The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis

Abstract

Background

The secondary cell wall is a defining feature of xylem cells and allows them to resist both gravitational forces and the tension forces associated with the transpirational pull on their internal columns of water. Secondary walls also constitute the majority of plant biomass. Formation of secondary walls requires co-ordinated transcriptional regulation of the genes involved in the biosynthesis of cellulose, hemicellulose and lignin. This co-ordinated control appears to involve a multifaceted and multilayered transcriptional regulatory programme.

Scope

Transcription factor MYB46 (At5g12870) has been shown to function as a master regulator in secondary wall formation in Arabidopsis thaliana. Recent studies show that MYB46 not only regulates the transcription factors but also the biosynthesis genes for all of the three major components (i.e. cellulose, hemicellulose and lignin) of secondary walls. This review considers our current understanding of the MYB46-mediated transcriptional regulatory network, including upstream regulators, downstream targets and negative regulators of MYB46.

Conclusions and Outlook

MYB46 is a unique transcription factor in that it directly regulates the biosynthesis genes for all of the three major components of the secondary wall as well as the transcription factors in the biosynthesis pathway. As such, MYB46 may offer a useful means for pathway-specific manipulation of secondary wall biosynthesis. However, realization of this potential requires additional information on the ‘MYB46-mediated transcriptional regulatory programme’, such as downstream direct targets, upstream regulators and interacting partners of MYB46.

INTRODUCTION

Vascular plants have evolved to have secondary cell wall structure between the plasma membrane and the primary cell wall in fibres and tracheid/vessel elements, which provide mechanical support for the growing body and serve as a conduit for long-distance transport of water and solutes, respectively. The secondary walls of these cells allow them to resist gravitational forces and the forces of the tension associated with the transpirational pull on their internal columns of water. Economically, secondary walls constitute the vast majority of plant biomass, which is of primary importance to humans for fibre, pulp and paper manufacture, and as an environmentally cost-effective renewable source of energy. Formation of secondary walls requires co-ordinated transcriptional regulation of the genes involved in the biosynthesis of the major secondary wall components (e.g. cellulose, hemicellulose and lignin). Complex transcriptional networks appear to be involved in the co-ordinated regulation of secondary wall biosynthesis (Demura and Ye, 2010; Ko et al., 2011, 2012; Wang and Dixon, 2011).

Many transcription factors (TFs) have been identified as central regulators of secondary wall biosynthesis (for recent reviews, see Yamaguchi and Demura, 2010; Zhang et al., 2010; Zhong et al., 2010a; Wang and Dixon, 2011; Zhao and Dixon, 2011; Ko et al., 2012; Pimrote et al., 2012; Hussey et al., 2013; Schuetz et al., 2013). Of these, MYB46 (At5g12870) and its paralogue, MYB83 (At3g08500), have been shown to function as a master switch for the secondary wall biosynthetic programme in Arabidopsis thaliana (Zhong et al., 2007a; Ko et al., 2009). Recent studies on this MYB46-mediated regulation have provided novel insights into the transcriptional control of secondary wall biosynthesis (Kim et al., 2013a, b, 2014). This review describes the current understanding of the MYB46-mediated transcriptional regulatory network and its implication on pathway-specific engineering of the properties and quantity of plant biomass.

MYB46-MEDIATED TRANSCRIPTIONAL REGULATION OF SECONDARY WALL BIOSYNTHESIS

The TF MYB46, a central regulator in secondary wall formation (Zhong et al., 2007a; Ko et al., 2009), is specifically expressed in both fibres and xylem cells undergoing secondary wall thickening (Zhong et al., 2007a). Promoter activity of MYB46 was also detected in both protoxylem and metaxylem (Nakano et al., 2010). Constitutive overexpression of either MYB46 or its close homologue MYB83 upregulates the genes involved in secondary wall biosynthesis (e.g. cellulose, hemicellulose and lignin biosynthesis genes), resulting in ectopic deposition of secondary walls even in epidermis, cortex and pith cells that are normally parenchymatous. On the other hand, dominant suppression of MYB46 significantly reduced secondary wall thickening in the fibres and vessels of the transgenic plants (Zhong et al., 2007a; Ko et al., 2009). Indeed, myb46myb83 double mutants show lack of secondary wall formation in both the vessels and fibres, and severe growth arrest in young seedlings followed by wilting and subsequent death (McCarthy et al., 2009). These observations clearly suggest that MYB46/MYB83 function as an essential regulator for secondary cell wall biosynthesis in arabidopsis. However, single myb46 or myb83 loss-of-function mutants do not produce any observable phenotype, while the myb46myb83 double mutant results in a ‘seedling-lethal’ phenotype, suggesting functional redundancy between the two TFs.

