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Plant Physiol. Nov 2010; 154(3): 1428–1438.
Published online Aug 31, 2010. doi:  10.1104/pp.110.162735
PMCID: PMC2971618

MYB75 Functions in Regulation of Secondary Cell Wall Formation in the Arabidopsis Inflorescence Stem1,[W]

Abstract

Deposition of lignified secondary cell walls in plants involves a major commitment of carbon skeletons in both the form of polysaccharides and phenylpropanoid constituents. This process is spatially and temporally regulated by transcription factors, including a number of MYB family transcription factors. MYB75, also called PRODUCTION OF ANTHOCYANIN PIGMENT1, is a known regulator of the anthocyanin branch of the phenylpropanoid pathway in Arabidopsis (Arabidopsis thaliana), but how this regulation might impact other aspects of carbon metabolism is unclear. We established that a loss-of-function mutation in MYB75 (myb75-1) results in increased cell wall thickness in xylary and interfascicular fibers within the inflorescence stem. The total lignin content and S/G ratio of the lignin monomers were also affected. Transcript profiles from the myb75-1 inflorescence stem revealed marked up-regulation in the expression of a suite of genes associated with lignin biosynthesis and cellulose deposition, as well as cell wall modifying proteins and genes involved in photosynthesis and carbon assimilation. These patterns suggest that MYB75 acts as a repressor of the lignin branch of the phenylpropanoid pathway. Since MYB75 physically interacts with another secondary cell wall regulator, the KNOX transcription factor KNAT7, these regulatory proteins may form functional complexes that contribute to the regulation of secondary cell wall deposition in the Arabidopsis inflorescence stem and that integrate the metabolic flux through the lignin, flavonoid, and polysaccharide pathways.

The allocation of carbon to different metabolic pathways in plants is a central feature of growth and development in plants (Bloom et al., 1985; Smith and Stitt, 2007). However, the molecular underpinnings regulating the sensing and signaling system(s) that are anticipated to link carbon assimilation to particular metabolic pathways have yet to be identified. Plant secondary cell walls represent a major carbon sink in plants (Brown et al., 2005; Pauly and Keegstra, 2008), and many proteins catalyzing deposition of secondary cell wall polysaccharides and lignin have been characterized (Zhong and Ye, 2007). For example, the synthesis of cellulose at the plasma membrane during both primary and secondary cell wall synthesis has been shown to be dependent on distinct Cellulose Synthase A (CESA) proteins. The deposition of cellulose in the secondary cell wall involves CESA4, 7, and 8 in Arabidopsis (Arabidopsis thaliana; Somerville et al., 2004; Brown et al., 2005; Persson et al., 2005), while primary cell wall cellulose formation is orchestrated by CESA3, 6, and 9 (Joshi and Mansfield, 2007). Additional polysaccharide components are synthesized by enzymes involved in hemicellulose production, primarily of the glycosyltransferase families (Persson et al., 2007; Brown et al., 2009). Secondary cell wall lignin biosynthesis requires the activity of both core phenylpropanoid pathway enzymes, such as l-Phe ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H), as well as enzymes more directly engaged with lignin biosynthesis, including caffeoyl-CoA O-methyltransferase (CCoAOMT; Do et al., 2007), ferulate 5-hydroxylase (F5H; Meyer et al., 1998), cinnamoyl-CoA reductase 1 (CCR1; Mir Derikvand et al., 2008), and cinnamoyl alcohol dehydrogenase (CAD; Sibout et al., 2005). The regulation of the corresponding genes is, in part, facilitated by a number of transcription factors that have been shown to regulate secondary cell wall formation in Arabidopsis and other plants and include members of the NAC domain (Kubo et al., 2005) and MYB families of transcription factors (Zhong and Ye, 2007, 2009).

Lignin biosynthesis represents a major terminal product of phenylpropanoid metabolism, a multibranched system of reactions that converts the carbon skeleton of l-Phe into a wide variety of phenolic plant metabolites. Another major branch of phenylpropanoid metabolism generates flavonoids, a diverse group of phenolics that includes flavonols, isoflavonoids, leucoanthocyanidins (tannins), and anthocyanins. Various transcription factors, including a number of MYB family proteins, have been shown to directly or indirectly regulate the activity of genes encoding enzymes involved in specific branches of the phenylpropanoid pathway (Davies and Schwinn, 2003; Dubos et al., 2005; Gonzalez et al., 2008; Zhong and Ye, 2009), but less is known about the transcriptional regulation of carbon partitioning across different branches of phenylpropanoid metabolism by individual transcription factors.

