• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plntcellLink to Publisher's site
Plant Cell. Oct 2008; 20(10): 2763–2782.
PMCID: PMC2590737

A Battery of Transcription Factors Involved in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis

Abstract

SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a master transcriptional switch activating the developmental program of secondary wall biosynthesis. Here, we demonstrate that a battery of SND1-regulated transcription factors is required for normal secondary wall biosynthesis in Arabidopsis thaliana. The expression of 11 SND1-regulated transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), was developmentally associated with cells undergoing secondary wall thickening. Of these, dominant repression of SND2, SND3, MYB103, MYB85, MYB52, MYB54, and KNAT7 significantly reduced secondary wall thickening in fiber cells. Overexpression of SND2, SND3, and MYB103 increased secondary wall thickening in fibers, and overexpression of MYB85 led to ectopic deposition of lignin in epidermal and cortical cells in stems. Furthermore, SND2, SND3, MYB103, MYB85, MYB52, and MYB54 were able to induce secondary wall biosynthetic genes. Direct target analysis using the estrogen-inducible system revealed that MYB46, SND3, MYB103, and KNAT7 were direct targets of SND1 and also of its close homologs, NST1, NST2, and vessel-specific VND6 and VND7. Together, these results demonstrate that a transcriptional network consisting of SND1 and its downstream targets is involved in regulating secondary wall biosynthesis in fibers and that NST1, NST2, VND6, and VND7 are functional homologs of SND1 that regulate the same downstream targets in different cell types.

INTRODUCTION

Plant cells can be classified into three basic types (i.e., parenchyma, collenchyma, and sclerenchyma) based on their wall characteristics. While parenchyma and collenchyma cells have only primary walls, sclerenchyma cells contain both primary and secondary walls. Biosynthesis of secondary walls in sclerenchyma cells, an ability acquired when vascular plants emerged during the Silurian period, enabled vascular plants not only to build strong xylem conduits for transport of water and minerals but also to attain mechanical support for the plant body (Raven et al., 1999). Elucidation of the mechanisms that plants evolved to produce secondary walls is undoubtedly an important issue in plant biology and will help us understand both the evolution of vascular plants and the development of different cell types. The importance of studying the biosynthesis of secondary walls is further exemplified by the fact that wood, which is primarily composed of secondary walls, constitutes the most abundant biomass on earth and is widely used for pulping and papermaking, construction, and potentially also for biofuel production.

It was proposed that cells developmentally programmed to become sclerenchyma receive specific signals that are further transduced to activate the secondary wall biosynthetic pathways (Zhong and Ye, 2007). Although the secondary wall biosynthetic pathways have been characterized biochemically and genetically in great detail, little is known about the signals and transcriptional regulators that are responsible for turning on the secondary wall biosynthetic program. Recent studies on NAC and MYB transcription factors have provided a first glimpse into the complex process of transcriptional regulation of secondary wall biosynthesis (Zhong and Ye, 2007). Evidence indicates that a group of closely related Arabidopsis thaliana NAC domain proteins, including NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1; also called NST3), VASCULAR-RELATED NAC-DOMAIN6 (VND6), and VND7, are key transcriptional regulators of secondary wall biosynthesis in various sclerenchyma cell types (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2007b; Ko et al., 2007). While SND1 and NST1 function redundantly in the activation of secondary wall biosynthesis in fibers (Zhong et al., 2006, 2007b; Mitsuda et al., 2007), VND6 and VND7 were proposed to regulate the differentiation of metaxylem and protoxylam, respectively, in primary roots (Kubo et al., 2005). NST1 and NST2 have been shown to function redundantly in regulating secondary wall thickening in endothecium cells of the anther (Mitsuda et al., 2005). Overexpression of all of these NAC genes leads to ectopic deposition of secondary walls in cells that are normally parenchymatous, and inhibition of their functions by knockout or dominant repression results in a reduction in secondary wall thickening (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2007b). These studies demonstrate that these secondary wall NACs are master switches capable of turning on the entire biosynthetic pathways of cellulose, xylan, and lignin, which leads to the deposition of secondary walls.

In addition to NACs, several MYB transcription factors have also been shown to be important regulators of secondary wall biosynthesis in Arabidopsis. MYB46, a direct target of SND1, is able to turn on the biosynthetic pathways of cellulose, xylan, and lignin (Zhong et al., 2007a). Similarly, overexpression of MYB26, which was thought to act upstream of NST1 and NST2, results in ectopic deposition of secondary walls (Yang et al., 2007). Other MYB genes, such as pine (Pinus taeda) MYB1 (Patzlaff et al., 2003b) and MYB4 (Patzlaff et al., 2003a) and eucalyptus (Eucalyptus grandis) MYB2 (Goicoechea et al., 2005), are able to bind to the AC elements present in the promoters of lignin biosynthetic genes and are proposed to regulate the biosynthesis of lignin.

It has been suggested that a network of transcription factors are involved in the regulation of secondary wall biosynthesis (Zhong and Ye, 2007). In this transcriptional network, the secondary wall NACs, including SND1, NST1, NST2, VND6, and VND7, are master switches turning on a subset of transcription factors, which in turn activate the secondary wall biosynthetic pathways. Previous studies have revealed that the expression of several transcription factors, including SND2, SND3, MYB103, MYB85, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), is regulated by SND1 (Zhong et al., 2006, 2007b).

In this study, we report the functional characterization of a battery of SND1-regulated transcription factors and provide evidence that they are part of the transcriptional network regulating secondary wall biosynthesis. We show that the expression of these SND1-regulated transcription factors is specifically associated with cells undergoing secondary wall thickening. Using dominant repression and overexpression studies, we demonstrate their involvement in regulating secondary wall biosynthesis. We further reveal that, in addition to MYB46, which is a known SND1 direct target, SND3, MYB103, and KNAT7 are also direct targets of SND1 and its close homologs, NST1, NST2, VND6, and VND7. Our study suggests that the secondary wall NACs, including SND1, NST1, NST2, VND6, and VND7, share common downstream targets in the activation of the secondary wall biosynthetic program.