Furthermore, several MYB46 orthologues from other plant species have also been shown to function as a master switch for secondary wall biosynthesis, including PtMYB4 from pine, EgMYB2 from eucalyptus, OsMYB46 from rice, PtrMYB2/3/20/21 from poplar and ZmMYB46 from maize (Table 1) (Patzlaff et al., 2003; Goicoechea et al., 2005; Zhong et al., 2011, 2013).

Table 1.

Regulators identified in the transcriptional network of secondary wall biosynthesis in plants

Protein name	ID	Plant	Function	Regulation	References
MYB family transcription factors
MYB46	At5g12870	Arabidopsis thaliana	A direct target of SND1 and regulates secondary wall biosynthesis	Activator	Zhong et al. (2007a); Ko et al. (2009)
MYB83	At3g08500	Arabidopsis thaliana	Act redundantly with MYB46	Activator	McCarthy et al. (2009)
MYB26/MS35	At3g13890	Arabidopsis thaliana	Regulates secondary wall thickening in the endothecium	Activator	Yang et al. (2007)
MYB52	At1g17950	Arabidopsis thaliana	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2008)
MYB54	At1g73410	Arabidopsis thaliana	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2008)
MYB85	At4g22680	Arabidopsis thaliana	Regulates lignin biosynthesis	Activator	Zhong et al. (2008)
MYB103	At1g63910	Arabidopsis thaliana	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2008)
MYB58	At1g16490	Arabidopsis thaliana	Activates lignin biosynthetic pathway	Activator	Zhou et al. (2009)
MYB63	At1g79180	Arabidopsis thaliana	Activates lignin biosynthetic pathway	Activator	Ko et al. (2009); Zhou et al. (2009)
MYB75/PAP1	At1g56650	Arabidopsis thaliana	Represses lignin biosynthetic pathway	Repressor	Bhargava et al. (2010)
MYB32	At4g34990	Arabidopsis thaliana	Represses SND1 and lignin biosynthesis	Repressor	Wang et al. (2011)
MYB4	At4g38620	Arabidopsis thaliana	Probably similar function to MYB32	Repressor	Wang et al. (2011)
MYB7	At2g16720	Arabidopsis thaliana	Probably similar function to MYB32	Repressor	Wang et al. (2011)
PtMYB1	AY356372	Pinus taeda	Regulates secondary wall biosynthesis	Activator	Bomal et al. (2008)
PtMYB4	AY356371	Pinus taeda	Regulates lignin biosynthesis	Activator	Patzlaff et al. (2003)
PtMYB8	DQ399057	Pinus taeda	Regulates secondary wall biosynthesis	Activator	Bomal et al. (2008)
PtrMYB2	Potri.001G258700	Populus trichocarpa	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2013)
PtrMYB3	Potri.001G267300	Populus trichocarpa	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2013)
PtrMYB20	Potri.009G061500	Populus trichocarpa	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2013)
PtrMYB21	Potri.009G053900	Populus trichocarpa	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2013)
PttMYB21a	AJ567345	P. tremula × tremuloides	Negatively regulates lignin biosynthesis	Repressor	Karpinska et al. (2004)
EgMYB1	AJ576024	Eucalyptus gunnii	Negatively regulates secondary wall formation	Repressor	Legay et al. (2010)
EgMYB2	AJ576023	Eucalyptus gunnii	Positvely regulates secondary wall formation	Activator	Goicoechea et al. (2005)
OsMYB46	Os12g0515300	Oryza sativa	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2011)
ZmMYB46	JN634085	Zea mays	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2011)
ZmMYB31	NM_001112479	Zea mays	Directly represses lignin biosynthesis	Repressor	Fornale et al. (2006, 2010)
ZmMYB42	NM_001112539	Zea mays	Represses lignin biosynthesis	Repressor	Sonbol et al. (2009)
TaMYB4	JF746995	Triticum aestivum	Negatively regulates lignin biosynthesis	Repressor	Ma et al. (2011)
PvMYB4	JF299185	Panicum virgatum	Negatively regulates lignin biosynthesis	Repressor	Shen et al. (2012)
NAC family transcription factors
NST1	At2g46770	Arabidopsis thaliana	Regulates secondary wall thickenings and required for anther dehiscence	Activator	Mitsuda et al. (2005)
NST2	At3g61910	Arabidopsis thaliana	Regulates secondary wall thickenings and required for anther dehiscence	Activator	Mitsuda et al. (2005)
NST3/ANAC012/SND1	At1g32770	Arabidopsis thaliana	Regulates secondary wall synthesis in fibres	Activator	Zhong et al. (2006); Ko et al. (2007)
SND2	At4g28500	Arabidopsis thaliana	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2008)
SND3	At1g28470	Arabidopsis thaliana	Regulates secondary wall biosynthesis	Activator	Zhong et al. (2008)
VND6	At5g62380	Arabidopsis thaliana	Promotes protoxylem differentiation	Activator	Kubo et al. (2005)
VND7	At1g71930	Arabidopsis thaliana	Promotes metaxylem differentiation	Activator	Kubo et al. (2005)
VNI2	At5g13180	Arabidopsis thaliana	Negatively regulates xylem vessel formation	Repressor	Yamaguchi et al. (2010)
XND1	At5g64530	Arabidopsis thaliana	Negatively regulates lignocellulose synthesis and PCD in xylem.	Repressor	Zhao et al. (2008)
MtNST1		Medicago truncatula	Regulates secondary wall biosynthesis	Activator	Zhao et al. (2010b)
Other transcription factors
ASL19/LBD30	At4g00220	Arabidopsis thaliana	Positively regulates xylem differentiation	Activator	Soyano et al. (2008)
ASL20/LBD18	At2g45420	Arabidopsis thaliana	Positively regulates xylem differentiation	Activator	Soyano et al. (2008)
AtC3H14	At1g66810	Arabidopsis thaliana	Regulates secondary wall biosynthesis	Activator	Ko et al. (2009)
KNAT7	At1g62990	Arabidopsis thaliana	Negatively regulates secondary wall synthesis	Repressor	Li et al. (2012)
OFP4	At1g06920	Arabidopsis thaliana	Forms a functional complex with KNAT7 to repress secondary cell wall formation	Repressor	Li et al. (2011)
SHN2	At5g25390	Arabidopsis thaliana	Co-ordinated activation of cellulose and repression of lignin biosynthesis	Activator	Ambavaram et al. (2011)
WRKY12	At2g44745	Arabidopsis thaliana	Represses secondary wall formation in pith	Repressor	Wang et al. (2010)
MtSTP	HM622067	Medicago truncatula	Represses secondary wall formation in pith	Repressor	Wang et al. (2010)
Ntlim1	AB079513	Nicotiana tabacum	Regulates lignin biosynthesis	Activator	Kawaoka et al. (2000)

