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Abstract

For implementation of a sustainable bioeconomy, biorefineries will play a crucial role in converting different biomasses into various platform molecules. For biorefineries using lignocellulose biomass, Miscanthus is one of the ideal sources because of high-yield potential, low-input requirements, and high energy outcome ratios. However, with respect to the lignin component, the demand for high-value products from isolated lignin requires lignin feedstocks with unique properties. Therefore, a better understanding of lignification and monolignol biosynthesis is mandatory. For several model plants (Arabidopsis, rice, poplar), the lignin biosynthesis pathway has been elucidated in extensive detail. In particular, the transcriptional regulatory network of lignin biosynthesis as well as of secondary cell wall formation has attracted attention of worldwide research. However, how transcriptional repressors are involved in regulating lignin biosynthesis in Miscanthus has remained largely unknown. In this study, two R2R3-MYB transcription repressors, MsMYB31 and MsMYB42 were identified from Miscanthus sinensis. Sequence and expression analysis revealed their close structural relationship with AtMYB4, ZmMYB31 and ZmMYB42, transcription factors which have been identified as negative regulators of lignin biosynthesis and the phenylpropanoid pathway. Further characterization of both repressors has been performed via subcellular localization and functional analysis, e.g. via dual-luciferase-assays (DLA) and electrophoretic mobility shift assays (EMSA) to confirm their mode of action and specificity of binding to certain cis-elements in target gene promoters, i.e. MsC4H, MsCCR and MsCAD. Inducible expression of MsMYB31 or MsMYB42 in wild type Arabidopsis Col-0 further confirmed repression of phenylpropanoid metabolic pathway by both repressor proteins. Additionally, transforming the Arabidopsis myb4 mutant with MsMYB31 or MsMYB42 under control of the AtMYB4 promoter revealed that both repressors do not complement the function of AtMYB4, indicating similar but different mechanisms of these repressors. Finally, while showing target redundancy, the differential developmental expression patterns of MsMYB31 and MsMYB42 indicate specific regulatory functions during lignification in planta. Possible physiological functions of both repressors are discussed.