This is the accepted version of the article: Soler, Marçal; Verdaguer, Roger; Fernández-Piñán, Sandra; [et al.]. Silenc- ing against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem. DOI 10.1016/j.plantsci.2019.110360 This version is avaible at https://ddd.uab.cat/record/218484 under the terms of the license Journal Pre-proof Silencing against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem Marçal Soler, Roger Verdaguer, Sandra Fernández-Piñán, Dolors Company-Arumı́, Pau Boher, Elsa Góngora-Castillo, Marc Valls, Enriqueta Anticó, Marisa Molinas, Olga Serra, Mercè Figueras PII: S0168-9452(19)31533-X DOI: https://doi.org/10.1016/j.plantsci.2019.110360 Reference: PSL 110360 To appear in: Plant Science Received Date: 12 June 2019 Revised Date: 23 November 2019 Accepted Date: 25 November 2019 Please cite this article as: Soler M, Verdaguer R, Fernández-Piñán S, Company-Arumı́ D, Boher P, Góngora-Castillo E, Valls M, Anticó E, Molinas M, Serra O, Figueras M, Silencing against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110360 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier. Silencing against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem Marçal Solera, Roger Verdaguera, Sandra Fernández-Piñána, Dolors Company-Arumía, Pau Bohera, Elsa Góngora-Castillob1, Marc Vallsc, Enriqueta Anticód, Marisa Molinasa, Olga Serraa and Mercè Figuerasa* a Laboratori del Suro, Biology Department, Universitat de Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain. b Department of Plant Biology, Michigan State University, East Lansing, MI48824, USA. of c Genetics Department, Universitat de Barcelona and Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB). Edifici CRAG, Campus UAB, 08193 Bellaterra, Catalonia, Spain. ro d Chemistry Department, Faculty of Sciences, University of Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain. *Corresponding author: merce.figueras@udg.edu -p re 1 Present address: CONACYT Research Fellow-Unidad de Biotecnología, Centro de Investigación Científica de Yucatán. Mérida, Yucatán, México. lP Graphical Abstract na ur Jo Highlights  . Conserved silencing to the NAC domain of StNAC103 increases suberin and wax load  . Conserved silencing has stronger effects on waxes than StNAC103 specific silencing  . Conserved silencing is correlated with upregulation of genes related with waxes  . StNAC103 silencing boosts lipid-regulators and inhibits polyamine enzymes Abstract Both suberin and its associated waxes contribute to the formation of apoplastic barriers that protect plants from the environment. Some transcription factors have emerged as regulators of of the suberization process. The potato StNAC103 gene was reported as a repressor of suberin polyester and suberin-associated waxes deposition because its RNAi-mediated downregulation ro (StNAC103-RNAi) over-accumulated suberin and associated waxes in the tuber phellem concomitantly with the induction of representative biosynthetic genes. Here, to explore if other genes of the large NAC gene family participate to this repressive function, we extended the -p silencing to other NAC members by targeting the conserved NAC domain of StNAC103 (StNAC103-RNAi-c). Transcript profile of the StNAC103-RNAi-c phellem indicated that StNAC101 re gene was an additional potential target. In comparison with StNAC103-RNAi, the silencing with StNAC103-RNAi-c construct resulted in a similar effect in suberin but yielded an increased load lP of associated waxes in tuber phellem, mainly alkanes and feruloyl esters. Globally, the chemical effects in both silenced lines are supported by the transcript accumulation profile of genes involved in the biosynthesis, transport and regulation of apoplastic lipids. In contrast, the genes na of polyamine biosynthesis were downregulated. Altogether these results point out to StNAC101 as a candidate to repress the suberin-associated waxes. ur Abbreviations: ABA, abscisic acid; ADC, arginine decarboxylase; AP2/ERWEBP, apetala2/ehtylene-responsive element binding proteins; APRT, adenine phosphoribosyl Jo transferase; ASFT, aliphatic suberin feruloyl transferase; AtNAP, NAC-like activated by AP3/PI; bHLH, basic helix loop helix; CD2/ANL2, cutin deficient 2/anthocyaninless 2; FHT, fatty ω- hydroxyacid/fatty alcohol hydroxycinnamoyl transferase; GC-FID, gas chromatography-flame ionization detector; GC-MS, gas chromatography-mass spectrometry; HD-ZIPIV, homeodomain leucine zipper; KCS, β-ketoacyl-CoA synthase; LTP, lipid transfer protein; NAC, NAM, ATAF and CUC2; PI4Kγ5, phosphatidylinositol 4-kinase; RD26, responsive to dessication 26; SPDS, spermidine synthase; SPMS, spermine synthase; TRR, transcription regulatory region, VLCFA, very long chain fatty acids; WOX4, WUSCHEL-related HOMEOBOX; WRI1, Wrinkled1. Keywords: Suberin, suberin-associated waxes, periderm, phellem, NAC transcription factors, Solanum tuberosum 1. Introduction The periderm is a protective organ consisting of three different tissues: the phellem, the phellogen or cork cambium and the phelloderm. The phellem is the external tissue responsible of of the protection against dehydration and pathogens [1]. The protective function of the phellem is achieved by the deposition of suberin and associated waxes in the cell wall providing a ro hydrophobic barrier in the apoplast. Suberin, also named aliphatic suberin, is an insoluble polyester mainly composed by oxygenated fatty acid derivatives (such as ω-hydroxy-fatty acids, -p α,ω-diacids) and in minor amounts by fatty acids, primary alcohols, glycerol and ferulic acid [2– 5]. Suberin-associated waxes are the organic solvent-soluble extract mostly consisting of linear very long chain fatty acid derived compounds, including alkanes, primary alcohols, fatty acids re and feruloyl esters of primary alcohols [6]. This fraction has been proposed to play a significant role in the apoplastic barrier of potato tuber