RESULTS

Identification of Transcription Factors That Are Regulated by SND1

To unravel the transcriptional network regulating secondary wall biosynthesis, we searched for transcriptional factor genes that are regulated by SND1. We used interfascicular fiber cells laser-microdissected from Arabidopsis inflorescence stems to identify transcription factors that are associated with secondary wall thickening in fibers (Zhong et al., 2006) and then selected those that were downregulated by RNA interference (RNAi) inhibition of SND1 and NST1. In addition to MYB46, which has been shown to be a direct target of SND1 (Zhong et al., 2007a), we found 11 additional transcription factors whose expression is downregulated by RNAi inhibition of SND1/NST1 (Figure 1A). These include two NACs (SND2 and SND3), eight MYBs (MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, and MYB20), and a homeodomain transcription factor (KNAT7). Among them, six transcription factors, including SND2, SND3, MYB103, MYB85, MYB20, and KNAT7, were previously shown to be regulated by SND1 (Zhong et al., 2006, 2007b); however, their roles in the regulation of secondary wall synthesis have not been characterized. Consistent with their downregulation of expression by SND1/NST1 RNAi inhibition, the expression of all of these transcription factors except for MYB69 was significantly upregulated by SND1 overexpression (Figure 1B). The expression of these transcription factors including MYB69 was also upregulated by NST1 overexpression (Figure 1C). These results indicate that these transcription factors are most likely involved in the SND1/NST1-mediated transcriptional regulation of secondary wall synthesis.

Figure 1.
SND1 Regulates the Expression of a Set of Transcription Factor Genes.

Expression Patterns of SND1-Regulated Transcription Factors

To investigate whether the expression of these SND1-regulated transcription factors is associated with secondary wall biosynthesis, we analyzed their expression in different cell types and organs by quantitative RT-PCR analysis. All 11 SND1-regulated transcription factors exhibited a high level of expression in interfascicular fibers and xylem cells undergoing secondary wall thickening, but not in parenchymatous pith cells (Figures 2 and and3).3). At the organ level, they were preferentially expressed in inflorescence stems in which interfascicular fibers and xylem cells were abundantly developed (Figures 2 and and33).

Figure 2.
Quantitative Real-Time PCR Analysis of the Expression of SND2, SND3, MYB103, MYB85, MYB52, and MYB54 Genes in Different Cell Types and Organs.
Figure 3.
Quantitative Real-Time PCR Analysis of the Expression of MYB69, MYB42, MYB43, MYB20, and KNAT7 Genes in Different Cell Types and Organs.

We next used the β-glucuronidase (GUS) reporter gene and in situ mRNA hybridization to examine the developmental expression patterns of eight of these transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, and KNAT7, which were found to cause secondary wall defects when their expression was dominantly repressed (see below). For the GUS reporter gene analysis, the entire exon and intron region of the transcription factor gene sequence, a 3-kb 5′ upstream sequence, and a 2-kb 3′ downstream sequence were included to ensure that the sequence used contains all of the cis-elements required for the endogenous gene expression. For in situ mRNA hybridization, gene-specific probes were used for detecting the gene expression patterns. In elongating stem internodes, all eight transcription factors were found to be expressed in developing protoxylem (Figures 4 and and5),5), the only secondary wall–containing cell type present (Ye et al., 2002). In addition to protoxylem, SND2, SND3, MYB52, and MYB54 were also expressed in the elongating interfascicular fiber cells, although at this stage, they do not yet have visible secondary wall thickening. In nonelongating internodes, in which both metaxylem cells and interfascicular fibers are undergoing secondary wall thickening (Ye et al., 2002), a high level of expression of all eight transcription factors was evident in developing metaxylem cells and interfascicular fibers (Figures 4 and and5).5). In roots, these genes were predominantly expressed in developing secondary xylem, in which secondary wall thickening occurs (Figures 4 and and5).5). These results demonstrate that the expression of these SND1-regulated transcription factors is developmentally associated with secondary wall biosynthesis in vessels and fibers.

Figure 4.
Expression Patterns of SND2, SND3, MYB103, and MYB85 Genes in Arabidopsis Stems and Roots.
Figure 5.
Expression Patterns of MYB52, MYB54, MYB69, and KNAT7 Genes in Arabidopsis Stems and Roots.

Consistent with their predicted function as transcription factors, yellow fluorescent protein (YFP)–tagged fusions of these transcription factors were found to be targeted to the nucleus when expressed in Arabidopsis leaf protoplasts (Figures 6A to 6L). Transcriptional activation analysis demonstrated that SND2, SND3, MYB103, MYB85, and MYB69 were able to activate the expression of the His3 and β-Gal reporter genes in yeast (Figure 6M), confirming that they are indeed transcriptional activators.

Figure 6.
Subcellular Localization and Transcriptional Activation Analysis of the Secondary Wall–Associated Transcription Factors.

Dominant Repression of SND1-Regulated Transcription Factors Leads to Defects in Secondary Wall Thickening

Because the expression of the SND1-regulated transcription factors is closely associated with secondary wall thickening, we reasoned that these transcription factors might be involved in regulation of secondary wall biosynthesis. To elucidate their potential roles in the regulation of secondary wall biosynthesis, we first attempted to use the RNAi approach to study the effects of their downregulation on secondary wall thickening. The RNAi approach has been effectively used to study the redundant functions of SND1 and NST1 in regulation of secondary wall biosynthesis (Zhong et al., 2007b). However, no changes on secondary wall thickening were observed when these transcription factors were downregulated by RNAi inhibition, probably due to the functional redundancy of their homologs. We next resorted to the dominant repression approach that has been successfully applied to study transcription factors involved in secondary wall biosynthesis (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2007a). The dominant repression approach was first developed by Hiratsu et al. (2004) to facilitate the analysis of functionally redundant transcription factors. It should be noted that transcription factors fused with the EAR (ethylene-responsive element binding factor-associated amphiphilic repression) dominant repression domain not only inhibit the function of transcription factors targeted for repression but also that of their homologs by competing with their binding to the same cis-elements or interacting proteins, thereby leading to dominant repression (Hiratsu et al., 2004).

To repress their functions, the genes encoding each of the SND1-regulated transcription factors were fused with the EAR repressor domain at the C terminus and expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. For each transcription factor analyzed by dominant repression, at least 96 first generation transgenic plants were examined for the thickening of secondary walls. Among the 11 SND1-regulated transcription factors, dominant repression of eight of them, including SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, and KNAT7, caused a severe reduction in secondary wall thickening in both interfascicular fibers and xylary fibers in inflorescence stems (Figures 7 and and8,8, Table 1). In these repressor lines, 20 to 40% of transgenic plants showed significant reduction in secondary wall thickness, and the severity of reduction correlated with the amount of the corresponding repressor transcript. To examine the highest potential impact of dominant repression of these transcription factors on secondary wall thickening, transgenic plants with the most severe phenotypes were selected for analysis. The secondary wall thickening in vessels was not significantly affected in any of these repressor lines (Table 1). It was noted that dominant repression of MYB85 caused deformation of vessels (Figures 7Q and 7R), probably due to the weakening of the vessels' secondary walls. These results suggest that dominant repression of SND1-regulated transcription factors leads to a defect in secondary wall biosynthesis, which was further confirmed by a cell wall composition analysis, which showed that the amount of glucose and xylose, the main sugars in cellulose and xylan, respectively, was significantly reduced in these repressor lines (Table 2).