Upstream regulators of MYB46/MYB83

Upstream direct regulators of MYB46 are largely unknown. Several studies using inducible expression and/or activation systems coupled with comparative transcriptome analyses demonstrated that secondary wall NAC (NAM, ATAT1/2 and CUC2) TFs, such as NST1, NST2, NST3/ANAC012/SND1, VND6 and VND7, are direct upstream regulators of MYB46/MYB83 (Demura and Ye, 2010; Ohashi-Ito et al., 2010; Yamaguchi et al., 2011). The NAC TF family proteins are characterized by a conserved NAC domain located at the N-terminal region and a highly divergent C-terminal activation domain (Olsen et al., 2005). These TFs are specific to plants and play diverse roles in plant defence, growth and development (Olsen et al., 2005).

Several VASCULAR RELATED NAC DOMAIN (VND1–VND7) TF genes were isolated by whole-genome microarray analysis of transdifferentiating tracheary elements (TEs) in arabidopsis cell culture (Kubo et al., 2005). Expression of these VND genes is spatially and temporally correlated with TE differentiation. Among these, overexpression of VND6 and VND7 induces ectopic metaxylem and protoxylem formation, respectively, even in highly specialized cell types such as epidermis, stomata, trichomes and root hairs of arabidopsis and poplar (Kubo et al., 2005; Yamaguchi et al., 2008, 2010a). On the other hand, dominant suppression of VND6 and VND7 resulted in defective metaxylem and protoxylem formation, respectively, in arabidopsis roots (Kubo et al., 2005). However, single VND loss-of-function mutants are apparently not defective in TE formation, which indicates the functional redundancy with other members of the VND family (Kubo et al., 2005). These results indicate that VND6 and VND7 function as key regulators in xylem vessel differentiation (Fig. 1; Table 1).

Fig. 1.

MYB46/MYB83 function as a central regulator of secondary wall biosynthesis. Secondary wall biosynthesis is controlled by a complex and multifaceted transcriptional network including positive and negative feedback/forward regulation. Secondary wall NACs are upstream regulators of MYB46/MYB83, while several transcription factors are downstream targets. MYB46/MYB83 regulate the secondary wall biosynthesis genes either directly or co-operatively with downstream target transcription factors. Negative regulators are highlighted in red.