phellem as the removal of wax fraction increased lP the water permeability by a factor of 100 [6]. Suberin deposition is located between the plasma membrane and the primary cell wall and is suggested to be bound to the latter by a lignin-like polymer, also known as the suberin polyphenolic domain, mainly consisting of hydroxycinnamic na acids [3,5]. Suberin deposition occurs naturally in external and internal tissues to prevent water, ion and gas movement, such as in root and shoot periderm, cotton fibres, abscission zones, endodermis, exodermis/hypodermis and bundle sheaths of monocots [4,5]. Suberin deposition ur can also be induced by wounding to protect the healing tissue from water loss and pathogens [7]. Jo The first reported transcription factor with ability to induce suberin deposition ectopically in leaf and activation of biosynthetic genes of suberin was AtMYB41 [8], a stress responsive gene only detectable under ABA (abscisic acid), desiccation and salt treatments [9]. Recently, new MYB transcription factors have been described as regulators of suberin biosynthesis. Specifically, overexpression of apple MdMYB93 in Nicotiana benthamiana leaves correlated with strong accumulation of suberin and induction of genes involved in suberin synthesis [10]. Moreover, Arabidopsis seeds of the myb9 and myb107 mutants showed significant reduction in suberin accumulation and downregulation of the corresponding suberin biosynthetic genes [11]. Additional evidences of Myb107 role in suberin regulation were reported by Gou et al. [12] by probing the interaction of this transcription factor with genes involved in suberin synthesis. The cork oak QsMYB1, putative ortholog of AtMYB68, was proposed as a master regulatory factor of cork formation and differentiation, due to its ability to bind to lignin and suberin biosynthetic genes [13]. In contrast, a repressor of suberin and associated waxes from the NAC family, StNAC103, was reported in potato [14]. The StNAC103 gene is expressed in the tuber phellem and is also induced by wound-healing and ABA. The knockdown of StNAC103 (StNAC103-RNAi) was correlated with an increased load of suberin and associated waxes, a modification of the chemical composition of both components and a strong induction of some genes related with of suberin and wax accumulation [14]. ro NAC proteins constitute one of the largest families of plant-specific transcription factors found in land plants [15] and they have been suggested to contribute to the adaptation of plants to the terrestrial environment [16]. In general, NAC proteins share a well-conserved N-terminal -p domain and a diversified C-terminal, designed as NAC domain and transcription regulatory region (TRR), respectively [15,17]. The NAC domain is divided in five subdomains: two involved re in the binding of DNA (C and D), one participating in the dimerization (A) and two implicated in the functional diversity (B and E), whereas the TRR domain can either activate or repress lP transcription [17]. NAC transcription factors have been involved in tolerance to abiotic stress, defence, development, senescence, auxin signalling and secondary cell wall biosynthesis [15]. Several NAC genes have been defined as master transcriptional switches for xylem na differentiation, and together with MYB genes, regulate lignin biosynthesis in harmony with plant development and environmental adaptation [18]. A total of 110 NAC genes coding for 135 proteins are found in potato [19]. StNAC103 belongs to the subgroup NAC-c, which contains ur genes responsive to ABA and abiotic stress such as StNAC017 and StNAC030 [19] and genes involved in organ development, like StNAC044, the putative ortholog to CUC3 [20]. Interestingly, Jo the Arabidopsis ortholog of StNAC103 is ANAC058, whose overexpression provides hypersensitivity to exogenously applied ABA, a germinating-inhibiting compound [21]. The fact that suberin-deficient seeds display the same phenotype concerning ABA hypersensitivity [22] is consistent with a repressive role of ANAC058 in suberization. It has been suggested that the enhanced sensitivity to ABA can be explained by the impaired apoplastic barrier in the seed, which results in a higher permeability to water and ABA [22]. Here, to further understand the involvement of NAC genes in the apoplastic barrier regulation, we report the extended silencing, by targeting the conserved NAC domain of StNAC103 (StNAC103-RNAi-c), and their effects in potato tuber phellem. StNAC103-RNAi-c lines and StNAC103 specifically silenced lines (StNAC103-RNAi) [14] show similar amounts and composition of suberin, whereas stronger accumulation of the suberin-associated waxes in the potato tuber phellem is observed in StNAC103-RNAi-c. Transcript accumulation analyses of different NAC genes and the putative targets of StNAC103 are presented, providing a further detail of molecular mechanisms underlying StNAC103 silencing. 2. Materials and Methods of 2.1. Plant Material, potato tissues and RNA extraction Potato plants (Solanum tuberosum Group Tuberosum) cv. Desirée were in vitro propagated as ro described previously by Serra et al. [23]. To produce tubers, in vitro plants were transferred to soil and grown for 3 months in a walk-in chamber. The periderm (skin) of potato tuber was -p manually dissected using sterile scalpels and immediately frozen in liquid nitrogen. Total RNA was extracted using guanidine hydrochloride [24] and the RNA fraction was loaded on an RNeasy MinElute (Qiagen) column to perform DNAse digestion and further purify the RNA. RNA integrity re and concentration was analysed by formamide-formaldehyde denaturing agarose gel electrophoresis and a Nanodrop spectrophotometer, respectively. lP 2.2. Cloning, binary vector construction and plant transformation Synthesis of first-strand cDNA was carried out using 2 µg of DNAse digested RNA, oligo(dT)18 na primers and Superscript