Research Article Norway spruce (Picea abies) laccases; characterisation of a laccase in a lignin-forming tissue culture

Running title: Norway spruce laccases Sanna Koutaniemi1†, # , Heli A. Malmberg1†, Liisa K. Simola2##, Teemu H. Teeri1, Anna Kärkönen1* 1

Department of Agricultural Sciences, University of Helsinki, Finland, 2Department of Biosciences,

University of Helsinki, Finland. #

Present address: Department of Food and Environmental Chemistry, University of Helsinki, Finland,

## †

Present address: Ritarikatu 9 B, 00170 Helsinki, Finland.

These authors contributed equally to this work.

* Correspondence: [email protected]

Edited by: Kurt Fagerstedt, Helsinki University, Finland

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1111/jipb.12333] This article is protected by copyright. All rights reserved. Received: November 5, 2014; Accepted: January 14, 2015

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Abstract

Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilising available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments and pseudogenes) were detected in the spruce genome. Out of the 112 gene models annotated as laccases, 79 are expressed at some level. We isolated five fulllength laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterised one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8-4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 µM; however, the laccase has a lower catalytic efficiency (Vmax/Km) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.

Keywords: Coniferyl alcohol oxidase; developing xylem; laccase; lignin biosynthesis; Norway spruce; Picea abies; tissue culture

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INTRODUCTION

Lignin is a phenolic polymer that is deposited in the secondarily thickened cell walls of water-conducting tracheids and vessels and support-giving schlerenchyma (fibers, stone cells). Lignin makes the cell walls rigid and water impermeable, and gives structural support to cell wall polysaccharides. In coniferous plants lignin is composed mainly of guaiacyl (G) lignin originating from coniferyl alcohol, and a small percentage of p-hydroxyphenyl (H) lignin derived from p-coumaryl alcohol (Boerjan et al. 2003). In dicot plants syringyl (S) lignin, derived from sinapyl alcohol, is also present. Lignin in grasses and cereals consists of G, S and H lignin, with some p-hydroxycinnamic acids (ferulic acid, p-coumaric acid) attached to lignin.

Whether peroxidases (class III; EC 1.11.1.7; donor:hydrogen peroxide oxidoreductase) or laccases (pdiphenol:dioxygen oxidoreductase, EC 1.10.3.2) function in the last step of lignin biosynthesis, i.e., oxidise monolignols to radicals that then couple and make the lignin polymer, has been under active evaluation. Both peroxidases and laccases can form dehydrogenation polymers (DHPs) from monolignols in vitro (Freudenberg 1959). Qualitatively the laccase- and peroxidase-made DHPs are indistinguishable (Bao et al. 1993; Sterjiades et al. 1993; Ranocha et al. 1999; Kärkönen et al. 2002). There is high laccase and peroxidase gene expression in developing xylem of many tree species, such as Populus (Sterky et al. 1998), Pinus taeda (Sato et al. 2001) and Picea abies (Norway spruce; Koutaniemi et al. 2007), and several isoenzymes of both enzyme types are found in the apoplast of lignin-synthesising cells (Sato et al. 2001; Ranocha et al. 2002; Kärkönen et al. 2002; Marjamaa et al. 2003, 2006; Berthet et al. 2011, Novo-Uzal et al. 2013). Antisense plants or knockout mutants of certain laccases (e.g. Liang et al. 2006; Berthet et al. 2011; Zhao et al. 2013) and peroxidases (e.g. Blee et al. 2003; Herrero et al. 2013, Shigeto et al. 2013) have reduced lignin contents and/or changes in its subunit composition. Recently, a complete loss of lignification in the vasculature was observed in a triple mutant of Arabidopsis thaliana (Arabidopsis) with simultaneous downregulation of LAC4, LAC17 and LAC11, suggesting that laccases have a crucial role in activation of monolignols for lignin polymerisation in Arabidopsis vasculature (Zhao et al. 2013). Interestingly, in developing tracheary elements of the protoxylem the spatial localization of LAC4 and LAC17 in the secondary cell walls seems to determine the sites where lignin is polymerised (Schuetz et al. 2014). Involvement of laccases in lignin formation has also been shown in Populus trichocarpa, as overexpression of Ptr-miR397a, a microRNA that is a negative regulator of several laccase genes, resulted in a clear decrease in laccase activity and reduced amount of Klason lignin in the wood as compared with control plants (Lu et al. 2013).