In addition, other NAC family TFs, such as, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2 and NST3/ANAC012/SECONDARY WALL-ASSOCIATED NAC-DOMAIN 1 (SND1), were identified as key regulators of secondary wall biosynthesis in fibre cells (Mitsuda et al., 2005, 2007; Zhong et al., 2006; Ko et al., 2006, 2007; Demura and Ye, 2010). Overexpression of these NAC genes resulted in ectopic deposition of secondary walls in non-vascular cell types, while their suppression reduced secondary wall thickness. For example, the nst1nst3 double knockout showed complete loss of secondary wall thickening in the fibres (Mitsuda et al., 2007; Zhong et al., 2007b). However, their double knockout or simultaneous RNAi (RNA interference) inhibition did not affect secondary wall biosynthesis in the xylem vessels (Mitsuda et al., 2007; Zhong et al., 2007b). NST3/ANAC012/SND1 is specifically expressed in fibres, while VND6 and VND7 are expressed in xylem vessels. Overexpression of any of the NST or VND genes is able to activate the entire secondary wall biosynthetic programme, indicating that NST1 and NST3/ANAC012/SND1 are responsible for secondary wall biosynthesis in fibres, and VND6 and VND7 are responsible in xylem vessels. NST2 was found to be responsible for the secondary wall thickening of endothecium in anther development (Mitsuda et al., 2005).

Members of the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) protein family were identified as a part of a positive feedback loop regulating VND6 and VND7 in the transcriptional network of secondary wall biosynthesis (Soyano et al., 2008). Overexpression of ASL19/LBD30 and ASL20/LBD18 genes transdifferentiated non-vascular tissues into TE-like cells in arabidopsis, similar to those induced by VND6 or VND7 overexpression, while their dominant suppression caused aberrant TEs (Soyano et al., 2008). Both ASL19 and ASL20 were expressed in immature TEs, and their expression depends on VND6 and VND7 (Soyano et al., 2008) (Table 2). Moreover, ectopic expression of VND7 was detected in plants overexpressing ASL20. Therefore, ASL20/LBD18 and ASL19/LBD30 function in a positive feedback loop that amplifies the expression of VND6 and VND7 (Soyano et al., 2008).

Table 2.

Transcriptional regulators of secondary wall biosynthesis and their downstream target genes

Protein name	Downstream target genes	Cis-acting element	References
MYB46/83	MYB4, MYB7, MYB32, MYB52, MYB54, KNAT7, MYB43, MYB58, MYB63, AtC3H14, CesA4, CesA7, CesA8	M46RE: (T/C)ACC(A/T)A(A/C) (T/C)	Ko et al. (2009)
			Zhong et al. (2007a)
	PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, CCR, CAD, FRA8, IRX8, IRX9, IRX14	SMRE: ACC(A/T)A(A/C) (T/C)	Kim et al. (2012)
			Zhong and Ye (2012)
VND6	MYB46/83, MYB5, MYB63, VND7, MYB103	SNBE: (T/A)NN(C/T) (T/C/G) TNNNNNNNA(A/C)GN(A/C/T) (A/T)	Demura and Ye (2010)
			Ohashi-Ito et al. (2010)
			Zhong et al. (2010b)
VND7	MYB46/83, MYB58, MYB63, BFN1, XCP1, XCP2, XSP1, LBD18, LBD30, MYB103, ASL19, ASL20, CesA4, CesA8	SNBE: (T/A)NN(C/T) (T/C/G) TNNNNNNNA(A/C)GN(A/C/T) (A/T)	Yamaguchi et al. (2011)
			Soyano et al. (2008)
			Zhong et al. (2010b)
NST1	MYB46/83, MYB58, MYB63	SNBE: (T/A)NN(C/T) (T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T)	Mitsuda et al. (2007)
			Zhong et al. (2010b)
SND1/NST3/ANAC012	MYB4, MYB7, MYB20, MYB32, MYB42, MYB43, MYB52, MYB54, MYB58, MYB69, MYB85, MYB46/83, SND2, SND3, MYB103, KNAT7, VND7	SNBE: (T/A)NN(C/T) (T/C/G) TNNNNNNNA(A/C)GN(A/C/T) (A/T)	Zhong et al. (2006)
			Zhong et al. (2007a)
			Ko et al. (2007)
			McCarthy et al. (2009)
			Zhong et al. (2010b)
MYB58/63	LAC4, PAL1, C4H, 4CL1, HCT, C3H1, CCoAOMT1, CCR1, COMT, CAD6	AC-I: ACCTACC, AC-II: ACCAACC, AC-III: ACCTAAC	Zhou et al. (2009)
			Zhong et al. (2008)
MYB85	4CL1	ND	Zhong et al. (2008)
KNAT7	CesA1, CesA3, CesA6, IRX8, IRX9, IRX10, FRA8, CesA4, CesA7, CesA8, PAL1, C4H, 4CL1, HCT, C3H1, CCoAOMT1, CCR1, F5H1, COMT1, CAD5	ND	Li et al. (2012)
MYB4	C4H	ND	Jin et al. (2000)

Bold and underlined genes are suggested as direct targets.

ND, not determined.

Taken together, it appears that the transcriptional network activated by the secondary wall NACs functions through MYB46/MYB83 (Ko et al., 2012; Schuetz et al., 2013) (Fig. 1). Yeast-one hybrid (Y1H) analysis using MYB46 as bait might lead to identification of additional upstream regulators of MYB46.