MYB75 (At1g56650), also known as PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), was earlier identified as a positive regulator of anthocyanin biosynthesis in Arabidopsis, based on the strong accumulation of anthocyanins in activation-tagged seedlings overexpressing the MYB75/PAP1 gene (Borevitz et al., 2000; Pourtau et al., 2006; Gonzalez et al., 2008). In addition to this impact of PAP1 overexpression on anthocyanin production, the activity of PAP1/MYB75 has been found to influence senescence (Bernhardt et al., 2003), Suc signaling (Teng et al., 2005), and lignin deposition in Arabidopsis (Borevitz et al., 2000). A loss-of-function MYB75 allele (myb75-1) harbors mutations inside the DNA-binding domain of the encoded protein (Teng et al., 2005), and myb75-1 plants show only a very weak anthocyanin accumulation response to elevated levels of Suc. The expression of the MYB75 gene is also responsive to a number of abiotic factors, including nitrogen deficiency, phosphate starvation, high temperature, and high light intensity (Misson et al., 2005; Tohge et al., 2005a, 2005b; Vanderauwera et al., 2005; Lea et al., 2007; Morcuende et al., 2007; Muller et al., 2007; Rowan et al., 2009), and this spectrum of metabolic and environmental sensitivities suggests that, in addition to modulating anthocyanin biosynthesis, MYB75 might play a more general role in regulating cellular metabolism.

Since the secondary cell wall represents one of the main carbon sinks in plants and its formation requires coordination of metabolic fluxes through both polysaccharide and phenylpropanoid biosynthetic pathways, we postulated that transcription factors, such as MYB75, could be involved in the allocation of photosynthetic carbon between these two dissimilar, yet highly important, end-products. The Arabidopsis inflorescence stem has a high developmental commitment to secondary wall formation, which makes this tissue particularly well suited to study the regulation of secondary cell wall by specific transcription factors. We show here that while MYB75 overexpression results in general up-regulation of anthocyanin accumulation, as previously reported (Borevitz et al., 2000), a myb75 loss-of-function mutant displays an overall increase in secondary cell wall formation in the inflorescence stem, accompanied by elevated expression of genes encoding enzymes integral to the biosynthesis of lignin and secondary cell wall polysaccharides. This suggests that MYB75 may be acting as a repressor of the lignin branch of the phenylpropanoid pathway.

RESULTS

MYB75 Expression in Wild-Type and Gain- and Loss-of-Function Mutants

To study the role of MYB75 in regulating cell wall biosynthesis and phenylpropanoid metabolism, we compared an activation-tagged MYB75 gain-of-function mutant (Supplemental Fig. S1A; pap1-D), referred to here as MYB75(o/x), with a loss-of-function transposon-tagged Ds insertion mutant (myb75-1; Supplemental Fig. S1A). Plants homozygous for both MYB75(o/x) and myb75-1 alleles were identified by PCR-aided genotyping, and the abundance of MYB75 transcripts was assessed using quantitative real-time PCR (qRT-PCR). As expected, MYB75 transcript abundance was higher in the overexpression line than in wild-type plants, while negligible MYB75 expression was detected in the loss-of-function mutant (Supplemental Fig. S1B). MYB75(o/x) seedlings showed elevated levels of anthocyanin accumulation under normal growth conditions, as reported earlier (Borevitz et al., 2000), while the anthocyanin content was slightly reduced from wild-type levels in myb75-1 seedlings (Supplemental Fig. S1C), a pattern that is consistent with MYB75 acting as a positive regulator of this branch of the phenylpropanoid pathway. No visible difference in growth or inflorescence stem morphology was observed when the mutants were compared with their corresponding wild-type controls (Supplemental Fig. S1D) and grown under normal growth conditions.

When MYB75 expression was assessed in various tissues of 6-week-old plants by qRT-PCR, transcript levels were found to be highest in the lower part of the inflorescence stem (Fig. 1A). Lower levels of MYB75 transcripts could be detected in flowers, leaves, and siliques, but none could be detected in roots (Fig. 1A).

Figure 1.
Expression pattern of the MYB75 in mature Arabidopsis plant and stems. A, qPCR analysis showing the predominant expression of MYB75 in stems. The expression level of MYB75 is relative to actin and is expressed in arbitrary units. Error bars represent ...

To obtain a spatially and developmentally better resolved picture of the expression of MYB75, transgenic Arabidopsis plants containing a MYB75pro::GUS transgene were examined using histochemical staining for GUS activity (Fig. 1B). It had been previously reported that GUS expression is seen in most parts of MYB75pro::GUS seedlings (Gonzalez et al., 2008), but in 6-week-old plants, we found that GUS activity was primarily localized in the vasculature of leaves and flowers (Fig. 1B, a and b) and in the epidermis of siliques (Fig. 1B, c), but not in roots (data not shown), a pattern consistent with the qRT-PCR data. Within the lower portion of the inflorescence stem, where the highest levels of MYB75 transcript had been detected, GUS activity was observed specifically in the cortex, vascular bundles, and fibers (Fig. 1B, d).