Figure 7.
Effects of Dominant Repression of SND2, SND3, MYB103, and MYB85 on Secondary Wall Thickening in Fibers.
Figure 8.
Effects of Dominant Repression of MYB52, MYB54, MYB69, and KNAT7 on Secondary Wall Thickening in Fibers.
Table 1.
Wall Thickness of Vessels and Fibers in the Stems of Wild-Type Plants and in Dominant Repressors and Overexpressors of SND1-Regulated Transcription Factors
Table 2.
Monosaccharide Composition of Cell Walls from the Stems of Wild Type Plants and of Dominant Repressors of SND1-Regulated Transcription Factors

Effects of Overexpression of SND1-Regulated Transcription Factors on Secondary Wall Thickening

We next studied the effects of overexpression of the eight transcription factors, of which dominant repression resulted in secondary wall defects (Figure 9). These transcription factors were placed under the control of the CaMV 35S promoter and expressed in wild-type Arabidopsis plants. Examination of leaves, stems, and roots of transgenic plants did not reveal any ectopic deposition of secondary walls. The only exception was that plants overexpressing MYB85 had ectopic deposition of lignin in the epidermal and cortical cells of stems (Figures 9Q and 9R). No ectopic deposition of cellulose and xylan was observed in these lignified cells by Calcoflour White staining and xylan antibody immunolocalization, indicating that overexpression of MYB85 specifically induces the lignin biosynthetic pathway. Although no ectopic deposition of secondary walls was seen in transgenic plants overexpressing the other transcription factors, it was found that overexpression of three of them, SND2, SND3, and MYB103, resulted in a noticeable increase in the secondary wall thickness of both xylary fibers and interfascicular fibers (Figures 9A to 9P). Quantitative analysis showed that the wall thickness of xylary fibers and interfascicular fibers was increased by at least 32 and 58%, respectively, in these overexpressors compared with the wild type (Table 1). No changes were observed in the secondary wall thickness of fibers and vessels in transgenic plants overexpressing MYB52, MYB54, MYB69, and KNAT7.

Figure 9.
Effects of Overexpression of SND2, SND3, MYB103, and MYB85 on Secondary Wall Deposition.

To test whether any of these transcription factors has the ability to induce the expression of secondary wall biosynthetic genes, we engineered reporter constructs by ligating the promoters of a cellulose synthase gene (CesA8; Taylor et al., 2004), a xylan biosynthetic gene (IRX9; Pena et al., 2007), and a lignin biosynthetic gene (4CL1; Boerjan et al., 2003) in front of the GUS reporter gene. These reporter constructs were then cotransfected with the transcription factor overexpression constructs into Arabidopsis leaf protoplasts for transactivation analysis. We found that SND2, SND3, and MYB103 significantly induced the GUS reporter gene expression driven by the CesA8 promoter, whereas MYB85 specifically induced GUS expression driven by the 4CL1 promoter (Figure 10). MYB52 and MYB54 appeared to induce GUS expression driven by all three promoters, albeit to various levels (Figure 10). No significant induction in GUS expression was observed for MYB69 and KNAT7. We confirmed that all of the transcription factor genes were highly expressed in the transfected protoplasts (Figure 10C). These results suggest that some of these SND1-regulated transcription factors have the ability to induce the expression of certain secondary wall biosynthetic genes.

Figure 10.
Induction of Secondary Wall Biosynthetic Genes by Secondary Wall–Associated Transcription Factors.

Expression of SND3, MYB103, and KNAT7 Is Directly Activated by SND1

Our findings that the SND1-regulated transcription factors are expressed in cells undergoing secondary wall thickening and are involved in regulation of secondary wall synthesis suggest that they are downstream targets of SND1. To investigate whether any of them are direct targets of SND1, we set out to test whether SND1 can directly activate the expression of these transcription factors using the steroid receptor-based inducible system. In this system, a transcription factor is fused with a steroid binding domain, which renders the transcription factor inactive in the cell, as it is sequestered in the cytoplasm via binding to a cytoplasmic complex. Binding of the steroid to the steroid binding domain disrupts the complex and thus allows the transcription factor to enter the nucleus and regulate the expression of downstream target genes. In the presence of a protein synthesis inhibitor, activation of the transcription factor by a steroid treatment should still induce the expression of the direct target genes, whereas the induction of further downstream genes is inhibited since it requires new protein synthesis. Therefore, the steroid receptor-based inducible system can be employed to search for direct targets of transcription factors and has been successfully applied to finding direct targets of transcription factors in plants (Sablowski and Meyerowitz, 1998; Wagner et al., 1999; Baudry et al., 2004).

To identify direct targets of SND1, we fused SND1 with the regulatory region of the human estrogen receptor (HER) (Figure 11A; Zuo et al., 2000) and constitutively expressed the SND1-HER fusion protein under the control of the CaMV 35S promoter in Arabidopsis leaf protoplasts. We first tested whether the SND1-HER chimeric protein is functional using protoplasts cotransfected with 35S:SND1-HER and MYB46P:GUS (Figure 11A). Activation of SND1 by estradiol treatment drastically induced the GUS reporter activity driven by the MYB46 promoter (Figure 11B), demonstrating that SND1-HER is functional in the induction of downstream genes. We next examined the effect of various concentrations of cycloheximide on the inhibition of new protein synthesis. The GUS activity induced by estradiol-activated SND1-HER was completely abolished when as little as 2 μM cycloheximide was added (Figure 11B), indicating that new protein synthesis is completely inhibited by cycloheximide.

Figure 11.
SND1 Directly Activates the Expression of the SND3, MYB103, and KNAT7 Genes.

To investigate whether the SND1-HER inducible system could be used to search for direct targets of SND1, we tested whether SND1-HER could directly activate the expression of the MYB46 gene, which was previously shown to be a direct target of SND1 (Zhong et al., 2007a). The expression of the MYB46 gene was drastically induced by estradiol-activated SND1-HER, and this induction still occurred when new protein synthesis was completely inhibited by cycloheximide (Figure 11C), indicating that the induction of MYB46 expression by SND1 does not require new protein synthesis and is thus direct. It should be noted that the induction level of MYB46 by SND1-HER in the presence of cycloheximide was lower than that without cycloheximide, which was most likely due to cycloheximide inhibition of the overall protein synthesis, including that of SND1-HER. These results established that it is feasible to use the SND1-HER–inducible system to identify additional direct targets of SND1.