Downstream targets of MYB46/MYB83

In order to study the downstream transcriptional network leading to secondary wall biosynthesis controlled by MYB46, a comprehensive time-course transcriptome profiling was performed with an inducible secondary wall thickening system in arabidopsis plants by overexpressing MYB46 under the control of a dexamethasone-inducible promoter (Ko et al., 2009). This study identified a total of 42 TFs whose expression either coincides with, or precedes, the induction of secondary wall biosynthetic genes. Subsequent transient transcriptional activation assays confirmed that MYB46 activates the expression of MYB4, MYB7, MYB32, KNAT7, MYB52, MYB54, MYB63 and AtC3H14 (Fig. 1; Tables 1 and 2). Among them, AtC3H14, MYB52 and MYB63 were shown to activate the genes involved in secondary wall biosynthesis (Ko et al., 2009) (Table 2).

Identification of cis-acting regulatory elements (i.e. TF-binding motifs) that are recognized by MYB46 may facilitate the search for direct downstream target genes of MYB46. Independent studies by Kim et al (2012) and Zhong and Ye (2012) identified such motifs. Kim et al. (2012) analysed the promoter region of the TF gene AtC3H14, a known direct target of MYB46 (Ko et al., 2009), and identified an eight-nucleotide core motif ([A/G][G/T]T[A/T]GGT[A/G]) named M46RE (MYB46-responsive cis-regulatory element) that is recognized by MYB46. Various in vitro and in vivo experimental approaches [e.g. electrophoretic mobility shift assay (EMSA), transient transcriptional activation assay and chromatin immunoprecipitation (ChIP) analysis] were used to confirm that M46RE is a ‘necessary and sufficient’ element for MYB46-mediated transcriptional activation of the target genes. Zhong and Ye (2012) also reported a cis-acting motif called SMRE (secondary wall MYB-responsive element, ACC[A/T]A[A/C][T/C]) that is recognized by MYB46. This motif was identified by MYB46 binding assays with serial deletions of the promoter of MYB63, another direct target of MYB46. These two cis-elements (i.e. M46RE and SMRE) are essentially identical except for the eighth nucleotide in M46RE that is absent in SMRE (Table 2).

Several TFs identified as putative direct target of MYB46 have these elements in their promoter region, including AtC3H14, MYB43, MYB58, MYB63 and KNAT7 (Kim et al., 2012; Zhong and Ye, 2012) (Table 2). The TFs MYB58 and MYB63, which are direct targets of MYB46, have been shown to function as direct transcriptional activators of lignin biosynthesis during secondary wall formation in arabidopsis (Ko et al., 2009; Zhou et al., 2009; Demura and Ye, 2010). MYB85 also regulates lignin biosynthesis by activating the lignin biosynthetic genes and causes ectopic lignin deposition when overexpressed (Zhong et al., 2008).

A bioinformatics survey of the arabidopsis genome using the M46RE motif as bait found that many of the cell wall biosynthesis genes (e.g. cellulose, xylan and lignin) or the genes involved in cell wall biosynthesis-related cellular processes (e.g. cytoskeletal organization and signal transduction) have the motif in their promoters (Kim et al., 2012). The list includes three secondary wall-associated cellulose synthase genes (IRX1/CESA8, IRX3/CESA7 and IRX5/CESA4) (Taylor et al., 2003), xylan biosynthesis genes (IRX8, IRX9, IRX14 and IRX15-L) (Peña et al., 2007; Jensen et al., 2010; Wu et al., 2010; Brown et al., 2011), three laccase genes (IRX12/LACCASE4, LACCASE10 and LACCASE11) involved in lignin biosynthesis (Brown et al., 2005), two cytoskeleton-related genes (MYOSIN5 and microtubule-associated protein) (Kaneda et al., 2010; Pesquet et al., 2010) and two DUF579 genes (At1g33800 and At4g09990) (Jensen et al., 2010; Brown et al., 2011). This result suggests that MYB46 may directly regulate not only TFs but also structural genes of secondary wall biosynthesis (Kim et al., 2012).

