A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism1[OPEN]

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AKeyRoleforApoplasticH2O2 in Norway Spruce Phenolic Metabolism1[OPEN]

Teresa Laitinen,a,2 Kris Morreel,b,c,2 Nicolas Delhomme,d,2 Adrien Gauthier,a,3 Bastian Schiffthaler,e Kaloian Nickolov,a,f Günter Brader,g,4 Kean-Jin Lim,a Teemu H. Teeri,a Nathaniel R. Street,e,5 Wout Boerjan,b,c,5 and Anna Kärkönena,h,5,6 aDepartment of Agricultural Sciences, Viikki Plant Science Centre, University of Helsinki, 00014 Helsinki, Finland bGhent University, Department of Plant Biotechnology and Bioinformatics, 9052 Ghent, Belgium cVIB Center for Plant Systems Biology, 9052 Ghent, Belgium dUmeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden eUmeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden fDepartment of Biology, University of Oulu, 90014 Oulu, Finland gDepartment of Biosciences, University of Helsinki, 00014 Helsinki, Finland hNatural Resources Institute Finland (Luke), Green Technology, 00790 Helsinki, Finland ORCID IDs: 0000-0002-3121-9705 (K.M.); 0000-0002-3053-0796 (N.D.); 0000-0002-1351-353X (A.G.); 0000-0002-9771-467X (B.S.); 0000-0002-5135-5691 (K.N.); 0000-0002-6682-7110 (G.B.); 0000-0003-2147-0215 (K.-J.L.); 0000-0002-3812-7213 (T.H.T.); 0000-0001-6031-005X (N.R.S.); 0000-0003-1495-510X (W.B.); 0000-0001-8870-3250 (A.K.).

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including fl (glycosylated) oligolignols, (glycosylated) avonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses fl revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon ux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.

Lignin, a phenolic polymer present in cell walls of it is largely consistent across different plant groups water-conducting vessels and tracheids and in support- (Mottiar et al., 2016). In Arabidopsis (Arabidopsis thaliana), giving sclerenchyma cells, constitutes 20% to 35% of the both laccases and peroxidases are required for mono- dry weight of wood, making it the second most abundant lignol oxidation in lignin polymerization, possibly acting terrestrial biopolymer after cellulose. Lignin has a high sequentially, and/or in different tissues (Berthet et al., economic impact, as it hinders the industrial processing of 2011; Lee et al., 2013; Novo-Uzal et al., 2013; Zhao et al., lignocellulosic biomass into pulp and fermentable sugars, 2013; Barros et al., 2015; Shigeto and Tsutsumi, 2016). leading to large economic costs to extract it from the bio- In Norway spruce, numerous peroxidase and laccase mass. On the other hand, lignin is increasingly considered isoenzymes have been reported in the cell walls of de- as a potentially valuable product, and new applications veloping xylem and in the culture medium of the lignin- and profit revenues for this biopolymer are being identi- forming cell culture utilized in this study (Kärkönen et al., fied (Ragauskas et al., 2014; Van den Bosch et al., 2015). In 2002; Fagerstedt et al., 2010; Koutaniemi et al., 2015). Norway spruce (Picea abies), an economically important Isolated peroxidases and laccases are able to mediate conifer in Europe, lignin is composed mainly of coniferyl high-molecular-weight dehydrogenation polymer forma- alcohol giving rise to G units, with a small proportion of tion from coniferyl alcohol in vitro (Kärkönen et al., 2002; p-coumaryl alcohol-derived H units (Boerjan et al., 2003). Koutaniemi et al., 2005; Warinowski et al., 2016). However, Although there is some degree of variation among species the relative contribution of these oxidative enzymes to in the architecture of the monolignol biosynthetic pathway, lignin synthesis in vivo and the specific isoenzymes that

Ò Plant Physiology , July 2017, Vol. 174, pp. 1449–1475, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved. 1449 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Laitinen et al. oxidize monolignols for lignin biosynthesis are not apoplast: one sensitive to diphenylene iodonium, an known. inhibitor of flavin-containing enzymes such as RBOHs, The high number of putative peroxidase and laccase and the other sensitive to a low concentration of azide, genes (over 200 and 100, respectively) in the Norway an inhibitor of heme-containing enzymes such as per- spruce genome (Nystedt et al., 2013) has hindered the oxidases (Kärkönen et al., 2009). identification of specific genes encoding lignification- The aim of this work was to investigate the effects of related isoenzymes. The origin of apoplastic hydrogen apoplastic H O level modulation on phenolic metabo- 2 fi2 peroxide (H2O2) required by peroxidases as an oxidant in lism and ligni cation in Norway spruce in order to cell wall cross-linking also is unclear, as is the contribution identify genes encoding isoenzymes (e.g. oxidative and of the apoplastic redox state in controlling lignin forma- ROS-producing enzymes as well as transcription factors) tion. Several plant cell wall- and plasma membrane- specific for lignin biosynthesis. Studying the role of the located sources for apoplastic reactive oxygen species apoplastic redox state in lignin biosynthesis in tree stems (ROS;superoxide,H2O2, and hydroxyl radical) exist during wood development is technically challenging, as (Kärkönen and Kuchitsu, 2015), including apoplastic per- differentiating xylem cells constitute a thin layer at the oxidases, various oxidases, and plasma membrane-located inner side of the cambium, with sampling-induced enzymes such as respiratory burst oxidase homologs wounding inducing apoplastic ROS production. There- (RBOHs; also called NADPH oxidases) and quinone fore, the extracellular lignin-forming cell culture of Nor- reductases. There are indications that RBOHs produce way spruce used here represents a model system for such ROS in zinnia (Zinnia elegans) during vascular xylem studies (Simola et al., 1992; Kärkönen and Koutaniemi, lignification (Ros Barceló, 1998) and in in vitro tracheary 2010). The cultured cells produce extracellular lignin element (TE) formation (Karlsson et al., 2005; Pesquet when transferred from solid medium into liquid cultures et al., 2013), with some participation of peroxidases (Simola et al., 1992; Kärkönen et al., 2002). Results of a (Karlssonetal.,2005).Inaddition,inArabidopsis,ROS comparative real-time reverse transcription-PCR analysis produced by AtRBOHF are essential for the formation of of the cultured cells and several lignin-forming tissues of lignin-composed Casparian strips in the root endoder- spruce indicated that the same genes encoding enzymes mis (Lee et al., 2013). In the tissue-cultured spruce cells, in the phenylpropanoid and monolignol biosynthesis at least two H2O2 generation systems are present in the pathways are induced in the culture system as in developing xylem (Koutaniemi et al., 2007). In addition, the linkage structure of the extracellular lignin resembled that 1 This work was supported by the Academy of Finland (grant of native wood lignin (Brunow et al., 1993; Koutaniemi nos. 251390, 256174, and 283245 to A.K.) and the Research Funds et al., 2005). The availability of the Norway spruce genome of the University of Helsinki (to A.K.), the Finnish Cultural Foun- sequence (Nystedt et al., 2013), in addition to those of other dation, Häme Regional Fund (to T.L.), the Trees and Crops for the conifer genomes (white spruce [Picea glauca] and loblolly Future project (to N.R.S.), the Knut and Alice Wallenberg Founda- pine [Pinus taeda]; De La Torre et al., 2014), now enables tion Norway spruce genome project (to N.D.), the Bijzonder more comprehensive genomic, transcriptomic, and pro- Onderzoeksfonds-Zware Apparatuur of Ghent University for the Fourier transform ion cyclotron resonance mass spectrometer (grant teomic analyses to investigate the process of lignin bio- no. 174PZA05 to K.M. and W.B.), and the Multidisciplinary Re- synthesis in conifers. Here, the lignin-forming cell culture of search Partnership Biotechnology for a Sustainable Economy of Norway spruce was used in a systems biology approach Ghent University (grant no. 01MRB510W). combining liquid chromatography-mass spectrometry- 2 These authors contributed equally to the article and share first based phenolic and RNA sequencing (RNA-Seq)-based authorship. transcriptome profiling over the course of lignin bio- 3 Current address: Unité AGRI’TERR, UniLaSalle, Campus de synthesis to investigate whether the scavenging of Rouen, 3 Rue du Tronquet, CS40118, 76134 Mont-Saint-Aignan, apoplastic ROS affects the coupling of monolignols into France. 4 lignin and intracellular aromatic metabolism. Differen- Current address: Austrian Institute of Technology GmbH, Centre tial expression and gene coexpression network analyses for Health and Bioresources, 3430 Tulln an der Donau, Austria. fi 5 These authors contributed equally to the article and share last identi ed candidate enzymes for monolignol oxidation authorship. and apoplastic H2O2 production, novel putative tran- 6 Address correspondence to anna.karkonen@luke.fi. scription factors, and potential H2O2 receptors. The results The author responsible for distribution of materials integral to the suggest that the apoplastic redox state is an important findings presented in this article in accordance with the policy de- regulatory factor for apoplastic reactions as well as whole scribed in the Instructions for Authors (www.plantphysiol.org) is: cellular metabolism. Anna Kärkönen (anna.karkonen@luke.fi). A.K., T.L., A.G., K.N., T.H.T., G.B., K.M., W.B., N.D., and N.R.S. conceived and designed the experiments; T.L., K.M., N.D., A.G., RESULTS K.N., G.B., and A.K. performed the research; T.L., K.M., N.D., A.G., B.S., K.N., K.-J.L., G.B., and A.K. analyzed the data; W.B. and N.R.S. Scavenging of H2O2 Inhibits Extracellular contributed reagents/materials/analysis tools; A.K., K.M., N.D., Lignin Production A.G., T.L., G.B, N.R.S., and W.B. wrote the article with contributions of all the authors. A cell culture of Norway spruce that produces ex- [OPEN] Articles can be viewed without a subscription. tracellular lignin in the culture medium was used to www.plantphysiol.org/cgi/doi/10.1104/pp.17.00085 investigate the effects of modulating apoplastic H2O2

1450 Plant Physiol. Vol. 174, 2017 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Apoplastic Redox State Affects Phenolic Metabolism levels on phenolic metabolism using phenolic and (Ihssen et al., 2014), the results suggest that peroxidases transcriptomic analyses (the experimental setup is are crucial for monolignol oxidation during extracel- presented in Fig. 1 and Supplemental Fig. S1). Spruce lular lignin formation. callus cells maintained on a solid culture medium were transferred into liquid cultures with a concomitant decrease in 2,4-dichlorophenoxyacetic acid concentration H2O2 Scavenging Affects Both the Intracellular and (by 20-fold), and the nutrient medium was supple- Apoplastic Phenolic Pools mented with potassium iodide (KI; 5 mM), an H2O2 scavenger (Nose et al., 1995). Control cells treated with To investigate the effects of apoplastic H2O2 scavenging on phenolic metabolism, a time-course sam- KCl (5 mM) or an equivalent volume of water (to ascertain that KI effects were due to iodide and not pling of cells and culture medium from the various potassium) produced extracellular lignin after 5 d in types of culture (Supplemental Fig. S1A) was analyzed liquid culture, visible as a fine, white precipitate in the for methanol-soluble phenolic compounds using ultra- culture medium (Figs. 1 and 2A). Cell weight did not HPLC-mass spectrometry (Morreel et al., 2004, 2010a, increase during this period in any of the treatments (Fig. 2010b, 2014). In cells and culture medium, 505 and 278 compounds were profiled, of which only 31 com- 2B). The addition of KI substantially decreased H2O2 concentration in the culture medium (Fig. 2C) and pre- pounds were in common, providing strong evidence that cross-contamination between the medium and cell vented extracellular lignin production (H2O2-scavenging conditions; Fig. 2A). The KI-treated cells also started to extracts was low or even absent. Subsequently, a can- divide after 3 weeks of culturing, observed as an increase didate substrate-product pair (CSPP) network (Morreel in cell fresh weight (Fig. 2B). KI was washed away after et al., 2014) was constructed using all 752 (505 + 278 2 20 d, and the cells were further cultured in the standard 31) compounds (Fig. 3). In the CSPP network, nodes nutrient medium. Four to 12 d after KI removal, extra- represent compounds while edges reflect mass differ- cellular lignin subsequently appeared in the culture ences corresponding with well-known metabolic con- medium (lignin reformation; Fig. 1; Supplemental Fig. versions (for the included types of metabolic conversions, S1), showing that the prevention of lignin formation see Supplemental Table S1). Hence, this method aids was reversible and that KI treatment did not impact cell structural characterization and provides insight into the viability. This is in contrast to the situation in tobacco various profiled biochemical compound classes. In total, (Nicotiana tabacum) BY-2 cells, which do not survive in 71 and 20 compounds were structurally elucidated in cells the presence of elevated levels of KI (Väisänen et al., and culture medium, respectively (Table I; Supplemental 2015). These results suggest an important role for apo- TableS2).IntheconstructedCSPPnetwork,thelargest plastic H2O2 in extracellular lignin production. More- subnetworks represented compounds that were either over, as iodide does not inhibit laccase activity in vitro almost exclusively present in the cells (circles, Fig. 3) or in

Figure 1. Setup for the experiments using the extracellular lignin-forming Norway spruce cell culture. Spruce cells were trans-

ferred at time 0 from the solid maintenance medium to liquid nutrient medium supplemented either with an H2O2 scavenger, KI (5 mM), or the corresponding volume of water (H2O). After 5 d (H2O_5d), extracellular lignin became visible in the culture medium of water-treated samples (lignin-forming conditions). In contrast, KI-treated cells did not form any visible extracellular

lignin in the culture medium at 5 d (KI_5d) up to 20 d (KI_20d; H2O2-scavenging conditions). After 20 d of culturing, KI was removed by washing the cells three times with fresh nutrient medium (2KI) and cultivated further in the fresh nutrient medium without extra KI supplementation (KI_20+nd; with n, number of days in the culture after the medium-change step; lignin- reforming conditions).