We next investigated the expression of SND1-regulated transcription factors in the SND1-HER–inducible system. Estradiol activation of SND1 resulted in a substantial induction in the expression of these transcription factors (Figure 11D). To find out whether any of them are direct targets of SND1, we applied the protein synthesis inhibitor cycloheximide before addition of estradiol and examined their induction by SND1. We found that the expression of SND3, MYB103, and KNAT7 was still highly induced upon estradiol activation of SND1 even when new protein synthesis was completely inhibited (Figure 11E), indicating that their induction by SND1 occurred without new protein synthesis and is therefore direct. By contrast, the expression of other transcription factors was not induced under the same conditions. Consistent with the finding that the expression of SND3, MYB103, and KNAT7 is directly activated by SND1, electrophoretic mobility shift assay (EMSA) showed that SND1 was able to bind to their promoter fragments and caused a mobility shift (Figure 11F). These results demonstrate that, besides MYB46, the SND3, MYB103, and KNAT7 transcription factors are additional direct targets of SND1.

Direct Induction of MYB46, SND3, MYB103, and KNAT7 by the SND1 Homologs NST1, NST2, VND6, and VND7

We previously showed that, like SND1, close homologs of SND1, namely, NST1, NST2, VND6, and VND7, were able to activate the expression of MYB46, suggesting that these secondary wall NACs might regulate common target genes involved in secondary wall synthesis (Zhong et al., 2007a). To further substantiate this hypothesis, we investigated whether these SND1 homologs were also able to directly activate the expression of other direct target genes of SND1. To do this, NST1, NST2, VND6, and VND7 were fused with the regulatory region of the human estrogen receptor and expressed under the control of the CaMV 35S promoter in Arabidopsis protoplasts (Figure 12A). Estradiol activation of these transcription factors greatly induced expression of MYB46, SND3, MYB103, and KNAT7 (Figures 12B to 12E). To test whether this induction was direct, new protein synthesis was inhibited by addition of cyclohexmide before addition of estradiol. We found that NST1, NST2, VND6, and VND7 still effectively activated the expression of MYB46, SND3, MYB103, and KNAT7, while expression of SND3 was no longer induced by NST1 (Figures 12B to 12E). The induction level of the target genes by NST1, NST2, VND6, and VND7 in the presence of cycloheximide was lower than that in the absence of cycloheximide, which is most likely due to the overall inhibition of protein synthesis by cycloheximide. This result suggests that SND1 and its close homologs, NST1, NST2, VND6, and VND7, activate the same set of direct targets, thereby regulating secondary wall biosynthesis.

Figure 12.
Direct Activation of the MYB46, SND3, MYB103, and KNAT7 Genes by SND1 Homologs.

VND6 and VND7 Genes Are Expressed in Vessels of Stems

SND1 is specifically expressed in fibers but not in vessels in Arabidopsis inflorescence stems, and it functions redundantly with NST1 to regulate secondary wall biosynthesis in fibers, suggesting that other SND1 homologs are involved in regulation of secondary wall biosynthesis in the vessels of stems (Zhong et al., 2006). RT-PCR analysis of the expression of SND1 homologs in laser-dissected interfascicular fibers, xylem, and pith cells from inflorescence stems revealed that VND6 and VND7 were expressed in the xylem but not in interfascicular fibers or pith cells in Arabidopsis inflorescence stems (Figure 13A). Further gene expression analysis in transgenic plants expressing the GUS reporter gene fused with the VND6 and VND7 genes, including a 3-kb 5′ upstream sequence, the entire exon and intron region, and a 2-kb 3′ downstream sequence, confirmed their expression in xylem but not in interfascicular fibers of inflorescence stems. Detailed examination of their expression in the xylem of stems revealed that while no expression of VND6 and VND7 was detected in xylary fibers, VND6 was specifically expressed in the vessels of metaxylem (Figures 13B and 13C), and VND7 was expressed in the vessels of both protoxylem and metaxylem (Figures 13E and 13F). It was previously reported that VND6 and VND7 were expressed in metaxylem and protoxylem, respectively, in primary roots (Kubo et al., 2005). VND7 was also found to be expressed in the developing vessels of the secondary xylem in roots undergoing secondary growth (Figure 13D). VND6 expression was not detected in the secondary xylem of roots. These results suggest that, in Arabidopsis inflorescence stems, SND1 and NST1 are master switches activating secondary wall biosynthesis in fibers, whereas VND6 and VND7 are responsible for activating secondary wall biosynthesis in vessels.

Figure 13.
Expression Patterns of the VND6 and VND7 Genes in Arabidopsis Inflorescence Stems.

DISCUSSION

The developmental program of secondary wall biosynthesis is activated at the end of cell elongation in some specialized cells, such as fibers and tracheary elements (Ye et al., 2002). Because fibers and tracheary elements are the principal components of wood, which is an important raw material for our daily lives, it is imperative to investigate the molecular mechanisms underlying the activation of the secondary wall biosynthetic program during wood formation. Previous studies have discovered that the secondary wall–associated NAC genes, such as SND1, NST1, NST2, VND6, and VND7, are master switches activating secondary wall biosynthesis, which opens an unprecedented avenue to uncover the transcriptional network regulating the secondary wall biosynthetic program (Zhong and Ye, 2007). Our findings that the SND1-regulated transcription factors are required for secondary wall biosynthesis and that three of them are direct targets of SND1 and its close homologs mark another important step toward our dissection of the transcriptional network regulating secondary wall biosynthesis.

SND3, MYB103, and KNAT7 Are Direct Targets of SND1

We have found that SND1 is able to directly activate the expression of SND3, MYB103, and KNAT7 without new protein synthesis, indicating that these three transcription factors are direct targets of SND1. This finding is consistent with the expression data, showing that their expression is regulated by SND1 and they are expressed in the same cell types as SND1. In addition, dominant repression of SND3, MYB103, and KNAT7 leads to a reduction in the secondary wall thickening of fibers. These results provide direct evidence that SND3, MYB103, and KNAT7, together with the previously characterized MYB46, are part of the SND1-mediated transcriptional network regulating secondary wall biosynthesis.