MYB46 directly regulates secondary wall-associated cellulose synthases

Cellulose, the most abundant biopolymer on Earth, is a central component of plant cell walls and highly abundant (up to 50 %) in the secondary walls (Somerville, 2006). Recently, conversion of cellulose from energy crops into biofuels (e.g. cellulosic ethanol) has attracted global attention as an alternative fuel source. In the secondary cell walls of arabidopsis, three cellulose synthases (CESA4, CESA7 and CESA8) are necessary for cellulose production (Turner and Somerville, 1997; Taylor et al., 1999, 2000, 2003; Doblin et al., 2002; Williamson et al., 2002). However, little was known about the transcriptional regulation of these CESA genes. Interestingly, all three of the secondary wall-associated CESA genes (CESA4, CESA7 and CESA8) have the M46RE motif in their promoters, suggesting that their expression may be directly regulated by MYB46. Kim et al. (2013a) reported several lines of experimental evidence in support of this hypothesis. First, all three of the CESA genes were highly upregulated in both constitutive and inducible overexpression of MYB46 in planta. Secondly, MYB46 directly activates the transcription of the three CESA genes in a steroid receptor-based inducible activation system. Thirdly, MYB46 protein directly binds the promoters of the three CESA genes both in vitro and in vivo, which was confirmed by EMSA and ChIP analysis, respectively. Fourthly, ectopic upregulation of MYB46 resulted in a significant increase of crystalline cellulose content in arabidopsis (Kim et al., 2013a). Taken together, the evidence is quite convincing that MYB46 is a direct regulator of all three secondary wall-associated CESA genes.

Since cellulose biosynthesis in the secondary wall is critical to the plant's survival, it is prudent to speculate that MYB46 is not the only direct regulator of the secondary wall cellulose synthases. Previously, it has been demonstrated that VND6 binds to the TE-specific cis-element TERE (Pyo et al., 2007) in the promoter of CESA4 (Ohashi-Ito et al., 2010). VND7 was also suggested as a direct transcriptional regulator of CESA4 and CESA8 (Yamaguchi et al., 2011) (Table 2). In addition, Y1H screening using the promoter sequences of CESA4, CESA7 and CESA8 as bait identified multiple TFs that bind to the promoter sequences (Kim et al., 2013a). The Y1H identified 13 TFs for the CESA4 promoter and one TF for the CESA7 promoter. However, none of them appears to be involved in the MYB46-mediated regulation pathway because their expression is not altered by MYB46 nor are they co-expressed with MYB46 (Ko et al., 2009; Kim et al., 2012). Thus, the presence of multiple regulators, independent of the MYB46-mediated regulatory pathway, supports the notion that the transcriptional regulation of cellulose biosynthesis is multifaceted and complex.

MYB46 is required for functional expression of the secondary wall cellulose synthases

Considering the elaborate nature of transcriptional control of secondary wall cellulose biosynthesis, one pertinent question is whether MYB46 is the necessary regulator for functional expression of the secondary wall CESA genes. To address this question, Kim et al. (2013b) used a series of genetic complementation experiments using cesa knockout mutants with the CESA coding sequence driven by either the native or the mutated promoter of the genes. The mutant promoters have two-nucleotide point mutations in the M46RE such that MYB46 cannot bind to the promoter, while the binding of other non-MYB-type secondary wall TFs is not affected. The results showed that MYB46 binding to the intact M46RE is essential to restore the normal phenotype of the cesa mutants (Kim et al., 2013b), suggesting that MYB46 is an obligate component of the transcriptional regulatory complex involved in the commitment to secondary wall cellulose synthesis in arabidopsis.

MYB46 directly regulates hemicellulose and lignin biosynthesis genes

The genome-wide survey of promoter sequences in arabidopsis revealed that many genes involved in hemicellulose and lignin biosynthesis have one or more M46RE motifs in their promoter region (Kim et al., 2012), leading to the hypothesis that the expression of these genes may be directly regulated by MYB46. For example, cellulose synthase-like A9 (CSLA9) is responsible for the majority of glucomannan synthesis in both primary and secondary walls of arabidopsis inflorescence stems (Liepman et al., 2005; Goubet et al., 2009). Both in vitro (EMSA) and in vivo (ChIP) binding assays clearly showed that MYB46 binds to the promoter of CSLA9 (Kim et al., 2014). Overexpression of MYB46 resulted in a significant increase in mannan content (Kim et al., 2014). Recently, we obtained experimental evidence for direct regulation of four xylan biosynthesis genes (FRA8, IRX8, IRX9 and IRX14) by MYB46 (W.-C. Kim and K.-H. Han, unpubl. res.). These four genes encode glycosyltransferases that are required for glucuronoxylan synthesis in secondary cell walls (Peña et al., 2007; Keppler and Showalter, 2010). FRA8 and IRX8 are involved in the reducing end synthesis of xylan chains (Scheller and Ulvskov, 2010), while IRX9 and IRX14 are responsible for the xylan backbone synthesis (Lee et al., 2012). Therefore, it appears that MYB46 directly regulates the biosynthesis of the xylan backbone. However, it is notable that the other four known xylan biosynthesis genes (PARVUS, IRX10, IRX15 and IRX15-L) do not seem to be directly regulated by MYB46, indicating the multifaceted nature of the regulation of xylan biosynthesis.