the culture medium (squares, Fig. 3), with very few nodes representing compounds that were detected in both cells and the medium (triangles, Fig. 3). Subnetworks for the compounds in the culture medium represented mainly lignin oligomers, termed oligolignols (Morreel et al., 2004), whereas those for the compounds in the cells were associated predominantly with hexosylated phenylpropanoids, hexosylated flavonoids, and proanthocyanidins (condensed tannins). Especially the latter compounds were much more prevalent in the cell-specific CSPP subnetworks. Upon a targeted search for the presence of any of the monolignols, either as aglycones or glycosylated, only coniferin was observed in the chromatograms and was connected as a cellular compound in the subnetwork of the hexosylated oligolignols (Fig. 3). The time course-associated abundance changes of metabolites were modeled by piece-wise regression and the metabolites with highly correlated abundances were grouped into cliques, which were further combined into clique clusters whenever they shared more than a defined number of metabolites (see “Materials and Methods”; Fig. 4; Supplemental Fig. S2). In agreement with the absence of any other detectable monolignol, coniferin could not be further grouped into a clique (Fig. 4). Coniferin was detected during the entire time course in the cells of cultures where H2O2 was initially scavenged (KI-treated and lignin-reforming cultures) but was never observed in the lignin- forming cultures. Likely, the coniferin formed in the H2O2-scavenging cultures remained stored in the cells after KI removal at day 20. When exploring the con- nection between CSPP network nodes and clique cluster membership, CSPP subnetwork nodes associated with cellular phenolic compounds were found to be- long almost exclusively to clique cluster 1 (filled blue circles and triangles, Figs. 3 and 4), whereas those associated with phenolic compounds in the culture medium belonged mainly to either clique cluster 1 (squares and triangles with blue border, Figs. 3 and 4) or clique cluster 4 (squares and triangles with yellow border, Figs. 3 and 4). In addition, two and one nodes related to

experiments with 25 mL of cell culture per 100-mL ﬂask. Mean 6 SE; n =

Figure 2. Scavenging of H2O2 by KI inhibits lignin formation in the 3 to 7 (treatment replicates). There was a statistically significant differ- Norway spruce cell culture. A, Amount of extracellular lignin formed ence between the groups (a, b, c, and d) as determined by one-way after culturing of Norway spruce cells in the culture medium supple- ANOVA [F(6,27) = 39.917, P = 3.4759E-12, Tukey’s honestly significant mented with water (H2O), KCl (5 mM), or KI (5 mM). Culture medium difference; SPSS software]. FW, Cell fresh weight. C, H2O2 concentra- was separated from cells by filtering through Miracloth. Extracellular tion (mM) in the culture medium supplemented with water or KI (5 mM). lignin was pelleted by centrifugation, washed two times with water, In this culturing experiment, extracellular lignin was clearly visible in lyophilized, and weighed. The experiment was repeated several times the culture medium of water-treated cultures at day 14. After 20 d, KI with a similar trend in results. Representative results are shown. Mean 6 was removed and cell cultivation was continued in the regular nutrient SE; n = 3 (treatment replicates). There was a statistically significant dif- medium without extra KI supplementation until extracellular lignin was 6 ference between the groups (a and b) as determined by one-way visible at KI_20+13d. Mean SE. *, KI significantly lowers H2O2 con- ANOVA [F(5,12) = 92.758, P = 3.6524E-9, Tukey’s honestly significant centration in the culture medium; two-tailed Student’s t test: 7 d, t(8) = difference; SPSS software]. B, Cell growth in the medium supplemented 5.384, P = 0.0013; 14 d, t(4.009) = 4.495, P = 0.011; 20 d, two-tailed with water or KI (5 mM). After 21 d, KI was removed and cell cultivation Mann-Whitney test: U = 45, nH2O =3,nKI = 15, P = 0.0025. #, After the was continued in the regular nutrient medium without extra KI sup- removal of extra KI supplementation, H2O2 levels resemble those in the plementation (KI_21+nd; with n, number of days of culture after the water treatment after 14 d: t(8) = 1.491, P = 0.174. H2O2 was measured medium-change step). The values show representative results for with the xylenol orange method (Ka¨rko¨nen and Fry, 2006).

1452 Plant Physiol. Vol. 174, 2017 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Figure 3. CSPP network. In the CSPP network, the nodes representing mass-to-charge ratio (m/z) features speciﬁc for cells and culture medium are shown as circles and squares, whereas triangles represent those found in both. The clique cluster (see Fig. 4) to which the m/z feature (node) belongs is indicated by a color. The node itself is colored whenever the clique cluster is derived from the cellular metabolites, whereas the node border color represents clique clusters derived from the culture medium-based metabolites. Only a few representative molecules for the various biochemical classes are shown, except for the CSPP subnetwork

Plant Physiol. Vol. 174, 2017 1453 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Laitinen et al. cellular phenolics of clique cluster 10 (filled yellow the culture medium. In addition to IDDDC hexoside squares and triangles, Figs. 3 and 4) and to an apoplastic and pinoresinol hexoside that were present in the cells phenolic compound of clique cluster 20 (squares with or- and the medium, several other dilignol hexosides were ange border, Figs. 3 and 4) were observed. In the culture detected exclusively in the cells of all cultures, such as medium and cells, 114 and 318 compounds, of which G(8-8)H hexoside, lariciresinol hexoside, G(8-5)G 10 and 44 were structurally elucidated, respectively, hexoside, G(8-O-4)G hexoside, and G(8-O-4)vanilloyl showed significantly different (P , 0.001) abundances hexose (mainly clique clusters 10 and 69; Fig. 4; between the lignin-forming and H2O2-scavenging cultures. Supplemental Fig. S2B). Whereas the former two are In agreement with the known structure of gymno- 8-8-linked lignan-type dilignols, the latter three are sperm lignin, the oligolignols in the medium of the either 8-5- or 8-O-4-linked neolignan-type dilignols. lignin-forming cell cultures contained units derived Furthermore, the latter three showed equal abundances from the incorporation of coniferyl alcohol (G units), in water-treated, lignin-forming cultures compared p-coumaryl alcohol (H units), coniferaldehyde (G9 with H2O2-scavenging cultures, with an increase ob- units), dihydro-p-coumaryl alcohol (DHH units), and served during lignin reformation. dihydroconiferyl alcohol (DHG units) that are con- Several types of flavonoids (e.g. dihydroflavonols nected via 8-8, 8-5, and 8-O-4 linkages (Boerjan et al., and flavonols), condensed tannins (proanthocyanidins 2003). Many of these oligolignols were also observed in with few subunits), stilbenes, and hydroxycinnamic the medium of H2O2-scavenging cultures, albeit often at acid glycosides also were identified in the cells but not much lower abundances (clique clusters 1, 4, 16, 28, 36, in the medium (clique clusters 1, 10, 23, 39, 52, and 69; 57, and 58; Supplemental Fig. S2A; exemplified for cli- Fig. 3; Supplemental Fig. S2B). Many of these com- que clusters 1 and 4 in Fig. 4). Upon KI removal, higher pounds appeared at higher abundance in the cells that abundances of these compounds were observed in the produced extracellular lignin (especially clique clusters medium of lignin-reforming cultures (especially in cli- 1, 23, 39, and 52; Fig. 4; Supplemental Fig. S2B). Among que clusters 4, 36, and 58), although these compounds them were many flavonoids with antioxidative capac- generally did not accumulate to the levels observed in ity (e.g. taxifolin, quercetin, and myricetin) as well as the medium of water-treated, lignin-forming cultures. condensed tannins. The variety of condensed tannins A few oligolignols were more abundant in the medium was partitioned mainly between clique clusters 1 and of H2O2-scavenging cultures compared with that of 10, with those of clique cluster 1 more frequently com- lignin-forming cultures (clique clusters 20, 50, 56, and prising units derived from (epi)catechin compared with fi 74; Supplemental Fig. S2A; exempli ed for clique those of clique cluster 10. cluster 20 in Fig. 4), which could be explained for clique clusters 20 and 56 by their consumption in further oligomerization reactions during lignin formation in H2O2 Scavenging Induces Remodulation of the lignin-forming cultures [compare the accumulation the Transcriptome pattern of G(8-O-4)DHG in clique cluster 20 with that of G(8-O-4)G(8-O-4)DHG in clique cluster 16 and that Given the large differences in intracellular metabolite of G(8-O-4)G9 in clique cluster 56 with that of G(8-O-4) pools in the contrasting cell cultures (lignin-forming G(8-O-4)G9 in clique cluster 57; Supplemental Fig. versus non-lignin-forming cultures), an RNA-Seq ex- S2A]. Remarkably, two hexosylated dilignols, iso- periment was conducted to ascertain whether apo- dihydrodehydrodiconiferyl alcohol (IDDDC) hexoside plastic H2O2 similarly affected gene expression. Gene and pinoresinol hexoside, were observed in the me- expression was compared between the lignin-forming dium. As IDDDC hexoside was the most abundant (including lignin-reforming) cultures and those where structurally characterized phenol in the cells (Table I; extracellular lignin formation was not induced (i.e. cells Supplemental Table S3), showing a similar accumula- harvested at time 0) or those under H2O2 scavenging tion pattern in the medium (clique cluster 1; Fig. 4) and (Fig. 1; Supplemental Fig. S1B). in the cells (clique cluster 1; Fig. 4), we cannot exclude A principal component analysis (PCA) revealed that that its presence in the culture medium might have the variable extracellular lignin formation explained the resulted from a minor cellular contamination during majority of the differential expression observed in the isolation. In contrast, the accumulation patterns of data (Fig. 5), with the first component clearly separating pinoresinol hexoside in the medium (clique cluster 74; lignin-forming and non-lignin-forming samples, rep- Supplemental Fig. S2A) and in the cells (clique cluster resenting 61% of the overall variance. Altogether, 10; Fig. 4) were entirely different, providing evidence 10,751 genes (approximately 21% of expressed genes) for distinct pools of pinoresinol hexoside in the cells and were differentially expressed between lignin-forming

Figure 3. (Continued.) comprising monolignols, oligolignols, and their glycosides. The latter subnetwork includes both cell- and culture medium- speciﬁc nodes. Nevertheless, both node types are clearly separated, as the left and the central/right part of the subnetwork represent the hexosides (present in cells) and the aglycones (present in medium) of the monolignols and oligolignols, respectively.

1454 Plant Physiol. Vol. 174, 2017 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Apoplastic Redox State Affects Phenolic Metabolism

Table I. Abundances of phenolic compounds in the cells and in the culture medium of Norway spruce cell culture

Column A shows lignin forming at 15 d, and column B shows H2O2 scavenging at 15 d. The mass spectrometry (MS) response represents the image current generated in the ion cyclotron resonance cell and could be arbitrarily expressed in charge units. See Supplemental Table S3 to compare with the abundances of a particular compound at other time points. Note that extrapolating concentration differences between compounds from their relative MS response is ﬂawed due to the highly varying ionization and transfer efﬁciencies of compounds by MS. NA, Not available. Cells Culture Medium Trivial Name ABAB p-Coumaroyl hexose 2.1E+05 2.3E+04 NA NA Caffeic acid hexoside 3.5E+05 1.8E+05 NA NA Benzoyl hexose 7.7E+05 4.7E+05 NA NA Coniferin NA 7.1E+05 NA NA Naringenin O-hexoside 2.4E+05 NA NA NA Naringenin-8-C-hexoside NA 2.0E+05 NA NA Aromadendrin hexoside 2.2E+05 1.2E+05 NA NA Aromadendrin hexoside 2.5E+05 1.2E+05 NA NA p-Coumaroyl quinoyl hexose 1.4E+05 3.0E+05 NA NA Methylmyricetin hexoside 1.4E+05 2.4E+04 NA NA Syringetin hexoside NA 6.9E+03 NA NA G(8-O-4)Vanilloyl hexose 1.1E+05 2.1E+05 NA NA G(8-O-4)G hexoside NA 1.7E+05 NA NA G(8-O-4)IDDDC 3.0E+05 1.7E+05 1.8E+06 2.5E+06 G(8-O-4)IDDDC 1.0E+06 1.5E+05 7.7E+06 1.4E+06 Myricetin dihexoside 2.4E+05 NA NA NA (Epi)gallocatechin 4.9E+05 2.9E+05 NA NA Caffeoyl shikimic acid 2.8E+05 NA NA NA Cinnamoyl hexose 7.1E+05 3.3E+05 NA NA Vanillyl alcohol hexoside 4.6E+04 9.7E+04 NA NA Transpiceatannol hexoside 1.9E+06 4.0E+05 NA NA Naringenin-6-C-hexoside NA 1.8E+05 NA NA Kaempherol hexoside 8.1E+05 2.7E+05 NA NA Myricetin deoxyhexoside 3.5E+05 NA NA NA Quercetin hexoside 1.8E+06 1.6E+05 NA NA Taxifolin hexoside 2.5E+05 NA NA NA Myricetin hexoside 1.2E+06 6.7E+04 NA NA Myricetin hexoside 6.2E+05 3.7E+04 NA NA G(8-8)H hexoside 1.2E+05 NA NA NA Methylmyricetin hexoside 1.9E+05 NA NA NA Pinoresinol hexoside 5.1E+06 4.6E+06 3.1E+04 1.1E+05 Lariciresinol hexoside 3.8E+05 5.3E+05 NA NA (Epi)catechin-4,8’/7’,2-(epi)catechin 1.1E+06 5.4E+05 NA NA (Epi)catechin-4,8’/7’,2-(epi)catechin 4.1E+05 2.5E+05 NA NA (Epi)catechin-4,8’-(epi)catechin 6.4E+06 1.5E+06 NA NA (Epi)catechin-4,8’-(epi)catechin 4.3E+06 9.9E+05 NA NA (Epi)catechin-4,8’-(epi)catechin 2.8E+06 NA NA NA (Epi)catechin 9.2E+06 2.7E+06 NA NA G(8-5)G hexoside 9.1E+05 1.4E+06 NA NA IDDDC hexoside 3.7E+07 1.4E+07 1.1E+06 4.1E+05 (Epi)gallocatechin-4,8’/7’,2-(epi)catechin 4.5E+05 3.8E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)catechin 7.0E+05 3.4E+05 NA NA (Epi)catechin-4,8’/7’,2-(epi)gallocatechin 8.9E+05 4.9E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)catechin 9.1E+05 3.0E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)catechin 3.3E+05 1.0E+05 NA NA (Epi)gallocatechin-4,8’-(epi)catechin 1.2E+06 8.7E+05 NA NA (Epi)catechin-4,8’-(epi)gallocatechin 2.0E+06 8.9E+05 NA NA (Epi)gallocatechin-4,8’-(epi)catechin 8.2E+05 3.2E+05 NA NA Hydroxybenzoic acid hexoside 2.0E+06 3.4E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)gallocatechin 2.2E+05 2.7E+05 NA NA (Epi)gallocatechin-4,8’-(epi)gallocatechin 8.2E+05 3.6E+05 NA NA Quercetin dihexoside 6.9E+05 2.8E+05 NA NA (Epi)catechin-4,8’-(epi)catechin-4’,8’’/7’’,2’-(epi)catechin 1.9E+06 3.6E+05 NA NA (Epi)catechin-4,8’/7’,2-(epi)catechin-4’,8’’-(epi)catechin 1.8E+06 5.0E+05 NA NA (Epi)catechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin 6.9E+06 7.6E+05 NA NA (Epi)catechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin 3.0E+06 3.0E+05 NA NA (Table continues on following page.)