III reverse transcriptase (Invitrogen). The design of primers used to amplify and clone the regions for StNAC103 silencing was performed using the information available at that time from the potato Expressed Sequence Tag assembly (TC143904) and the ur putative orthologs of cork oak (EE743827) [25], Arabidopsis (At3g18400) and Petunia x hybrid (GI:21389167). The conserved silencing (StNAC103-RNAi-c) was triggered using a conserved Jo fragment of 229 bp in the NAC domain. Amplification products were first cloned into the pENTR/D TOPO vector (Life Technologies) and then transferred into the binary destination vector pBIN19RNAi [23] using LR clonase II (Invitrogen). Sequence of the primers used for the cloning are reported in Table S1. Agrobacterium tumefaciens (GV2260) was transformed with the recombinant plasmids according to Höfgen and Willmitzer [26] and afterwards potato leaves were infected with these A. tumefaciens cells. Kanamycin-resistant plants were regenerated in vitro by organogenesis following the protocol of Banerjee et al. [27]. 2.3. Real-time PCR analysis cDNA synthesis was performed using the high capacity cDNA reverse transcription kit (Life Technologies) and random primers. Real-time PCR was done in an optical 96-well plate with an ABI PRISM 7300 Sequence Detector System (Applied Biosystems) synthesis. The gene-specific PCR primers were designed with Primer 3 [28] and checked with Primer Express 3.0 (Applied Biosystems). PCR reactions contained 1 x Power SYBR Green Master Mix reagent (Applied Biosystems), 300 nM of the respective primers and 5 µl of a 25-fold dilution of the corresponding cDNA in a final volume of 20 µl. Wild-type plants were used to standardize the results and APRT, encoding an adenine phosphoribosyl transferase, was used as a housekeeping gene [29,30]. The amplification conditions for all PCRs were as follows: 95 °C for 10 min; 40 cycles of 95 °C for 15 of s and 60 °C for 1 min. After amplification, a dissociation step was performed to confirm the presence of a single amplicon. All data were processed and analysed with 7300 SDS 1.3.1 ro software (Applied Biosystems). For microfluidics quantitative PCR (qPCR; Fluidigm), 1.25 μl of the synthesized cDNA was pre-amplified and then purified with exonuclease treatment. Pre- amplified and purified cDNAs were diluted to 1:5 and used for real-time qPCR amplification by -p the BioMark™ system (Fluidigm). The primer specificity for each amplification reaction also was confirmed by a dissociation curve. Data collection and analysis was performed using Fluidigm re Real-Time PCR analysis software 3.0.2 (Fluidigm). The amplification efficiency was determined for each primer pair by performing standard curves with a 5-fold dilution series (1/1, 1/5, 1/25, lP 1/125, 1/625) of template. The geometric mean of two validated housekeeping genes, APRT and Cyclophilin [29,30], was used as reference to normalize data. The mRNA abundance was calculated as relative transcript abundance = (Etarget)ΔCt target (control- na sample) /(Ereference)ΔCt reference (control-sample) [31]. The controls used were a mix with equal amounts of different biological replicates of untransformed lines. The absence of genomic DNA contamination was confirmed by non-retrotranscriptase controls (RT-), whereas the lack of ur environmental contamination was checked with non-template controls (NTC). Sequence of primers used during Real-time PCR is shown in Table S1. MeV [32] was used for expression to Jo generate a heatmap. 2.4. Suberin and wax chemical analyses For aliphatic suberin and wax chemical analyses, the periderm membranes were isolated. Periderm membranes are obtained by enzymatic digestion of suberin-free cell walls as detailed by Schreiber et al. [6]. Hence, although designated as periderm, this tissue consists of the phellem or cork. Briefly, tuber skin was separated from the inner flesh with a cork-borer and treated with a 2% (v/v) cellulase and a 2% (v/v) pectinase solution. When digestion was completed, the remaining phellem membranes were washed, dried and stored at room temperature. All membranes were obtained from tubers after 39 days’ storage at room temperature to permit the periderm maturation. First the wax fraction was extracted from 1-2 dry isolated periderms (2-6 mg dry weight) using a chloroform and methanol (1:1 v/v) solution for 18 h. 2 µg C24 alkane was added to the solution as internal standard. The periderm was then removed and rinsed two times with new chloroform: methanol solution. The cleaning solutions were mixed together with the overnight extract and the solvent evaporated to dryness. The residue obtained was then used for wax analysis without further purification. Aliphatic suberin was depolymerised by transesterification of immersing the dry wax-free periderms in ~10% of boron trifluoride in methanol (BF3/MeOH) and incubating the samples at 70ºC for 16-18 hours in a teflon-sealed screw-cap tube [33]. Then, ro 10 µg of C32 alkane were added as internal standard and the periderm removed. The methanolysate was transferred to a new vial containing 2 ml of saturated NaHCO3 aqueous solution. The aqueous: methanol phase was then extracted two times with chloroform and, after -p phase separation, the chloroform extract was rinsed with ultrapure water. The organic phase was dried with anhydrous sodium sulphate powder and the solvent evaporated to dryness. The re dry residues from both chloroform/methanol extracts (waxes) and the monomers obtained after depolymerisation (suberin) were derivatized using N, O-bis (trimethylsilyl) trifluoroacetamide lP and derivative products were identified by GC-Mass Spectrometry (MS) and quantified by GC- Flame Ionization Detector (FID). GC-MS analyses were performed with a selective mass detector with ion trap (Trace GC 2000 na series coupled to a Thermo Scientific Polaris Q mass spectrometer), using a BPX-5 capillary column (30 m length, 0.25 mm i.d., 0.25 µm film thickness (SGE, Australia & Pacific Region)) with a split/splitless injector in the splitless injection mode (splitless time 1 min), with the injector ur temperature at 280ºC and using helium as the carrier gas at a constant flow rate of 1 ml/min. The oven temperature program for wax analyses was as follows: initially the temperature was Jo 120ºC, then increasing by 15ºC min-1 up to 240ºC, hold 1 min at this temperature, then increasing by 3ºC min-1 up to 340ºC and hold for 26 minutes. For suberin, the oven temperature was initially set at 140ºC, then increasing by 3ºC min-1 up to 310ºC, and finally hold for 3.3 min at 310ºC. Electron impact ionization was operated at 70 electronvolts and the transfer line was held at 290ºC. The ion source was set at 225ºC. MS acquisition was carried out in full-scan mode, with a scan range of 50-700 amu (atomic mass unit). For each peak, the mass spectrum was compared with data reported in literature [34–36]. GC-FID analyses were performed in a Shimadzu GC-2010 Plus, using a TRB-1ht capillary column (30 m length, 0.25 mm i.d., 0.1 µm film thickness, Teknokroma), with a split/splitless injector in the splitless injection mode (splitless time 1 min), and using helium as the carrier gas at a constant flow rate of 1 ml/min. The injector temperature was 280ºC. The oven temperature program for wax analyses was as follows: initially the temperature was 120ºC, then increasing by 15ºC min-1 up to 240ºC, then by 3ºC min-1 up to 380ºC and hold for 5.33 minutes. The detector temperature was maintained at 380ºC. For suberin trimethylsilylderivatives obtained after N,O- Bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatisation, the initial oven temperature program started at 140ºC, followed by 3ºC min-1 increases up to 310ºC, where it was held for 5 min. The detector temperature was maintained at 320ºC. Chromatograms were processed using GC Solution software (version 2.41) from Shimadzu. of 2.5. Statistical analyses ro Results from wild-type and transformed periderms were statistically analysed using one-way Analysis of Variance (ANOVA). One-way ANOVA was performed to assess differences among -p means. When variances were found to be heterogeneous using Levene’s test, Welch modification was used to robustly assess differences among means. Posthoc analysis was used to compare groups showing statistical differences, using Tukey HSD test if there is homogeneity re of variances, and Games-Howell test when variances are heterogeneous. lP 3. Results 3.1. Transgenic RNAi potato lines show repression of StNAC103 and other NAC members in the phellem na To determine the contribution of the NAC family regulators to suberization, we performed silencing using the conserved NAC domain of StNAC103 to trigger the mechanism of RNA ur interference. These lines were named as StNAC103-RNAi-c (Fig. 1A). Transcript abundance of StNAC103 was evaluated in these lines and compared with those obtained with gene specific silencing reported as StNAC103-RNAi [14], in which the highly variable region corresponding to Jo the TRR domain was targeted (Fig. 1A). The three StNAC103-RNAi-c lines showing the lowest StNAC103 expression (Fig. 1B) were selected for further analyses together with the two lines with the StNAC103-RNAi construct previously characterized [14]. In all these lines, StNAC103 was strongly downregulated, with transcript abundances 3 to 15 times lower than wild-type lines. To uncover the StNAC family members putatively silenced in StNAC103-RNAi-c, we performed BLASTN analyses against the potato transcript database using as a query the region used for producing the StNAC103-RNAi-c (Table I) and the StNAC103-RNAi (Table II) lines. We found 26 putative targets of StNAC103-RNAi-c silencing construct, including StNAC103 and one gene not annotated as NAC (Table I). Amongst the identified genes, even some of them displayed a complete match in 21 consecutive nucleotides with the StNAC103-RNAi-c construct, others presented one or two mismatches (Fig. S1). In contrast, only two NAC genes other than StNAC103 were identified as putative targets of the StNAC103-RNAi region, and they contained 1 or 2 mismatches to the silencing construct (Table II). Transcript accumulation of all 26 genes showing significant similarity to the silencing sequences of (Tables I and II) was analysed in the phellem of StNAC103-RNAi and StNAC103-RNAi-c lines (Fig. 2). We confirmed again that StNAC103 transcript accumulation was similar in both types of ro silencing lines. In addition, StNAC072, StNAC017 and StNAC096, which were predicted as silenced only in the StNAC103-RNAi-c lines, showed a decrease in transcript levels in both silencing lines, suggesting that their repression is a consequence of StNAC103 downregulation. -p StNAC032 exhibited lower gene expression in StNAC103-RNAi-c lines and slight downregulation in StNAC103-RNAi. In contrast, StNAC101 showed a strong reduction in transcript accumulation re specifically in StNAC103-RNAi-c lines, suggesting that it was a direct target of the hairpin RNAi together with StNAC103. lP To further characterize all the genes showing significant similarity to the silencing constructs, in silico expression analyses were performed using RNA-seq data reported for different potato tissues and conditions (Fig. S2) [37,38]. Only StNAC103 was preferentially expressed in the na potato tuber peel (including phellem), whereas StNAC101, StNAC032, StNAC072, StNAC017 and StNAC096 were also expressed in a large variety of tissues and conditions in addition to potato tuber peel. Thus, based on their transcript abundance, a putative role of these genes in potato ur suberin is possible. Jo Overall, considering all these results, the StNAC103-RNAi-c hairpin presumably targeting StNAC101 and StNAC103 causes the phenotype described below. 