Both peroxidases and laccases are abundant in cell walls of coniferous tree species (e.g. Sato et al. 2001; Kärkönen et al. 2002; Marjamaa et al. 2003, 2006; Nystedt et al. 2013). Similarly as for peroxidases (Fagerstedt et al. 2010), there are only indirect indications that laccases and/or other coniferyl alcohol-

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oxidising enzymes have a role in lignin biosynthesis in conifers (Table 1). One laccase capable of forming small molecular weight DHPs from coniferyl alcohol has been purified from developing xylem of Pinus taeda at the time of active lignification (Bao et al. 1993). Altogether, eight laccase genes have a prominent expression in developing xylem of this Pinus species (Sato et al. 2001). High laccase gene expression occurs also in developing xylem of Norway spruce (Koutaniemi et al. 2007), and two laccase-like isoenzymes with the ability to form DHPs have been found in a lignin-forming cell culture of spruce (Kärkönen et al. 2002; Koutaniemi 2007). In some conifers, lignin formation has also been reported to correlate spatially and temporally with O2-requiring coniferyl alcohol oxidase (CAO) activities (Savidge and Udagama-Randeniya 1992; Udagama-Randeniya and Savidge 1994; Savidge et al. 1998; Table 1). These enzymes are firmly attached to the cell wall, and can be extracted only after a treatment with cell wall-degrading enzymes. CAO isolated from actively lignifying xylem of Pinus strobus is able to form dimers and higher molecular weight DHPs from coniferyl alcohol in vitro (Savidge and Udagama-Randeniya 1992). CAOs have been characterized as catechol oxidases (o-diphenol:oxygen oxidoreductase, E.C.1.10.3.1) that are tetrameric enzymes and have a single copper atom per polypeptide (Udagama-Randeniya and Savidge 1995).

For lignin biosynthesis studies we use a tissue culture of Norway spruce that produces extracellular lignin into the culture medium (Simola et al. 1992; Kärkönen and Koutaniemi 2010). The spent culture medium of the extracellular lignin-producing cell culture of Norway spruce contains low coniferyl alcohol oxidase activity, which is less than 1% of the total coniferyl alcohol peroxidase activity in vitro (Kärkönen et al. 2002). Isoelectric focusing (IEF) gels show that the activity is due to a single isoenzyme with an isoelectric point (pI) of 8.4 (Kärkönen et al. 2002). Two oxidase isoenzymes are detected in a protein fraction that is bound to the extracellular lignin (Koutaniemi 2007). Interestingly, the laccase genes are expressed at a markedly lower level in the lignin-forming cultured cells than in developing xylem, whereas some peroxidase genes have a moderate expression level during extracellular lignin formation (Koutaniemi et al. 2007). As scavenging of apoplastic H2O2 significantly decreases the formation of extracellular lignin in this cell culture, peroxidases seem to be essential in activation of monolignols for lignin polymerisation (Kärkönen et al. 2002). Interestingly, dilignols and oligolignols accumulate in both the cells and the culture medium after H2O2 scavenging (T. Laitinen, K. Morreel, N. Delhomme, K. Nickolov, G. Brader, K.J. Lim, N. Street, W. Boerjan, T.H. Teeri, A. Kärkönen, under preparation) suggesting that laccases are involved in the synthesis of soluble, small molecular weight coupling products from coniferyl alcohol.

Norway spruce genome has recently been published (Nystedt et al. 2013). To study the prevalence and the type of laccase genes in Norway spruce, we searched for all laccase genes present in the genome. RNA sequencing data from published databanks and from our own project (unpublished) were utilised in mining for the expressed laccase genes. For five of the identified genes, full-length cDNA sequences were cloned from total RNA isolated from the lignin-forming cell culture and from developing xylem. Furthermore, we purified one laccase from the spent culture medium of the lignin-producing tissue culture, and studied its

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biochemical properties. The properties are discussed in context of the information already known for laccases/CAOs in conifers.

RESULTS

Sequence analysis

Spruce laccase sequences were mined from the Congenie database (http://congenie.org/; Nystedt et al. 2013). Using laccase amino acid sequences from arabidopsis, maize and available gymnosperm species, the Picea abies ver 1.0 (Pabies1.0) genome sequences were searched for sequences encoding similar polypeptides. We found some 50 sequences with 85% or better coverage of the best scoring query sequence, 100 with ca. 50% coverage and altogether 292 models with some coverage at a bit score >50. This represents an upper limit of laccase genes in spruce and includes in addition to pseudogenes and gene fragments, also genes that span several scaffolds (which get counted separately). The gene models of Pabies1.0 contain “laccase” in annotation of 112 gene models, of which 79 are expressed at some level in the transcriptome analysis (Nystedt et al. 2013; ftp://congenie.org/ConGenIE/Nystedt_2013/).

Cloning of spruce laccases

Using total RNA isolated from developing xylem of adult trees and from the lignin-forming cultured cells, five full-length cDNAs for Norway spruce laccases were isolated and sequenced (Table 2; GenBank JX500685-JX500692). As there were slight amino acid variations in some of the sequences probably due to allelic variation, all variants were submitted to GenBank and numbered as PaLAC3a-c and PaLAC5a,b. PaLAC1 and PaLAC2 were isolated from developing xylem, PaLAC4 and PaLAC5 from lignin-forming cultured cells and PaLAC3 from both sources (Table 2). We started numbering of the full-length laccase genes from 1. Laccase EST sequences published in Koutaniemi et al. (2007) have overlapping names, but since the sequences are partial, their match with full-length sequences reported here is ambiguous. The ESTs were submitted to GenBank with their EST code numbers only. Alignment of the full-length laccase sequences and the EST sequences are found in Figure S1. All spruce laccase cDNAs characterised in the current study had highest homology to Pinus taeda laccases PtLAC2 (AF132120) and PtLAC4 (AF132122). They had a putative signal sequence suggesting apoplastic localisation (Table 2). The calculated pIs were cationic (8.8-9.7) for laccases PaLAC2, PaLAC3a-c and PaLAC5a,b, whereas PaLAC1 and PaLAC4 had a neutral pI (7.9 and 7.1, respectively).