Table I. (Continued from previous page.) Cells Culture Medium Trivial Name ABAB (Epi)catechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin 2.6E+06 9.9E+05 NA NA (Epi)catechin-4,8’-(epi)gallocatechin-4’,8’’/7’’,2’-(epi)catechin 5.9E+05 4.2E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)catechin-4’,8’’-(epi)catechin 2.0E+06 3.4E+05 NA NA (Epi)gallocatechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin 8.9E+05 3.5E+05 NA NA (Epi)gallocatechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin 3.6E+06 4.5E+05 NA NA (Epi)catechin-4,8’-(epi)gallocatechin-4’,8’’-(epi)catechin 1.4E+06 5.2E+05 NA NA (Epi)catechin-4,8’-(epi)gallocatechin-4’,8’’-(epi)catechin 1.4E+06 3.1E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)gallocatechin-4’,8’’-(epi)catechin 3.7E+05 3.2E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)gallocatechin-4’,8’’-(epi)catechin 3.6E+05 3.3E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)gallocatechin-4’,8’’-(epi)catechin 7.9E+05 2.2E+05 NA NA (Epi)gallocatechin-4,8’/7’,2-(epi)gallocatechin-4’,8’’-(epi)catechin 4.4E+05 1.4E+05 NA NA (Epi)gallocatechin-4,8’-(epi)gallocatechin-4’,8’’-(epi)catechin 6.5E+05 3.6E+05 NA NA (Epi)catechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin-4’’,8’’’-(epi)catechin 3.3E+06 3.2E+05 NA NA (Epi)catechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin-4’’,8’’’-(epi)catechin 1.6E+06 NA NA NA (Epi)catechin-4,8’-(epi)catechin-4’,8’’-(epi)catechin-4’’,8’’’-(epi)catechin 2.3E+06 NA NA NA DDDC NA NA 1.5E+06 5.9E+04 G(8-O-4)DHG NA NA 5.1E+06 5.5E+06 G(8-O-4)DHH NA NA 6.3E+05 NA G(8-O-4)G NA NA 1.4E+06 2.3E+06 G(8-O-4)G NA NA 4.2E+05 5.2E+05 G(8-O-4)G’ NA NA 5.1E+04 1.2E+05 G(8-O-4)G(8-5)DHH NA NA 1.2E+05 NA G(8-O-4)G(8-8)G(4-O-8)G NA NA 1.7E+06 1.1E+06 G(8-O-4)G(8-O-4)DHG NA NA 5.5E+06 4.7E+06 G(8-O-4)G(8-O-4)G NA NA 1.9E+06 8.9E+05 G(8-O-4)G(8-O-4)G’ NA NA 1.2E+06 1.6E+05 G(8-O-4)G(8-O-4)G(8-O-4)G NA NA 2.3E+06 3.8E+05 G(8-O-4)G(8-O-4)H NA NA 1.5E+06 2.9E+06 G(8-O-4)G(8-O-4)Homovanillyl alcohol NA NA 1.2E+05 5.4E+05 G(t8-O-4)G(8-5)G NA NA 1.3E+06 6.8E+05 IDDDC NA NA 1.7E+06 8.7E+04 and non-lignin-forming conditions (P , 0.001; cyclin genes except one were up-regulated in non-lignin- adj , Supplemental Table S4). As KI was used as an H2O2 forming conditions [Padj 0.001]; Supplemental Fig. S4B). scavenger, it is possible that part of the identified dif- Together with microscopic evaluation, these observations ferential expression resulted from KI-induced effects suggest that the observed increase in cell fresh weight rather than from the difference between lignin forma- (Fig. 2B) was due to cell division rather than cell en- tion and not. To address this concern, we performed a largement. These GO over-enrichment results were in range of comparisons the results of which indicate that agreement with other findings from the study. KI did not induce a significant transcriptional response (Fig. 6; Supplemental Table S5; Supplemental Fig. S3; Shikimate, Phe, and Phenylpropanoid Pathways Supplemental Protocol S1). Are Induced in Lignin-Forming, But Not in In the comparison of lignin-forming versus non- H O -Scavenging Conditions lignin-forming conditions, only 5.8% (619) of the genes 2 2 were affected by KI, and as a caution, these were removed To understand the role of apoplastic H2O2 in mono- from subsequent analyses. Gene Ontology (GO) category lignol biosynthesis, the expression of genes putatively enrichment testing of the differentially expressed genes encoding enzymes in the shikimate, aromatic amino revealed that large differences in cellular metabolism acid, phenylpropanoid, and monolignol biosynthesis were seen in lignin-forming cells as compared with non- pathways was investigated. Examination of the set of lignin-forming cells (Supplemental Fig. S4). In lignin- annotated gene models (high, medium, and low confi- forming conditions, genes involved in carbohydrate, dence; Nystedt et al., 2013) identified gene models for amine, and lipid metabolism and response to stress were all enzymes of these pathways. p-Coumaroyl shikimate up-regulated, for example (Supplemental Fig. S4A). Also, 3-hydroxylase is a P450 enzyme the annotation of genes encoding enzymes in the shikimate pathway all the which is problematic, as enzyme activity cannot be way down to monolignol biosynthesis were strongly in- inferred from the gene sequence. Additionally, there are duced (see below). In non-lignin-forming conditions, a considerable number of P450s present in the genome genes involved in cell division, DNA replication, and re- annotation, and these all share high sequence similarity sponse to various stimuli were enriched (e.g. all putative yet will represent a broad potential range of activities.

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Figure 4. Phenolic time-course accumulation proﬁles. The symbol color and the shape of the inserts in the separate plots are used to indicate the clique cluster to which each node in the CSPP network (Fig. 3) belongs. Time-course proﬁles are shown for clique clusters for which representative compounds are present in the CSPP network (Fig. 3). Conditions are as follows: lignin-forming

conditions (+H2O; dashed lines, days 0–20), H2O2-scavenging conditions (+KI; continuous lines, days 0–20); and lignin- reforming conditions (2KI; continuous lines, days 20+n; with n, number of days in the culture after the medium-change step). , To constrain the analysis, only candidate genes (Padj O-METHYLTRANSFERASE (COMT)wereup-regulated, 0.001) with at least a 2-fold expression difference even though sinapyl alcohol is not present in spruce wood between lignin-forming and non-lignin-forming con- lignin or in this cell culture. Several genes with similar- ditions were considered. Out of 127 putative genes ity to the recently discovered CAFFEOYLSHIKIMATE encoding enzymes in the shikimate and aromatic amino ESTERASE (Vanholme et al., 2013) were identified, many acid pathways, 26 were up-regulated in lignin-forming of which had a moderate expression in the tissue-cultured conditions (Fig. 7; Supplemental Table S6). The four cells. However, none of these were induced over 2-fold most highly induced genes putatively encode arogen- , in lignin-forming conditions (Padj 0.001). Transcripts ate dehydratase/prephenate dehydratase, enzymes for many enzymes of the flavonoid pathway also were fi catalyzing the nal steps in Phe biosynthesis. Only four strongly induced during lignin formation (Fig. 7; genes in the pathway from chorismate to Trp had a Supplemental Table S6). These results support those fi signi cant increase in expression, although it was of a obtained from the phenolic analysis, where elevated , small magnitude (1.3- to 1.5-fold; Padj 0.001). This abundances of various flavonoids and proanthocyani- observation suggests that H2O2 scavenging did not dins were observed in lignin-forming cells (Fig. 3; have a major effect on the pathway leading to Trp but Supplemental Fig. S2B). that it strongly affected that leading to Phe (Fig. 7). Subsequently, the effects of H2O2 scavenging on the phenylpropanoid and monolignol biosynthesis path- Several LACCASE, PEROXIDASE, and DIRIGENT Genes ways were evaluated. In lignin-forming conditions, Were Differentially Expressed between Lignin-Forming several genes encoding enzymes belonging to these and Non-Lignin-Forming Conditions pathways were up-regulated (Fig. 7; Supplemental Table S6). Typically, one or few members of each gene Next, the expression of genes encoding oxidative family were induced. Interestingly, several genes anno- enzymes (class III peroxidases and laccases) that are tated as CAFFEATE/5-HYDROXYCONIFERALDEHYDE specifically involved in monolignol oxidation was

Apoplastic H2O2 Production and Signaling The inhibition of extracellular lignin formation under H2O2 scavenging suggests that peroxidases oxidize monolignols for extracellular lignin formation in the cell culture, requiring sources of apoplastic ROS, such as RBOHs, to be present. RBOHs are plasma membrane-located enzymes that produce superoxide in the apoplast (Ros Barceló, 1998; Suzuki et al., 2011; Kärkönen and Kuchitsu, 2015). Remarkably, five putative RBOH genes were significantly up-regulated in non-lignin-forming conditions but none in conditions of lignin formation (Fig. 8; Supplemental Table S7). Other sources of apoplastic H2O2 have been reported, including a number of cell wall-located oxidases, such as polyamine oxidases, copper-containing amine oxi- Figure 5. PCA of the RNA-Seq data. The largest variance, 61%, in the dases, and germins with oxalate oxidase activity data is explained by the difference between lignin-forming samples (Bernier and Berna, 2001; Cona et al., 2006; Kärkönen (H2O_5d, KI_20+4d, and KI_20+12d) and non-lignin-forming samples (time 0, KI_5d, and KI_20d). Three biological replicates are represented and Kuchitsu, 2015). Plasma membrane-located qui- for each treatment. For treatment abbreviations, see the legend for none reductases also may contribute to superoxide Figure 1. production into the apoplast (Lüthje et al., 2013). In line with these findings, the expression of several genes encoding amine oxidases, germin-like proteins, and examined. Thirty-nine of 281 PEROXIDASE genes (in- quinone reductases was strongly induced during lignin cluding full-length and partial sequences) were induced formation in the spruce cell culture (Fig. 8C; Supplemental in lignin-forming conditions (Fig. 8A; Supplemental Table S7). Table S7), whereas 18 were up-regulated in non-lignin- Cys-rich receptor-like kinases (CRKs) have been forming conditions. Phylogenetic sequence comparison shown to be involved in signaling during biotic and of these 57 genes did not reveal any condition-specific abiotic stresses that generate apoplastic ROS, and CRKs sequence clade (data not shown). LACCASE genes be- are potential candidates for apoplastic ROS perception haved similarly to PEROXIDASEs, with nine and eight and signaling (Bourdais et al., 2015). Among genes full-length LACCASE genes induced in lignin-forming coding for CRKs, 47 and nine were up-regulated in and non-lignin-forming cells, respectively (Fig. 8B; lignin-forming conditions and when H2O2 was scavenged, Supplemental Table S7). In some species, such as weeping forsythia (Forsythia suspensa), dirigent proteins guide a stereoselective coupling of two coniferyl alcohol radicals to an optically active lignan, (+)-pinoresinol (Davin et al., 1997). The participation of dirigent proteins in lignin biosynthesis is controversial. In Norway spruce, there are 78 annotated putative DIRIGENT genes, of which 12 were strongly induced (up to 72-fold) in lignin- forming conditions and four in non-lignin-forming conditions (Fig. 8B; Supplemental Table S7). Two LACCASE and two DIRIGENT genes were constitu- tively expressed in all conditions.

