SND1 is a higher order activator expressed in xylem cells that activates the biosynthesis of cellulose, matrix polysaccharides, and lignin (Zhong et al. 2006). Repression of SND1 leads to abnormal Arabidopsis plants lacking vascular and interfascicular fibers; whereas, overexpression lines display ectopic expression of genes involved in secondary wall biosynthesis. Zhao et al. (2010) also found that SND1 directly regulates the expression of F5H, one of the key enzymes in lignin biosynthesis. The genes encoding MYB46, MYB83, MYB103, MYB32, SND3 and KNAT7 possess the Secondary wall NAC Binding Element (SNBE) cis-element and appear to also be direct targets of SND1 (Zhong et al. 2007; Zhong et al. 2011; Zhong et al. 2012).

Other NAC transcription factors, including NST1/2, VND6, and VND7, also play a key role in regulation of secondary cell wall synthesis. They all positively regulate similar downstream targets compared with SND1, including MYB46, MYB83, MYB103, MYB58, and SND3 (Zhong et al. 2010). NST1 and NST2 are involved in regulating secondary wall thickening in anther walls as well as stems (Mitsuda et al. 2007). NST2 especially is strongly expressed in anther tissue. VND6 and VND7 act as a key regulator of xylem differentiation. Overexpression of the VNDs prompts the differentiation of non-vascular tissues into treachery elements (Kubo et al. 2005). VND6 physically binds to the Trachery Element Regulating cis-Element (TERE), which is possessed by a number of genes involved in tissue-specific trachery cell wall biosynthesis and programmed cell death. VND7 is negatively regulated by VNI2 (VND-INTERACTING2 NAC PROTEIN2), which is another recently characterized NAC domain transcription factor (Yamaguchi et al. 2010). The secondary wall regulatory network that functions in xylem differentiation also includes ASL19 (ASSYMETRIC LEAVES2-LIKE19) and ASL20. Expression of these proteins is activated by VND6 and VND7 and also forms a positive feedback loop in turn up-regulating expression of the VND genes (Soyano et al. 2008).

MYB46 and MYB83, which are controlled by NACs, act as positive regulators of secondary cell wall synthesis (Zhong et al. 2012). Among the transcription factors downstream of these MYB proteinss, MYB52, MYB54, MYB58, and MYB63, are important for secondary cell wall synthesis. Promoter deletion coupled with transactivation analysis revealed the Secondary wall MYB Responsive Element (SMRE), a cis-element that is enriched in the promoters of known targets of MYB46 and MYB83. In a further regulatory layer, MYB58 and MYB63, controlled by both NACs and MYB46/83, are implicated in regulating lignin biosynthesis. These proteins target AC-rich elements, which are enriched in the promoters of at least some lignin biosynthesis genes (Lois et al. 1989; Zhong et al. 2012).

More recently a handful of transcriptional regulators in protein families other than NAC and MYB R2R3 have also been determined to have central roles in regulating secondary wall biosynthesis. For example, Wang et al. found that WRKY12 appears to function as a high level negative regulator of secondary cell wall biosynthesis in stems. Medicago and Arabidopsis wrky12 mutants have thickened cell walls in stem pith cells and increased biomass with abnormal deposition of lignin, xylan, and cellulose (Wang et al. 2010). Another example is KNAT7 which is a KNOX-type homeodomain transcription factor that also negatively regulates cell wall thickening and lignin biosynthesis (Li et al. 2011). Loss-of-function knat7 mutants exhibit increased cell wall synthesis gene expression. Besides SND1, MYB75 physically interacts with KNAT7 to restrain cell wall biosynthesis (Bhargava et al. 2010).

A number of secondary wall regulators in grasses have been characterized via heterologous expression in Arabidopsis, but very few have been examined in situ. Heterologous overexpression of ZmMYB31 and ZmMYB42 in Arabidopsis leads to reduced lignin content (Fornale et al.

2010) . Arabidopsis lines that overexpress ZmMYB42 exhibit reduced plant stature, leaf size, tertiary venation, and S-unit lignin content. ZmMYB31 directly interacts with an element similar to the AC-element present in the ZmCOMT promoter (Sonbol et al. 2009). The orthologs of the Arabidopsis SWNs and MYB46 from rice and maize are able to activate secondary wall biosynthesis in Arabidopsis (Zhong et al. 2011). Consistent with conservation of regulatory mechanisms, the promoters of OsMYB46 and ZmMYB46 contain SNBE cis-elements and the rice and maize SWNs directly bind these elements to activate gene expression (Zhong et al. 2011). Another example is expression of an AP2 transcription factor from Arabidopsis, AtSHN2, which in rice significantly enhances cellulose content while reducing lignin content and resulting in improved saccharification yields (Ambavaram et al. 2011). Promoter analysis and binding assays suggest that AtSHN2 may repress expression of the rice orthologs of SND1, NST1/2 and VND6, and activate expression of MYB20 and MYB43.

Dixon and colleagues recently reported that the switchgrass protein, PvMYB4A, an ortholog to the Arabidopsis MYB4 and the maize MYB31 proteins, acts as a repressor of lignin biosynthesis in switchgrass (Shen et al. 2012). Overexpression of PvMYB4 in switchgrass reduced the total lignin content and the amount of cell wall ester-linked p-coumarate. The efficiency of sugar release from transgenic biomass was increased by almost 3-fold. An element similar to the AC-element found in dicots is also the probable binding site of PvMYB4. This discovery demonstrates that manipulating transcription factors that control enzymes that function in cell wall biosynthesis is a good alternative way to reduce the recalcitrance of switchgrass and improve lignocellulosic biomass. Still, the differences in cell wall content between grasses and dicots might be consistent with some divergence in the factors that regulate cell walls. Certainly, given the high ploidy level of switchgrass (4n, 6n, or 8n) and its outcrossing nature, heterozygosity that could have functional consequences has evolved. For example, five distinct, but closely related PvMYB4 sequences were identified from a single switchgrass genotype (Shen et al. 2012; Shen personal communication).

Independent of the possibility of grass-diverged mechanisms of regulation, the continual flow of new publications about cell wall regulators suggests that all factors, and certainly the interactions among them, have yet to be uncovered. Due to space constraints we are not able to elaborate on posttranscriptional and posttranslational regulatory mechanisms in cell wall synthesis, the study of which is still in its infancy (Humphrey et al. 2007; Wolf et al. 2012).