3.2. Wide silencing has a stronger effect on suberin-associated waxes in potato phellem Total suberin content was measured in the phellem, showing a total load 1.5 times higher in both StNAC103-RNAi and StNAC103-RNAi-c than in wild-type tissues (Fig. 3A), in agreement with higher accumulation of primary alcohols, ω-hydroxyacids, diacids and ferulic acid (Fig. 3B). When inspecting the single monomer abundances included in each substance class, no major differences were detected between StNAC103-RNAi and StNAC103-RNAi-c lines (Fig. S3B). Therefore, StNAC103-RNAi-c showed a phenotype nearly identical to StNAC103-RNAi concerning suberin content. In contrast, when suberin-associated waxes were quantified in the tuber phellem, StNAC103-RNAi-c resulted in a stronger phenotype than StNAC103-RNAi, showing twice the total wax load of wild-type lines. These levels were about one third higher than those of the StNAC103-RNAi lines (Fig. 4A). When the main waxes compounds were analysed in detail, it was apparent that the levels of alkanes and feruloyl esters of primary alcohols were all significantly higher in the StNAC103-RNAi-c phellem than in StNAC103-RNAi (Fig. 4B). Notably, alkane content was three times more abundant in StNAC103-RNAi-c lines and twice in StNAC103-RNAi than wild-type lines (Fig. 4C). In contrast, feruloyl esters were only more abundant in StNAC103-RNAi-c, being nearly equally abundant in StNAC103-RNAi and in wild- of type lines. The precise feruloyl esters that increased in the StNAC103-RNAi-c phellem were the major compounds, specifically C23, C28 and C30 feruloyl esters (Fig. 4D). Other waxes ro compounds (primary alcohols and fatty acids) were also analysed in detail and no important differences among lines were detected (Fig. S3A). -p 3.3. NAC silencing alters regulatory genes and biosynthetic genes involved in suberin, waxes and polyamines re To determine which genes were transcriptionally altered in StNAC103-RNAi and StNAC103-RNAi- c as a consequence of gene silencing, we analysed the transcript abundance pattern of some lP genes involved in the biosynthetic pathway of suberin and associated waxes, including the synthesis of very long chain fatty acids (VLCFA) and feruloyl-CoA, the formation of suberin monomers and waxes compounds, and their transport to the apoplast. Overall, all these steps na were induced in transgenic lines compared to wild-type, excepting those genes involved in feruloyl-CoA accumulation (Fig. 5). The KCS (β-ketoacyl-CoA synthase) gene, involved in VLCFA biosynthesis [23], was upregulated in StNAC103-RNAi-c lines. The orthologs to CER1, CER3 and ur CYPB5, which are related with the biosynthesis of alkanes [39-40], were not differentially expressed between silenced and wild-type lines. Regarding FHT, which is involved in the Jo biosynthesis of feruloyl esters of primary alcohols and ω-hydroxyacids in both waxes and suberin [41–43], was upregulated in both silenced lines. Other genes related with waxes, such as the At5g02890 [44] and WSD1 [45], were clearly upregulated in StNAC103-RNAi-c but not in StNAC103-RNAi. Concerning suberin genes, CYP86A33 and GPAT5 [46, 47] showed upregulation in both silenced lines, and CYP86B1 [48] only in StNAC103-RNAi-c. Also, two LTPs (lipid transfer proteins), putatively involved in the transport of waxes and suberin monomers across the cell wall [49], were only upregulated in StNAC103-RNAi-c. In addition, there are two BAHD genes annotated as benzoyltransferases with unknown function that are preferentially induced in potato peel [37,38] and upregulated in StNAC103-RNAi-c. Transcript accumulation of regulatory genes that could control the phellem composition was also analysed (Fig. 6). These included transcription factors related to the synthesis of acyl-lipids, cutin and waxes (Wrinkled1 or WRI1 and Cutin deficient 2/Anthocyaninless 2 or CD2/ANL2) [50– 52], controlling cambium activity (WUSCHEL-related HOMEOBOX or WOX4) [53] and cell differentiation (ALCATRAZ) [54]. As shown in Fig. 6, WOX4 did not show significant differences in any of the silenced lines compared to the wild-type, whereas ALCATRAZ and CD2/ANL2 were upregulated in both StNAC103-RNAi-c and StNAC103-RNAi lines and WRI was upregulated only in StNAC103-RNAi-c. All these results agree with the induction of suberin and wax biosynthetic of genes and with the higher amounts of suberin and waxes detected in StNAC103 silencing lines. Since a NAC family member represses polyamine biosynthesis [55] and polyamine levels ro increased in a suberin-deficient potato mutant [41], we also analysed the expression pattern of some polyamine biosynthetic genes. The genes encoding enzymes that produce putrescine, -p spermine and spermidine from arginine (arginine decarboxylase, spermine synthase and spermidine synthase; ADC, SPMS and SPDS, respectively) were downregulated in the transgenic lines compared to wild-type, and the level of repression was similar in StNAC103-RNAi-c and re StNAC103-RNAi (Fig. 6). Altogether suggests a cross-talk between suberin and polyamine biosynthesis and reinforces a putative role of polyamines in phellem formation. lP 4. Discussion StNAC103-RNAi-c lines showed an obvious increase of suberin and associated waxes in the na potato tuber phellem in comparison to the wild type. Similar effects on suberin amount and composition were also observed in StNAC103-RNAi lines [14] (Fig. 3), but the boost in suberin- associated waxes was higher in StNAC103-RNAi-c, specially enhancing the alkanes and feruoyl ur esters content (Fig. 4). Since NACs are transcription factors, the chemical changes observed in knockdown lines were compared with the gene expression of biosynthetic genes of suberin and Jo suberin-associated waxes (Fig. 5). KCS enzyme yielding to VLCFAs [23] was only upregulated in StNAC103-RNAi-c lines suggesting that it may trigger a higher accumulation of VLCFA to be channelled to the downstream pathways, hence contributing