Pinoresinol Formed in the Medium Was a Racemic Mixture To ascertain whether dirigent proteins are functional in dilignol formation in the spruce cell culture, pinoresinol was isolated from the culture medium of Figure 6. Venn diagram of the comparisons of three differential ex- KI-treated cultures, and its optical activity was deter- pression analyses. Venn intersections identify the speciﬁcity of ex- mined. The optical rotation [a]22D was less than +3°, pression changes to the different conditions, including genes with a potential KI response. The striped gene set represents genes affected by indicating that pinoresinol was a racemic mixture of – KI in a stable manner, which were removed from the subsequent both (+)- and ( )-forms in almost equal amounts, as the analyses. All other responses are unstriped. See Supplemental Table S5 optically active enantiomer (–)-pinoresinol shows an for the respective gene identiﬁers and Supplemental Protocol S1 for 23 optical rotation [a] Dof–42.1° (Ishii et al., 1983). further details.

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Figure 7. Representation of the shikimate, aromatic amino acid, general phenylpropanoid, monolignol, and flavonoid pathways and the expression levels of corresponding genes in lignin-forming and non-lignin-forming conditions. Heat map color boxes show the gene family members of corresponding genes up-regulated by 2-fold or greater in lignin-forming conditions (scale, , yellow-red) and in non-lignin-forming conditions (scale, blue; Padj 0.001). For the shikimate pathway and aromatic amino acid pathways (according to Maeda and Dudareva, 2012): ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; CM, chorismate mutase; CS, chorismate synthase; DAHP synthase,3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DHD-SDH, 3-dehydroquinate dehydratase-shikimate dehydrogenase; DHQS, 3-dehydroquinate synthase; EPSP synthase,5-enolpyruvylshikimate 3-phosphate synthase; HPP-AT, 4-hydroxyphenylpyruvate aminotransferase; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; PPA-AT, prephenate aminotransferase; PPY-AT, phenylpyruvate aminotransferase; SK, shikimate kinase. For the flavonoid pathway (according to Hammerbacher et al., 2014): ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone-3-hydroxylase; F39H, flavonol-39-hydroxylase; F395H, flavonol-39,59-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase. For the general phenylpropanoid pathway: CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; C3H, p-coumaroyl shikimate 3-hydroxylase; C4H,cinnamate 4-hydroxylase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; PAL, Phe ammonia lyase; 4CL, 4-coumarate-CoA ligase. For the monolignol pathway: CAD,cinnamylalcoholdehydrogenase;CCR, cinnamoyl-CoA reductase; COMT, caffeate/5-hydroxyconiferaldehyde O-methyltransferase. For gene identifiers, see Supplemental Table S6. respectively (Fig. 8D; Supplemental Table S7), suggesting catalases, and glutathione S-transferases were induced, a potential role in H2O2 signaling. suggesting that the antioxidant system was activated and that the synthesis of glutathione conjugates was increased during lignin formation (Fig. 9B; Supplemental Table S8). The Expression of Antioxidant and Related Genes Suggests That Spruce Cells Experience an Oxidative Stress during Lignin-Forming Conditions A Broad Array of Transcription Factor Families Were Induced during Lignin Formation In order to evaluate the effectiveness of KI as an H2O2 scavenger, the expression of antioxidant protein- To gain insight into the transcriptional regulation of encoding genes and genes of related metabolic func- extracellular lignin biosynthesis, the expression of pu- tions was analyzed. In conditions of H2O2 scavenging tative transcription factor genes was assessed. In lignin- (KI_5d and KI_20d), several genes annotated as ascorbate forming samples, the expression of 167 transcription oxidases were up-regulated (Fig. 9A; Supplemental Table factor/regulator genes was up-regulated by 2-fold or , S8). In contrast, in lignin-forming cultures, several genes greater (Padj 0.001), while that of 138 transcription encoding putative monodehydroascorbate reductases, factor genes was more abundant in non-lignin-forming

Figure 8. Expression profiles of genes encoding putative enzymes involved in monolignol oxidation, further modification of dilignols, apoplastic ROS production, and signaling. Expression profiles are shown for PEROXIDASE (PRX; A), DIRIGENT (DIR), LACCASE (LAC), PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE (PCBER), and PINORESINOL REDUCTASE (PRR; B), genes of putative apoplastic ROS-generating enzymes: RESPIRATORY BURST OXIDASE (RBOH), AMINE OXIDASE (AM. OX.), GERMIN-LIKE, QUINONE REDUCTASE (Q. R.), and GAL OXIDASE (G. OX.; C), and CYS-RICH RECEPTOR-LIKE KINASE (CRK; D) genes in non-lignin-forming conditions (time 0, KI_5d, and KI_20d) and in lignin-forming conditions (KI_20+4d, KI_20+12d, $ , and H2O_5d). The heat map was generated using normalized gene expression with differential (fold change 2, Padj 0.001) or constant, considerable expression (genes marked with asterisks). Blue lines show genes up-regulated in non-lignin-forming conditions, and red lines show genes up-regulated in lignin-forming conditions. For gene identifiers, see Supplemental Table S7. samples (Supplemental Table S9). MYB and WRKY more naive clustering based on expression patterns. transcription factor families were enriched among the This inference analysis helped circumvent the paucity differentially expressed genes (Supplemental Table S9). of functionally annotated genes in the initial release of NAC (NAM, ATAF1/2, and CUC2) transcription fac- the genome by using a guilt-by-association approach to tors are important for secondary cell wall development infer the role of genes of unknown function based on in Arabidopsis (Nakano et al., 2015; Zhong and Ye, coexpression with genes having functional annotations. 2015) and are likely also involved in the regulation of After stringent filtering (see “Materials and Methods”), secondary cell wall biosynthesis in conifers (Duval the network consisted of 4,485 genes (Supplemental et al., 2014; Raherison et al., 2015; Lamara et al., 2016). Table S10), with a main subcluster containing 3,472 Ten NAC genes were up-regulated during lignin for- nodes (representing genes) that, as expected, showed a mation, whereas six NAC genes were down-regulated strong polarity separating the lignin-forming and non- (Supplemental Table S9). Similar observations were lignin-forming conditions (Fig. 10A). The network in- made for the R2R3-MYB transcription factor family, cluded 13 out of 196 gene models of the pathway members of which act as second-level master regulators starting from phosphoenolpyruvate and D-erythrose-4- and/or direct regulators of secondary cell wall bio- phosphate and leading to Phe, 10 out of 111 general synthesis (Nakano et al., 2015; Zhong and Ye, 2015; phenylpropanoid, and 11 out of 117 monolignol-specific Supplemental Table S9). In addition to NACs and pathway genes. This set of genes was used as a bait to MYBs, several members of the APETALA2/ethylene gather all of their first and second degree neighbors, which response element-binding protein (AP2/EREBP), LBD, are likely to be involved in similar biological processes. The WRKY, C2H2 zinc finger, and Tify transcription factor network obtained (Supplemental Fig. S5) was further re- families showed up-regulation in lignin-forming condi- duced of the excess-KI-affected genes (Supplemental Table tions. In non-lignin-forming conditions, transcripts of S5). The resulting 399 nodes (Fig. 10, B and C) were several homeobox, bHLH, and AUX-IAA transcriptions enriched in the GO categories secondary metabolism, in- factors were more abundant (Supplemental Table S9). cluding phenylpropanoid metabolism, transmembrane receptor kinase signaling pathway, and protein modifica- Gene Network Inference Analysis tion (Supplemental Fig. S6; Supplemental Table S11). Of these, 203 genes also were differentially expressed in the A gene coexpression network was inferred, an anal- lignin-forming versus non-lignin-forming comparison , ysis that has acknowledged limitations due to the al- (Padj 0.001). This subnetwork contained many genes with most binomial state of the experimental setup (i.e. a putative link to lignin biosynthesis (Table II). Addition- lignin-forming versus non-lignin-forming conditions) ally, six flavonoid biosynthesis genes were present. Of the but that was deemed to be more comprehensive than a 26 genes encoding transcription factors, the MYB family

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Figure 9. Expression profiles of putative antioxidant and related proteins. Differential expression is shown for the lignin-forming $ , (H2O_5d and KI_20+4/12d) and H2O2-scavenged (KI_5d and KI_20d) samples (fold change 2, Padj 0.001). A, Transcripts more abundant under H2O2-scavenging conditions (KI_5d and KI_20d). B, Transcripts more abundant in lignin-forming conditions (H2O_5d and KI_20+4/12d). White bars indicate genes affected by KI (removed from the other analyses). AAO, ASCORBATE OXIDASE; GST, GLUTATHIONE S-TRANSFERASE; MDHA-REDUCTASE, MONODEHYDROASCORBATE REDUCTASE. For gene identifiers, see Supplemental Table S8. was most represented with seven members (Tables II indicate those that were associated with values of P , 0.1 and III). and P , 0.05, respectively. Among the most discriminating metabolites were putrescine, oxalic acid, ribonic acid, 2-keto-L-gluconic acid, glycerol-3-phospate, pal- Gas Chromatography-Mass Spectrometry mitylglycerol, malic acid, Glu, pyro-Glu, and tartaric The results from the phenolic profiling and tran- acid, which were all higher in abundance in lignin- scriptome analyses suggested oxidative stress in water- forming cells compared with non-lignin-forming cells, treated, lignin-forming cells. Further insight was gained whereas discriminating compounds with a lower by analyzing primary metabolism with gas chromatography- abundance in lignin-forming cells were represented b mass spectrometry (GC-MS). The profiling of cellular by GalUA, 2-O-glycerol- -D-galactoside, threonic acid, and hydroxylamine (Supplemental Fig. S7; extracts from 5-d-old lignin-forming and H2O2-scavenged cultures yielded 67 metabolites, including various or- Supplemental Table S12). ganic acids, amino acids, sugars, amines, and lipids (Supplemental Table S12). Comparative analysis via PCA revealed that the largest proportion of explained DISCUSSION variation in the data set (33%) was associated with the The Antioxidant System Is Induced during Lignin difference between the lignin-forming and the H2O2- Formation in Cultured Spruce Cells scavenged cell cultures (Supplemental Fig. S7, left plot). To reveal metabolites that contributed most to the sep- In this study, the effects of modulated apoplastic ROS aration between lignin-forming and non-lignin-forming cell levels on phenolic metabolism and its transcriptional cultures, a loading plot was constructed (Supplemental regulation in a cell culture of Norway spruce that pro- Fig. S7, right plot). Metabolites that show up on the left duces extracellular lignin were studied. In this cell and on the right side of the loading plot were more culture, extracellular lignin becomes visible a few days abundant in lignin-forming (H2O_5d) and in non-lignin- after the transfer of cells from the solid maintenance forming (KI_5d) cells, respectively. To verify whether medium into liquid cultures (Simola et al., 1992; these trends also were apparent in univariate analyses, Kärkönen et al., 2002). The reduction in auxin concen- Student’s t test was performed independently on the tration at this stage was the factor proposed to stimulate metabolite abundances. Metabolites in orange and red lignin production in liquid conditions (Simola et al.,

1992). Even though the majority of cultured spruce cells (;97%) remain undifferentiated (Kärkönen and Koutaniemi, 2010), the structure of the extracellular lignin is more similar to that of native wood lignin than that of any other currently available, artificially produced lignin (Brunow et al., 1993; Koutaniemi et al., 2005; Warinowski et al., 2016). Furthermore, the same gene family members of the phenylpropanoid and monolignol biosynthesis pathways are induced in the tissue-cultured cells during lignin formation as in developing xylem (Koutaniemi et al., 2007). During culturing in liquid medium, micromolar levels of H2O2 are detectable in the culture medium (Kärkönen and Fry, 2006; Kärkönen et al., 2009). The substantial scavenging of H2O2 from the medium by KI (Huwiler et al., 1985; Nose et al., 1995; Fig. 2C) at a concentration of 5 mM prevented extracellular lignin formation (Fig. 2A; Supplemental Fig. S1). The inhibition was similar to that detected in a loblolly pine cell suspension culture where extracellular lignin formation had been induced by a high-osmoticum treatment (8% Suc; Nose et al., 1995). This observation suggests that peroxidases and not laccases are essential for the oxidative monolignol activation during lignin biosynthesis in the spruce cell culture. Although we cannot exclude the possibility that some of the genes we identified as differentially expressed represent off-target KI effects, our analyses suggest that any such effects were minimal (Fig. 6; Supplemental Protocol S1). In support of this, KI repressed lignin formation, and KI-treated cells started to divide actively without showing any symptoms of toxicity (Fig. 2). Furthermore, the results are in accordance with those obtained in transgenic tobacco with a modified apoplastic redox state (Pignocchi et al., 2006) and are supported by the phenolic analyses presented here. The observations corroborate that the detected effects were due to H2O2 scavenging. The induced expression of genes encoding antioxidant and related

Figure 10. Inference analysis networks. A, Inference analysis with stringent filtering resulted in one big network and several small clusters (at right). The big network shows high polarity. Red squares indicate positive and blue circles indicate negative fold change. Fold-change values are positive and negative when the gene expression is up-regulated in lignin-forming conditions and in non-lignin-forming conditions, respectively. Yellow triangles indicate the shikimate and phenylpropanoid pathway genes (no cutoff), and gray hexagons indi- . cate nonsignificant (Padj 0.05) or excess-KI-affected genes. B, Sub- networks of the stringent network created by selecting nodes to the second degree with shikimate pathway genes leading to Phe and with phenylpropanoid pathway genes. Differentially expressed pathway genes are named. Symbols are the same as in A. For abbreviations of the pathway genes, see the legend for Figure 7. C, Subnetworks highlighted , for the transcriptional regulators (purple diamonds; Padj 0.05). The pathway genes (triangles) are colored for fold change: red, positive; blue, negative; yellow, nonsignificant. For gene identifiers, see Table II and Supplemental Tables S10 and S11.