Although overexpression of MYB46 leads to activation of the entire secondary wall biosynthetic program, including the biosynthesis of cellulose, xylan, and lignin (Zhong et al., 2007a), overexpression of SND3, MYB103, and KNAT7 was unable to induce ectopic secondary wall deposition in transgenic plants. Based on the observations that dominant repression of SND3, MYB103, and KNAT7 causes a reduction in secondary wall thickening (Figure 7) and that overexpression of SND3 and MYB103 results in an increase in secondary wall thickening (Figure 9), it is tempting to propose that SND3, MYB103, and KNAT7 are part of a transcriptional complex required for activation of secondary wall biosynthetic genes. Dominant repression or overexpression of one of the components in the complex may lead to a reduction or an increase, respectively, in the activity of the transcriptional complex, thus resulting in an alteration in the level of activation of secondary wall biosynthetic genes. Because activation of the secondary wall biosynthetic program may require all the components in the transcriptional complex, expression of one transcription factor in the absence of other components is thus insufficient to induce ectopic secondary wall deposition. The involvement of a transcriptional complex consisting of multiple transcription factors synergistically binding to the promoters of target genes to activate their expression has been considered to be a general characteristic of eukaryotic transcriptional regulation (Ogata et al., 2003). Although it is currently unclear how SND3, MYB103, and KNAT7 function in the transcriptional regulation of secondary wall biosynthesis, identification of SND3, MYB103, and KNAT7 as direct targets of SND1 moves us a step closer to elucidating the cascade of transcription factors involved in regulation of secondary wall biosynthesis.

Transcriptional Network Regulating the Secondary Wall Biosynthetic Program

We have demonstrated that SND1 regulates the expression of a number of transcription factors that are important for secondary wall biosynthesis, and among them, SND3, MYB103, and KNAT7 together with MYB46 are direct targets of SND1 (Figures 1 and and11).11). These findings support the hypothesis that a transcription network comprising a hierarchy of transcription factors is involved in regulation of secondary wall biosynthesis (Figure 14). In this network, SND1 acts as a master switch at the top because it is able to activate the entire developmental program of secondary wall biosynthesis (Zhong et al., 2006). MYB46, a direct target of SND1, apparently also functions as a key regulator albeit at another level since it is also capable of activating the whole secondary wall biosynthetic program (Zhong et al., 2007a). However, the SND1-mediated transcriptional cascade is not solely through MYB46; SND1 also directly activates the expression of at least three other transcription factors, SND3, MYB103, and KNAT7. Therefore, SND1 exerts its functions through activating several direct target genes in the transcriptional cascade leading to secondary wall biosynthesis (Figure 14).

Figure 14.
Diagram of the Transcriptional Network Regulating Secondary Wall Biosynthesis in Different Cell Types.

The other SND1-regulated transcription factors appear not to be direct targets of SND1 and are likely positioned further downstream in the transcriptional network (Figure 14). Overexpression of these transcription factors does not induce ectopic deposition of secondary walls except that MYB85 overexpression results in ectopic lignin deposition (Figure 9). It is likely that some of these transcription factors are involved in enhancing or fine-tuning the level of expression of secondary wall biosynthetic genes, whereas others may be part of a transcriptional complex directly involved in regulating the expression of genes in a particular biosynthetic pathway. This possibility is consistent with the finding that, whereas SND2 is able to induce the expression of cellulose biosynthetic genes, MYB85 can induce the expression of lignin biosynthetic genes. In addition, MYB52 and MYB54 appear to slightly induce the expression of genes in all three major secondary wall biosynthetic pathways. Although individually these SND1-regulated transcription factors are not sufficient to induce ectopic deposition of secondary walls, some of them are likely involved in the normal regulation of secondary wall biosynthesis because dominant repression of SND2, MYB85, MYB52, MYB54, and MYB69 causes a significant reduction in secondary wall thickening in fibers (Figures 7 and and8).8). Elucidation of the positions of these transcription factors in the SND1-mediated transcriptional cascade will undoubtedly contribute to our understanding of how the expression of secondary wall biosynthetic genes is coordinately regulated.

It is intriguing to find that in addition to SND1 and its homologs, many transcription factors are involved in the regulation of secondary wall biosynthesis. Although some of these transcription factors are shown to be required for secondary wall biosynthesis, we are still far from understanding how they function in the transcriptional network and why they are all needed. Considering that a few hundred genes are probably involved in the biosynthesis of secondary walls in wood formation (Mellerowicz et al., 2001), it is reasonable to predict that many transcription factors are required to coordinately regulate the expression of these secondary wall biosynthetic genes. It is possible that the regulation of individual biosynthetic pathways of secondary wall components, such cellulose, xylan, and lignin, may require common and unique transcription factors. It has been suggested that some MYB (Patzlaff et al., 2003a, 2003b; Goicoechea et al., 2005) and LIM (Kawaoka et al., 2000) genes are specifically involved in regulating the expression of lignin biosynthetic genes. Among the SND1-regulated transcription factors, MYB85 overexpression leads to specific induction of lignin biosynthetic genes and ectopic lignin deposition (Figures 9 and and10),10), suggesting that it is involved in regulation of lignin biosynthesis. This finding is consistent with a previous report showing that pine MYB1, a homolog of MYB85, is expressed in secondary xylem of wood and its recombinant protein is able to bind to the AC element commonly present in the promoters of monolignol biosynthetic genes (Patzlaff et al., 2003b). Other SND1-regulated transcription factors, such as SND2, SND3, and MYB103, may play a role in regulation of cellulose biosynthesis, since they are able to induce the expression of cellulose biosynthetic genes when overexpressed (Figure 10). It is anticipated that further studies of secondary wall–associated transcription factors will reveal how the biosynthetic pathways of secondary wall components are coordinately regulated. It should be noted that although SND2, SND3, MYB103, and MYB85 differentially regulate the expression of secondary wall biosynthetic genes, dominant repression of these transcription factors all leads to a reduction in the amount of cellulose and xylan (Table 2). This reduction could be due to a defect in the biosynthesis of one wall component, which may result in impediment of the overall secondary wall biosynthesis and assembly. It has been shown that a reduction in any one of the three major secondary wall components (i.e., cellulose, xylan, and lignin) can cause a decrease in the overall secondary wall biosynthesis (Zhong et al., 1998, 2005; Taylor et al., 2004), suggesting that the biosynthesis of secondary wall components is a highly coordinated process.

SND1 and Its Close Homologs, NST1, NST2, VND6, and VND7, Activate the Same Set of Direct Targets

We have demonstrated that NST1, NST2, VND6, and VND7, close homologs of SND1, are able to activate the same set of direct target genes as SND1, including MYB46, SND3, MYB103, and KNAT7 (Figure 12). This finding suggests that these NACs execute their functions in the same way as SND1. Because these secondary wall NACs are expressed in different types of secondary wall–containing cells and all of them are able to turn on the entire secondary wall biosynthetic program (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2007b), we propose that they are functional homologs acting as master switches that regulate secondary wall biosynthesis in different cell types (Figure 14). Whereas SND1 and NST1 activate the secondary wall biosynthetic program in fibers, VND6 and VND7 specifically regulate secondary wall biosynthesis in vessels. NST1 and NST2 act together in regulating secondary wall biosynthesis in endothecium of anthers. It is apparent that plants evolved to have cell type–specific, functionally redundant NAC genes to regulate secondary wall biosynthesis in different types of secondary wall–containing cells.