PaLAC1-4 are all represented in the Pabies1.0 genome by single gene models that have close to 100% sequence identity to the cloned cDNAs at the nucleotide level, whereas PaLAC5 sequence is split into two non-overlapping gene models. According to the Congenie database, PaLAC1 (MA_208475g0010), PaLAC2

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(MA_8866650g0010), PaLAC3 (MA_67291g0010) and PaLAC4 (MA_10427033g0010) are expressed in wood tissues (phloem, cambium and xylem) and young shoots, PaLAC3 and PaLAC4 additionally in buds and cones. PaLAC5 (MA_97119g0010 + MA_97119g0020) has a low expression level in wood, but is expressed at a higher level in young shoots, buds and cones.

Purification of a laccase from the lignin-forming spruce tissue culture

The culture medium oxidase was purified to homogeneity (Figure 1) using ammonium sulphate precipitation, hydrophobic interaction, lectin affinity, gel filtration and cation exchange chromatography (Table 3). Based on SDS-PAGE, the molecular weight of the culture medium oxidase was ca. 89 kDa (Figure 1). The protein had a bright blue colour. The N-terminal sequence was determined to be EIQTHTFVLQSTSVKKLXGTHN, where X is either C or a glycosylated N. The sequence matches exactly with the N-terminal sequence of PaLAC4 after removal of the signal sequence, with C as the non-determined amino acid. It is also 72% identical to the N-terminal sequence of PtLAC4, where a conserved C exists in the position of X. Based on the sequence homologies and the blue colour, the culture medium oxidase was designated as a laccase.

Kinetic parameters

The optimal pH of the purified culture medium laccase for coniferyl alcohol oxidation was 3.8-4.2, and only little activity was detected above pH 5.5 (Figure 2). The kinetic parametres were determined at pH 4.0 (Table 4). The laccase had a high affinity for coniferyl alcohol with an apparent Km value of 3.5 μM. DISCUSSION

Properties of the laccase from the spruce tissue culture

The purified culture medium laccase had a high affinity towards coniferyl alcohol with an apparent Km value of 3.5 µM; this is lower than those estimated for the culture medium peroxidases (Table 4; Koutaniemi et al. 2005). However, the lowest coniferyl alcohol concentration used for the determination was 5 µM, and therefore, it is possible that the true K m value deviates somewhat from the determined value. The laccase isolated from Populus euramericana xylem has high affinity for monolignols with Km values in the micromolar range (Ranocha et al. 1999), whereas the Km values of the laccase purified from developing xylem of Pinus taeda are in the millimolar range (12±1.3 mM for coniferyl alcohol and 25.4±5.5 mM for sinapyl alcohol; Bao et al. 1993). The Norway spruce laccase characterised in the present study, however, is much less efficient in coniferyl alcohol oxidation than the culture medium peroxidases as shown by the low catalytic efficiency (Vmax/Km) (Table 4). The specific activity (14.2 nkat mg-1) is, nevertheless, considerably higher than that of CAO purified from Pinus strobus (85 pkat mg-1; Udagama-Randeniya and Savidge 1995).

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Although the low Km value indicates that the culture medium laccase is specialised in monolignol oxidation, the low catalytic efficiency makes it unlikely that this laccase has a major role in coniferyl alcohol oxidation in the Norway spruce tissue culture.

Published conifer laccases/laccase-like oxidases have molecular weights of 59-120 kDa, whereas those of CAOs are 107.5 kDa (Table 1). The difference in the observed size (89 kDa, Figure 1) and the calculated size (59 kDa; Table 2) of the protein product encoded by PaLAC4, likely to encode the culture medium laccase based on the N-terminal sequence, suggests that the enzyme is heavily glycosylated. All spruce laccase genes characterised in the present study were predicted to have several N-glycosylation sites (Table 2). Also the observed pI (8.4) of the culture medium laccase differs from the calculated value (7.1). This could be due to glycosylation, as N-glycans of some laccases contain positively charged hexosamines (Fitchette-Lainé et al. 1997; Ranocha et al. 1999).

The optimal pH of the purified culture medium laccase for coniferyl alcohol oxidation was in the acidic range (pH 3.8-4.2). This pH optimum is in the pH range present in the culture medium during lignin formation (pH 4.0-4.6; Kärkönen et al. 2002), but somewhat more acidic than those (pH 5.0-6.0) reported for two Pinus taeda laccases (Sato and Whetten 2006). Interestingly both Pinus taeda laccases catalyse the oxidation of sinapyl alcohol, the non-natural substrate for conifers, more efficiently than their native substrate coniferyl alcohol (Sato and Whetten 2006). Laccase-like oxidases isolated from the compression wood and the opposite (non-compression) wood of Picea sitchensis also prefer sinapyl alcohol over coniferyl alcohol in vitro (McDougall 2000).

