Distinct and Overlapping Functions of Miscanthus sinensis MYB Transcription Factors SCM1 and MYB103 in Lignin Biosynthesis.

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.

The in vivo impact of MsLAC1, a Miscanthus laccase isoform, on lignification and lignin composition contrasts with its in vitro substrate preference.

BACKGROUND: Understanding 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. RESULTS: Using 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. CONCLUSIONS: In 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.

Regulation of secondary cell wall biosynthesis by a NAC transcription factor from Miscanthus.

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 SECONDARY 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 double mutant, and ectopic expression of MsSND1 in tobacco leaves was sufficient to trigger patterned deposition of cellulose, hemicellulose, and lignin reminiscent of xylem elements. Transgenic studies in Arabidopsis thaliana plants revealed that MsSND1 regulates, directly and indirectly, the expression of a broad range of genes involved in secondary cell wall formation, including MYB transcription factors which regulate only a subset of the SCW differentiation program. Together, our findings suggest that MsSND1 is a transcriptional master regulator orchestrating secondary cell wall biosynthesis in Miscanthus.

Botrytis cinerea can import and utilize nucleosides in salvage and catabolism and BcENT functions as high affinity nucleoside transporter.

Nucleotide de novo synthesis is an essential pathway in nearly all organisms. Transport processes as well as salvage and catabolism of nucleotides and pathway intermediates are required to balance nucleotide pools. We have analysed the genome of the fungal plant pathogen Botrytis cinerea for genes involved in nucleotide metabolism and found a complete set of genes necessary for purine and pyrimidine uptake and salvage based on homology of the gene products to corresponding proteins from Aspergillus nidulans. Candidate genes required for a complete purine catabolic sequence were identified in addition. These analyses were complemented by growth tests showing functional transport and salvage activity for pyrimidines. Growth of B. cinerea mycelium in nitrogen free medium could be restored by addition of purines, indicating the presence of a functional purine catabolism, whereas pyrimidines did not support growth. Bcin07g05490 (BcENT) was identified as sole member of the equilibrative nucleoside transporter (ENT) family. The protein synthesized in Saccharomyces cerevisiae revealed high affinity transport of adenosine (KM = 6.81 µM) and uridine (KM=9.04 µM). Furthermore, a BcENT knockout mutant was generated and tested in a range of growth and infection assays. These results provide detailed insight in the use of externally supplied nucleobases and nucleosides by B. cinerea.