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, Table II. Gene identiﬁcations and annotations of discussed genes of the subnetworks with Padj 0.05 The fold-change value is positive and negative when the gene expression is up-regulated in lignin-forming conditions and in non-lignin-forming conditions, respectively. Lignin-Forming versus Non-Lignin- Annotation Gene Identiﬁer Forming

Fold Change Padj Shikimate pathway 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase MA_6189912g0010 2.7 4.6E-02 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase MA_10142969g0010 1.4 2.6E-03 3-Dehydroquinate dehydratase/shikimate dehydrogenase MA_10436080g0010 2.3 8.0E-12 Chorismate synthase MA_2539599g0010 2.4 1.1E-03 Putative prephenate aminotransferase MA_17674g0010 1.5 9.8E-03 Arogenate dehydratase/prephenate dehydratase MA_7514745g0010 39.3 1.9E-18 Arogenate dehydratase/prephenate dehydratase MA_864553g0010 27.2 2.1E-20 Arogenate dehydratase/prephenate dehydratase MA_387748g0010 1.8 4.3E-07 Arogenate dehydratase MA_864553g0020 32.4 4.3E-19 Arogenate dehydrogenase MA_42232g0010 6.8 7.5E-08 Phenylpropanoid pathway Phe ammonia lyase MA_5862513g0010 4.8 4.1E-05 Cinnamate 4-hydroxylase MA_10159692g0010 5.0 1.6E-04 Cinnamate 4-hydroxylase MA_10435536g0010 61.7 2.9E-38 4-Coumarate-CoA ligase-like MA_166573g0010 2.9 3.3E-05 Cinnamoyl-CoA reductase MA_137109g0010 2.7 3.3E-02 Caffeate/5-hydroxyconiferaldehyde O-methyltransferase MA_10430251g0010 24.1 6.8E-08 Caffeate/5-hydroxyconiferaldehyde O-methyltransferase MA_10375504g0010 4.7 3.2E-03 Caffeate/5-hydroxyconiferaldehyde O-methyltransferase MA_630428g0010 25.1 5.7E-03 Caffeate/5-hydroxyconiferaldehyde O-methyltransferase MA_10426580g0010 17.0 4.1E-07 Caffeate/5-hydroxyconiferaldehyde O-methyltransferase MA_86438g0010 9.1 3.1E-04 Flavonoid pathway Flavonoid 39-hydroxylase MA_10434848g0010 12.8 2.6E-06 Flavonoid 39-hydroxylase MA_158072g0010 4.0 8.6E-05 Dihydroﬂavonol-4-reductase MA_110462g0010 3.2 9.2E-06 Leucoanthocyanidin dioxygenase MA_169203g0010 33.5 9.0E-21 Anthocyanidin 3-O-glucosyltransferase MA_156209g0010 2.5 3.3E-08 Transcription factors Abscisic stress-ripening protein, ASR MA_60383g0010 2.4 2.2E-02 Abscisic stress-ripening protein, ASRa MA_773806g0010 3.8 1.7E-03 Abscisic stress-ripening protein, ASR MA_89246g0010 7.7 4.7E-05 bHLH transcription factor MA_895755g0010 2.6 2.4E-08 C2H2 zinc ﬁnger transcription factor MA_756968g0010 8.2 7.5E-06 Ethylene-responsive transcription factor MA_4072g0020 22.7 4.3E-11 Ethylene-responsive transcription factor MA_98464g0010 8.2 7.1E-09 GRAS transcriptional regulator MA_99004g0010 22.1 2.7E-02 MYB family transcription factora MA_139448g0010 20.3 1.5E-13 MYB family transcription factor MA_937875g0010 9.3 3.3E-04 MYB family transcription factor MA_10225049g0010 8.3 1.7E-04 MYB family transcription factor MA_51173g0010 2.5 1.5E-05 MYB family transcription factor MA_37058g0010 2.7 2.0E-03 MYB family transcription factor MA_316475g0010 34.1 1.5E-28 NAC transcription factor MA_139896g0010 1.9 1.3E-06 NAC transcription factor MA_18958g0010 6.5 3.0E-08 NAC transcription factor MA_16595g0010 1.9 4.9E-04 Probable WRKY transcription factor MA_76002g0010 17.1 8.1E-07 Tify family transcription factora MA_10427283g0030 11.7 1.0E-08 Tify family transcription factora MA_10436587g0020 4.7 1.3E-02 Two-component response regulator ARR MA_8059983g0010 54.9 2.8E-17 Other ABC transporter G family member MA_18507g0010 23.2 6.1E-04 Cys-rich receptor-like protein kinase MA_874084g0010 22.1 3.7E-02 Cys-rich receptor-like protein kinase MA_9111704g0010 3.9 8.8E-22 Gal oxidase MA_812690g0010 5.6 2.1E-03 Dirigent protein MA_10428143g0020 19.5 5.9E-12 Dirigent protein MA_6385864g0010 10.5 8.2E-06 (Table continues on following page.)

Table II. (Continued from previous page.) Lignin-Forming versus Non-Lignin- Annotation Gene Identiﬁer Forming

Fold Change Padj Dirigent protein MA_10428143g0010 8.5 1.4E-05 Dirigent protein MA_362458g0010 30.9 6.1E-21 Laccase MA_414479g0010 11.2 3.2E-12 Laccase MA_67291g0010 10.4 1.1E-20 Peroxidase MA_10432865g0020 8.1 2.2E-23 Peroxidase MA_10433564g0010 30.8 4.2E-64 Peroxidase MA_179122g0010 23.5 4.3E-17 Pinoresinol reductase homolog MA_445017g0010 18.5 6.2E-16 Pinoresinol reductase homolog MA_222809g0010 10.9 3.4E-09 aSwitch or regulation position in the subnetwork. proteins indicated that the cells were subjected to oxi- conditions under KI treatment, ascorbate would prefer- dative stress during extracellular lignin formation and entially exist in a reduced form. Ascorbate oxidases reg- that this oxidative condition was alleviated by KI ulate the redox state of apoplastic ascorbate by oxidizing treatment (Fig. 9). Likewise, various flavonoids and ascorbate to monodehydroascorbate without generating tannins accumulated preferentially in lignin-forming ROS (Pignocchi and Foyer, 2003). However, not all cells (e.g. clique clusters 1, 10, 39, and 52; Supplemental proteins encoded by genes annotated as ascorbate oxi- Fig. S2B), with some, such as taxifolin, quercetin, and dase have ascorbate oxidase activity (Ueda et al., 2015), myricetin, being efficient radical scavengers and/or an- and further studies are needed to resolve the function of tioxidants for lipid peroxidation, albeit sugar moieties can the proteins encoded by these genes in spruce. GC-MS influence their antioxidant activity (Hopia and Heinonen, analysis additionally indicated altered ascorbate me- 1999; Willför et al., 2003). The strong up-regulation of tabolism, with tartaric acid (a precursor for ascorbate genes encoding glutathione S-transferases (Fig. 9B) and biosynthesis) as well as oxalic acid and threonic acid UDP-Glc/GlcA transferases in lignin-forming conditions (ascorbate breakdown products) belonging to the most suggests that the synthesis of glutathione and sugar discriminating metabolites between lignin-forming and conjugates was increased during lignin formation, further non-lignin-forming cell cultures (Supplemental Fig. S7; supporting that cells under lignin-forming conditions Supplemental Table S12). were subjected to oxidative stress. The up-regulation of similar transferase genes together with those of cyto- chrome P450s has been shown to be typical for a catalase- fi Various Low-Molecular-Weight Phenolic Compounds de cient Arabidopsis mutant that suffers from oxidative Were Present in Cells and in the Culture Medium in Both stress (Queval et al., 2012; Noctor et al., 2015). Glutathione Lignin-Forming and Non-Lignin-Forming Conditions S-transferases, for example, catalyze the conjugation of oxidized lipid compounds to glutathione (Farmer and The most apparent change in the cultures where Mueller, 2013; Noctor et al., 2015). In conditions of H2O2 H2O2 was scavenged was the abolition of extracellular scavenging, several genes encoding putative ascorbate lignin formation (Fig. 2A; Supplemental Fig. S1). In the oxidases were induced (Fig. 9A). In the less oxidative lignin-forming and H2O2-scavenging cultures, a large

Table III. Transcription factor families found in the inference subnetworks, and occurrence in cell wall studies Enrichment in differential expression of lignin-forming versus non-lignin-forming conditions also is presented (compare with Supplemental Table S9). Transcription Factor Family Enrichment in the Differential Taylor-Teeples Cassan-Wang in the Subnetworks of Spruce (No. of Genes) Expression Analysis (Supplemental Table S9) et al. (2015) et al. (2013) Shi et al. (2017) MYB (7) Yes – Yes Yes ASR (5) – – – – NAC (3) – – Yes Yes AP2/EREBP (2) – Yes Yes Yes bHLH (2) – Yes Yes Yes Tify (2) – – – – bZIP (1) – – Yes Yes C2H2 zinc ﬁnger (1) – Yes Yes Yes GRAS (1) – Yes Yes Yes ARR (1) – – – – WRKY (1) Yes – Yes Yes

1464 Plant Physiol. Vol. 174, 2017 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Apoplastic Redox State Affects Phenolic Metabolism variety of low-molecular-weight phenolic compounds that flavonoids, some of which were abundant in cells, accumulated within the cells and in the culture me- were not detected in the medium also indicates that the dium. Several glycosylated dilignols were present in medium was not contaminated with cellular content. cells during H2O2 scavenging (Supplemental Fig. S2B; In the spruce cell culture, the expression of several clique cluster 69), with further accumulation after KI putative dirigent-encoding genes was induced signifi- removal (lignin reformation; clique clusters 10 and 69). cantly in lignin-forming cells and some in non-lignin- In addition, several types of flavonoids, phenolic alco- forming conditions (Fig. 8B). In order to ascertain hols, and hydroxycinnamic acids were identified in- whether dirigent proteins contributed to dilignol syn- tracellularly. Many of these compounds also were thesis, we isolated pinoresinol from the culture medium detected in cells of the lignin-forming cultures, but with of KI-treated cultures. The isolated pinoresinol was a contrasting accumulation patterns. The observation racemic mixture of both (+)- and (–)-forms, suggesting that spruce cells contain various dilignol glycosides is in that dirigent proteins did not participate in pinoresinol accordance with the data of Dima et al. (2015), who synthesis when H2O2 was scavenged. All putative detected glycosylated dilignols, trilignols, and tetralig- DIRIGENT genes of spruce (at least all those for which a nols in vacuoles of Arabidopsis leaves. It has been full-length sequence could be obtained) encode an demonstrated that vacuole-located oligolignols are N-terminal signal peptide, suggesting that they enter generated by protoplasmic combinatorial radical cou- the secretory pathway. If a dirigent protein was re- pling of monolignols similar to that observed in cell sponsible for pinoresinol synthesis, its concentration in walls, likely due to photooxidative stress (Dima et al., the culture medium may have been too low for the 2015). Also in lignifying tissues, intracellular oxidative protein to catch the conifer alcohol radicals. Hence, cross-coupling of monolignols was suggested to occur purification of pinoresinol from the apoplastic fluid of based on the detection of cysteinylated 8-O-4-type developing xylem is needed to evaluate the role of dilignols in the xylem of transgenic poplar (Populus dirigent proteins in dilignol formation in the cell wall. spp.) with a diminished phenylcoumaran benzylic Usually, conditions leading to apoplastic ROS forma- ether reductase (PCBER) activity (Niculaes et al., 2014). tion (e.g. wounding and pathogen infection) induce The latter aryl ether dilignols are formed when Cys DIRIGENT expression (Ralph et al., 2006). Their exact rather than water acts as a nucleophile in the rear- role in defense has not yet been elucidated. omatization of quinone methides formed by the 8-O-4 Reduced structures [IDDDC hexoside and G(8-O-4) cross-coupling of monolignol radicals. As Cys is not IDDDC] were identified in the culture medium in both present in the apoplast, this observation implied that conditions (clique clusters 1 and 74; Supplemental Fig. these quinone methides were formed inside the cells. S2A). IDDDC is formed from dehydrodiconiferyl al- Intracellular radical-radical cross-coupling of mono- cohol [G(8-5)G] by a PCBER that, in poplar, is one of the lignols is further supported in this study, as the time- most abundant proteins present in xylem (Niculaes course profiles of pinoresinol hexoside inside the cells et al., 2014). Since the PCBER is located in the cytoplasm and in the culture medium were clearly different (Gang et al., 1999; Vander Mijnsbrugge et al., 2000a, (Supplemental Fig. S2). In contrast to Arabidopsis leaf 2000b; Niculaes et al., 2014), the data suggest that these cells, in which the radical-radical cross-coupling-based reduced dilignols are produced intracellularly and trans- scavenging mechanism yields only oligolignol glyco- ported into the apoplast. The spruce genome contains sides (Dima et al., 2015), the condensed tannins within 10 sequences of high similarity to Populus trichocarpa the spruce cells also are supposedly formed by such a PtrPCBER, several of which have good expression in de- mechanism. Indeed, condensed tannin polymerization veloping xylem (Nystedt et al., 2013). In the spruce cell has been suggested to proceed via the intermediate culture, four putative PCBER-encoding genes had a formation of quinone methides with the involvement of considerable, constant expression across all conditions, polyphenol oxidases and/or peroxidases (Pourcel et al., whereas one PCBER gene was induced in lignin-forming 2007). In agreement with such a mechanism, an in- and one in non-lignin-forming conditions (Fig. 8B). creased abundance of condensed tannins was observed Heterologous production of the proteins and biochemi- in the lignin-forming and lignin-reforming cultures cal enzyme activity assays are needed to investigate (Supplemental Fig. S2B; clique clusters 1 and 10). which of these genes encodes the enzyme contributing to The presence of pinoresinol hexoside in the culture the biosynthesis of IDDDC. The observation that IDDDC medium (Table I) suggests that glycosylated dimers are hexoside was exported whereas many other hexosylated transported to the apoplast. Indeed, UDP-glycosyl phenolics remained intracellular suggests an as yet transferases and activated sugars required for the gly- unknown biological role for IDDDC hexoside in the cosylation reaction are considered to locate intracellu- apoplast. larly (Le Roy et al., 2016). An alternative hypothesis for Lariciresinol hexoside, another reduced dilignol the presence of pinoresinol hexoside in the culture glycoconjugate, was detected with a good response by medium is that some of the cells had ruptured and re- mass spectrometry in the extracts of spruce cells in both leased their cytoplasmic content. This hypothesis is not lignin-forming and H2O2-scavenging conditions, but supported by the data, as the accumulation profiles of unlike IDDDC hexoside, it was not detected in the pinoresinol hexoside inside and outside the protoplast medium (Table I; Supplemental Table S2). This com- were clearly different (Supplemental Fig. S2). The fact pound is formed from pinoresinol via the action of