MYB75 Is Nuclear Localized and Acts as a Transcriptional Activator

We used an Arabidopsis protoplast transient expression system to assay the subcellular localization of a MYB75-yellow fluorescent protein (YFP) fusion, which was found to accumulate in the nucleus (Fig. 2A). A protoplast transfection system (Wang et al., 2007, 2008) was also used to assess the transcriptional repression or activation activity of MYB75. Cotransfection of a GAL4:GUS reporter construct with an effector construct containing the MYB75 open reading frame fused to the GAL4 DNA-binding domain (GD; Fig. 2B) revealed that MYB75 could weakly activate expression of the GUS reporter gene when recruited to the promoter region of the reporter gene by GD (Fig. 2B). To test the possibility that MYB75 might also be able to act as a transcriptional repressor, we coexpressed a construct containing the GUS gene driven by the 35S promoter of Cauliflower mosaic virus supplemented with both LexA and Gal4 DNA binding sites. When cotransfected with both the MYB75-GD effector construct and the transactivator LD-VP16, the MYB75-GD gene product failed to suppress activation of the GUS reporter by LD-VP16. As a positive control, we also coexpressed the target reporter with a KNAT7-GD construct that had been previously shown function as a repressor (E. Li, S. Wang, J.-G. Chen, and C.J. Douglas, unpublished data). As expected, KNAT7 expression strongly reduced GUS expression from the 35S/LexA/Gal4 promoter::GUS reporter (data not shown). Taken together, these data suggest that MYB75 may act as a weak transcriptional activator but not as a repressor.

Figure 2.
Nuclear localization and transcriptional activity of MYB75. A, Protoplast transfected with 35S:MYB75-YFP. Left, DIC image; middle, YFP channel; right, merged images. B, Effector and reporter constructs used in the transfection assays. C, Transcriptional ...

Loss of MYB75 Function Affects Secondary Cell Wall Structure and Composition

The RIKEN line pst16228 (myb75-1 loss of function) has a transposon inserted in the third exon of MYB75/PAP1 (Supplemental Fig. S1), and this Ds insertion event is tightly linked to the phenotype of myb75-1 (Teng et al., 2005). No other loss-of-function alleles appear to be available, but a MYB75/PAP1-RNA interference line has been reported to display an anthocyanin-deficient phenotype similar to that of pst16228 seedlings (Gonzalez et al., 2008), confirming that this pigment phenotype is due to loss of MYB75/PAP1 function.

When the basal portion of the inflorescence stem in myb75-1 plants was examined in Toluidine Blue-stained cross sections (Fig. 3A), and by transmission electron microscopy (TEM; Fig. 3B), the secondary cell walls of the interfascicular fibers appeared to be thicker, compared with wild-type plants, while no change in vessel wall thickness or cell morphology was apparent (Fig. 3, A and B). Measurements taken from TEM micrographs confirmed that the interfascicular fiber wall thickness was increased in myb75-1 plants, while little or no change was observed in vessel or xylary fiber wall thickness (Fig. 3C). No obvious differences were observed in the primary cell walls.

Figure 3.
Secondary wall thickening in fibers and vessels in myb75-1 plants. The bottom inflorescence stem of 8-week-old plants were used for examination of secondary walls in fibers and vessels. A, Toluidine Blue staining of the cross sections in mutants and the ...

To determine if these changes in interfascicular fiber wall thickness might be associated with changes in cell wall chemistry, we assayed the Klason lignin content in mature inflorescence stems of both loss-of-function and gain-of-function mutant plants. Klason lignin content was significantly greater in stems of myb75-1 plants but remained unaffected in the MYB75(o/x) genotype (Table I). Thioacidolysis was used to estimate the relative amounts of syringyl (S) and guaiacyl (G) monomers in the inflorescence stem lignin. This analysis revealed that the S/G monomer ratio was lower in myb75-1 plants compared to the wild type, due primarily to lower levels of S subunits released by thioacidolysis (Table II). In the MYB75(o/x) genotype, the S/G ratio was higher than in the wild type, due both to an increase in released S units and a decrease in released G units (Table II). When we assayed changes in cell wall carbohydrate content in both myb75-1 and MYB75(o/x) lines, no significant changes in Glc content were observed nor were significant differences detected in Gal, rhamnose, Man, or Ara content in either mutant (Table III).

Table I.
Lignin content in the lower stems of the wild type and mutants as determined by Klason analysis
Table II.
Lignin composition in the lower stems of the wild type and mutants as determined by thioacidolysis
Table III.
Cell wall composition in the lower stems of the wild type and mutants determined by Klason analysis

Loss of MYB75 Function Affects Expression of Genes Associated with Secondary Cell Wall Formation

Since manipulation of MYB75 function led to changes in lignin content, we used qRT-PCR to examine the expression of genes encoding enzymes associated with phenylpropanoid metabolism and lignin monomer biosynthesis. This gene expression analysis focused on the lower inflorescence stems of the myb75-1 and MYB75(o/x) lines and compared these with the respective wild-type backgrounds. Many of the genes examined were found to be up-regulated in the myb75-1 stems (Fig. 4A), while their expression was generally unaffected in the MYB75 overexpression background. We also assayed the expression of genes associated with cellulose and hemicellulose metabolism in secondary cell wall deposition or remodeling (Fig. 4B). While expression of primary cell wall-associated cellulose synthase genes (CesA3, CesA5, and CesA6) did not show any change in expression in either the loss-of-function or gain-of-function mutant, genes encoding the cellulose synthase isoforms (CesA4, 7, and 8) believed to be specifically responsible for biosynthesis of secondary cell wall cellulose microfibrils (Taylor et al., 2004) were strongly up-regulated in the myb75-1 plant stems (Fig. 4B). In contrast to this impact on wall synthesizing systems, the expression of IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis, or of FRA8, which encodes a putative glucuronyltransferase (essential for normal secondary wall synthesis) did not show any changes relative to wild-type stems. Interestingly, however, expression of two xylan biosynthetic genes, IRX8 and IRX9 (Pena et al., 2007; Persson et al., 2007), was increased in myb75-1 (Fig. 4B). No reciprocal pattern of reduced gene expression of either phenylpropanoid or CesA genes was observed in the MYB75 overexpression plants, relative to wild-type plants (Fig. 4).

