A rice MYB transcription factor, OsMYB58/63, was found to directly upregulate the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 ( OsCesA7 ); in contrast, the Arabidopsis putative orthologs AtMYB58 and AtMYB63 have been shown to specifically activate lignin biosynthesis. Although indirect evidence has shown that grass plants are similar to but partially different from dicotyledonous ones in transcriptional regulation of lignocellulose biosynthesis, little is known about the differences. This study showed that a rice MYB transcription factor, OsMYB58/63, directly upregulated the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 (OsCesA7). Gene co-expression analysis showed that, in rice, OsMYB58/63 and several rice MYB genes were co-expressed with genes encoding lignocellulose biosynthetic enzymes. The expression levels of OsMYB55/61, OsMYB55/61-L, OsMYB58/63, and OsMYB42/85 were commonly found to be high in culm internodes and nodes. All four MYB transcription factors functioned as transcriptional activators in yeast cells. OsMYB58/63 most strongly transactivated the expression of OsCesA7 in rice protoplasts. Moreover, recombinant OsMYB58/63 protein was bound to two distinct cis-regulatory elements, AC-II and SMRE3, in the OsCesA7 promoter. This is in sharp contrast to the role of Arabidopsis orthologs, AtMYB58 and AtMYB63, which had been reported to specifically activate lignin biosynthesis. The promoter analysis revealed that AC elements, which are the binding sites for MYB58 and MYB63, were lacking in cellulose and xylan biosynthetic genes in Arabidopsis, but present in cellulose, xylan, and lignin biosynthetic genes in rice, implying that the difference of transcriptional regulation between rice and Arabidopsis is due to the distinct composition of promoters. Our results provide a new insight into transcriptional regulation in grass lignocellulose biosynthesis.

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five Arabidopsis thaliana (Arabidopsis) and one Oryza sativa (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice (O. sativa L. ssp. japonica cv. Nipponbare). Among them, three Arabidopsis MYBs (AtMYB55, AtMYB61, and AtMYB63) in transgenic rice T 1 lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in AtMYB61-overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of AtMYB61 in rice increased lignin content mainly by enriching syringyl units as well as p-coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as Sorghum, switchgrass, Miscanthus and Erianthus.

Lignin is a major component of the secondary cell walls of vascular plants, and an obstacle in the conversion of plant cell wall polysaccharides into biofuels. Erianthus spp. are large gramineous plants of interest as potential energy sources. However, lignocelluloses of Erianthus spp. have not been chemically characterized. In this study, we analysed lignins, related compounds, enzymatic saccharification efficiencies, and minerals in the ash of the inner and outer parts of the internode, leaf blade and leaf sheath of Erianthus arundinaceus. Lignins in four organs consisted of guaiacyl, syringyl, and p-hydroxyphenyl units. The ratios of syringyl to guaiacyl lignins and lignin contents ranged from 0.43 to 0.79 and 20 to 28%, respectively, with values highest in the outer part of the internode. The amounts of ferulic acid were similar (7.3-11.8 mg g −1 dry weight of cell-wall material) in all four organs, while there was more p-coumaric acid in the inner part of the internode (44.7 mg g −1 dry weight of cell-wall material) than in other organs (25.7-28.8 mg g −1 dry weight of cell-wall material). The enzymatic saccharification efficiency (24 h reaction time) of the leaf blade was 21.6%, while those of the other organs ranged from 10.0 to 15.2%. The leaf blade had the highest ash content (17.1%); the main inorganic element was silicon. This paper provides the first fundamental knowledge of E. arundinaceus lignins.

Biosynthesis of plant secondary cell walls is controlled by several master transcription factors. Ubiquitin ligases, which mediate ubiquitination of proteins, including transcription factors in the protein degradation pathway, are also believed to regulate secondary wall biosynthesis; however, the exact ubiquitin ligases involved in secondary wall formation have not yet been identified. We conducted a gene co-expression network analysis and found that ATL54, annotated as a RING-finger protein, was highly co-expressed with several transcription factor and enzyme genes involved in secondary wall formation. A recombinant ATL54 protein showed ubiquitin ligase activity. The expression of several biosynthetic genes of cellulose, lignin, and xylan in apical portions of inflorescence stems was up-regulated by ATL54 knock-out. The expression of Xylem Cysteine Peptidase1 (XCP1), which participates in the programmed cell death process of xylem tracheary elements, was down-regulated in middle stem portions of both ATL54-knock-out and ATL54-overexpressed mutants. Alteration of ATL54 expression levels did not, however, affect lignin and polysaccharide content and composition in whole mature stems. Our results suggest that ATL54 is an E3 ubiquitin ligase involved in secondary wall biosynthesis and programmed cell death during xylogenesis.