Plant Physiol. Vol. 174, 2017 1465 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Laitinen et al. pinoresinol reductase (Fujita et al., 1999). Two genes obtained with transgenic tobacco with modified as- encoding putative pinoresinol reductase were induced corbate oxidase levels in the apoplast: the oxidation in lignin-forming conditions, and one gene had a con- state of the apoplast had an effect on gene expression stant expression in both conditions (Fig. 8B). This enzyme and hormone signaling, affecting growth and suscep- catalyzes the reduction of pinoresinol to lariciresinol and, tibility to pathogens (Pignocchi et al., 2006). It is in some species, also the further reduction of lariciresinol tempting to speculate that H2O2 present at micromolar to secoisolariciresinol (Fujita et al., 1999). Native Norway concentrations in the culture medium of lignin-forming spruce lignin contains pinoresinol and secoisolariciresinol cultures (Kärkönen and Fry, 2006; Kärkönen et al., 2009) structures (Zhang et al., 2003). Secoisolariciresinol units is perceived by plasma membrane-located receptors can originate via dehydrogenative coupling of secoiso- (e.g. CRKs); the consequent signal transduction would lariciresinol into lignin polymer (Zhang et al., 2003). In lead to the up-regulation of pathways leading to ex- this work, lariciresinol was not detected in the culture tracellular lignin and flavonoid biosynthesis. Under medium of lignin-forming cells, neither as such nor as H2O2-scavenging conditions (i.e. higher reducing condi- part of an oligolignol. tion in the apoplast), on the contrary, different metabolic pathways were operating, with cells dividing actively. In cell cultures of zinnia that differentiate into TEs, the lig- The Apoplastic Redox State Influences nification of TEs initiates only after TEs undergo pro- Cellular Metabolism grammed cell death, even if the monolignols are already present (Pesquet et al., 2013). The authors hypothesized The RNA-Seq data analysis revealed that the phe- that ROS released from cells by programmed cell death nylpropanoid pathway was up-regulated in lignin- could have a role in triggering lignification. forming conditions compared with non-lignin-forming During lignin formation, the most highly induced conditions (Fig. 7; Supplemental Table S6), which is genes of the shikimate and aromatic amino acid pathways consistent with the accumulation of extracellular lignin putatively encode arogenate dehydratase/prephenate in water-treated and in lignin-reforming cultures after KI dehydratase, enzymes catalyzing the final steps in Phe removal. The data support that a mechanism exists, biosynthesis (Fig. 7; Supplemental Table S6). Certain possibly mediated by apoplastic H2O2, sensing a reduced isoenzymes of arogenate dehydratase have been shown need for monolignol biosynthesis under H2O2-scavenging to profoundly and differentially modulate carbon flux conditions when monolignols cannot polymerize, into the downstream lignin biosynthesis pathway in whereas under more oxidative conditions, the synthesis Arabidopsis (Corea et al., 2012a, 2012b). It seems likely of monolignols is up-regulated. Alternatively, the sig- that the induced genes in spruce encode isoenzymes nal can be a phenolic compound (Bonawitz et al., 2014). responsible for the synthesis of Phe destined for mon- The differential expression analysis revealed several olignol and/or flavonoid biosynthesis, while some CRK-encoding genes that were up-regulated in lignin- other isoenzymes are responsible for the formation of forming conditions (Fig. 8D). CRK expression has been Phe utilized for protein synthesis. shown to be modulated in response to ROS- and cellular redox state-related stresses (Lehti-Shiu et al., 2009; Wrzaczek et al., 2010; Bourdais et al., 2015). Moreover, Both Peroxidases and Laccases Are Likely to Have a Role CRKs are thought to be involved in cellular redox and in Phenolic Coupling in the Spruce Cell Culture apoplastic ROS sensing by the presence of conserved Cys residues in their apoplastic domain. Thus, CRKs Both PEROXIDASEs and LACCASEs showed differ- are potential candidates for ROS signaling and regula- ential expression in lignin-forming and non-lignin- tion (Idänheimo et al., 2014; Bourdais et al., 2015). forming conditions: many genes were either up- or The cellular redox state has been shown to affect cell down-regulated (Fig. 8, A and B). In addition, the coex- proliferation in animals (Diaz-Vivancos et al., 2015), pression subnetwork contained three PEROXIDASE and and this is likely also to be the case in plants (Schippers two LACCASE genes that were expressed similarly to et al., 2016). Differences in redox buffering (i.e. anti- monolignol and shikimate pathway genes (Table II; oxidative capacity) in cellular compartments, with the Supplemental Table S11). All these genes were up-regulated apoplast being more oxidizing compared with the in lignin-forming conditions (Supplemental Table S7), and highly redox-buffered cytosol, suggest that plant cells all but one have some expression in early wood or in veg- can utilize redox-sensitive signal transduction as a etative shoots in spruce (Nystedt et al., 2013), making them powerful mechanism to integrate apoplastic stimuli to strong candidates for further focus. One PEROXIDASE in adjust cellular metabolism (Foyer and Noctor, 2016). the subnetwork (MA_10432865g0020; Table II) encodes During plant defense responses, transient oxidative PaPX2, a cationic peroxidase shown to be expressed in dif- bursts have been shown to induce major changes in ferentiating tracheids of developing xylem (Marjamaa et al., plant metabolism (Wrzaczek et al., 2013). Interestingly, 2006). One subnetwork LACCASE (MA_67291g0010), on in spruce cells, the modulation of the longer term redox the other hand, is PaLAC3c (Koutaniemi et al., 2015) and status of the apoplast had a profound influence, not has considerable expression in early wood and stems of only on apoplastic metabolism but on whole cellular vegetative shoots (absolute expression values of 6.98 and metabolism. Our results are in accordance with those 9.68, respectively; Nystedt et al., 2013).

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An analogous situation to that in the spruce cell (also at time 0; Fig. 8C). Although these data do not in- culture has been observed in flax (Linun usitatissimum) dicate a role for RBOHs, it has to be remembered that plants, whose outer stem tissues contain hypolignified the activity of RBOH enzymes is efficiently regulated bast fibers with increased levels of glycosylated oligo- posttranslationally by calcium and phosphorylation lignols compared with the inner stem, which contains (Kärkönen and Kuchitsu, 2015). lignified xylem and a higher content of less polar oli- Apoplastic isoenzymes of polyamine oxidases and golignols (Huis et al., 2012). The data obtained from copper-containing amine oxidases produce H2O2 in the flax mutants with lignified bast fibers suggest that, cell wall using apoplastic diamines and polyamines as similar to that in the KI-treated spruce cell cultures substrates (Cona et al., 2006). Oxalate oxidases (also called (Fig. 2A), insufficient polymerization of monolignols germins), on the other hand, use oxalic acid/oxalate as a leads to hypolignification in the wild-type bast fibers substrate; alternatively, some have SOD activity (Bernier (Chantreau et al., 2014). In the flax lignified bast fiber1 and Berna, 2001). In our experiment, several genes anno- mutants, which have ectopic lignin formation in bast tated as COPPER-CONTAINING AMINE OXIDASE or fibers, PEROXIDASE expression is increased in com- GERMIN were strongly up-regulated in lignin-forming parison with the wild type, suggesting that wild-type conditions(Fig.8C).However,externallyaddedoxalate, flax stems have insufficient peroxidase action, leading putrescine, spermidine, or Arg (the precursor of the latter fi fi to the hypoligni cation of bast bers. According to the compounds) did not increase apoplastic H2O2 levels in current opinion in the field, laccases function in the the lignin-forming spruce cell culture, suggesting that initial coupling of monolignols to oligolignols, while these enzymes do not produce H2O2 into the culture peroxidases contribute to lignin formation at later medium (Kärkönen et al., 2009). Interestingly, higher stages (Sterjiades et al., 1993; Zhao et al., 2013). Addi- amounts of putrescine and oxalate were detected in tionally, some isoenzymes of them probably participate lignin-forming cells compared with non-lignin-forming in the polymerization of proanthocyanidins (condensed cells (Supplemental Fig. S7; Supplemental Table S12). tannins) detected in cells (Fig. 3; Supplemental Fig. S2B; Levels of these metabolites in the culture medium, a site Pourcel et al., 2005, 2007). As peroxidase action is es- of apoplastic H2O2 formation, were not determined. sential in the formation of high-molecular-weight lignin Other possible apoplastic ROS-producing enzymes in- polymer in this cell culture and polymers were not clude plasma membrane-located quinone oxidoreductases formed in the presence of KI, the involvement of an- (Schopfer et al., 2008; Lüthje et al., 2013; Kärkönen et al., other type of oxidative enzyme is likely. This observa- 2014). In the cell culture, two quinone oxidoreductase- tion supports the role of laccases in the synthesis of encoding genes (FLAVOPROTEIN WrbA) were induced dilignols and oligolignols (Fig. 3). However, H2O2 during lignin formation. Both of these genes also have scavenging by KI seemed to be incomplete (Fig. 2C), strong expression (absolute expression values greater allowing for some peroxidase action during the KI than 10.5) in developing xylem (Nystedt et al., 2013). treatment. Thus, the possibility exists that there was less The inference analysis revealed novel enzyme can- peroxidase action in the KI-supplemented cultures due didates, Gal oxidases, for apoplastic H2O2 production to reduced H2O2 concentration; this would lead to (Table II; Supplemental Table S11). Furthermore, dif- shorter polymers. Hence, the data from this work can- ferential expression analysis revealed additional GAL not exclude that only peroxidases participate in mon- OXIDASE genes that were induced in lignin-forming , olignol oxidation in the spruce cell culture. conditions or in non-lignin-forming conditions (Padj 0.001; Fig. 8C). There have been reports suggesting the involvement of carbohydrate oxidases in defense-related Apoplastic ROS Generation H2O2 production in plants (Custers et al., 2004). The detection of GalUA as a main discriminating metabolite Since peroxidases are involved in monolignol acti- between the two types of cell cultures with higher abun- vation for lignin biosynthesis in the spruce cell culture, dance in non-lignin-forming cells (Supplemental Fig. S7; an aim of this study was to identify the source(s) of Supplemental Table S12) may support such a hypothesis, apoplastic H2O2. Candidates include isoenzymes of as galacturonaldehyde, the Gal oxidase product, should apoplastic peroxidases that can form H2O2 if there is a be readily oxidized to GalUA during GC-MS analysis. reductant present (Daudi et al., 2012); however, the Alternatively, GalUA may have been released from pec- native apoplastic reductant is unknown. Other candi- tins during cell wall modifications, followed by uptake dates include plasma membrane-located RBOH en- into the cells for reactivation for cell wall biosynthesis. zymes (Ros Barceló, 1998; Suzuki et al., 2011; Kärkönen Clearly, more work is needed to elucidate the role of ROS- and Kuchitsu, 2015). RBOHs generate apoplastic su- producing enzymes in plant development and, specifi- peroxide that dismutates to H2O2 either nonenzymati- cally, in lignin biosynthesis. cally in the acidic pH present in the cell wall or enzymatically via a superoxide dismutase (SOD)- catalyzed reaction (Karlsson et al., 2005). Surprisingly, Transcriptional Regulation none of the putative RBOH genes were induced in lignin-forming conditions, but the expression of several Similar to that in Arabidopsis, in white spruce, a RBOH genes was high in all non-lignin-forming conditions number of NAC domain transcription factors regulate