Several laccases isolated from Pinus sp. are able to produce dimers and small molecular weight oligomers from coniferyl alcohol in vitro. Higher oligomer formation, however, has been observed only in small amounts (Bao et al. 1993; Sato and Whetten 2006), except with the CAO isolated from Pinus strobus (Savidge and Udagama-Randeniya 1992). The laccase present in the culture medium of Norway spruce purified in the current study is able to produce native-like DHP from coniferyl alcohol in vitro (Kärkönen et al. 2002). Also the extracted protein fraction that contains the lignin-bound oxidase (Koutaniemi 2007) was able to produce high molecular weight DHPs from coniferyl alcohol (data not shown).

Possible role of laccases in lignin formation

In the extracellular lignin-producing Norway spruce tissue culture, peroxidases seem to have a major role in activation of monolignols for lignin formation, as hindering the peroxidase action by scavenging H2O2 reduced the amount of extracellular lignin (Kärkönen et al. 2002). There are, however, numerous laccase genes present in the spruce genome, and several laccases are expressed in developing xylem (Nystedt et al.

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2013), and in the lignin-forming tissue culture during lignin formation (under preparation). It remains to be determined whether Norway spruce laccases participate in lignin biosynthesis in the native situation, i.e., during xylem development. Recent data in Arabidopsis shows that the developmental lignin formation in vascular tissues is mediated by a collaborative action of several laccase isoenzymes (Berthet et al. 2011; Zhao et al. 2013). It is possible that laccases are required, for example, in the initiation of lignin biosynthesis, and peroxidases contribute to lignin formation at later stages (Zhao et al. 2013). In Arabidopsis, knockout mutants of certain cell wall peroxidases contain reduced lignin amount and/or changes in its subunit composition (Herrero et al. 2013; Shigeto et al. 2013); observations that support the idea that both laccases and peroxidases are needed for developmental lignification. There is also evidence for the tissue-specific division of labor between peroxidases and laccases: during lignification of the Casparian strips in root endodermal cells a certain peroxidase has the main role in monolignol activation (Lee et al. 2013). Also the LAC triple mutants that lack lignin in the vascular tissues have normally lignified Casparian strips (Zhao et al. 2013).

Dean et al. (1998) hypothesised that the presence of several laccase isoenzymes with differing redox potentials may be linked to the synthesis of lignins with different monolignol compositions in xylem vessels, and xylem and phloem fibers, respectively. This idea is supported by the observation that different isoenzymes of laccase-like oxidase are predominant in the compression wood and the opposite (noncompression) wood of Picea sitchensis (McDougall 2000). Compression wood develops in the underside of horizontally oriented branches or leaning stems in conifers, and has elevated lignin content with more H units than the normal wood (Timell 1986). Interestingly, protein extracts from compression wood have a lower ability to oxidise coniferyl alcohol in vitro than extracts from the opposite wood (McDougall 2000).

Norway spruce has numerous laccase and peroxidase genes in the genome. Many of them, like the purified laccase isoenzyme, are able to oxidise monolignols and form native-like DHP in vitro. Solving the actual roles of these oxidative enzymes requires genetic evidence, i.e., silencing of the genes of interest and evaluation of the effects. As Norway spruce transformation is laborious and time-consuming, this still awaits to be done; however, successful silencing of the monolignol biosynthetic genes has already been reported (e.g. Wadenbäck et al. 2008). Availability of the genome sequence is a useful resource that enables us to compare coniferous laccase and peroxidase genes with those of angiosperms shown to be involved in lignin biosynthesis; also the co-expression pattern of laccases and peroxidases with other lignin biosynthesis genes will enable us to select the genes of primary interest for further studies.

MATERIAL AND METHODS

In silico analysis of conifer laccases

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Scaffolds, gene models and expression patterns for the Picea abies genome ver 1.0 (Nystedt et al. 2013) were downloaded from http://congenie.org/. Search for additional laccase genes was done with TBLAST search (Altschul et al. 1997) of the scaffolds with a set of gymnosperm and angiosperm laccase amino acid sequences.

Cloning of spruce laccase cDNAs

The tissue culture line A3/85 (Simola et al. 1992) was maintained and propagated on a solid medium and transferred into liquid culture for increased lignin production as described in Kärkönen et al. (2002) and Koutaniemi et al. (2005). Suspension cultures were cultivated on a shaker (100 rpm) at 20°C in an 18 h light / 6 h dark cycle (30-50 µmol m-2 s-1, Osram warm white). After four days in liquid culture the cultured cells were washed briefly with cold water in a Büchner funnel, weighed, frozen in liquid nitrogen and stored at -80˚C. Developing xylem from a mature (ca. 40 years old) Norway spruce tree (clone E8504) was collected in late June at Ruotsinkylä forest area in southern Finland under the permission of the Finnish Forest Research Institute. After felling of a tree the bark was removed and the developing xylem tissues were scraped from the outer surface of the xylem by using knives and razor blades. The developing xylem was frozen immediately in liquid nitrogen and stored at -80˚C.

