Boerjan, Wout

Bulone, Vincent

De Rycke, Riet

Hertzberg, Magnus

Kim, Hoon

Lu, Fachuang

McKee, Lauren

Morreel, Kris

Niculaes, Claudiu

Polle, Andrea

Ralph, John

Vanholme, Bartel

English

Ghent University Academic Bibliography

See	discussions,	stats,	and	author	profiles	for	this	publication	at:https://www.researchgate.net/publication/301198101Proceedings	of	the	IUFROTree	Biotechnology	2015Conference:	"Forests:	theimportance	to	the	planet	andsociety"Research	·	April	2016DOI:	10.13140/RG.2.1.4603.68824	authors,	including:Cristina	VettoriItalian	National	Research	…Available	from:	Cristina	VettoriRetrieved	on:	11	April	2016IUFRO Tree Biotechnology 2015 Conference“Forests: the importance to the planet and society”8-12 June 2015Florence, ItalyProceedings3Cristina VettoriInstitute of Bioscience and BioResoucesNational Research CouncilSesto Fiorentino (FI)ItalyGiovanni Giuseppe VendraminInstitute of Bioscience and BioResoucesNational Research CouncilSesto Fiorentino (FI)ItalyDonatella PaffettiDepartment of Agriculture, Food and Environmental SciencesUniversity of FlorenceFlorenceItalyDavide TravagliniDepartment of Agricultural, Food and Forestry SystemsUniversity of FlorenceFlorenceItalyEditorsCite these articles as in this example:Vettori et al. 2015. Gene expression profiling in response to UV-B radiation in different Populus alba clones. In Proceedings of the IUFRO Tree Biotechnology 2015 Conference: “Forests: the importance to the planet and society”. Eds: Vettori C, Vendramin G.G., Paffetti D., Travaglini D. - doi: 10.13140/RG.2.1.4603.6882; ID: S1.P2DOI 10.13140/RG.2.1.4603.6882 2Discovery of the biological role of PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE (PCBER), one of the most abundant proteins in poplar xylem Claudiu Niculaesa,b,c, Kris Morreela,b, Hoon Kimd, Fachuang Lud, Lauren McKeee, Bartel Vanholmea,b, Riet De Ryckea,b, Magnus Hertzbergf, Vincent Bulonee, Andrea Polleg, John Ralphd and Wout Boerjana,b a Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium. b Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium. c Current address: Lehrstuhl für Genetik, Wissenschaftszentrum Weihenstephan, Technische Universität München, 85354 Freising, Germany d Department of Biochemistry and the DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA. e Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden f  SweTree Technologies AB, P.O Box 4095,  SE-904 03 Umeå, Sweden g Forstbotanik und Baumphysiologie, Büsgen-Institut, Georg-August-Universität Göttingen, Göttingen, Germany Claudiu Niculaes niculaes@wzw.tum.de Kris Morreel krmor@psb.vib-ugent.be Hoon Kim hoonkim@wisc.edu Fachuang Lu fachuanglu@wisc.edu Lauren McKee mckee@kth.se Bartel vanholme bahol@psb.vib-ugent.be Riet De Rycke riryc@ugent.be Magnus Hertzberg Magnus.Hertzberg@SweTree.com Vincent Bulone bulone@kth.se Andrea Polle  apolle@gwdg.de  John Ralp Jralph@wisc.edu Wout Boerjan woboe@psb.vib-ugent.be Proceedings of the IUFRO Tree Biotechnology 2015 ConferenceFlorence, Italy – 8/12 June, 2015Oral Communication Abstract – ID: S3.O2DOI 10.13140/RG.2.1.4603.6882      ____________________________________________________________________________117Background Wood is the most abundant renewable natural material on earth and a prime source for timber, pulp and paper. In addition to these traditional sectors, wood is increasingly considered as an alternative for the production of second generation biofuels such as  bioethanol. Given the worldwide importance of wood, it is striking that the biological role of one of the most prominent proteins in poplar xylem, Phenylcoumaran Benzylic Ether Reductase (PCBER), has remained obscure. PCBER was discovered 15 years ago as one of the most abundant proteins in 2-dimensional gels made from poplar xylem (Vander Mijnsbrugge 2000a; 2000b). PCBER belongs to a large class of NADPH dependent oxido-reductases. Davin et al (1999) showed that both the poplar and the pine PCBER can reduce dehydrodiconiferyl alchol (DDC) in vitro. DDC is a dimer of coniferyl alcohol, one of the main lignin monomers. Given that PCBER is a cytoplasmic protein and that monolignol coupling and further polymerization to lignin are believed to occur in the cell wall, the question raised what the in vivo substrate of PCBER could be, why PCBER is so abundant, and what its biological role is.   