to the boost in wax amount of StNAC103-RNAi-c lines. Concerning genes involved in suberin biosynthesis, the similar upregulation in both silenced lines is consistent with the analogous increase in suberin load in both transgenic lines. Regarding wax genes, the orthologs to the WSD1 and At5g02890 [44,45] were only upregulated in StNAC103-RNAi-c lines again in agreement with the higher accumulation of suberin-associated waxes in these lines. Concerning FHT, it synthesizes feruloyl esters to be incorporated mainly in suberin (96% of the apoplastic lipids [46]) but also in waxes [41], and as a consequence, the FHT transcript accumulation cannot be used as a readout to evaluate the solely increase of feruloyl esters in waxes of StNAC103-RNAi-c. In agreement, FHT was upregulated in both transgenic lines consistent with the increase in suberin of ferulic acid together with ω-hydroxyacids and primary alcohols in StNAC103-RNAi and StNAC103-RNAi-c. The genes related with the biosynthesis of alkanes such as CER1, CER3 and CYPB5 [39,40], were not correlated with the boost of alkanes in both silencing lines. But interestingly, the upregulation in both silenced lines of the transcription factor CD2/ANL2, not detected in wild- type samples (Fig. 6), may explain the alkanes increase because cd2/anl2 mutants display a strong reduction in alkanes of cuticular waxes and a global drop of cutin monomers [51]. Even, of the overall increase of suberin monomers in silenced lines also can be due to the upregulation of CD2/ANL2. Finally, two LTP genes, that may be involved in the transport of suberin and waxes ro compounds through the apoplast [49], were only upregulated in StNAC103-RNAi-c lines and therefore, they could also promote a higher accumulation of suberin-associated waxes in these -p lines. As a whole, StNAC103 downregulation was correlated with a strong upregulation of the aliphatic metabolism associated to the suberin polyester and waxes biosynthesis, while the re phenolic metabolism was not affected (Fig. 5). In the same line of evidence, differential regulation of aliphatic and phenolic metabolisms was also observed during wound-induced suberization in potato tuber [56]. lP Three out of the four regulatory genes analyzed in the phellem of the silencing lines showed statistically significant differences. As explained above, the gene CD2/ANL2 was strongly na upregulated in both StNAC103 transgenic lines in agreement with the increase of suberin and wax compounds. It has been reported that CD2/ANL2 is a class IV homeodomain Leucine zipper (HD-ZIP IV) whose mutation in addition to the effects in cutin and waxes, gives rise to an increase ur in cuticle water permeability [51,57] and also lower cell wall extensibility [58]. ALCATRAZ is a basic helix loop helix (bHLH) transcription factor involved in fruit dehiscence by promoting cell Jo differentiation [59] and was also upregulated in both NAC silencing lines. The activation of ALCATRAZ in the phellem of silencing lines could be related to the regulation of processes that also occurs in the abscission zone such as cell wall modification or the formation of apoplastic barriers [60]. WRI1 was upregulated only in StNAC103-RNAi-c lines and this result is consistent with the higher accumulation of waxes because WRI is a transcription factor of the AP2/ERWEBP (Apetala2/Ehtylene-responsive element binding proteins) class involved in the induction of fatty acid biosynthetic genes [50,61,62] and the accumulation of the most abundant cutin monomers [52]. Regarding the genes involved in polyamines biosynthesis (ADC, SPMS and SPDS), they are similarly downregulated in both silencing lines. Interestingly, the putative ortholog of ANAC072 in trifoliate orange, PtrNAC72, is able to repress the biosynthesis of putrescine by binding to the promoter of PtADC (arginine decarboxylase) [55]. In potato phellem, StNAC072, the putative ortholog of ANAC072, is similarly downregulated in both silencing lines like the polyamines biosynthetic genes, suggesting that StNAC072 does not have a repressive role in potato or that other transcription factors are responsible for polyamine repression. The specific increase in suberin-associated waxes in the StNAC103-RNAi-c phellem, suggests co- silencing of other NAC genes that may repress the biosynthesis of waxes compounds by targeting some of the genes involved in wax biosynthesis, KCS or LTPs (lipid transport protein). of This stronger phenotype was explained because to trigger RNA interference in these lines we used a region of the conserved NAC domain, which according to BLASTN analyses was found in ro 26 genes (Table I). StNAC057 was the most similar gene after StNAC103 to both silencing constructs, but surprisingly it was not downregulated neither using an RNAi construct containing a fragment of the TRR domain nor the one containing a region of the NAC domain. In contrast, -p other genes with less similarity, such as StNAC072, StNAC017 and StNAC069, showed a clear downregulation in both types of silencing lines (Fig. 2). The lack of significant similarity of these re three genes with the sequence used for silencing StNAC103-RNAi lines suggests that they are not directly downregulated by the artificial RNAi but probably indirectly downregulated due to lP StNAC103 silencing. Regarding StNAC032, although there is downregulation in StNAC103-RNAi- c phellem in comparison to the wild-type, the transcript levels in StNAC103-RNAi are similar to both the wild-type and the StNAC103-RNAi-c, thus it is unclear if the gene is directly na downregulated by StNAC103-RNAi-c. Conversely, StNAC101 is downregulated only in StNAC103- RNAi-c, rendering it the preferred candidate to participate in the biosynthesis of suberin- associated waxes. ur In contrast to StNAC103, StNAC101 did not have a preferential expression in the potato tuber peel. Instead, it showed a broad expression