The genes involved in monolignol biosynthesis have been identified (Boerja et al., 2003). Nine out of ten monolignol biosynthesis genes (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, CCR and CAD) are directly regulated by MYB46 (W.-C. Kim and K.-H. Han, unpubl. res.) (Table 2). Previously, two TFs, MYB58 and MYB63, were identified as master regulators of lignin biosynthesis (Zhou et al., 2009). These TFs directly control the expression of seven monolignol biosynthesis genes (PAL, 4CL, C3H, CCoAOMT, CCR and CAD), but not of F5H, a key gene in syringyl (S) lignin biosynthesis (Raes et al., 2003; Zhou et al., 2009). The TF MYB46 directly regulates F5H as well as MYB58 and MYB63 (W.-C. Kim and K.-H. Han, unpubl. res.). In Medicago truncatula, a secondary wall master switch SND1 directly regulates F5H but not the other monolignol genes (i.e. C4H, COMT, CCoAOMT and 4CL) (Zhao et al., 2010a, b). Whether SND1 regulates F5H in arabidopsis is not known. Recently, Ohman et al. (2013) showed that a loss-of-function mutation of MYB103 substantially reduced F5H expression, resulting in a 70–75 % decrease in S-lignin, while it did not transactivate F5H expression. Taken together, these observations further support the hypothesis that a multifaceted regulatory network exists for the control of lignin biosynthesis, and MYB46 is a key regulator in the network.

Interacting partners of MYB46/MYB83

In eukaryotes, gene expression is frequently controlled by multiprotein complexes. The formation of protein complexes enables the combinatorial action of TFs on the basis of both specific protein–DNA and protein–protein interactions, which facilitate the complex regulatory networks found in higher eukaryotes (Du et al., 2009).

The physical interaction and regulatory synergy between particular sub-classes of MYB and bHLH (basic helix–loop–helix) family TFs is well known in plant gene regulation (Du et al., 2009). In addition, members of the MYB and bHLH families also interact with a number of other regulatory proteins, forming complexes that either activate or repress the expression of sets of target genes (Feller et al., 2011). Examples of such complexes include the PAP1 (MYB)–GL3/EGL3/TT8 (bHLH)–TTG1 (WD40) complex in anthocyanin production, WER (MYB)–GL3/EGL3–TTG1 in root hair development, MYB61–TT8–TTG1 in seed coat mucilage production and GL1 (MYB)–GL3–TTG1 in trichome development (Petroni and Tonelli, 2011). To date, no interacting partners of MYB46 have been identified. However, it is probable that MYB46 can interact with bHLH family TFs. To test this hypothesis, we carried out yeast two-hybrid (Y2H) screening using MYB46 as bait and the bHLH TF library as prey. Our preliminary results indicate that two bHLH TFs strongly interact with MYB46 in yeast (J.-H. Ko and K.-H. Han, unpubl. res.). In planta interaction with MYB46 and the functional significance of the bHLH TFs remain to be elucidated.

Negative regulators of secondary wall biosynthesis

In terms of adaptation to the changing environmental and developmental contexts, negative regulation of secondary wall biosynthesis may be required for tissue-type fine-tuning of secondary wall deposition. Two NAC TFs, VNI2 and XND1, were identified as negative regulators of xylem formation in arabidopsis (Zhao et al., 2008; Yamaguchi et al., 2010b). VND-INTERACTING2 (VNI2) can bind to VND proteins and has been shown to function as a transcriptional repressor of VND7-mediated gene transcription (Yamaguchi et al., 2010b). During xylem differentiation, VNI2 protein is targeted for degradation to unleash VND7 that is required for xylem differentiation, while VNI2 expression precedes that of VND7 in procambial cells (Yamaguchi et al., 2010b). However, VNI2 expression persists in neighbouring xylary parenchyma cells, suggesting that VNI2 functions as a negative regulator of xylem differentiation (Yamaguchi et al., 2010b). Overexpression of XYLEM NAC DOMAIN1 (XND1) causes the complete suppression of xylem vessel secondary wall biosynthesis and programmed cell death (PCD), but not phloem marker gene expression, suggesting that XND1 negatively regulates xylem differentiation (Zhao et al., 2008). Interestingly, both VNI2 and XND1 appear to be targeted for proteasomal degradation by the 20S proteasome (20SP) (Zhao et al., 2008; Yamaguchi et al., 2010b; Han et al., 2012). The 20SP is thought to be a part of the ubiquitin–26SP proteolytic system, possessing caspase-3-like activity, which is a characteristic of animal cell apoptosis (Han et al., 2012). This fact suggests that 20SP may degrade VNI2 and XND1 to induce xylem differentiation in arabidopsis and Populus (Han et al., 2012).

Mutation of the arabidopsis WRKY12 gene caused secondary cell wall thickening in pith cells associated with ectopic deposition of lignin, xylan and cellulose by upregulation of downstream genes encoding NST2 and AtC3H14 TFs that activate secondary wall synthesis (Wang et al., 2010). Direct binding of WRKY12 to the NST2 gene promoter and repression of NST2 and AtC3H14 were confirmed by in vitro assays and in planta transgenic experiments (Wang et al., 2010). The WRKY12 gene is expressed in both pith and cortex that do not have secondary wall thickening. These results suggest that WRKY12 controls the parenchymatous nature of pith cells by acting as a negative regulator of secondary wall NACs.