Isolation of total RNA was done from the tissue-cultured cells and the developing xylem as described in Chang et al. (1993). For RNA extraction the tissues were ground to fine powder in liquid nitrogen in a mortar with pestle. cDNA was synthesised from total RNA by using a dT 29VN primer and M-MLV Reverse Transcriptase (Promega). Partial laccase cDNA sequences were amplified with the polymerase chain reaction (PCR) using Phusion DNA polymerase (Finnzymes) together with the dT 29VN primer and degenerate primers (LacAForward, LacBForward; Table S1) that were designed based on the conserved sequences in Pinus taeda laccases and the laccase unigenes found in the Norway spruce EST collection (Koutaniemi et al. 2007). 5’ Rapid amplification of cDNA ends (5’RACE) was performed with the classic RACE procedure according to Scotto-Lavino et al. (2006) except that Superscript III Reverse Transcriptase (Invitrogen) and Phusion polymerase were used in reverse transcription and PCR, respectively. For some genes that had a high homology to published EST sequences, sequence information from DFCI Spruce Gene Index database (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=spruce, Release 5.0, March 30, 2011) was utilised for designing of the primers. Gene specific forward primers were designed based on the 5’ untranslated regions of the genes and used in 3’RACE with the dT 29VN primer to obtain full-length sequences.

For

primers

see

Table

S1.

Five

full-length

cDNAs

were

recovered.

SIG-Pred

(http://bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html) was used to predict putative N-terminal signal peptides, EMBOSS Pepstats (http://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/) was used to predict the

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properties of the mature peptides and NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) to predict putative N-glycosylation sites.

Purification of a Norway spruce laccase from the tissue culture

The cells were transferred into liquid culture for increased lignin production as described in the previous chapter. The culture medium was collected four days later. From each 4 to 9 L culture, the secreted proteins were isolated from the spent culture medium as described in Koutaniemi et al. (2005). The concentrated culture medium proteins from 38 L of suspension culture were fractionated with 2 M ammonium sulphate and centrifuged. The supernatant was loaded into a Phenyl Sepharose 6 Fast Flow column (high sub., Amersham Biosciences) that was equilibrated with 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.5 supplemented with 2 M ammonium sulphate and 1 mM CaCl2. Proteins were eluted with a liner gradient to the same buffer without ammonium sulphate. The fractions that contained oxidase activity were loaded into a ConA-Sepharose column (Amersham Biosciences), equilibrated with 20 mM MES, pH 6.5, 1 M ammonium sulphate, 1 mM MnCl2, MgCl2 and CaCl2 each. Proteins were eluted with 500 mM methyl-α-mannopyranoside (Fluka) in the same buffer. Eluted proteins were concentrated using an Omega 10K stirred cell system (Pall Life Sciences) and loaded into a Superdex 75 gel filtration column (Amersham Biosciences) equilibrated with 20 mM MES, pH 6.5, 50 mM NaCl, 1 mM CaCl2. The culture medium oxidase activity was finally purified to homogeneity in SP-Sepharose High Performance column (Amersham Biosciences) that was equilibrated with 20 mM Na-acetate, pH 5.0, supplemented with 1 mM CaCl2. Oxidase activity was eluted with a linear gradient to 500 mM NaCl in the same buffer. Phenylmethylsulphonyl fluoride (PMSF) was added during purification to all fractions that contained ABTS-oxidase activity.

Enzyme activity and protein assays

During the purification process, the fractions were screened for oxidase activity at 405 nm in a microtiter plate format using 600 µM 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) in 100 mM Naacetate buffer, pH 5.0. Protein contents were determined with the Bio-Rad microassay procedure (Bradford 1976) using bovine γ-globulin (Bio-Rad) as a standard. The optimum pH and kinetic parameters (K m, Vmax) for coniferyl alcohol oxidation were determined for the purified laccase. For pH optimum the enzyme activity was determined using 100 µM coniferyl alcohol in 50 mM Na-acetate (pH range 3.6 to 6.0) and 50 mM Bis-Tris (pH range 6.5 to 8.0) supplemented with catalase (125 U µL-1; Sigma-Aldrich). The oxidation of coniferyl alcohol was followed at 262 nm. The enzyme assays for K m and Vmax determination were done with 5 to 50 µM coniferyl alcohol in 100 mM Na-acetate, pH 4.0, supplemented with catalase.

Isoelectric focusing and SDS-PAGE

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The purified culture medium laccase was run in an IEF gel as described in Koutaniemi et al. (2005). Approximately 10 pkat of coniferyl alcohol oxidase activity was loaded to the gel, and after the run the gel was stained for activity with 600 µM ABTS in 100 mM Na-acetate buffer, pH 5.0. The molecular weight and the purity of the laccase were estimated with a 10% denaturing SDS-PAGE gel (Laemmli 1970) stained with Coomassie Brilliant Blue.