Material and methods Transgenic poplars (Populus tremula x P. tremuloides) were made that were downregulated for PCBER by an RNAi approach. Young developing xylem was scraped (~0.5 cm deep) with a scalpel while frozen, from 40 cm long debarked stems harvested from the greenhouse grown poplars. The material was then ground to a fine powder in a mortar cooled with liquid nitrogen, and immediately processed or stored at -70 °C. Immunodetection was according to standard procedures using a polyclonal antibody raised against recombinant poplar PCBER (Vander Mijnsbrugge et al., 2000b). Phenolic profiling was done according to Niculaes et al. (2014). PCBER enzymes used for enzyme activity tests were heterologous produced by PSF (http://www.vib.be/en/research/services/Protein-production-purification-and-analysis/Pages/default.aspx). Results and conclusion Transgenic poplars downregulated for PCBER by an RNAi approach were first analyzed by Western blotting, which revealed the strong downregulation of PCBER protein abundance. Lignin amount and composition, as analyzed by acetylbromide, thioacidolysis and NMR, and cellulose amount, were similar between transgenic and wild type trees, indicating that PCBER had no major role in cell wall biosynthesis. Comparative metabolite profiling of the transgenic poplars downregulated for PCBER revealed the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8–5-coupling product between sinapyl alcohol and guaiacylglycerol and the product as its benzyl-reduced form (Figure 1). This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These 118compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. From these data we deduce that apparently, some of the monolignols that are biosynthesized in the cytoplasm are oxidized to radicals in the cytoplasm, upon which they spontaneously couple to form dimers (and higher order oligolignols). The coumaran type of dimers are substrates for PCBER and are reduced to molecules that become themselves radical scavengers. In the absence of PCBER, the cell makes less of the reduced dimers (thus less scavengers) and the cytoplasm accumulates products derived from the increased oxidative conditions in the cytoplasm. Thus, the data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against the oxidative damage associated with lignifying cells. In addition to unveiling the catalytic activity of one of the most abundant enzymes in wood, our data provide the first experimental evidence for an in planta antioxidant role of a monolignol coupling product.  Figure 1. In vivo substrate and product of PCBER  Acknowledgments JR, FL, and HK were funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). We also acknowledge the Hercules program of Ghent University for the Synapt Q-Tof (grant AUGE/014); the Bijzonder Onderzoeksfonds-Zware Apparatuur of Ghent University for the Fourier transform ion cyclotron resonance mass spectrometer (174PZA05); and the Multidisciplinary Research Partnership Biotechnology for a Sustainable Economy (01MRB510W) of Ghent University. CN was funded by Flanders Research Foundation (FWO) grant number G.0637.07N and by the European collaborative project ENERGYPOPLAR (FP7-211917). We thank Catherine Lapierre and Brigitte Pollet (INRA-Orléans) for pilot wood chemistry analyses and Andras Gorzsas for FTIR analysis of wood samples. References 119Gang D.R., Kasahara H., Xia Z.-Q., Vander Mijnsbrugge K., Boerjan W., Van Montagu M., Davin L.B. and Lewis N.G. (1999). Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductase. J. Biol. Chem. 274, 7516-7527. Niculaes C., Morreel K., Kim H., Lu F., McKee L., Ivens B., Haustraete J., Vanholme B., De Rycke R., Hertzberg M., Fromm J., Bulone V., Polle A., Ralph J. and Boerjan W. (2014). Phenylcoumaran benzylic ether reductase (PCBER) prevents accumulation of compounds formed under oxidative conditions in poplar xylem. Plant Cell 26, 26(9):3775-91 Vander Mijnsbrugge K., Meyermans H., Van Montagu M., Bauw G. and Boerjan W. (2000a). Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins. Planta 210, 589-598. Vander Mijnsbrugge K., Beeckman H., De Rycke R., Van Montagu M., Engler G. and Boerjan W. (2000b). Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells. Planta 211, 502-509.  120

Discovery of the biological role of PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE (PCBER), one of the most abundant proteins in poplar xylem

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Discovery of the biological role of PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE (PCBER), one of the most abundant proteins in poplar xylem

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