Physiologia Plantarum 152: 17–31. 2014 © 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317 A model system to study the lignification process in Eucalyptus globulus Pedro Ara ´ ujo a , Igor Cesarino a,b,c , Juliana Lischka Sampaio Mayer a , Ilse Fernanda Ferrari a , Eduardo Kiyota a , Alexandra Christine Helena Frankland Sawaya a , Adriana Franco Paes Leme d and Paulo Mazzafera a,∗ a Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil b Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium c Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium d Laborat ´ orio Nacional de Bioci ˆ encias, CNPEM/ABTLuS, Campinas, Brazil Correspondence *Corresponding author, e-mail: pmazza@unicamp.br Received 1 December 2013 doi:10.1111/ppl.12152 Recalcitrance of plant biomass is closely related to the presence of the phenolic heteropolymer lignin in secondary cell walls, which has a negative effect on forage digestibility, biomass-to-biofuels conversion and chemical pulping. The genus Eucalyptus is the main source of wood for pulp and paper industry. However, when compared to model plants such as Arabidopsis thaliana and poplar, relatively little is known about lignin biosynthesis in Eucalyptus and only a few genes were functionally characterized. An efficient, fast and inexpensive in vitro system was developed to study lignification in Eucalyptus globulus and to evaluate the potential role of candidate genes in this biological process. Seedlings were grown in four different conditions, in the presence or absence of light and with or without sucrose in the growth medium, and several aspects of lignin metabolism were evaluated. Our results showed that light and, to a lesser extent, sucrose induced lignin biosynthesis, which was followed by changes in S/G ratio, lignin oligomers accumulation and gene expression. In addition, higher total peroxidase activity and differential isoperoxidase profile were observed when seedlings were grown in the presence of light and sucrose. Peptide sequencing allowed the identification of differentially expressed peroxidases, which can be considered potential candidate class III peroxidases involved in lignin polymerization in E. globulus. Introduction Lignin is a phenolic heteropolymer deposited in the secondary thickened walls of specialized cells to con- fer mechanical support and strength for the plant to stand upright and hydrophobicity for the xylem Abbreviations – CAPrx, coniferyl alcohol peroxidase; D, dark; DS, dark with sucrose; DTT, dithiothreitol; EST, expressed sequence tag; GPrx, guaiacol peroxidase; IDH, isocitrate dehydrogenase; L, long-day photoperiod; LC-MS, liquid chromatography-mass spectrometry; LS, long-day photoperiod with sucrose; MW, molecular weight; PVPP, polyvinylpolypyrrolidone; RT-PCR, reverse transcription polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; SYRPrx, syringaldazine peroxidase; UPLC-MS/MS, ultrahigh performance liquid chromatography-tandem mass spectrometry. elements to enable long-distance water transport (Weng and Chapple 2010). Moreover, owing to its distinc- tive polymeric structure and consequent recalcitrance to chemical and enzymatic degradation, lignin is an effec- tive defensive barrier against herbivores and pathogens Physiol. Plant. 152, 2014 17