Protein sequencing

The purified culture medium laccase was separated on an SDS-PAGE gel and blotted on a PVDF membrane for N-terminal sequencing via Edman degradation using Procise 494 A sequencer (Perkin-Elmer Applied Biosystems Division).

ACKNOWLEDGEMENTS We thank Maaret Mustonen for help in the enzyme assays and in subculturing spruce cells. We also thank Md. Sharif Iqbal and Teresa Laitinen for help in cloning PaLAC5. Dr. Nicolas Delhomme, Umeå Plant Science Centre, is thanked for his advice related to Congenie database. This work was supported by University of Helsinki Research Funds (to A.K.), Academy of Finland (grant 251390 to A.K.) and Societas pro Fauna et Flora Fennica (to H.A.M.).

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REFERENCES

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Webb Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402

Bao W, O'Malley DM, Whetten R, Sederoff RR (1993) A laccase associated with lignification in loblolly pine xylem. Science 260: 672-674

Berthet S, Demont-Caulet N, Pollet B, Bidzinski P, Cézard L, Le Bris P, Borrega N, Hervé J, Blondet E, Balzergue S, Lapierre C, Jouanin L (2011) Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell 23: 1124-1137

Blee KA, Choi JV, O'Connell AP, Schuch W, Lewis NG, Bolwell GP (2003) A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification. Phytochemistry 64: 163–176

Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54: 519-546

Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254

Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113-116

Dean JFD, LaFayette PR, Rugh C, Tristram AH, Hoopes JT, Eriksson KEL, Merkle SA (1998) Laccases associated with lignifying vascular tissues, In: Lewis NG, Sarkanen S, eds. Lignin and lignan biosynthesis. ACS Symposium Series 697, Oxford University Press. pp. 96-108

Fagerstedt KV, Kukkola EM, Koistinen VVT, Takahashi J, Marjamaa K (2010) Cell wall lignin is polymerised by class III secretable plant peroxidases in Norway spruce. J Integr Plant Biol 52: 186–194

Fitchette-Lainé AC, Gomord V, Cabanes M, Michalski JC, Saint Macary M, Foucher B, Cavelier B, Hawes C, Lerouge P, Faye L (1997) N-glycans harbouring the Lewis a epitope are expressed at the surface of plant cells. Plant J 12: 1411-1417

12

Freudenberg K (1959) Biosynthesis and constitution of lignin. Nature 183: 1152-1155

Herrero J, Fernández-Pérez F, Yebra T, Novo-Uzal E, Pomar F, Pedreño MÁ, Cuello J, Guéra A, EstebanCarrasco A, Zapata JM (2013) Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis. Planta 237: 1599-1612

Kärkönen A, Koutaniemi S (2010) Lignin biosynthesis studies in plant tissue cultures. J Integr Plant Biol 52: 176-185

Kärkönen A, Koutaniemi S, Mustonen M, Syrjänen K, Brunow G, Kilpeläinen I, Teeri TH, Simola LK (2002) Lignification related enzymes in Picea abies suspension cultures. Physiol Plant 114: 343-353

Koutaniemi S (2007) Lignin biosynthesis in Norway spruce: From a model system to the tree. Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki 28/2007. http://ethesis.helsinki.fi

Koutaniemi S, Toikka MM, Kärkönen A, Mustonen M, Lundell T, Simola LK, Kilpeläinen IA, Teeri TH (2005) Characterization of basic p-coumaryl and coniferyl alcohol oxidizing peroxidases from a ligninforming Picea abies suspension culture. Plant Mol Biol 58: 141-157

Koutaniemi S, Warinowski T, Kärkönen A, Alatalo E, Fossdal CG, Saranpää P, Laakso T, Fagerstedt KV, Simola LK, Paulin L, Rudd S, Teeri TH (2007) Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR. Plant Mol Biol 65: 311-328

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685

Lee Y, Rubio MC, Alassimone J, Geldner N (2013) A mechanism for localized lignin deposition in the endodermis. Cell 153: 402-412

Liang M, Davis E, Gardner D, Cai X, Wu Y (2006) Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta 224: 1185-1196

Lu S, Li Q, Wei H, Chang MJ, Tunlaya-Anukit S, Kim H, Liu J, Song J, Sun YH, Yuan L, Yeh TF, Peszlen I, Ralph J, Sederoff RR, Chiang VL (2013) Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa. Proc Natl Acad Sci USA 110: 10848-10853

13

Marjamaa K, Hildén K, Kukkola E, Lehtonen M, Holkeri H, Haapaniemi P, Koutaniemi S, Teeri TH, Fagerstedt KF, Lundell T (2006) Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies). Plant Mol Biol 61: 719-732