Figure 4.
Secondary cell wall-associated gene expression in lower stems of myb75-1 plants. A, qRT-PCR analysis of the expression of lignin biosynthetic genes in lower inflorescence stems of MYB75 overexpresser [MYB75(o/x)] and loss-of-function mutants (myb75-1 ...

MYB75 Physically Interacts with Other Transcription Factors Involved in Secondary Cell Wall Regulation

Several transcription factors whose expression in the Arabidopsis inflorescence stem is correlated with secondary cell wall deposition were identified in an earlier microarray study (Ehlting et al., 2005). Among these, MYB63 (Zhou et al., 2009) and KNAT7 (Brown et al., 2005; Zhong et al., 2008; Li, 2009) have since been demonstrated to be regulators of secondary wall formation. Since transcription factors are believed to often exert their regulatory activity through participation in multiprotein complexes, we asked whether any protein-protein interactions could be detected in directed yeast two-hybrid assays between MYB75 and the candidate regulators identified earlier by Ehlting et al. (2005). Within this assay matrix, positive yeast two-hybrid interactions were observed between MYB75 and KNAT7 and between TT8 and MYB63 (Fig. 5). MYB75 and TT8 have previously been shown to interact (Zimmermann et al., 2004), and this interaction therefore served as a positive control for these assays. All positive interactions detected were subsequently reconfirmed using different reporter genes (Supplemental Fig. S2), and the strength of the interactions was assayed using a chlorophenol red-β-d-galactopyranoside colorimetric reporter (Supplemental Fig. S3). The bimolecular florescence complementation (BiFC) assay using split YFP was used to demonstrate that the MYB75-KNAT7 interaction could also be observed in vivo in Arabidopsis protoplasts (Supplemental Fig. S4).

Figure 5.
In vitro protein-protein interactions among potential secondary cell wall-associated transcription factors and MYB75 as determined by yeast two-hybrid assay. Known interactions (shown in dark-gray box; Zimmermann et al., 2004) were used as a positive ...

Photosynthetic Machinery and Cell Wall Modification Genes Are Up-Regulated in the myb75-1 Loss-of-Function Mutant

To obtain a broader perspective on the possible role of MYB75 in carbon redistribution during inflorescence stem development in Arabidopsis, we compared the gene expression profiles of wild-type and myb75-1 plants, with a specific focus on transcriptional activity in the maturing inflorescence stem. This analysis revealed that loss of MYB75 function resulted in both up-regulation and down-regulation of different gene sets in this tissue (Supplemental Table S2). Application of stringent cutoff values (P < 0.05 and fold change >2) excluded some secondary cell wall genes that had been earlier shown by qRT-PCR to be up-regulated in the myb75-1 genotype (Fig. 4), but these lists included a number of up-regulated genes whose products are either predicted to be involved in cell wall modification or are important in other aspects of carbon metabolism, such as photosynthesis (Table IV). These include glycosyl hydrolases, a putative arabinogalactan-protein, several members of the light-harvesting protein complexes, LHCA1 (At3g54890), LHCB1 (At1g29910), LHCA3 (At1g61520), and LHCB3 (At5g54270), and genes encoding ribulose-bisphosphate carboxylase small subunit (At5g38420 and At1g67090). Differential expression of these genes was subsequently validated by qRT-PCR analysis (Supplemental Fig. S5).

Table IV.
Expression changes of genes related to the photosynthetic machinery and cell wall modification in the inflorescence stem of myb75-1 plants

DISCUSSION

Secondary cell wall deposition in plants is an important and dynamic phenomenon. Individual transcription factors involved in directly regulating secondary cell wall formation have been identified in previous studies (Zhong and Ye, 2007), but little is known about the common factors involved in secondary cell wall synthesis and in different carbon distribution pathways. MYB75 is a known regulator of anthocyanin accumulation in addition to its involvement in many other metabolic and environmental responses. It was shown here to act as a transcription factor that influences secondary cell wall formation in the maturing Arabidopsis inflorescence stems, where it impacts the lignin branch in particular. The regulation of cell wall deposition is particularly relevant for the inflorescence stems in Arabidopsis because of the prominence in the mature stem of interfascicular fibers and xylem vessels that possess lignified secondary cells walls.

MYB75 was originally characterized as a transcriptional regulator promoting anthocyanin biosynthesis (Gonzalez et al., 2008) and much of that earlier work focused on phenotypes in seedlings and other juvenile tissues that display only limited commitment to secondary wall formation. In this study, we demonstrate a unique contribution of MYB75 to secondary cell wall biogenesis through its influence on lignin deposition, specifically in the inflorescence stem. It appears from our MYB75 expression data that as the plant matures, the ubiquitous MYB75 expression pattern reported previously in juvenile vegetative tissues becomes restricted to specific tissues. This observation is also consistent with the MYB75 expression data from the AtGenExpress database (Schmid et al., 2005).

