction and was presented as:

530

푠푎푚푝푙푒 퐴515 (1 − ) × 100% 푐표푛푡푟표푙 퐴515 531

532 Hydrogen peroxide assay

533 50 mg of four-week-old plant green tissue ground in liquid nitrogen were used for the analysis. Each 534 sample was supplemented with 200 μl of 20 mM phosphate buffer pH 6.5, mixed and centrifuged 535 (4°C, 12000×g, 10 min). 50 μl of the supernatant was then used for the assay with Amplex Red Assay 536 (Life Technologies) according to the producer’s instruction. The absorbance was measured at λ=560

537 nm, fluorescence at λex = 571 nm and λem = 585 nm (Wang et al., 2017).

538

539 Treatment of plants with L-DOPA

540 Four-week-old potato plants from in vitro culture were submitted to 0.1 mg/ml L-DOPA treatment. 541 The plants grown in glass jars (5 plants per a jar) were sprayed with 3 ml of L-DOPA and collected in 542 3, 6 and 12 hours after the treatment.

543

544 Determination of resistance to pathogen

545 Phytophtora infestans (pathotype 33-IHAR PIB) was grown on solid PDA medium (3%) at 18°C for 14 546 days in darkness. Next, rye caryopses sterilized by autoclave, were placed onto the well grown 547 mycelium (30 per a Petri dish) and were left for 7 days under the same conditions. The inoculum 548 prepared in this manner was applied to a leave of individual plant. The plants were grown in 549 hydroponic culture in a grow chamber (12 hour photoperiod with 15°C at day and 10°C at night) on 550 liquid B5 medium (diluted 10 times) (Gamborg et al., 1968). The experiment was conducted for 14 551 days and the degree of infection was assessed according to a bonitation scale (1-1.5 – high 552 resistance; 2-2.5 – mediocre resistance; 3-3.5 – weak resistance).

553

554 Statistical analysis

555 All experiments were performed in at least 3 biological repeats. The results were presented as mean 556 values ± standard deviation. For significance of changes evaluation one-way ANOVA with Tukey post- 557 hoc or Kruskal-Wallis test was used (Statistica, Statsoft). Independent Component Analysis (ICA) for

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558 the data obtained from GC-MS analysis was performed with MetaGeneAnalyse software (v1.7.1; 559 http://metagenealyse.mpimpgolm.mpg.de) (Scholz et al., 2005).

560

561 Acknowledgements

562 This study was supported by grant No. 2017/24/C/NZ1/00393 from National Science Centre (NCN, 563 Poland). We wish to thank to the group of Prof. Joachim Kopka (MPI Molecular Plant Physiology, 564 Golm, Germany) for useful advices and great help in GC–MS experiments.

565 Figure and Table captions

566 Fig. 1. Independent Component Analysis on data obtained from GC-MS analysis of transgenic HTZ 567 lines and the control. Colored areas cover agglomerations of the same sample’s datapoints.

568 Fig. 2. Levels of catecholamine group metabolites in HTZ transgenic lines relatively to the control 569 (non-transgenic potato) obtained with GC-MS technique presented as means of 6 biological 570 replicates ± standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

571 Fig. 3. Amounts of free phenolic compounds in HTZ transgenic lines relatively to the control (CTRL – 572 non-transgenic potato) obtained with LC-MS technique presented as means of 6 biological replicates 573 ± standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

574 Fig. 4. Antioxidant potential expressed as percent of quenched DPPH radical measured in methanol 575 extracts (A) and extracts after hydrolysis (B) from transgenic HTZ lines compared to the non- 576 transgenic potato (CTRL), presented as means of 6 biological replicates ± standard deviation. 577 Statistically significant changes (p<0.05) are marked with asterisks.

578 Fig. 5. The level of H2O2 in HTZ potato lines compared to the non-transgenic potato (CTRL) presented 579 as means of 6 biological replicates ± standard deviation. Statistically significant changes (p<0.05) are 580 marked with asterisks.