Marjamaa K, Lehtonen M, Lundell T, Toikka M, Saranpää P, Fagerstedt KF (2003) Developmental lignification and seasonal variation in β-glucosidase and peroxidase activities in xylem of Scots pine, Norway spruce and silver birch. Tree Physiol 23: 977-986

McDougall GJ (2000) A comparison of proteins from the developing xylem of compression and noncompression wood of branches of Sitka spruce (Picea sitchensis) reveals a differentially expressed laccase. J Exp Bot 51: 1395-1401

Novo-Uzal E, Fernandez-Perez F, Herrero J, Gutierrez J, Gomes-Ros LV, Bernal MA, Diaz J, Cuello J, Pomar F, Pedreno MA (2013) From Zinnia to Arabidopsis: Approaching the involvement of peroxidases in lignification. J Exp Bot 64: 3499-3518

Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin YC et al. (2013) The Norway spruce genome sequence and conifer genome evolution. Nature 497: 579-584

Ranocha P, Chabannes M, Chamayou S, Danoun S, Jauneau A, Boudet AM, Goffner D (2002) Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar. Plant Physiol 129: 145-155

Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabenes-Macheteau M, Boudet AM, Goffner D (1999). Biochemical characterization, molecular cloning and expression of laccases-a divergent gene family-in poplar. Eur J Biochem 259: 485-495

Richardson A, Stewart D, McDougall GJ (1997) Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis). Planta 203: 35-43

Richardson A, Duncan J, McDougall GJ (2000) Oxidative activity in lignifying xylem of taxonomically diverse range of trees. Tree Physiol 20: 1039-1047

Sato Y, Bao W, Sederoff R, Whetten R (2001) Molecular cloning and expression of eight laccase cDNAs in loblolly pine (Pinus taeda). J Plant Res 114: 147-155

14

Sato Y, Whetten R (2006) Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells. J Plant Res 119: 581-588

Savidge R, Udagama-Randeniya P (1992) Cell wall-bound coniferyl alcohol oxidase associated with lignification in conifers. Phytochemistry 31: 2959-2966

Savidge R, Udagama-Randeniya PV, Xu Y, Leinhos V, Förster H (1998) Coniferyl alcohol oxidase: a new enzyme spatio-temporally associated with lignifying tissues, In: Lewis NG, Sarkanen S, eds. Lignin and lignan biosynthesis. ACS Symposium Series 697, Oxford University Press. pp. 109-130

Schuetz M, Benske A, Smith RA, Watanabe Y, Tobimatsu Y, Ralph J, Demura T, Ellis B, Samuels AL (2014) Laccases direct lignification in the discrete secondary cell wall domains of protoxylem. Plant Physiol 166: 798-807 Scotto-Lavino E, Du G, Frohman MA (2006) 5’ end cDNA amplification using classic RACE. Nature Protocols 1: 2555-2562

Shigeto J, Kiyonaga Y, Fujita K, Kondo R, Tsutsumi Y (2013) Putative cationic cell-wall-bound peroxidase homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, are involved in lignification. J Agric Food Chem 61: 3781-3788

Simola LK, Lemmetyinen J, Santanen A (1992) Lignin release and photomixotrophism in suspension cultures of Picea abies. Physiol Plant 84: 374-379

Sterjiades R, Dean JFD, Gamble G, Himmelsbach DS, Eriksson KEL (1993). Extracellular laccases and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension cultures. Planta 190: 75-87

Sterky F, Regan S, Karlsson J, Hertzberg M, Rohde A, Holmberg A, Amini B, Bhalerao R, Larsson M, Villarroel R, Montagu MV, Sandberg G, Olsson O, Teeri TT, Boerjan W, Gustafsson P, Uhlén M, Sundberg B, Lundeberg J (1998) Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags. Proc Natl Acad Sci USA 95: 13330-13335

Timell TE (1986) Compression wood in gymnosperms. Springer-Verlag, Berlin

15

Udagama-Randeniya PV, Savidge RA (1994) Electrophoretic analysis of coniferyl alcohol oxidase and related laccases. Electrophoresis 15: 1072-1077

Udagama-Randeniya PV, Savidge RA (1995) Coniferyl alcohol oxidase-a catechol oxidase? Trees 10: 102107

Wadenback J, von Arnold S, Egertsdotter U, Walter MH, Grima-Pettenati J, Goffner D, Gellerstedt G, Gullion T, Clapham D (2008) Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR). Transgenic Res 17: 379-392

Zhao Q, Nakashima J, Chen F, Yin Y, Fu C, Yun J, Shao H, Wang X, Wang ZY, Dixon RA (2013) LACCASE is necessary and nonredundant with PEROXIDASE for lignin polymerization during vascular development in Arabidopsis. Plant Cell 25: 3976-3987

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SUPPORTING INFORMATION

Additional supporting information can be found in the online version of this article:

Figure S1. Sequence alignment of the full-length laccases characterised in the present work (PaLac1-5) with the EST sequences of laccases published in Koutaniemi et al. (2007).

Table S1. Primers used to clone five full-length Norway spruce laccase cDNAs.

