Cell wall recalcitrance is a major limitation for the sustainable exploitation of lignocellulosic biomass as a renewable resource. Species and hybrids of the genus Miscanthus have emerged as candidate crops for the production of lignocellulosic feedstock in temperate climates, and dedicated efforts are underway to improve biomass yield. However, nothing is known about the molecular players involved in Miscanthus cell wall biosynthesis to facilitate breeding efforts towards tailored biomass. Here, we identify a Miscanthus sinensis transcription factor related to SEC-ONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1), which acts as a master switch for the regulation of secondary cell wall formation and lignin biosynthesis.MsSND1 is expressed in growth stages associated with secondary cell wall formation, together with its potential targets. Consistent with this observation, MsSND1 was able to complement the secondary cell wall defects of the Arabidopsis snd1 nst1

BackgroundUnderstanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo.ResultsUsing a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4–2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate.ConclusionsIn summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.

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38 Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as 39 renewable resource for energy and bio-based products. Transcriptional regulators of the lignin 40 biosynthetic pathway represent promising targets for tailoring lignin content and composition in 41 plant secondary cell walls. A wealth of research in model organisms has revealed that 42 transcriptional regulation of secondary cell wall formation is orchestrated by a hierarchical 43 transcription factor (TF) network with NAC TFs as master regulators and MYB factors in the lower 44 tier regulators. However, knowledge about the transcriptional regulation of lignin biosynthesis in 45 lignocellulosic feedstocks, such as Miscanthus, is limited. Here, we characterized two Miscanthus 46 MYB TFs, MsSCM1 and MsMYB103, and compared their transcriptional impact with that of the 47 master regulator MsSND1. In Miscanthus leaves MsSCM1 and MsMYB103 are expressed at 48 growth stages associated with lignification. Ectopic expression of MsSCM1 and MsMYB103 in 49 tobacco leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin 50 composition. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 51 and MsMYB103 overexpression showed extensive overlap with the response to MsSND1, but 52 were distinct from each other, underscoring the inherent complexity of secondary cell wall 53 formation. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of 54 lignin content and composition in Miscanthus towards tailored biomass. 55 56 157 158 Tissue Staining and Microscopy 159

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

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