Recently, a homeodomain TF KNAT7 was described as a negative regulator of secondary wall formation despite being a direct downstream target of both MYB46 and NST3/ANAC012/SND1 (Zhong et al., 2008; Ko et al., 2009; Li et al., 2012). KNAT7 is specifically expressed in vascular tissues and shown to function as a transcriptional repressor. In a transient activation assay using protoplasts, the expression of secondary wall biosynthetic genes was increased in the absence of KNAT7 function (Li et al., 2012). Overexpression of KNAT7 resulted in thinner secondary cell walls in interfascicular fibres, while its loss-of-function mutant knat7 forms thicker secondary cell walls (Li et al., 2011, 2012).

MYB4, MYB7 and MYB32, further downstream targets of both SND1 and MYB46/MYB83, were identified as negative regulators in secondary wall biosynthesis (Ko et al., 2009; Zhong et al., 2010b; Wang et al., 2011). As potent transcriptional repressors, these MYBs can reduce both their target gene expression and the expression of the SND1 upstream regulator (Ko et al., 2009; Zhong et al., 2010b; Wang et al., 2011) (Fig. 1). MYB4 and MYB32 were shown to regulate general phenylpropanoid biosynthesis genes negatively (Preston et al., 2004). MYB4 specifically suppresses the expression of the C4H gene, which encodes the first committed step in the phenylpropanoid pathway (Jin et al., 2000).

Taken together, negative regulators identified so far may function to fine-tune the expression of other TFs or genes involved in secondary wall biosynthesis. This mechanism provides a potential negative feedback regulation that may be a critical homeostatic control within the MYB46-mediated transcriptional regulation network (Fig. 1; Table 1).

Functional homology between MYB46 and its homologues in other plant species

The poplar genome has four close homologues of arabidopsis MYB46: PtrMYB2, PtrMYB3, PtrMYB20 and PtrMYB21 (McCarthy et al., 2010). Both PtrMYB3 and PtrMYB20 have been shown to function as a master switch for secondary wall biosynthesis in poplar (McCarthy et al., 2010). These MYB TFs are directly activated by PtrWND2, an activator of secondary wall biosynthesis in the wood tissue of poplar (Zhong et al., 2010a). Based on these findings and the high degree of collinearity between arabidopsis and Populus, it is hypothesized that the MYB46-mediated transcriptional regulatory programme may control the biosynthesis of secondary walls in poplars. This hypothesis was supported by the results from our EMSA analyses, clearly showing that arabidopsis MYB46 binds to the promoters of Populus secondary wall CESA genes and PtrMYB21 to the promoters of both arabidopsis and poplar secondary wall CESA genes (W.-C. Kim, J.-Y. Kim and K.-H. Han, unpubl. res.). In addition, rice and maize MYB TFs, OsMYB46 and ZmMYB46, were suggested as functional orthologues of MYB46 (Zhong et al., 2011). Both OsMYB46 and ZmMYB46 were directly regulated by the secondary wall NAC TFs OsSWNs and ZmSWNs, respectively, and were able to activate the secondary wall biosynthetic programme when they were overexpressed in arabidopsis (Zhong et al., 2011). These observations, along with the reciprocal binding of arabidopsis MYB46 and PtrMYB21 to the promoters of secondary wall CESA genes, suggest that the MYB46-mediated regulation of secondary wall biosynthesis may be functionally conserved among plant species.

PERSPECTIVES

Multiple observations in the literature indicate that a complex transcriptional regulatory programme appears to be involved in the control of secondary wall biosynthesis. MYB46 and its functional paralogue MYB83 play a central role in the regulatory programme, evidenced by the fact that myb46myb83 double knockout mutants show severe growth arrest in the early seedling stage. Recent studies have shown that MYB46 regulates not only the TFs in the secondary wall biosynthesis pathway but also the biosynthesis genes for all three of the major components (i.e. cellulose, hemicellulose and lignin) of secondary walls. Having the ability to regulate directly the biosynthesis genes for the major components, MYB46 may be useful in pathway-specific manipulation of secondary wall biosynthesis. For example, upregulation of MYB46 can increase the biosynthesis of cellulose and hemicellulose, while lignin biosynthesis is reduced. In order to realize this potential fully, additional information on the upstream regulators, downstream targets and interacting partners of MYB46 is critical. Considering the high degree of functional homology between arabidopsis MYB46 and its homologues in other plant species, the knowledge gained from the model arabidopsis plant on MYB46-mediated transcriptional regulation can be applicable in economically important crop species for production of biofuel and bioproducts.