Cellulose synthesis in primary and secondary cell walls in Arabidopsis is believed to rely upon distinct members of the CesA gene family (Brown et al., 2005). A MYB75 loss-of-function mutant displayed no obvious defects in primary cell wall formation but instead showed changes in the thickness of interfascicular fiber secondary cell walls and in cell wall chemistry of inflorescence stems in which secondary walls predominate. Consistent with a specific role for MYB75 in the regulation of secondary cell wall biosynthesis, we observed MYB75-dependent regulation of a set of lignin biosynthetic genes (Fig. 4A) as well as those CesA genes thought to be dedicated to secondary cell wall synthesis (Fig. 4B). The role of MYB75 in inflorescence stem development appears to be restricted to secondary cell wall formation rather than more general regulation of tissue development within this organ, but we cannot exclude the possibility that MYB75 is involved in regulating other aspects of development not explored here.

Some secondary cell wall-associated MYB transcription factors, such as Arabidopsis MYB4 and MYB32, have been previously shown to act as negative transcriptional regulators of at least some steps in the phenylpropanoid pathway (Preston et al., 2004). Although loss of MYB75 function in the myb75-1 mutant resulted in activation of genes encoding enzymes involved in lignin and cellulose biosynthesis, accompanied by an increase in interfascicular fiber cell wall thickness, our test of MYB75 trans-activation activity in a protoplast reporter system demonstrated that MYB75 displays only weak transcriptional activator activity and does not have repressor activity by itself within this assay system (data not shown). The weak transcriptional activation activity, or lack of transcriptional repression activity, of MYB75 in the trans-action reporter assays employed in our study could be due to the absence from mesophyll protoplasts of appropriate interaction partners that affect MYB75 function. In addition, KNAT7 repression observed in this system could be due to steric hindrance. Alternatively, MYB75 could be required for the activation of unknown downstream regulators that themselves function to repress expression of genes encoding secondary cell wall biosynthetic genes. The effect of altered MYB75 transcript levels on the relative amounts of syringyl and guaiacyl monomer subunits released by thioacidolysis of inflorescence stem lignin is consistent with earlier evidence for the contribution of caffeic acid O-methyltransferase (COMT) and CCoAOMT activities to phenolic ring methylation (Do et al., 2007). In myb75-1, CCoAOMT is up-regulated, while COMT is unchanged, a pattern that could have resulted in synthesis of more G subunits and less S-type lignin (Table II).

In contrast to our findings, Borevitz et al. (2000) reported that MYB75 overexpression in whole Arabidopsis plants resulted in increased expression of core phenylpropanoid genes with no impact on the S/G ratio of the lignin. This discrepancy in lignin monomer ratios might be related to the differences in lignin analysis methodology (thioacidolysis versus derivatization) used in the two studies. More importantly, the two studies examined very different types of tissue, with the Borevitz et al. (2000) analyses being carried out on whole plants, while our study focused on the mature inflorescence stem. Consistent with a profound developmental effect on the function of MYB75, we observed that gene expression changes induced by MYB75 overexpression in MYB75(o/x) seedlings differed markedly from the patterns observed in inflorescence stems and more closely resembled those reported for whole plants by Borevitz et al. (2000) and Tohge et al. (2005b; Supplemental Fig. S6). It is clear, therefore, that the regulatory influence of transcription factors in plant tissues can be spatially and temporally conditioned.

The biosynthesis of secondary cell wall components is thought to be a highly integrated and coordinated process in which changes in the biosynthesis or regulation of an individual component can compromise the overall assembly or composition of the wall. For example, there is experimental evidence that a reduction in any of the three major secondary wall components, cellulose, xylan, or lignin, can result in a reduction in secondary wall thickening (Zhong et al., 1998, 2005; Taylor et al., 2004; Pena et al., 2007). The increased secondary wall thickening phenotype observed in the myb75-1 mutants could therefore be an indirect effect associated with increased lignin deposition.

The data presented here suggest that one role of MYB75, in addition to regulating aspects of phenylpropanoid metabolism, is in more generally regulating secondary cell wall formation in the Arabidopsis stem. Loss of MYB75 function results in the channeling of carbon toward the lignin pathway, generating increased lignin accumulation in secondary cell walls, whereas constitutive overexpression of MYB75 leads to activation of anthocyanin biosynthesis-related genes and enhanced carbon flow into the flavonoid pathway. It remains possible, however, that the visible increase in anthocyanin production (Borevitz et al., 2000) associated with overexpression of MYB75 does not reflect its endogenous function, since overexpression of transcription factors can potentially generate artefactual pleiotropic phenotypes. Nevertheless, several MYBs have been shown earlier to be involved in activation of anthocyanin biosynthesis (Gonzalez et al., 2008), and MYB75 may be contributing to this overall regulatory activity. In addition, transcript profiles (Table IV) from the myb75-1 inflorescence stems reflect carbon flux redistribution within the branches of phenylpropanoid metabolism as well as into other metabolic pathways.