in several organs, tissues and conditions of potato Jo plants, including the tuber peel (Fig. S2) [37,38]. Hence, despite the role of StNAC101 is not probably restricted to the potato tuber phellem, it can participate in the repression of suberin- associated waxes in potato tuber phellem. StNAC101 is highly induced by abiotic stress and ABA treatments [19], similarly to StNAC103 [14]. The putative Arabidopsis ortholog of StNAC101 is ANAC072 (also named responsive to dessication 26, RD26), however it is worth emphasizing that this gene has two orthologs in potato: StNAC072 and StNAC101 [19]. ANAC072 is also strongly induced by dehydration, ABA and high-salinity and it was suggested that the capacity of ANAC072 to provide tolerance to drought was achieved by detoxification of toxic aldehydes [63]. It has also been proposed that ANAC072 has a central role in ABA signalling and desiccation stress [64]. Recently it has been shown that RD26 controls multiple catabolic genes to rearrange metabolism giving rise to senescence [65]. The ortholog of StNAC032 in Arabidopsis, ANAC029/AtNAP (NAC-like activated by AP3/PI), was involved in leaf senescence by stomata closure as a result of inhibiting the ABA pathway [66,67]. The NAC genes downregulated unambiguously in both types of silencing are StNAC072, StNAC017, and StNAC096, and their putative Arabidopsis orthologs are ANAC072 and ANAC055, of ANAC100 and ANAC078, respectively [19]. As explained above, ANAC072 was associated with abiotic stress [63], similarly to ANAC055 and this drought tolerance provided by ANAC055 is ro concomitant with proline accumulation and activation of the corresponding biosynthetic gene [68]. In contrast, very little is known about ANAC100, besides the fact that it is targeted by miRNA164 [69] and that it can bind to the promoters of some genes involved in the degradation -p of chlorophyll and leaf senescence [70]. Concerning StNAC096, its putative ortholog in Arabidopsis, ANAC078, displays many different functions, including regulation of flavonoid re biosynthesis under high light [71], regulation of the proteasomes [72,73], and interaction with an atypical type II phosphatidylinositol 4-kinase (PI4Kγ5), to modulate cell proliferation and leaf lP development by negatively regulating auxin synthesis [74]. As a result, the decrease in the transcript level of StNAC103, StNAC101, StNAC032, StNAC072, StNAC017 and StNAC096 can have different physiological and molecular effects, in most cases related to ABA and abiotic na stress, two factors inducing suberin deposition. ABA and dehydration have been long associated to the suberization process in potato tuber phellem [75,76]. More recently, it has been widely reported that abiotic stresses such as drought and salt stress induce suberin accumulation in ur roots [4] and activate both suberin and wax biosynthesis [77], which in turn provides increased tolerance to drought and higher water use efficiency [78–80]. It has also been reported that ABA Jo induces activation of suberin genes [75,81–85], leading to suberin deposition [85,86], which in endodermis only occurs when the ABA signalling pathway is active [85]. In agreement with this, biosynthesis of suberin-associated waxes is also induced by salt stress in Arabidopsis taproots that already contained periderm [87]. The fact that suberin and their associated waxes share fatty acid precursors [23,88] suggests that a similar ABA regulation trend is expected also for waxes. Conversely ethylene decreases suberin biosynthesis and it is suggested to trigger degradation of suberin lamellae in Arabidopsis endodermis, evidencing that plasticity of suberin is necessary to allow a better adaptation of roots to different soil environments [85]. Taken together, these observations support again a strong connection between ABA/abiotic stress and the suberization process and that NAC repressors aid to the proper regulation of suberin- associated waxes in the apoplastic barrier. However, the precise contribution of each NAC gene in the process is still unclear and deserves further investigation. Acknowledgments The authors are grateful to Professor S. Prat (CNB, Madrid) for providing the silencing vectors and helpful advices along this work. The authors thank to S. Gómez (Departament de Biologia, UdG, Girona) for their valuable assistance in carrying out the laboratory work and taking care of plants. We also thank to Professor V. Salvadó (Departament de Química, UdG, Girona) and Professor C. Pla (Departament de Biologia, UdG, Girona) for kindly lending the GC-MS and of Thermocycler respectively. This work was supported by the Ministerio de Educación y Ciencia (AGL2006-07342, FPI grant to O.S.), the Ministerio de Innovación y Ciencia [AGL2009-13745, FPI grant to P.B.], the Ministerio de Economía y Competitividad [AGL2012-36725; AGL2015-67495- ro C2-1-R], FEDER funding, Departament d’Universitats, Investigació i Societat de la Informació of Catalonia [PhD grant to R.V. and M.S.], the University of Girona [PhD grant to D.C.-A. and S.F.-P. and the grant SING11/1]. -p re lP na ur Jo REFERENCES [1] R.L. Peterson, W.G. 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Pederson, Regulatory involvement of abscisic acid in potato tuber wound-healing, J. Exp. Bot. 59 (2008) 1175–1186. [87] D.K. Kosma, I. Molina, J.B. Ohlrogge, M. Pollard, Identification of an Arabidopsis fatty alcohol: caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes, Plant Physiol. 160 (2012) 237–248. [88] Y. Li, F. Beisson, J. Ohlrogge, M. Pollard, Monoacylglycerols are components of root waxes and can be produced in the aerial cuticle by ectopic expression of a suberin- associated acyltransferase, Plant Physiol. 