Close homologs of SND1 and SND1-regulated transcription factors are found in tree species, such as poplar (Populus trichocarpa), pine, eucalyptus, and spruce (Picea glauca), and some of them were shown to be expressed in developing wood (Patzlaff et al., 2003a, 2003b; Karpinska et al., 2004; Goicoechea et al., 2005; Bedon et al., 2007), suggesting that they may also regulate secondary wall biosynthesis during wood formation. Therefore, it is likely that the transcriptional network regulating secondary wall biosynthesis is conserved between herbaceous Arabidopsis and woody tree species. Further studies on the transcriptional regulation of secondary wall biosynthesis in Arabidopsis will undoubtedly provide a foundation for elucidating the molecular mechanisms underlying wood formation in tree species. Knowledge gained from such studies will potentially allow us to manipulate the biosynthetic pathways of cellulose, xylan, and lignin by altering the expression of certain transcription factor genes.

METHODS

Gene Expression Analysis

For analysis of gene expression in different cell types, interfascicular fiber cells, xylem cells, and pith cells were isolated from inflorescence stems of 6-week-old Arabidopsis thaliana plants using the PALM microlaser system (PALM Microlaser Technologies), and the isolated cells were used for RNA isolation and subsequent gene expression analysis as described (Zhong et al., 2006).

For analysis of gene expression in different organs, total RNA was isolated from Arabidopsis seedlings, leaves, stems, flowers, and roots using a Qiagen RNA isolation kit. The seedlings used were 2 weeks old. Mature leaves were from 6-week-old plants. Mature roots were from 8-week-old plants. Stems from 6-week-old plants were divided into top, middle, and bottom parts, which represent the rapidly elongating internodes, internodes near cessation of elongation, and nonelongating internodes, respectively.

For quantitative PCR analysis, total RNA was treated with DNase I and used for first-strand cDNA synthesis. The first-strand cDNA was then used as template for real-time quantitative PCR analysis with the QuantiTect SYBR Green PCR Kit (Clontech). The relative mRNA levels were determined by normalizing the PCR threshold cycle number of each gene with that of the EF1α reference gene. The expression level of each gene in the wild-type control or in the sample with the lowest expression level was set to 1, and the data reported were the average of three biological replicates.

GUS Reporter Gene Analysis

The developmental expression patterns of the transcription factors were studied using the GUS reporter gene. Genomic DNA fragments containing a 3-kb 5′ upstream sequence, the entire coding region, and a 2-kb 3′downstream sequence were used to ensure the sequences used contained all the cis-elements required for the endogenous gene expression. The GUS reporter gene was inserted in frame right before the stop codon of the transcription factor genes and then cloned into the binary vector pBI101 (Clontech) to create the GUS reporter constructs. The constructs were transformed into wild-type Arabidopsis plants by means of Agrobacterium tumefaciens–mediated transformation. The first generation of transgenic plants was selected on kanamycin and used for expression analysis of the GUS reporter gene as described previously (Zhong et al., 2005).

In Situ mRNA Localization

Wild-type Arabidopsis inflorescence stems were fixed in 2.5% formaldehyde and 0.5% glutaraldehyde and embedded in paraffin. Sections (10 μm thick) were cut, mounted onto slides, and hybridized with digoxigenin-labeled gene-specific antisense RNA probes synthesized using the DIG RNA labeling mix (Roche). The hybridization signals were detected with alkaline phosphatase-conjugated antibodies against digoxigenin and subsequent color development with alkaline phosphatase substrates. As a negative control, sense RNA probes were hybridized with the stem sections, and no signals were observed under the same hybridization and detection conditions.

Subcellular Localization of Transcription Factors

The full-length cDNAs of transcription factors were fused in frame at the C terminus with the YFP cDNA and ligated between the CaMV 35S promoter and the nopaline synthase terminator in pBI221 (Clontech). The constructs were introduced into Arabidopsis leaf protoplasts by polyethylene glycol–mediated transfection (Sheen, 2001). After a 20-h incubation period, the transfected protoplasts were examined for yellow fluorescence signals using a Leica TCs SP2 spectral confocal microscope (Leica Microsystems). Images were saved and processed with Adobe Photoshop Version 7.0 (Adobe Systems).

Transcriptional Activation Analysis in Yeast

The full-length cDNAs of transcription factors were fused in frame with the GAL4 DNA binding domain in the pAS2-1 vector (Clontech). The constructs were transformed into the yeast strain CG-1945 containing the His3 and LacZ reporter genes. The transformed yeast cells were grown on SD (synthetic defined) plates with or without histidine and subjected to β-galactosidase activity assay.

Dominant Repression and Overexpression of Transcription Factors

The transcription factor dominant repression constructs were created by fusing the full-length cDNAs in frame with the dominant EAR repression sequence (Hiratsu et al., 2004), which was ligated downstream of the CaMV 35S promoter in pBI121 (Clontech). The transcription factor overexpression constructs were produced by ligating the full-length cDNAs downstream of the CaMV 35S promoter in pBI121. The constructs were introduced into wild-type Arabidopsis plants by Agrobacterium-mediated transformation (Bechtold and Bouchez, 1994). Transgenic plants were selected on kanamycin (50 mg/L), and the first generation was used for examination of phenotypes.

Microscopy

The lower part of inflorescence stems was fixed in 2% formaldehyde and embedded in Low Viscosity (Spurr's) resin (Electron Microscopy Sciences) as described (Burk et al., 2006). One-micrometer-thick sections were cut with a microtome and stained with toluidine blue for light microscopy. For transmission electron microscopy, 85-nm-thick sections were cut, poststained with uranyl acetate and lead citrate, and observed using a Zeiss EM 902A transmission electron microscope (Carl Zeiss). Wall thickness was measured in metaxylem vessels and in the interfascicular fibers next to the endodermis. For lignin staining, 50-μm-thick sections of stems were stained with phloroglucinol-HCl, which was shown as bright red color. For each repression and overexpression construct, at least 10 transgenic plants with the most severe phenotypes were examined.