Many positive yeast two-hybrid interactions of different strengths were observed among the potential transcription factors (Fig. 5; Supplemental Figs. S2 and S3) tested from different transcription factor families. In particular, the protein-protein interaction of MYB75 with other transcription factors involved in secondary cell wall regulation is consistent with a model in which MYB75 acts as a member of one or more transcriptional regulatory complexes. Whether it might serve as an activator or repressor of transcriptional targets within such putative complexes in planta remains to be clarified. It is noteworthy that the interaction of MYB75 with TT8, a bHLH protein (Zimmermann et al., 2004), was previously shown to involve a multiprotein complex that regulates anthocyanin production (Gonzalez et al., 2008). Our observation that MYB75 also interacts with KNAT7 (Fig. 5; Supplemental Figs. S2 and S4), a transcription factor shown to play a role in secondary cell wall formation in Arabidopsis (E. Li, S. Wang, J.-G. Chen, and C.J. Douglas, unpublished data; Brown et al., 2005; Li, 2009) and also nuclear localized (Li, 2009), suggests a scenario in which multiple complexes might share specific sets of transcription factors, thus providing a rich palette of combinatorial diversity for cross-regulating different metabolic pathways as both activators and repressors of transcription.

MATERIALS AND METHODS

Plant Material

The Arabidopsis (Arabidopsis thaliana) loss-of-function allele of MYB75 (myb75-1; pst16228) is the result of a Ds insertion at the MYB75 locus, in the Nossen ecotype background (Kuromori et al., 2004) and was obtained from the RIKEN Bioresource Centre (http://rarge.gsc.riken.jp/dsmutant/index.pl This allele has been genetically characterized earlier and demonstrated to possess a Ds insertion tightly linked to the MYB75 phenotype (Teng et al., 2005). We further confirmed this insertion in the MYB75 locus and identified plants homozygous for this insertion by PCR screening. Primer sequences for amplification of the flanking fragments and genotyping of the insertion lines were as follows: left primer (LP), 5′-TGGTTTTGTAGGGCTAAACCG-3′, and right primer (RP), 5′-AAACACCGGATACATACCTTTTTC-3′. To amplify the flanking fragment, LP was combined with Ds5-3 (5′-TACCTCGGGTTCGAAATCGAT-3′), and RP was combined with Ds3-2a (5′-CCGGATCGTATCGGTTTTCG-3′). For genotyping the insertion, primers LP, RP, and Ds 3-2a were used. The wild-type line produces a PCR product of approximately 900 bp (from LP to RP) on a 1% agarose gel. Lines carrying the homozygous insertion produce an approximate 500-bp band (from RP to Ds 3-2a), while heterozygous lines produce both bands. The PCR amplification program was as follows: (1) one cycle of 94°C for 2 min; (2) 30 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 3 min; and (3) 72°C for 10 min. The gain-of-function mutant for MYB75 [MYB75(o/x); Borevitz et al., 2000] is an activation-tagged mutant (pap-1D) in the Columbia background and was obtained from the Arabidopsis Biological Resource Center. Homozygous plants of each genotype were used for all experiments, along with appropriate wild-type plants (myb75-1 versus Nossen and pap1-D versus Columbia) as comparison controls. Seeds were surface sterilized using 20% commercial bleach, cold treated at 4°C in the dark for 2 d, and plated on half-strength MS agar medium (2.16 g/L MS salts, 1% Suc, and 1% Bacto-agar pH 6.0 adjusted with 1 m KOH; Murashige and Skoog, 1962). Ten-day-old seedlings were grown in 5 × 5-cm pots containing a moistened Sunshine Mix #1 (Sun Gro Horticulture Canada),with a 16/8-h (light/dark) photoperiod at approximately 120 μmol m−2 s−1 and a temperature of 23°C, unless specified otherwise. Growth comparisons were performed on 6-week-old plants grown at the above-described conditions. For TEM, microscopy, and qRT-PCR experiments, inflorescence stems were harvested from 8-week-old plants, and the lower half of the stem was used for analysis. For chemical analyses, whole stems were dried in a 50°C oven overnight and ground in a Wiley mill to pass a 40-mesh screen.

Analysis of Anthocyanin Content

Seedling anthocyanin content was determined using a procedure modified from that of Neff and Chory (1998). In short, at least two groups (50 seedlings each) of 8-d-old seedlings from each genotype were extracted overnight in 150 μL of methanol acidified with 1% HCl. After the addition of 100 μL of distilled water and 250 μL of chloroform, anthocyanins were separated from chlorophylls by solvent partitioning. Total anthocyanin content in the aqueous phase was determined spectroscopically by measuring the A530 and A657. By subtracting the A657 from the A530, the relative amount of anthocyanin per seedling was calculated [(530 − A657) × 50 seedling−1].

GUS Reporter Gene Analyses

Transgenic Arabidopsis plants (T2) generated earlier by Gonzalez et al. (2008) were employed in this study. They express a MYB75pro::GUS construct derived from the 2.2-kb genomic DNA region upstream of the MYB75 coding sequence in the Columbia ecotype, fused with the GUS coding sequence. Histochemical analysis of the GUS reporter gene expression was performed as described previously (Malamy and Benfey, 1997) using different plant organs, as well as transverse hand sections of inflorescence stems of 6- to 8-week-old T2 MYB75pro::GUS transgenic plants.