581 Fig. 6. The level of transcript of genes involved in free radical processing in the HTZ lines presented as 582 relative quantification (RQ) in relation to the non-transgenic control (horizontal line at RQ = 1). 583 Elongation factor gene was used as a reference gene. The results were obtained with RT-qPCR 584 method on cDNA matrix as mean values of 3 biological repeats ± standard deviation. Statistically 585 significant changes (p<0.05) are marked with asterisks.

586 Fig. 7. The level of H2O2 in wild type potato plants treated with 0.1 mg/ml L-DOPA (brighter bars) 587 compared to the non-treated control (darker bars) presented as means of 6 biological replicates ± 588 standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

589 Fig. 8. The level of transcript of genes involved in free radical processing in wild type potato plants 590 treated with 0.1 mg/ml L-DOPA presented as relative quantification (RQ) in relation to the non- 591 treated control (horizontal line at RQ = 1). Elongation factor gene was used as a reference gene. The 592 results were obtained with RT-qPCR method on cDNA matrix as mean values of 3 biological repeats ± 593 standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

594 Fig. 9. Degree of infection of transgenic HTZ lines with P. infestans in comparison to the non- 595 transgenic potato (CTRL) (A). Sample photos of infected HTZ lines (B-E).

596

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597 Table. 1. Total phenolic content assayed in the transgenic HTZ lines in the methanolic extracts (free 598 phenolics) and after hydrolysis (cell wall bound phenolics) compared to the control (CTRL).

599

600 Supplementary File S1. Consecutive selection steps of potato plants transformed with HT gene from 601 R. norvegicus. A – sample PCR product electrophoresis separation with use of primers specific to a 602 486-nt fragment of HT cDNA from R. norvegicus. P – positive control, N – negative control (non- 603 transgenic potato); B – sample Northern blot of RNA samples from transgenic potato plants bearing 604 HT gene from R. norvegicus. P – positive control, N – negative control (wt potato); C – Western blot 605 analysis of proteins isolated from transgenic HT potato plants. N – negative control (non-transgenic 606 potato).

607 Supplementary File S2. Levels of soluble sugars in HTZ transgenic lines relatively to the control (non- 608 transgenic potato) obtained with GC-MS technique presented as means of 6 biological replicates ± 609 standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

610 Supplementary File S3. Levels of organic acids in HTZ transgenic lines relatively to the control (non- 611 transgenic potato) obtained with GC-MS technique presented as means of 6 biological replicates ± 612 standard deviation. Statistically significant changes (p<0.05) are marked with asterisks.

613 Supplementary File S4. Levels of soluble amino acids in HTZ transgenic lines relatively to the control 614 (non-transgenic potato) obtained with GC-MS technique presented as means of 6 biological 615 replicates ± standard deviation. Statistically significant changes (p<0.05) are marked with red color.

616 Supplementary File S5. Levels of soluble (A) and cell wall bound (B) phenolics’ derivatives in HTZ 617 transgenic lines relatively to the control (non-transgenic potato) obtained with LC-MS technique 618 presented as means of 6 biological replicates ± standard deviation. Statistically significant changes 619 (p<0.05) are marked with red color.

620 Supplementary File S6. The levels of transcripts of cinnamyl-alcohol dehydrogenase (CAD) and 621 shikimate/quinate hydroxy-cinnamoyl transferase (HCT) genes in the HTZ lines presented as relative 622 quantification (RQ) in relation to the control (horizontal line at RQ = 1). Elongation factor gene was 623 used as a reference gene. The results were obtained with RT-qPCR method on cDNA matrix as mean 624 values of 3 biological repeats ± standard deviation. Statistically significant changes (p<0.05) are 625 marked with asterisks.

626 627 628 Literature

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Parsed Citations This study was supported by grant No. 2017/24/C/NZ1/00393 from National Science Centre (NCN, Poland). We wish to thank to the group of Prof. Joachim Kopka (MPI Molecular Plant Physiology, Golm, Germany) for useful advices and great help in GC–MS experiments.