144 (2007) 1267–1277. Figure legends of ro -p re lP Fig. 1. StNAC103 silencing in potato tuber phellem. (A) Scheme of the constructs designed to StNAC103 silencing (StNAC103-RNAi targets the variable TRR region and StNAC103-RNAi-c the conserved NAC domain, specifically subdomains C and D). (B) StNAC103 relative transcript abundance in potato tuber phellem of wild-type and silencing lines. Transcript amounts were na expressed relative to the reference gene. Relative mRNA expression levels were expressed as mean ± SD of three replicates. ur Jo of ro Fig. 2. Effects of StNAC103 silencing in the transcript abundance of the NAC genes predicted as targets of StNAC103-RNAi-c in tuber phellem. Results were obtained by microfluidics qPCR and were represented as relative transcript abundance for wild-type, StNAC103-RNAi and StNAC103- -p RNAi-c lines. Values are the mean ±SD of biological replicates of the wild-type (n=4) and two independent transformation events including each a minimum of four biological replicates for StNAC103-RNAi (line 43 n=5, line 45 n=4) and for StNAC103-RNAi-c (line 1 n=4, line 31 n=4). re Statistical significant differences (P < 0.05) are indicated by lowercase letters. Arrows indicate genes downregulated in at least one transgenic line. lP na ur Jo of ro -p Fig. 3. Effects of StNAC103 silencing in suberin chemical composition of potato tuber phellem. re (A) Total suberin amount in tuber phellem of wild-type and silencing lines shown as the amount per surface area from 39-day-stored tubers. (B) Composition of suberin substance classes in wild-type and silenced lines. Values are the mean ± SD of n biological replicates of wild-type lP (n=5), two independent transformation events for StNAC103-RNAi (n=4, n=3) and three for StNAC103-RNAi-c (n=4, n=4, n=4). Statistically significant differences (P < 0.05) are indicated by lowercase letters. na ur Jo of ro -p re lP na Fig. 4. Effects of StNAC103 silencing in wax chemical composition of potato tuber phellem. (A) Total wax load in tuber phellem of wild-type and silencing lines shown as the amount per surface ur area from 39-day-stored tubers. (B) Total amount of different wax substance classes of tuber phellem in wild-type and silencing lines. (C) Profile of alkanes and (D) feruloyl esters of primary alcohols. Values are the mean ± SD of n biological replicates of wild-type (n=5), two independent Jo transformation events for StNAC103-RNAi (n=4, n=3) and three for StNAC103-RNAi-c (n=4, n=4, n=4). Statistical significant differences (P < 0.05) are indicated by lowercase letters. of Fig. 5. Heatmap of expression values for suberin and wax related genes in tuber phellem of StNAC103-RNAi and StNAC103-RNAi-c lines. Units are fold change values of RTA (relative ro transcript abundance) in StNAC103-RNAi and StNAC103-RNAi-c divided by RTA values in wild- type. These values were obtained by microfluidics qPCR and the values come from the mean -p ±SD of two independent transformation events and a minimum of four biological replicates (wild-type: n=4; StNAC103-RNAi line 43 n=5, line 45 n=4; StNAC103-RNAi-c line 1 n=4, line 31 n=4). Significant differences are denoted with one asterisk (P < 0.05) or two asterisks (P < 0.01). re Most genes are annotated according the Arabidopsis orthologs, excepting FHT and CYP86A33 because its role in potato is known. lP na ur Jo of ro -p re lP na Fig. 6. Effects of StNAC103 silencing in the transcript abundance of regulatory and polyamine biosynthetic genes in tuber phellem. Results were obtained by microfluidics qPCR and were represented as relative transcript abundance for wild-type, StNAC103-RNAi and StNAC103- ur RNAi-c lines. Values are the mean ±SD of the wild-type (n=4) and two independent transformation events for StNAC103-RNAi (line 43 n=5, line 45 n=4) and for StNAC103-RNAi-c (line 1 n=4, line 31 n=4). Statistically significant differences (P < 0.05) are indicated by lowercase Jo letters. Table I. List of the putative targets of StNAC103-RNAi-c construct. Results of the BLASTN analysis in potato transcript database using the StNAC103-RNAi-c construct as a query. Table II. List of the putative targets of StNAC103-RNAi construct. Results of the BLASTN analysis in potato transcript database using the StNAC103-RNAi construct as a query. Table I. Putative genes co-silenced with the StNAC103-RNAi-c construct. Gene identifier Gene name NAC subgroup Mismatch in Blastn e-value consecutive (RNAi sequence as 21nt fragment a query) PGSC0003DMG400005384 StNAC103 NAC-c 0 1.00E-125 PGSC0003DMG400028779 StNAC057 NAC-c 0 2.00E-64 of PGSC0003DMG400012403 StNAC069 NAC-c 0 2.00E-33 ro PGSC0003DMG400019294 StNAC072 NAC-f 0 1.00E-12 PGSC0003DMG400029593 StNAC078 NAC-c 0 2.00E-11 PGSC0003DMG400015342 PGSC0003DMG400002824 StNAC101 StNAC032 NAC-f NAC-f -p 0 0 3.00E-07 2.00E-05 PGSC0003DMG400031266 StNAC046 NAC-i 0 1.00E-03 re PGSC0003DMG400001338 StNAC017 NAC-c 1 3.00E-20 PGSC0003DMG400009920 StNAC044 NAC-c 1 2.00E-14 lP PGSC0003DMG400019523 StNAC030 NAC-c 1 4.00E-13 PGSC0003DMG400014845 StNAC076 NAC-c 1 6.00E-12 na PGSC0003DMG400000887 StNAC038 NAC-q-TNAC 1 8.00E-05 PGSC0003DMG400022134 StNAC074 NAC-b 1 1.00E-03 PGSC0003DMG400019092 StNAC110 NAC-n 1 2.00E-02 ur PGSC0003DMG400007130 StNAC062 NAC-e 1 2.00E-02 PGSC0003DMG400032555 StNAC033 NAC-f 1 3.20E-01 Jo PGSC0003DMG400033047 StNAC058 NAC-c 2 9.00E-14 PGSC0003DMG400026079 StNAC055 NAC-a 2 5.00E-05 PGSC0003DMG400004740 StNAC082 NAC-a 2 2.00E-02 PGSC0003DMG400004332 StNAC108 NAC-e 2 2.00E-02 PGSC0003DMG400031071 StNAC096 NAC-j 2 8.10E-02 PGSC0003DMG400006051 translocase sub secA - 2 8.10E-02 PGSC0003DMG400039898 StNAC073 NAC-m 2 3.20E-01 PGSC0003DMG400025031 StNAC088 NAC-f 2 3.20E-01 PGSC0003DMG400021474 StNAC006 NAC-e 2 3.20E-01 of ro -p re lP na ur Jo Table II. Putative genes co-silenced with the StNAC103-RNAi construct. Gene identifier Gene name NAC subgroup Mismatch in Blastn e-value consecutive (RNAi sequence 21nt fragment as a query) PGSC0003DMG400005384 StNAC103 NAC-c 0 1.00E-122 PGSC0003DMG400028779 StNAC057 NAC-c 1 1.00E-03 PGSC0003DMG400009920 StNAC044 NAC-c 2 6.10E-02 of ro -p re lP na ur Jo