Plant Physiol. Vol. 174, 2017 1467 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Laitinen et al. the expression of secondary transcription factors that, 7-PHOSPHATE SYNTHASE gene, encoding the first en- in turn, regulate the expression of lignin and cell zyme in the shikimate pathway. Furthermore, the MYB wall polysaccharide biosynthesis genes; other NACs gene was induced 20-fold in lignin-forming conditions , directly regulate some secondary cell wall biosynthe- (Padj 0.001). These observations make this MYB an in- sis genes (Duval et al., 2014; Raherison et al., 2015; teresting candidate regulator of events leading to phe- Lamara et al., 2016). This suggests conservation of the nylpropanoid biosynthesis. Apart from monolignol hierarchical transcriptional regulatory cascade net- biosynthesis, MYB transcription factors are important in workofsecondarycellwallsynthesisbetweenangio- regulating, for example, flavonoid biosynthesis, with sperms and gymnosperms. PgNAC7 is most likely a different members important in distinct processes (Bomal master switch regulating secondary cell wall formation et al., 2014). in white spruce (Duval et al., 2014). It is also the most In recent years, secondary cell wall and lignin bio- connected hub gene in the coexpression gene network synthesis studies have revealed new members of the of secondary cell wall-related genes (Lamara et al., transcriptional gene regulatory network (Cassan-Wang 2016). PgNAC7 anditshomologinNorwayspruce et al., 2013; Taylor-Teeples et al., 2015; Shi et al., 2017). (MA_6777g0010) are highly expressed during wood Using a yeast one-hybrid approach on Arabidopsis formation in stems (Nystedt et al., 2013; Raherison et al., root xylem-expressed transcription factors, Taylor- 2015). In the cultured spruce cells, the expression of Teeples and colleagues (2015) revealed a highly inter- MA_6777g0010 was highest in H2O2-scavenging con- connected gene regulatory network enabling subtle and ditions and at time 0 (cells on the solid maintenance highly focused regulation of cell wall biosynthesis. medium), with expression down-regulated in condi- AP2/EREBP, bHLH, C2H2 zinc finger, and GRAS tions of lignin formation. In addition, genes annotated transcription factor families showed enrichment in their as MYB transcription factors (MA_139238g0010 and studies. In addition, transcription factors from bZIP MA_62361g0010), which are putative orthologs of those and WRKY families coexpressed with cell wall bio- regulating secondary cell wall and/or monolignol bi- synthesis genes in xylem of Populus trichocarpa (Shi osynthesis, PgMYB8/PtMYB8 and PgMYB1/PtMYB1 et al., 2017). Previously, genes of the same transcription (Bedon et al., 2007; Bomal et al., 2008), respectively, had factor families were identified in a search for regulators the highest expression at time 0, with a decrease in of secondary cell wall biosynthesis in Arabidopsis KI-supplemented conditions and an even lower ex- (Cassan-Wang et al., 2013). Several genes belonging to pression in lignin-forming conditions. The majority of these same transcription factor families had similar lignin-forming spruce cells remain undifferentiated. expression patterns to the shikimate and monolignol Therefore, up-regulation of the genes coding for tran- pathway genes (Table III) and also were differentially , scription factors that regulate the entire process of sec- expressed (Padj 0.001). In the present study, enrich- ondary cell wall biosynthesis would not be expected ment analysis (Taylor-Teeples et al., 2015) showed sta- when extracellular lignin is synthesized. However, tistically significant overrepresentation of the members some callus cells (;3%) differentiate to tracheids on the of AUX-IAA, homeobox, MYB, and WRKY transcrip- solid maintenance medium (Kärkönen and Koutaniemi, tion factor families within the differentially expressed 2010), an observation that may explain the moderate genes (hypergeometric probability; Supplemental levels of expression of secondary cell wall-regulating Table S9). Members of the MYB, Tify, and ABSCISIC transcription factors at time 0. Interestingly, a recent STRESS-RIPENING PROTEIN (ASR) transcription fac- transcriptomic study with coexpression network analy- tor families also were present in the subnetwork sis over cambium and developing xylem up to mature (Tables II and III; Supplemental Table S11), some of xylem suggested that NAC transcription factors are not which have expression in developing xylem (Nystedt central regulators for secondary cell wall formation in et al., 2013). A putative ASR1 gene was found in a Norway spruce (Jokipii-Lukkari et al., 2017). switch position in the subnetwork (Fig. 10C; Table II). In Arabidopsis, AtMYB58 and AtMYB63 directly ac- In tomato (Solanum lycopersicum), ASR1 has been found tivate lignin biosynthesis genes by binding to AC elements to act as a transcription factor also targeting cell wall- presentinthepromotersofmostofthegenesinthephe- related genes (Ricardi et al., 2014). In addition, rice nylpropanoid and monolignol pathways (Zhou et al., 2009). (Oryza sativa) ASR1 may directly scavenge H2O2 (Kim A few putative spruce MYB genes (MA_10430220g0010, et al., 2012). Also, two putative TIFY genes were located MA_1201g0010, and MA_137934g0010) with similarity in possible switch positions in the subnetwork (Fig. to these Arabidopsis MYB genes showed significant 10C; Table II). The Tify transcription factor family has up-regulation during lignin formation. Furthermore, been connected to a variety of abiotic and biotic stresses MYB family transcription factors were enriched within and also to development (Zhang et al., 2015). In maize the differentially expressed genes (Supplemental Table (Zea mays), ZML2, a Tify family member, and MYB11 S9), and MYB was the most abundant transcription together repress COMT expression (Vélez-Bermúdez factor family in the inference analysis subnetwork (Table et al., 2015). III; Supplemental Table S11). A putative MYB gene The differential gene expression analysis identified (MA_139448g0010) was located in the subnetwork (Fig. AP2/EREBP transcription factors as the second most 10C; Table II). This MYB was a first-degree neighbor abundant group of transcription factors up-regulated of a putative 3-DEOXY-D-ARABINO-HEPTULOSONATE during lignin formation (Supplemental Table S9). The

1468 Plant Physiol. Vol. 174, 2017 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Apoplastic Redox State Affects Phenolic Metabolism ethylene response transcription factors (ERFs) mediate medium. At the transfer from solid to liquid medium, the auxin (2,4-dichloro- m the responses to ethylene, but some are involved in phenoxyacetic acid) concentration is reduced from 10 to 0.5 M, whereas that of cytokinin (kinetin) remains unchanged (2.5 mM). In addition, some changes in other hormonal (e.g. jasmonic acid and abscisic acid) macronutrients occur (e.g. total nitrogen concentration is reduced from 35 to signaling pathways (Vahala et al., 2013). In hybrid as- 30 mM; Simola and Santanen, 1990). In order to ensure a uniform cell popula- pen (Populus tremula 3 Populus tremuloides), over- tion, a large-volume cell suspension was initiated and divided into 100-mL expression of certain ERFs affects cambial growth and aliquots (approximately 2 g of cells per 100 mL of medium in a 500-mL flask). wood chemistry (lignin and carbohydrate content; Time 0 samples were taken from the solid maintenance medium immediately before the cells were transferred into liquid cultures. Cell suspensions were Vahala et al., 2013). During compression wood forma- cultivated on a shaker (100 rpm) at 20°C in an 18-h-light/6-h-dark cycle (30–50 2 2 tion in maritime pine (Pinus pinaster), the ethylene- mmol m 2 s 1; Osram warm white) and sampled at the indicated time points forming enzyme 1-aminocyclopropane-1-carboxylate (Supplemental Fig. S1). After 20 d of culturing, the culture medium of oxidase accumulates strongly (Plomion et al., 2000). In KI-treated cultures was decanted away. To remove any residual KI from the addition to eccentric growth, compression wood of apoplast of cells, 100 mL of fresh nutrient medium containing the normal amount of KI (20 mM; as a micronutrient) was added onto the cells, and cells conifers contains higher levels of lignin compared with were incubated for 15 to 20 min on a shaker, after which the medium was normal vertical wood. In Norway spruce and radiata decanted again. This was repeated three times, after which the culturing was pine (Pinus radiata), ethylene stimulates cambium ac- continued as described above. tivity (Barker, 1979; Eklund and Tiltu, 1999). However, no changes in lignin amount were detected in radiata Oligolignol Profiling pine wood formed under ethylene exposure, although the amount of water-soluble extractives increased Sample Preparation (Barker, 1979). To reduce variation between replicates, 100-mL suspension cultures in 500-mL flasks were used with four biological replicates; these were sampled throughout the time series (Supplemental Fig. S1A). The cultures with or CONCLUSION without KI supplementation were initiated as described above. At each time point, 2-mL samples containing both cells and the culture medium were The lignin-forming cell culture of spruce proved to be aseptically collected. Once the cells had settled down, culture medium a valuable system for lignin biosynthesis studies, as it (;1.5 mL) was transferred into a clean tube and centrifuged to remove any allows modification of the apoplastic redox state, an precipitates (5 min, 17,000g, and +4°C). Supernatant was transferred into a new fi objective difficult to investigate in the developing wood tube and the air space was lled with nitrogen gas, after which the samples were frozen in liquid nitrogen. From the cells, the remaining medium was of intact trees. The global analyses presented here pipetted away, and the cells were washed once with 1.8 mL of water. As soon as revealed a major role for the apoplastic redox state in the cells had settled again, water was pipetted out, the air space was filled with cellular metabolism. Cells with oxidative conditions in nitrogen gas, and the cells were frozen in liquid nitrogen. Usually, it is im- the apoplast supply large amounts of carbon into the possible to cultivate cells of this spruce cell culture line in liquid cultures for longer periods, as the cells die a few days after extracellular lignin becomes synthesis of various phenylpropanoids (dilignols and fi fl visible in the culture medium. In this speci c tissue culture experiment, water- oligolignols, lignin, and avonoids), some of which treated cells remained alive even when they produced extracellular lignin; have antioxidant properties. Under more reducing hence, the lignin-forming cultures were cultured with sampling up to 20 d. The conditions in the apoplast, other metabolic pathways samples were stored at 280°C before the analyses. Phenolic compounds were operate and cells proliferate actively. The isolation of extracted following the procedure described by Morreel et al. (2014). Two replicate samples of cells and the medium of the lignin-forming and non-lignin- apoplastic compounds separately from the cytoplasmic forming cultures were analyzed at time points 0, 5, 10, 15, and 20 d, and two ones revealed that spruce cells are able to transport replicates of the lignin-reforming cultures were analyzed at time points 20 + 4, glycosylated dilignols into the apoplast. CSPP net- 20 + 8, 20 + 12, 20 + 16, and 20 + 20 d, making a total of 60 samples. fi works enabled the identi cation of several phenolic fi compounds based on their biochemical modifications. Phenolic Pro ling Gene inference network analysis combined with dif- Phenolic profiling was performed by analyzing 10 mL of the extract by ferential expression revealed candidate enzymes for reverse-phase ultra-HPLC (Accela UHPLC system; Thermo Electron)- monolignol oxidation and apoplastic H O production, electrospray ionization-Fourier transform-ion cyclotron resonance-mass spec- 2 2 trometry (LTQ FT Ultra; Thermo Electron). Instrument conditions were as novel putative transcription factors and potential H2O2 described previously (Morreel et al., 2014) with some minor modifications: the receptors related to lignin biosynthesis. The results water content in the mobile phase was decreased from 99% (0 min) to 55% obtained are important for advancing understanding of (30 min), and the electrospray ionization values for the spray voltage, capillary the processes involved in lignin biosynthesis and have temperature, sheath gas, and auxiliary gas were set at 24 kV, 275°C, 20 (arbi- provided potential targets for future studies on the trary units), and 10 (arbitrary units), respectively. Preprocessing of the chromatograms, including peak integration, alignment, and the grouping of peaks utilization of lignocellulosic materials. representing the same compound, was performed as described previously (Morreel et al., 2014). Profiling of the cells and the medium yielded 968 and 551 m/z features that could be reduced further to 430 and 225 m/z peak groups, MATERIALS AND METHODS respectively. However, 75 and 53 m/z features could not be grouped and remained as singletons (Broeckling et al., 2014; Morreel et al., 2014). Statistical Plant Material analyses were performed on the pseudomolecular ion from each peak group and on the singleton ions (i.e. on 505 and 278 m/z features, respectively). Because Norway spruce (Picea abies) cells (line A3/85) were maintained on a solid 31 m/z features were in common between the 505 and 278 m/z features, a total of nutrient medium as described by Kärkönen et al. (2002). For experiments, cells 751 unique m/z features were considered as putative nodes in the construction (approximately 2.5 weeks after subculturing) were transferred into liquid me- of a CSPP network (Morreel et al., 2014). Metabolic conversion types on which dium 59 (Simola and Santanen, 1990), and freshly prepared KI (final concen- the edges were based, and their corresponding number of CSPPs, are shown in tration, 5 mM), KCl (5 mM), or an equivalent volume of water was added to the Supplemental Table S1. Metabolic conversion types were selected from the list