Protoplast Isolation, Transfection, and GUS Activity Assay

Leaves from Columbia wild-type plants approximately 3 to 4 weeks old were used for protoplast isolation and subsequent transfection and GUS activity assays, as described previously (Wang et al., 2007). For GUS activity assays, the plasmid DNAs for reporter and effector genes were isolated using Endofree Plasmid Maxi Kits (Qiagen). Additional GD plasmid DNA was used to equalize the amount of DNA in each plasmid preparation. A 10-μg aliquot of each effector plasmid and 10 μg of reporter plasmid were used in cotransfection assays. Each transfection assay was performed in triplicate, and each experiment was repeated at least twice

Localization of MYB75-YFP

To examine the localization of MYB75-YFP in protoplasts, the Gateway recombination cassette (Invitrogen) from pEARLYGATE104 (Earley et al., 2006) was used as a destination vector for generation of an N-terminal YFP fusion of MYB75. The resulting plasmid was transfected into freshly prepared Arabidopsis leaf mesophyll protoplasts and incubated for 20 to 22 h (Wang et al., 2007). YFP fluorescence was visualized using a Leica DM-6000B upright fluorescence microscope with phase and differential interference contrast (DIC) and photographed with a Leica FW4000 digital image acquisition and processing system (Leica Microsystems).

BiFC Using YFP

For generation of N-terminal YFP-tagged constructs, the appropriate entry clone was transferred into BiFC expression vector pCL112 (pBATL) to produce nYFP-vectors. The same procedure was used for C-terminal YFP-tagged constructs using pCL113 (pBATL) to produce cYFP vectors. The resulting plasmids were cotransfected into freshly prepared Arabidopsis leaf mesophyll protoplasts and incubated for 20 to 22 h (Wang et al., 2007). YFP fluorescence was examined and photographed using a Leica DM-6000B upright fluorescence microscope with phase and DIC equipped with a Leica FW4000 digital image acquisition and processing system (Leica Microsystems).

qRT-PCR

For organ-specific expression analyses, total RNA was isolated from different organs of 6-week-old Arabidopsis (Nossen wild type) plants (three biological replicates, each consisting of pooled tissues from 8–10 plants), and qPCR was performed as described below. Relative values are arbitrary units and were calculated as described previously (Gutierrez et al., 2008). For the secondary cell wall-specific and lignin-specific gene expression study, total RNA for real-time PCR was isolated from the lower half of inflorescence stems [wild type, myb75-1, and MYB75(o/x)] with three biological replicates (each consisting of pooled stems from 8–10 plants) using the RNeasy Mini Protocol (Qiagen). To eliminate residual genomic DNA, the RNA was treated with RNAse-free DNAseI according to the manufacturer’s instructions (Qiagen). The concentration of RNA was quantified using the A260, and the quality of the sample preparation was assessed using the A260/A280 ratio. Total RNA (2 μg) was reverse transcribed using the SuperScript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. cDNA was diluted (1:20), and 2 μL was used in each reaction in a 20-μL reaction volume. PCR amplification was performed with gene-specific primers for secondary cell wall or lignin-specific genes (Supplemental Table S1) using Actin8 as a normalization control (Supplemental Table S1). The cDNA was amplified using the PerfeCta qPCR FastMix (Quanta Biosciences) on the DNA Engine Opticon 2 (Bio-Rad). Differences in gene expression, expressed as fold change relative to control, were calculated using the [Δ][Δ]Ct = 2 [Δ]Ct,Actin-[Δ]Ct,gene method. Each measurement was carried out in triplicate, and the error bars represent se of the mean of fold changes for the three biological replicates.

Bright-Field and TEM

Tissue for light microscopy and TEM was harvested from the inflorescence stem (5 cm from the base) of 8- to 10-week-old plants and fixed with glutaraldehyde. Fixed stems were vacuum infiltrated in 1% osmium tetroxide and 0.05 m sodium cacodylate (pH 6.9) for 30 min, rinsed twice, and then dehydrated through an aqueous alcohol series (30%–100%; 15 min for each dilution). Dehydrated stems were soaked twice in anhydrous acetone before embedding in low viscosity Spurr’s resin. Sections (0.5 μm) were cut using a Leica Ultracut T and Druuker diamond Histoknife and stained with Toluidine Blue for bright-field microscopy (using an Olympus AX70 microscope) or with uranyl acetate for TEM (viewed on a Hitachi H7600 PC-TEM).

For cell wall thickness measurements, the width of the secondary cell wall in micrographs obtained from TEM was quantified in 75 cells for each genotype using ImageJ software (http://rsbweb.nih.gov/ij/).