Cell Wall Composition Analysis

Inflorescence stems were ground into a fine powder in liquid nitrogen and extracted in 70% ethanol at 70°C. The resulting cell wall residues were dried in a vacuum oven at 60°C and used for analysis of monosaccharide composition following the procedure described by Hoebler et al. (1989). Briefly, cell walls were incubated with 70% sulfuric acid at 37°C for 60 min, followed by addition of inositol as the internal standard and dilution with water to 2 n sulfuric acid. After heating for 120 min at 100°C, the solution was cooled and treated with 25% ammonium solution. After reduction with sodium borohydride in dimethyl sulfoxide, the solution was heated for 90 min at 40°C, followed by sequential treatment with glacial acetic acid, acetic anhydride, 1-methylimidazole, dichloromethane, and water. The organic layer containing the alditol acetates of the hydrolyzed cell wall sugars was washed three times with water, and sugars were analyzed on an Agilent 6890N gas-liquid chromatography equipped with a 30 m × 0.25 mm (i.d.) silica capillary column DB 225 (Alltech Associates).

Transactivation Analysis in Arabidopsis Leaf Protoplasts

For testing the ability of transcription factors to induce the expression of secondary wall biosynthetic genes, the full-length cDNAs of transcription factors were ligated between the CaMV 35S promoter and the nopaline synthase terminator in the pBI221 vector (Clontech) to create the effector constructs. The reporter constructs were created by placing 1-kb promoter fragments (located between −1 to −1000 bp relative to the start codon) of CesA8, IRX9, and 4CL1 genes in front of the GUS reporter gene in pBI221 (Clontech). Arabidopsis leaf protoplasts were cotransfected with the reporter and effector constructs together with a construct containing the firefly luciferase gene driven by the CaMV 35S promoter for determination of the transfection efficiency. After a 20-h incubation, transfected protoplasts were lysed and the soluble extracts were used for analysis of GUS and luciferase activities as described (Gampala et al., 2001) or for real-time quantitative PCR analysis of the effector mRNA level. The GUS activity was normalized against the luciferase activity in each transfection, and the data were the average of three biological replicates.

Estrogen-Inducible System for Identification of Direct Targets of SND1

The full-length cDNA of SND1 was fused to the C terminus of the regulatory region of HER (Zuo et al., 2000) and ligated between the CaMV 35S promoter and the nopaline synthase terminator in the pBI221 vector (Clontech). The SND1-HER expression construct was introduced into Arabidopsis leaf protoplasts alone or together with the MYB46P:GUS construct (Sheen, 2001). To activate SND1, the protoplasts were treated with 2 μM estradiol (Sigma-Aldrich) for 6 h. The control protoplasts were mock-treated with the same concentration (0.1%) of ethanol used to dissolve estradiol. To inhibit new protein synthesis, the protein synthesis inhibitor cycloheximide (2 μM) was added 30 min before addition of estradiol. Under this condition, new protein synthesis was completely inhibited, as tested by the GUS reporter activity analysis. After treatments, the protoplasts were harvested and subjected to total RNA isolation and subsequent quantitative RT-PCR analysis or used for GUS activity analysis. The expression level of each gene in the control protoplasts without addition of estradiol was set to 1, and the data were the average of three biological replicates.

EMSA

The NAC domain of SND1 fused with MBP was used in an EMSA with the promoter fragments (−600 to −1 from the start codon) of SND3, MYB103, and KNAT7 (Zhong et al., 2007a). The promoter fragments were PCR-amplified and biotin-labeled at the 3′ end using the Biotin 3′ end DNA labeling kit (Pierce). The biotin-labeled DNA fragments were incubated for 30 min with 100 ng of SND1-MBP in the binding buffer [10 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl2, 0.05% Nonidet P-40, and 100 ng/μL poly(dI-dC)]. The SND1-bound DNA fragments were separated from the unbound ones by polyacrylamide gel electrophoresis. The DNA was electroblotted onto nitrocellulose membrane and detected by the chemiluminescent method.

Statistical Analysis

The data in the experiments of quantitative PCR, transcriptional activation of the GUS reporter gene, and measurement of cell wall thickness were subjected to statistical analysis using the Student's t test program (http://www.graphpad.com/quickcalcs/ttest1.cfm). The quantitative differences between two groups of data for comparison in all of these experiments were shown to be statistically significant (P < 0.001).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: MYB46 (At5g12870), SND1 (At1g32770), SND2 (At4g28500), SND3 (At1g28470), MYB20 (At1g66230), MYB85 (At4g22680), MYB103 (At1g63910), MYB42 (At4g12350), MYB43 (At5g16600), MYB20 (At1g66230), MYB52 (At1g17950), MYB54 (At1g73410), MYB69 (At4g33450), KNAT7 (At1g62990), NST1 (At2g46770), NST2 (At3g61910), VND6 (At5g62380), VND7 (At1g71930), ATHB8 (4g32880), and IFL1 (At5g60690).

Acknowledgments

We thank N.H. Chua for providing the pER8 vector and the editor and reviewers for their constructive suggestions and comments. This work was supported by grants from the U.S. Department of Energy-Bioscience Division (DE-FG02-03ER15415) and the National Science Foundation (ISO-0744170).

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instruction for Authors (www.plantcell.org) is: Zheng-Hua Ye (zhye/at/plantbio.uga.edu).