given by Morreel et al. (2014) based on their prevalence in CSPP networks and successive elution with hexane and ethyl acetate and finally purified by thin- presumed presence in gymnosperms. Structural elucidation of the multiple- layer chromatography (Merck silica gel 60, F254, 0.25 mm) using CHCl3: stage mass spectrometry fragmentation MSn spectra was based on Eklund et al. methanol (97:3, v/v), to yield 3.9 mg of pinoresinol. The structure of pinoresinol (2008) and Morreel et al. (2004, 2010a, 2010b) for the (neo)lignans/oligolignols, was identified by 1H- and 13C-NMR recorded on a Bruker Avance III 400 in on Cuyckens and Claeys (2004), Fabre et al. (2001), Hughes et al. (2001), Morreel CDCl3 (Merck Uvasol; 99.8% D), and optical rotation was measured on a et al. (2006), and Hvattum and Ekeberg (2003) for the flavonoids, on Zhou et al. polarimeter (Perkin Elmer 341). (2013) and Abu-Reidah et al. (2014) for the C-glycosylated flavanones, on Morreel et al. (2014) for the phenylpropanoids/benzenoids, and on Gu et al. Spruce Transcriptomics (2003) and Jaiswal et al. (2012) for the condensed tannins. Shorthand names for the oligolignols are as explained by Morreel et al. (2004). The average abun- For transcriptomic analysis, cell suspension cultures supplemented with dances at each time point for the structurally characterized compounds are water or KI were initiated as described above (100 mL of culture per 500-mL fl shown in Supplemental Table S3. ask). A total of 12 replicate cultures were initiated for each treatment. Cells for RNA isolation were collected at time points 0 and 5 d, and KI-treated cells were Data Mining of Phenolic Profiles additionally collected at time points 20, 20 + 4, and 20 + 12 d (Supplemental Fig. S1B). Approximately 7 mL of the culture was aseptically decanted into 15-mL The metabolite data were modeled by piece-wise linear regression (lm tubes. After cell sedimentation (0.5–1 min), the supernatant was removed, and function) and the final model was reanalyzed by piece-wise robust linear re- the cells were washed briefly with water (5 mL). After cell sedimentation, water gression (rlm function, MASS library) in R version 3.1.3. Compared with classical was pipetted out and the cells were weighed and frozen in liquid nitrogen. To regression, robust regression is much less biased by outliers. Therefore, the reduce variation between the biological replicates, cells from four separate reliability of the classical regression coefficients can be judged by comparing flasks treated similarly were pooled at each time point for RNA isolation (hence, them with those obtained via robust regression. Missing data were replaced by three biological replicates for each treatment were analyzed for each time the mean at the particular time point whenever available or by the threshold point). Total RNA from approximately 1 g of cultured cells was isolated fol- value (100) in the whole phenolic profile data set. The full model contained knots lowing the procedure of Chang et al. (1993). The integrity of the isolated total at times 10, 15, 20, 20 + 4, 20 + 8, 20 + 12, 20 + 16, and 20 + 20 d. Model reduction RNA was visualized on a 1.2% (w/v) agarose gel, and total RNA concentrations occurred by iteratively removing the coefficient with the highest P value based were determined on a GeneQuant 1300 spectrophotometer (GE Life Sciences). fi on the Wald test and recalculation of the model. The nal model contained only Genomic DNA was removed by DNase I treatment (Qiagen) according to the fi , coef cients with P 0.05 (for culture medium and cell data, see Supplemental manufacturer’s instructions. Sample preparation for paired-end Illumina HiSeq fi , Tables S13 and S14, respectively). Signi cant regression models (P 0.001) sequencing was conducted in the Biomedicum Functional Genomics Unit, were obtained for 251 and 475 m/z features of the set of 278 and 505 m/z features University of Helsinki, using the NEBNext mRNA sample prep master mix set present in the ultra-HPLC-mass spectrometry chromatograms of extracts from (version 3.0 6/12). The mRNA was purified using the NEBNext poly(A) fi culture media and cells. Of those m/z features having a signi cant regression mRNA Magnetic Isolation Module (version 1.1 8/12) modified protocol. The fi , model, 114 and 318 m/z features differed signi cantly in abundance (P 0.001) sequencing was done at the Institute for Molecular Medicine Finland (Plat- between lignin-forming and non-lignin-forming cultures at one or more time form HiSeq 2000; number of lanes, two; paired-end sequencing, 2 3 100 bp; points during the experiment. cycles, 101 + 7 + 101; TruSeq version 3 [TruSeq PE Cluster Kit version 3, To cluster m/z features having covarying abundances over time, a correlation TruSeq SBS Kit version 3-HS, 200 cycles]) with 20 6 3 million paired-end se- analysis was performed. For each m/z feature, the mean abundance at each time quences per sample. point was calculated and pairwise Pearson correlations (cor function) for all m/z features were computed in R version 3.1.3. Pairs of m/z features having corre- Preprocessing of RNA-Seq Data and Differential lations above 0.75 were selected (Supplemental Table S15) and a correlation Expression Analyses network was constructed (graph.edgelist function, igraph library). In this correlation network, groups (called cliques) were searched for (maximal.cliques The quality of the raw sequence data was assessed, and reads were filtered to function, igraph library) in which the nodes (representing m/z features) were all remove adapters and trimmed for quality according to the Biomedicum mutually connected (i.e. having highly correlated abundances). However, a Functional Genomics Unit sequencing facility standard procedure. Briefly, node can be a member of multiple cliques. Therefore, to allow further clustering, FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was cliques that had more than 50% of their nodes in common were grouped (called used to assess the quality of the raw data, from which standard Illumina clique clusters). All m/z features represented in the whole set of maximal cliques adapters were removed using cutadapt (version 1.2rc2, with settings -m 15 -O 4; were retained in the clique clusters. Only clique clusters containing more than Martin, 2011), and reads were trimmed for quality using fastq_quality_trimmer fi ve m/z features are included in Supplemental Figure S2. (with settings -Q33 -t 16 -l 15) from the fastx_toolkit suite version 0.0.13.2 (http://hannonlab.cshl.edu/fastx_toolkit/). Filtered reads were aligned to the Analysis of Primary Metabolites Norway spruce genome (version 1.0, retrieved from the ConGenIE resource; Sundell et al., 2015) using STAR (version 2.3.1; Dobin et al., 2013; nondefault Cells grown for 5 d under either lignin-forming (H O_5d) or non-lignin- 2 settings:–outSAMstrandField intronMotif–readFilesCommand zcat–out- forming (KI_5d) conditions (25 mL of cell suspension per 100-mL flask, three ReadsUnmapped Fastx–alignIntronMax 70000). The annotations obtained from replicates for both conditions) were harvested and stored as described above for the Norway spruce version 1.0 GFF file contain only one transcript per gene oligolignol profiling. Extraction, GC-MS profiling, and the subsequent pro- model and contain all high-, medium-, and low-confidence gene models cessing of GC-MS data were conducted as described previously (Kaplan et al., fl fi 2004; Damiani et al., 2005; Desbrosses et al., 2005; Dauwe et al., 2007) using an (Nystedt et al., 2013). Brie y, high-con dence gene models are well supported HP6890 series mass spectrometry system coupled to a 5973 mass selective de- and predicted (greater than 70% coverage by either ESTs or UniProt proteins), fi tector (Agilent Technologies). The identification of primary metabolites was medium-con dence gene models have less support (less than 70% and greater fi based on the quadrupole mass spectral and retention time index library (i.e. than 30% coverage), while low-con dence gene models have little support (less fi Q_MSRI; Schauer et al., 2005). Sixty-seven metabolites were annotated and their than 30%). This GFF le and the STAR read alignments were used as an input to abundances analyzed by Student’s t test (t.test function in R version 3.1.3.). In the HTSeq (Anders et al., 2015) htseq-count python utility to calculate exon- addition, the metabolite abundances were centered and unit variance was based read count values. The htseq-count utility takes only uniquely mapping scaled prior to PCA (prcomp function in R version 3.1.3.). reads into account. Statistical analysis of single-gene differential expression between conditions was performed in R (version 3.2.3; R Core Team, 2015) Isolation of Pinoresinol and Its Chirality Analysis using the Bioconductor (version 3.2; Gentleman et al., 2004) DESeq2 package (version 1.10.1; Love et al., 2014). False discovery rate (FDR)-adjusted P values Liquid cultures (4.2 L) containing 5 mM KI were initiated as described above. were used to assess significance; thresholds of 0.001, 0.01, and 0.05 were used After culturing for 5 d, the culture medium was filtered through Miracloth and for the analyses. For the data quality assessment and visualization, the read centrifuged (15,000g, 20 min, and +4°C). The medium was concentrated by counts were normalized using a variance-stabilizing transformation as imple- freeze drying to roughly 1 L and extracted in a separation funnel portion wise mented in DESeq2. The biological relevance of the data (e.g. biological replicate

(500 mL) three times with equal volumes of CHCl3. The combined CHCl3 similarity) was assessed by PCA and other visualization tools (e.g. heat maps) fractions were evaporated to dryness with a rotavapor (40°C, water pump using custom R scripts. For all subsequent expression analyses, which also were vacuum) and separated by column chromatography (Merck silica gel 60) by performed in R, the normalized read counts obtained from DESeq2 were used.

1470 Plant Physiol. Vol. 174, 2017 Downloaded from on July 12, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Apoplastic Redox State Affects Phenolic Metabolism

An overview of the data, including raw and post-quality assessment read Supplemental Figure S2. Accumulation pattern of low-molecular-weight counts and alignment rates, is given in Supplemental Table S16. phenolic compounds in the Norway spruce cell culture during lignin ﬁ Transcription factor families were compiled following the classi cation in formation and when lignin formation was inhibited by H2O2 scavenging. planttfdb.cbi.pku.edu.cn and plntfdb.bio.uni-potsdam.de/v3.0/ by combining Supplemental Figure S3. REVIGO treemap of the KI-affected genes from it with the UniProt and pfam annotation from ConGenIE.org. Gene family Figure 6. enrichment in the differentially expressed genes was determined using the hypergeometric distribution online tool (www.geneprof.org; Taylor-Teeples Supplemental Figure S4. REVIGO treemaps of differentially expressed , et al., 2015). Success is a number of genes differentially expressed in lignin- transcripts (Padj 0.001). forming versus non-lignin-forming comparison (P , 0.001) from the popu- adj Supplemental Figure S5. Network nodes to the second degree pulled with lation of genes in the spruce genome. Sample is the size of the transcription the shikimate, Phe, phenylpropanoid, and monolignol pathway genes. factor family in the genome, and success in the sample is the number of family members in the differentially expressed genes. Supplemental Figure S6. REVIGO treemap of the subnetwork genes with the KI-affected genes removed. Extracting Excess-KI-Affected Genes Supplemental Figure S7. PCA of GC-MS-proﬁled compound abundances. Three independent differential expression analyses were conducted [H O_5d 2 Supplemental Table S1. CSPP conversions. versus time 0, KI_5d versus time 0, and KI_(5 and 20d) versus time 0], and their results intersected (Supplemental Protocol S1). The intersection of KI_(5 and Supplemental Table S2. Phenolic analysis and mass spectrometry data:

20d) versus time 0 and KI_5d versus time 0, excluding H2O_5d versus time 0, culture medium and cells. contains genes that are differentially expressed in response to either the pres- Supplemental Table S3. Time course-based raw data means of structurally ence of KI (aspeciﬁc effect, bias) or the absence of H O (biologically relevant). 2 2 characterized phenolic compounds. As these two effects could not be disentangled and in order to reduce the false- , positive rate at the likely expense of increasing the false-negative rate, this gene Supplemental Table S4. Differentially expressed genes (Padj 0.001). set (Supplemental Table S5) was removed from further analysis. Supplemental Table S5. Venn diagram gene lists. GO Enrichment Supplemental Table S6. Biosynthesis pathway genes.

Due to the low GO term coverage (approximately 40%; source, ConGenIE), Supplemental Table S7. Genes encoding putative enzymes involved in GO enrichment was conducted using GO terms from the Arabidopsis (Arabi- monolignol oxidation, further modiﬁcation of dilignols, apoplastic dopsis thaliana) homologs (coverage of approximately 84%). agriGO software ROS production, and signaling. (Du et al., 2010) was used with Arabidopsis homologs obtained from Con- Supplemental Table S8. Putative antioxidant and related protein genes. GenIE.org (Complete GO, Fisher’s exact test with Yekutieli FDR under de- pendency correction, Q , 0.05). GO treemaps were generated using REVIGO Supplemental Table S9. Differentially expressed transcription factor/ (Supek et al., 2011; FDR , 0.05, medium similarity) and custom R scripts. regulator families.

Gene Coexpression Network Inference Supplemental Table S10. Stringent network genes. Supplemental Table S11. Subnetwork genes. Eight gene network inference methods were run: ANOVA (Küffner et al., 2012), CLR (Faith et al., 2007), GeneNet (Opgen-Rhein and Strimmer, 2007), Supplemental Table S12. GC-MS data. GENIE3 (Huynh-Thu et al., 2010), NARROMI (Zhang et al., 2013), Pearson, Supplemental Table S13. Culture medium, piece-wise regression, and cli- fi Spearman, and a modi ed implementation of TIGRESS (Haury et al., 2012); que cluster analysis. their results were aggregated into a consensus network using an unweighted Borda counting method as suggested by Marbach et al. (2012). The resulting Supplemental Table S14. Cultured cells, piece-wise regression, and clique network was then analyzed using RmtGeneNet (Luo et al., 2007) to determine cluster analysis. the weight threshold for which its structure deviated from a Poisson to a Supplemental Table S15. Liquid chromatography-mass spectrometry- x2 fi Gaussian distribution, as assessed by a goodness-of- t test. This allowed for based clique analysis. the selection of the core of the network. Similarly, an assessment of the scale-free property of the consensus network, fitting a heavy-tailed distribution using a Supplemental Table S16. Summary of RNA-Seq sequencing reads and log-log linear model, led to the selection of two additional cutoffs, one stringent, alignments. where the r2 linear regression value was maximized, and one more lenient, Supplemental Protocol S1. Identifying and removing the putative KI effect where the weight was minimized, for an r2 greater than or equal to 0.8. These on gene expression: rationale. two cutoffs allowed for the selection of the stringent and lenient networks. These networks were further visualized and processed using the Cytoscape (Shannon et al., 2003) tool. Subsets of the stringent network were created by selecting the first- and second-degree neighbor nodes of the shikimate pathway, ACKNOWLEDGMENTS the Phe pathway (Maeda and Dudareva, 2012), and the phenylpropanoid and We thank Tuija Niinimäki for subculturing spruce cells. monolignol pathway genes, except that the genes determined as excess-KI- affected genes by the Venn comparison (Fig. 6; Supplemental Table S5) were Received January 20, 2017; accepted May 16, 2017; published May 18, 2017. not used. The resulting subsets (Supplemental Fig. S5) were further reduced to subnetworks (Fig. 10B) by removing the KI-affected genes. LITERATURE CITED

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