FIGURE LEGENDS:

Figure 1. Purified Norway spruce culture medium laccase separated in an SDS-PAGE gel stained with Coomassie brilliant blue (A) and in an isoelectric focusing gel stained for ABTS-oxidising activity (B). (A) 0.2, 2 and 10µg protein was applied in the SDS-PAGE gel. Numbers beside the SDS-PAGE gel indicate the sizes of the molecular weight markers (in kDa), (B) numbers above the IEF gel indicate the determined pH gradient in the gel.

Figure 2. The optimum pH of the purified Norway spruce culture medium laccase for coniferyl alcohol oxidation pH optimum was determined by using 100 µM coniferyl alcohol in 50 mM Na-acetate (pH range 3.6 to 6.0) and 50 mM Bis-Tris (pH range 6.5 to 8.0) supplemented with catalase (125 U µL-1). Oxidation of coniferyl alcohol was followed at 262 nm.

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Table 1. Some properties of oxidases associated with lignin formation in conifers. CAO = coniferyl alcohol oxidase, LAC = laccase. In case the enzyme is not characterised, it is marked as a LAC-like oxidase ______________________________________________________________________________________________________________________________ Plant species Enzyme Molecular pI Reference and organs weight (kDa) ______________________________________________________________________________________________________________________________ Picea abies lignin-forming cell culture

LAC in culture medium

89

Picea abies lignin-forming cell culture

LAC-like oxidase unknown bound to extracellular lignin

Picea sitchensis developing xylem in branches

LAC-like oxidase 84 ionically bound to cell wall

Richardson et al. 1997

Picea sitchensis developing xylem in branches

LAC-like oxidase 73 ionically bound to cell wall

Richardson et al. 2000

Picea sitchensis developing xylem in branches

LAC-like oxidase ionically bound to cell wall * in compression wood 120 * in non-compression wood 85

McDougall 2000

Pinus strobus developing xylem in stem

CAO

107.5a 67b 426c

8.4

Kärkönen et al. 2002, this study

~ 9.0

Koutaniemi 2007

7.6

Udagama-Randeniya and Savidge 1994 Savidge et al. 1998

Pinus taeda LAC 90 9.0 Bao et al. 1993 developing xylem in stem ionically bound to cell wall 70a _______________________________________________________________________________________________________________________________ a in SDS-PAGE, b Deglycosylated form in SDS-PAGE, c Native form in the native PAGE

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Table 2. The tissue origin and properties of Norway spruce laccase genes and their protein products characterised in this work.

Gene

Tissue 3’UTR

Coding

Signal

Mature

Molecular

sequence

sequence

peptide

weight, kDa

(nucleotides) (amino acids) (amino acids) (amino acids) (calculated)

Putative pI

N-glycosylation

(calculated)

sites

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PaLAC1

Developing xylem

188

575

1-33

542

59.1

7.9

6

PaLAC2

Developing xylem

277

570

1-28

542

59.5

8.8

9

PaLAC3a-c

Developing xylem, cultured cells

125

570

1-25

545

60.5

8.9

8

PaLAC4

Cultured cells

223

574

1-33

541

59.1

7.1

12

PaLAC5a,b

Cultured cells

365

573

1-26

547

61.5

9.7

8

SIG-Pred (http://bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html) was used to predict putative signal peptides, EMBOSS Pepstats (http://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/) to predict the properties of the mature peptides and NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) to predict putative N-glycosylation sites.

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Table 3. Purification of the culture medium laccase from 38 litres of the spent culture medium of Norway spruce suspension cultures ______________________________________________________________________________________________________________ CAO activity Protein

Specific activity

Purification

Yield

(nkat)

(nkat/mg protein)

fold

(%)

(mg)

______________________________________________________________________________________________________________ Culture medium

61.7

432

0.14

1

100

Ammonium sulphate precipitation

73.7

353

0.21

1.5

119

Hydrophobic interaction

110.0

208

0.53

3.8

178

Lectin affinity

50.9

9.6

5.30

38

82

Gel filtration

56.2

6.0

9.44

67

91

Cation exchange

16.8

1.3

12.9

92

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Oxidation of coniferyl alcohol (CAO) and protein concentrations were measured after each purification step as described in Material and Methods.

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Table 4. Km and Vmax values for coniferyl alcohol oxidation of the purified Norway spruce culture medium laccase ___________________________________________________________________________________ Enzyme Km (μM) Vmax (nkat/mg protein) Vmax/Km ___________________________________________________________________________________ Picea culture laccase

3.5

13.7

3.9

PAPX4

16.7 ± 2.4

2470 ± 138

150 ± 14

PAPX5

23.2 ± 1.9

2290 ± 85

99.0 ± 4.7

Values were determined from activity measurements at pH 4.0 using 5 to 50 μM coniferyl alcohol, and averaged from Lineweaver-Burk, Hanes, Eadie-Hofstee and Woolf plots. For comparison, values for soluble culture medium peroxidases PAPX4 and PAPX5 (Koutaniemi et al. 2005) are tabulated.

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Figure 1

22

Figure 2

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