Sugarcane Cell Wall Structure and Lignin Distribution Investigated by Confocal and Electron Microscopy CELSO SANT’ANNA, 1,2 * LILIAN T. COSTA, 1,3 YURI ABUD, 1,2 LUCAS BIANCATTO, 1,2 FL  AVIO COSTA MIGUENS, 4 AND WANDERLEY DE SOUZA 1,2,5 1 Laborat orio de Biologia Estrutural, Diretoria de Programa, Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO, RJ, Brazil 2 Instituto Nacional de Ci^ encia e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, UFRJ, RJ, Brazil 3 Polo de Xer em, Universidade Federal do Rio de Janeiro, UFRJ, RJ, Brazil 4 Laborat orio de Biologia Celular e Tecidual, Universidade Estadual Norte Fluminense Darcy Ribeiro, UENF, RJ, Brazil 5 Laborat orio de Ultraestrutura Celular Hertha Meyer, Instituto de Biof  ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, UFRJ, RJ, Brazil KEY WORDS sugarcane; plant cell wall; biomass; lignin; cellulose ABSTRACT Lignocellulosic plant cell wall is considered a potential source for second genera- tion biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a pre- requisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron mi- croscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and sur- rounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lig- nin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO 4 cytochemistry revealed that lignin was predominantly dis- tributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production. Microsc. Res. Tech. 76:829–834, 2013. V C 2013 Wiley Periodicals, Inc. INTRODUCTION Sugarcane is a perennial monocotyledon that origi- nated in Asia and is characterized by segmented stems, blade-like leaves, and reproduction by seed (Khanna et al., 1950). The six varieties (S. spontaneum, S. robus- tus, S. officinarum, S. barberi, S. sinense, and S. edule) of sugarcane belong to the genus Saccharum (Arceneaux, 1967). Sugarcane monoculture is found in tropical and subtropical countries and produces approxi- mately 1.6 billion tons annually (Chandel et al., 2012). A remarkable feature of this plant is its high concentration of sucrose, approximately 0.7 M (Moore, 1995), which is favorable for bioethanol production. Sugarcane is gener- ally used for sugar production, generating approximately two-thirds of the world’s supply of sugar. Ethanol derived from sugarcane represents a renewable source of biofuel with low cost and low pollutant emission (Lakshmanan et al., 2005). According to the World Trade Organization [www.wto.org], sugar is an agricultural commodity; etha- nol is an energy commodity; and sugarcane is an impor- tant global source of plant-based energy. The plant cell wall is a dynamic and highly complex 3D matrix that is mainly composed of polysaccharides (cellulose, hemicelluloses, and pectins), lignin, and gly- coproteins. Although the composition of the plant cell wall is well known, the organization of different mole- cules within the structure has not yet been fully eluci- dated. The mechanical and functional properties of the cell wall are thought to rely on its molecular architec- ture, which is related to cell growth, morphogenesis, and the recalcitrance of biomass to deconstruction (Lee et al. 2011). The plant cell wall is composed of three major layers: (i) the middle lamella (ML), which surrounds each cell and is responsible for adhesion to the neighboring *Correspondence to: Celso Sant’anna, Instituto Nacional de Metrologia, Quali- dade e Tecnologia- Inmetro, Diretoria de Programa – DIPRO, Laborat orio de Biotecnologia – LABIO, Av. Nossa Senhora das Grac ¸as, 50, pr edio 27- Xer em, CEP: 25250-020, Duque de Caxias, RJ, Brazil. E-mail: cbfilho@inmetro.gov.br Received 18 February 2013; accepted in revised form 1 May 2013 Contract grant sponsor: Centro de Pesquisas da Petrobras/Cenpes, Minist erio de Ci^ encia e Tecnologia/Financiadora de Estudos e Projetos (Finep), Conselho Nacional de Desenvolvimento Cient  ıfico e Tecnol ogico (CNPq), Fundac ¸~ ao Carlos Chagas Filho de Amparo  a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸~ ao de Aperfeic ¸oamento de Pessoal de N ıvel Superior (CAPES) Brazilian programs. DOI 10.1002/jemt.22235 Published online 3 June 2013 in Wiley Online Library (wileyonlinelibrary.com). V V C 2013 WILEY PERIODICALS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 76:829–834 (2013)