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Switchgrass is a C4 perennial forage grass native to most areas of the North American grasslands. Since switchgrass has high biomass yield and is well adapted to marginal lands, it has been selected as a dedicated lignocellulosic feedstock for bioenergy production in the United States (McLaughlin & Adams Kszos, 2005; Bouton, 2007; Schmer et al., 2008). The biomass yield of switchgrass varies according to precipitation during the growing season, annual temperature, nitrogen fertilization, and the type of cultivar (Fuentes & Taliaferro, 2002; Wullschleger et al., 2010). The estimated annual biomass yield of switchgrass in the United States is projected to average 12.9 metric tons per hectare for lowland ecotypes and 8.7 metric tons for upland ecotypes (Wullschleger et al., 2010).
The major barrier to efficient conversion of lignocellulose to liquid transportation fuels is the recalcitrant nature of the cell wall. The cellulose microfibrils are embedded in a matrix of hemicelluloses that are covalently linked with lignin and other aromatic phenolic compounds. These linkages, and the masking effect of lignin itself, increase the pretreatment costs and block the access of enzymes to the polysaccharide chains (Akin, 2007; Himmel et al., 2007; Pauly and Keegstra, 2008). Reduction of lignin content through transgenic approaches improves fermentable sugar yield and saccharification efficiency of alfalfa stems (Chen & Dixon, 2007; Jackson et al., 2008), and chemical analysis of field-grown switchgrass stems indicated that the saccharification efficiency is negatively correlated with lignin content and ester-linked p-CA : FA ratio (Shen et al., 2009a). Therefore, genetic modification of lignin and cell wall phenolic ester synthesis in switchgrass would be predicted to serve as an efficient method for reducing recalcitrance and thus improving bioenergy production efficiency (Hisano et al., 2009; Keshwani & Cheng, 2009).
Lignin is formed by the oxidative polymerization of three monolignols, p-coumaryl, coniferyl and sinapyl alcohols, to give the hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units of the mature lignin polymer. Although the monolignol biosynthetic pathway is now well understood (Supporting Information, Fig. S1), and a picture is beginning to emerge of its complex regulatory architecture, to date only a few of the lignin biosynthetic and regulatory genes from switchgrass have been functionally characterized or annotated by comparative bioinformatics analysis. This situation will soon be changed with the ongoing efforts in switchgrass genetics and genomics (Okada et al., 2010).
Common regulatory cis-elements have been identified within the promoters of the genes in the lignin biosynthesis pathway (Hatton et al., 1995; Raes et al., 2003; Legay et al., 2007). A detailed analysis of the bean phenylalanine ammonia-lyase 2 (PAL2) promoter identified three AC elements together with a G-box involved in xylem-specific expression (Hatton et al., 1995). Mutation of the AC-I (ACCTACC), AC-II (ACCAACC) and AC-III (ACCTAAC) elements resulted in a decrease in xylem-associated expression (Hatton et al., 1995). Bioinformatic analysis of the promoters of all lignin biosynthetic genes in Arabidopsis thaliana indicated that such AC elements are present in the majority of the genes except for the promoters of ferulate 5-hydroxylase (F5H) and caffeic acid 3-O-methyltransferase (COMT) (Raes et al., 2003). However, examination of 2 kb of a maize COMT gene promoter region identified a putative ACIII box (aACCTAAC) 200 bp upstream of the transcription start site (Fornaléet al., 2006).
The lignin biosynthesis pathway is coregulated with the secondary cell wall biosynthesis program through a master switch system which includes a group of NAC and R2R3-MYB transcription factors (TFs) (Mitsuda et al., 2007; Zhong et al., 2010). The Arabidopsis genome contains at least 114 NAC genes (Olsen et al., 2005; Shen et al., 2009b) and 126 members of the R2R3-MYB family (Romero et al., 1998; Stracke et al., 2001). The R2R3-MYB family members are classified into at least 22 subfamilies based on their conserved C-terminal motifs (Romero et al., 1998; Stracke et al., 2001). A group of R2R3-MYB transcriptional activators have been shown to bind directly to AC elements in vitro. These activators include PtMYB1 and PtMYB4 from pine (Patzlaff et al., 2003; Bomal et al., 2008), EgMYB2 from Eucalyptus (Goicoechea et al., 2005), VvMYB5a from Vitis vinifera (Deluc et al., 2006), NtMYBJS1 from tobacco (Gális et al., 2006), PtrMYB21a from poplar (Karpinska et al., 2004; Bylesjo et al., 2008) and AtMYB61, AtMYB68 and AtMYB63 from Arabidopsis (Zhou et al., 2009; Wang et al., 2010). It has been suggested that AtMYB68, AtMYB63 and their close homolog PtrMYB28 from poplar are lignin-specific TFs, whereas PtMYB4 and EgMYB2 also regulate the biosynthesis of cellulose and xylan and therefore serve as secondary cell wall master switches (Zhong et al., 2010).
By contrast, members from subfamily 4 of the R2R3-MYB family have been shown to act as transcriptional repressors of monolignol biosynthetic genes. This was first demonstrated for AmMYB308 and AmMYB330 from Antirrhinum majus (Tamagnone et al., 1998). Overexpression of AmMYB308 and AmMYB330 in tobacco causes reduced plant growth and a white lesion phenotype on leaves (Tamagnone et al., 1998). Similar phenotypes were observed on overexpression of AtMYB4 in Arabidopsis (Jin et al., 2000). AtMYB32 (Preston et al., 2004), a close homolog of AtMYB4, Eucalyptus gunnii EgMYB1 (Legay et al., 2007) and maize ZmMYB31 and ZmMYB42 have also been identified as lignin repressors, and ZmMYB31 was proposed to be a good candidate for biotechnological applications (Fornaléet al., 2006, 2010; Sonbol et al., 2009). However, no genetic manipulation of lignin repressors in monocot plants has yet been reported.
In the present study, we have identified and characterized the lignin repressor PvMYB4 from switchgrass. Effects of overexpression of PvMYB4 in transgenic tobacco and switchgrass suggest that the gene is functionally orthologous to AtMYB4 and ZmMYB31. PvMYB4 binds directly to AC-I (preferred), AC-II and AC-III elements, both in vitro and in a yeast transcription system. Lignin biosynthetic genes are significantly down-regulated in PvMYB4-overexpressing plants, associated with reductions in lignin content and ester-linked p-CA : FA ratio. Overexpression of PvMYB4 in transgenic switchgrass increases saccharification efficiency threefold.
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Fig. S1 The phenylpropanoid and monolignol biosynthesis pathways.
Fig. S2PvMYB4 gene variants in switchgrass.
Fig. S3 PtMYB4 binds to AC elements in a yeast transcription system.
Fig. S4 The PQ-rich motif does not possess transcriptional activation activity.
Fig. S5 Overexpression of PvMYB4 alters phenylpropanoid metabolism in transgenic tobacco plants.
Fig. S6 Genomic DNA PCR and qRT-PCR analysis of PvMYB4-OX transgenic switchgrass.
Fig. S7 The S : G ratio of PvMYB4-OX transgenic switchgrass.
Fig. S8 PvMYB4-OX transgenic switchgrass has smaller vascular bundles and thinner tillers.
Fig. S9 Estimation of total dry biomass yield for PvMYB4-OX transgenic switchgrass.
Fig. S10 qRT-PCR analysis of flavonoid biosynthetic gene transcripts in PvMYB4-OX transgenic tobacco.
Table S1 Sequences and references for the gene-specific primers used in this work
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