Metabolite Profiling of Sugarcane Genotypes and Identification of Flavonoid Glycosides and Phenolic Acids Isabel D. Coutinho,* ,† John M. Baker, § Jane L. Ward, § Michael H. Beale, § Silvana Creste, # and Alberto J. Cavalheiro † † Instituto de Quı ́ mica, Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP), Rua Prof. Francisco Degni 55, CEP 14800-060 Araraquara, Sã o Paulo, Brazil § Plant Biology and Crop Science Department, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom # Instituto Agronô mico - Centro de Cana, Rodovia Antonio Duarte Nogueira Km 321, CP 206, CEP 14032-800 Ribeirã o Preto, Brazil * S Supporting Information ABSTRACT: Sugarcane is an important agricultural crop in the economy of tropical regions, and Brazil has the largest cultivated acreage in the world. Sugarcane accumulates high levels of sucrose in its stalks. Other compounds produced by sugarcane are currently not of economic importance. To explore potential coproducts, we have studied the chemical diversity of sugarcane genotypes, via metabolite profiling of leaves by NMR and LC-DAD-MS. Metabolites were identified via in-house and public databases. From the analysis of 60 HPLC-fractionated extracts, LC-DAD-MS detected 144 metabolites, of which 56 were identified (MS-MS and 1 H NMR), including 19 phenolics and 25 flavones, with a predominance of isomeric flavone C-glycosides. Multivariate analysis of the profiles from genotypes utilized in Brazilian breeding programs revealed clustering according to sugar, phenolic acid, and flavone contents. KEYWORDS: sugarcane, amino acids, phenolic acid, flavones, metabolite profiling ■ INTRODUCTION The Saccharum genus and Sclerostachya, Narenga, Erianthus, and Miscanthus genera constitute a closely related interbreeding group known as the “Saccharum complex”. 1 With the exception of sugarcane, most genera belonging to this group generally have low sugar content and differing stalk morphology. 2 Saccharum has unique physiology and produces extremely high biomass yields while also accumulating high concentrations (>600 mM) of sucrose in its culm. 3 Modern sugarcane varieties are derived mainly from interspecific crosses between Saccharum officinarum L. and Saccharum spontaneum. 4,5 Hybridizations emerged as a solution to losses caused by diseases and the need to obtain more resistant plants. 5 Beyond being an established source of sugar, sugarcane is the current benchmark first-generation feedstock for efficient biofuel production, as well as animal feed, sugarcane spirit, sugarcane syrup, and other products. The first reports concerning secondary metabolites from sugarcane leaf extracts were published in the 1950s. 6,7 Later, Williams et al. carried out taxonomic character- ization of genera from Saccharum complex leading to the identification of eight flavone C- and O-glycosides. 8 In the past 10 years, further studies have reported the presence of flavones in Saccharum. 9-16 In addition, phenolic compounds and triterpenes have also been described in sugarcane leaves. 17-20 Despite the key role of sugarcane in the economy of Brazil, there have been few comprehensive studies of the metabolite and genetic diversity of the genotypes developed by Brazilian breeding programs (IAC-IACSP, SP, and RB). The genetic variability of modern varieties has been investigated by target region amplification polymorphism (TRAP), 21 whereas tentative metabotype discrimination among RB varieties has been proposed by 1 H NMR in solution and in solid matrices by high-resolution magic angle spinning (HR-MAS), 22 but this study focused on the content of primary metabolites, mainly sucrose. In this paper, we provide a phytochemical catalog of sugarcane leaves derived from metabolite screening of sugarcane genotypes. The applicability of identified metabolites is demonstrated in a study of metabolomic profiling of 13 sugarcane genotypes by 1 H NMR and LC-DAD to provide metabolite markers for future breeding programs. ■ MATERIALS AND METHODS Plant Material. Sugarcane genotypes (RB966928, IACSP955000, IACSP933046, IACSP974039, SP803280, RB92579, RB835486, IAC912218, IAC911099, IACSP962042, IACSP977569, RB867515, and CB49260) belong to different Brazilian breeding programs: IAC (Instituto Agronô mico de Campinas), SP (Copersucar), RB (Repú blica do Brasil), and CB (Campos Brasil) were cultivated in a greenhouse in Ribeirã o Preto, SP, Brazil (21°11′ S, 47°48′ W) in 50 L pots containing a 3:1:1 mixture of soil, sand, and pine and coconut bark substrate (Tropstrato) and fertilized according to the method of Van Raij et al. 23 Leaf samples (leaf +1) of first-cut plants were collected between 8:30 and 9:00 a.m. when plants were 9 months old and used for metabolomics screening. For phytochemical analysis, leaves of seedling IAC955000 were collected at 2 months of age. After harvest, plant material was immediately frozen under liquid nitrogen and stored at -80 °C. Prior to extraction, the samples were lyophilized and milled in a cryogenic mill using a first step for sample freezing (1 min) followed by Received: March 15, 2016 Revised: May 4, 2016 Accepted: May 6, 2016 Published: May 6, 2016 Article pubs.acs.org/JAFC © 2016 American Chemical Society 4198 DOI: 10.1021/acs.jafc.6b01210 J. Agric. Food Chem. 2016, 64, 4198-4206