www.plantcell.org/cgi/doi/10.1105/tpc.108.061325

References

  • Baudry, A., Heim, M.A., Dubreucq, B., Caboche, M., Weisshaar, B., and Lepiniec, L. (2004). TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 39 366–380. [PubMed]
  • Bechtold, N., and Bouchez, D. (1994). In planta Agrobacterium-mediated transformation of adult Arabidopsis thalianaplants by vacuum infiltration. In Gene Transfer to Plants, I. Potrykus, and G. Spangenberg, eds (Berlin: Springer-Verlag), pp. 19–23.
  • Bedon, F., Grima-Pettenati, J., and Mackay, J. (2007). Conifer R2R3-MYB transcription factors: Sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca). BMC Plant Biol. 7 17. [PMC free article] [PubMed]
  • Boerjan, W., Ralph, J., and Baucher, M. (2003). Lignin biosynthesis. Annu. Rev. Plant Biol. 54 519–546. [PubMed]
  • Burk, D.H., Zhong, R., Morrison, W.H.I.I.I., and Ye, Z.-H. (2006). Disruption of cortical microtubules by overexpression of green fluorescent protein-tagged α-tubulin 6 causes a marked reduction in cell wall synthesis. J. Integr. Plant Biol. 48 85–98.
  • Gampala, S.S., Hagenbeek, D., and Rock, C.D. (2001). Functional interactions of lanthanum and phospholipase D with the abscisic acid signaling effectors VP1 and ABI1-1 in rice protoplasts. J. Biol. Chem. 276 9855–9860. [PubMed]
  • Goicoechea, M., Lacombe, E., Legay, S., Mihaljevic, S., Rech, P., Jauneau, A., Lapierre, C., Pollet, B., Verhaegen, D., Chaubet-Gigot, N., and Grima-Pettenati, J. (2005). EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant J. 43 553–567. [PubMed]
  • Hiratsu, K., Mitsuda, N., Matsui, K., and Ohme-Takagi, M. (2004). Identification of the minimal repression domain of SUPERMAN shows that the DLELRL hexapeptide is both necessary and sufficient for repression of transcription in Arabidopsis. Biochem. Biophys. Res. Commun. 321 172–178. [PubMed]
  • Hoebler, C., Barry, J.L., David, A., and Delort-Laval, J. (1989). Rapid acid-hydrolysis of plant cell wall polysaccharides and simplified quantitative determination of their neutral monosaccharides by gas-liquid chromatography. J. Agric. Food Chem. 37 360–367.
  • Karpinska, B., Karlsson, M., Srivastava, M., Stenberg, A., Schrader, J., Sterky, F., Bhalerao, R., and Wingsle, G. (2004). MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen. Plant Mol. Biol. 56 255–270. [PubMed]
  • Kawaoka, A., Kaothien, P., Yoshida, K., Endo, S., Yamada, K., and Ebinuma, H. (2000). Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis. Plant J. 22 289–301. [PubMed]
  • Ko, J.H., Yang, S.H., Park, A.H., Lerouxel, O., and Han, K.H. (2007). ANAC012, a member of the plant-specific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana. Plant J. 50 1035–1048. [PubMed]
  • Kubo, M., Udagawa, M., Nishikubo, N., Horiguchi, G., Yamaguchi, M., Ito, J., Mimura, T., Fukuda, H., and Demura, T. (2005). Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 19 1855–1860. [PMC free article] [PubMed]
  • Mellerowicz, E.J., Baucher, M., Sundberg, B., and Boerjan, W. (2001). Unravelling cell wall formation in the woody dicot stem. Plant Mol. Biol. 47 239–274. [PubMed]
  • Mitsuda, N., Iwase, A., Yamamoto, H., Yoshida, M., Seki, M., Shinozaki, K., and Ohme-Takagi, M. (2007). NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19 270–280. [PMC free article] [PubMed]
  • Mitsuda, N., Seki, M., Shinozaki, K., and Ohme-Takagi, M. (2005). The NAC transcription factors NST1 and NST2 of Arabidopsis regulates secondary wall thickening and are required for anther dehiscence. Plant Cell 17 2993–3006. [PMC free article] [PubMed]
  • Ogata, K., Sato, K., and Tahirov, T. (2003). Eukaryotic transcriptional regulatory complexes: cooperative from near and afar. Curr. Opin. Struct. Biol. 13 40–48. [PubMed]
  • Patzlaff, A., McInnis, S., Courtenay, A., Surman, C., Newman, L.J., Smith, C., Bevan, M.W., Mansfield, S., Whetten, R.W., Sederoff, R.R., and Campbell, M.M. (2003. a). Characterization of a pine MYB that regulates lignification. Plant J. 36 743–754. [PubMed]
  • Patzlaff, A., Newman, L.J., Dubos, C., Whetten, R.W., Smith, C., McInnis, S., Bevan, M.W., Sederoff, R.R., and Campbell, M.M. (2003. b). Characterization of PtMYB1, an R2R3-MYB from pine xylem. Plant Mol. Biol. 53 597–608. [PubMed]
  • Pena, M.J., Zhong, R., Zhou, G.-K., Richardson, E.A., O'Neill, M.A., Darvill, A.G., York, W.S., and Ye, Z.-H. (2007). Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell 19 549–563. [PMC free article] [PubMed]
  • Raven, P.H., Evert, R.F., and Eichhorn, S.E. (1999). Biology of Plants, 6th ed. (New York: W.H. Freeman and Company).
  • Sablowski, R.W.M., and Meyerowitz, E.M. (1998). A homolog of NO APICAL MERISTEM is an immediate target of the floral homeotic genes APETALA3/PISTILLATA. Cell 92 93–103. [PubMed]
  • Sheen, J. (2001). Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 127 1466–1475. [PMC free article] [PubMed]
  • Taylor, N.G., Gardiner, J.C., Whiteman, R., and Turner, S.R. (2004). Cellulose synthesis in the Arabidopsis secondary cell wall. Cellulose 11 329–338.
  • Wagner, D., Sablowski, R.W.M., and Meyerowitz, E.M. (1999). Transcriptional activation of APETALA1 by LEAFY. Science 285 582–584. [PubMed]
  • Yang, C., Xu, Z., Song, J., Conner, K., Vizcay-Barrena, G., and Wilson, Z.A. (2007). Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in the endothecium and is essential for anther dehiscence. Plant Cell 19 534–548. [PMC free article] [PubMed]
  • Ye, Z.-H., Freshour, G., Hahn, M.G., Burk, D.H., and Zhong, R. (2002). Vascular development in Arabidopsis. Int. Rev. Cytol. 220 225–256. [PubMed]
  • Zhong, R., Demura, T., and Ye, Z.-H. (2006). SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 18 3158–3170. [PMC free article] [PubMed]
  • Zhong, R., Morrison, W.H., Negrel, J., and Ye, Z.-H. (1998). Dual methylation pathways in lignin biosynthesis. Plant Cell 10 2033–2046. [PMC free article] [PubMed]
  • Zhong, R., Pena, M.J., Zhou, G.-K., Nairn, C.J., Wood-Jones, A., Richardson, E.A., Morrison, W.H., Darvill, A.G., York, W.S., and Ye, Z.-H. (2005). Arabidopsis Fragile Fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis. Plant Cell 17 3390–3408. [PMC free article] [PubMed]
  • Zhong, R., Richardson, E.A., and Ye, Z.-H. (2007. a). The MYB46 transcription facgor is a direct target of SND1 and regulates secondary wall biosynthesis in Arabidopsis. Plant Cell 19 2776–2792. [PMC free article] [PubMed]
  • Zhong, R., Richardson, E.A., and Ye, Z.-H. (2007. b). Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis. Planta 225 1603–1611. [PubMed]
  • Zhong, R., and Ye, Z.-H. (2007). Regulation of cell wall biosynthesis. Curr. Opin. Plant Biol. 10 564–572. [PubMed]
  • Zuo, J., Niu, Q.-W., and Chua, N.-H. (2000). An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24 265–273. [PubMed]

Articles from The Plant Cell are provided here courtesy of American Society of Plant Biologists

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...