Chemical Analysis

Lignin content was determined by a modified Klason method, according to Coleman et al. (2008), in which solvent-extracted ground Arabidopsis stem tissue (0.1 g) was treated with 3 mL 72% H2SO4 for 2 h at room temperature and then diluted to 3% H2SO4 and autoclaved for 60 min. The concentrations of different monosaccharides in the acid hydrolysate were determined using HPLC (DX-500; Dionex) equipped with an anion-exchange PA1 (Dionex) column, a pulsed amperometric detector with a gold electrode, and a SpectraAS3500 autoinjector (Spectra-Physics). Each analysis was run in duplicate. The monosaccarides were separated on the PA1 column with water at a flow rate of 1 mL/min, and the eluate received a postcolumn addition of 200 mm NaOH (0.5 mL/min) prior to detection.

Thioacidolysis was performed as described by Robinson and Mansfield (2009), and the reaction products were analyzed by gas chromatography.

Microarray Analysis

Total RNA was extracted from Arabidopsis inflorescence stems (Nossen wild type and myb75-1) using a Qiagen Plant Mini RNA extraction kit. The quantity and quality of total RNA were assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies) using the Agilent RNA 6000 Nano kit and reagents. Samples of total RNA (10 μg) for six wild-type and six myb75-1 biological replicates were reverse transcribed using a SuperScript II RT kit (Invitrogen) and the appropriate 3DNA primers (cyanine5- or cyanine3-specific capture sequences) to achieve dye balance with two technical replicates for each of three biological replicate pairings. The 3DNA Array 350 kit (Genisphere) was used according to manufacturer specifications for cDNA hybridizations, and subsequent 3DNA (dendromer) fluorescent probe hybridizations onto custom-made full-genome (30 K) Arabidopsis 70-mer oligo arrays (Douglas and Ehlting, 2005; Ehlting et al., 2005) printed at the Prostate Centre Microarray Facility, Vancouver. Hybridizations were carried out using a Slidebooster SB401 (Advalytix) according to Array 350 specifications, and the hybridized slides were scanned with a ScanArray Express (Perkin-Elmer). Scanned images were quantified using Imagene software (BioDiscovery), and the resulting data were analyzed in the R package using Bioconductor tools and custom scripts. For background correction, the mean of the dimmest 5% of spots in a particular subgrid (grouping of 26 × 27 spots) was used as the background value for the spots in that subgrid. Background-corrected spot intensities were then normalized on each array using the robust local-linear regression algorithm LOWESS (or LOESS) included in the R package, with a span of 0.7 (Yang et al., 2002). The relative expression ratio for each gene represents the average of three biological replicates, where P value significance estimates were computed using a two-tailed Student’s t tests (α = 0.05) and adjusted for false discovery rate using a q-value correction based upon Storey (2002).

Yeast Two-Hybrid Assays

The ProQuest yeast two-hybrid system (Invitrogen) was used with full-length transcription factors in pDEST32 (bait vector) or pDEST-22 (prey vector) and introduced into the yeast strain MaV203 in different combinations. Positive clones were isolated on the basis of three selectable markers: HIS3, URA3, and LacZ. Positive interactions were indicated by activation of HIS3 or URA3, according to the manufacturer’s instructions. To compare the strength of the protein-protein interactions, quantitative assays for β-galactosidase activity in liquid cultures were performed using chlorophenol red-β-d-galactopyranoside as a substrate according to the manufacturer’s instructions.

GenBank database accession numbers for the genes investigated in this study are MYB75 (At1g56650), MYB63 (At1g79180), MYB20 (At1g66230), PAL1 (At2g37040), C4H (At2g30490), 4CL1 (At1g51680), HCT (At5g48930), C3H1 (At2g40890), CCoAOMT1 (At4g34050), CCR1 (At1g15950), F5H1 (At4g36220), COMT (At5g54160), CAD5 (At4g34230), CesA4 (At5g44030), CesA7 (At5g17420), CesA8 (At4g18780), IRX8 (At5g54690), IRX9 (At2g37090), CesA1 (At4g32410), CesA3 (At5g05170), CesA6 (At5g64740), FRA8 (At2g28110), and IFL1 (At5g60690).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. Activation-tagged and Ds insertion mutants of MYB75, anthocyanin content and growth, and inflorescence stem phenotype.
  • Supplemental Figure S2. Confirmation of yeast two-hybrid interactions by different reporter genes.
  • Supplemental Figure S3. Quantification of strength of yeast two-hybrid interactions by the CPRG assay.
  • Supplemental Figure S4. Confirmation of yeast two-hybrid interactions by BiFC assay in Arabidopsis protoplasts
  • Supplemental Figure S5. qRT-PCR validation of expression of candidate genes identified by microarray analysis as up-regulated (>2-fold; P = 0.05; Table I) in the lower inflorescence stem of myb75, relative to Nossen-wild type.
  • Supplemental Figure S6. Real-time quantitative PCR analysis of the expression of lignin biosynthetic genes in MYB75 overexpresser [MYB75(o/x)] seedlings compared with the wild type.
  • Supplemental Table S1. PCR primers used in the study.
  • Supplemental Table S2. Genes up-regulated or down-regulated in the wild type versus myb75-1 microarray study (P < 0.05 and fold change >2).

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Drs. Vicki Maloney and Andrew Robinson (University of British Columbia) for their input in chemical analyses, Brad Ross and the University of British Columbia Bioimaging Facility for TEM support, Dr. Shucai Wang (University of British Columbia) for help with protoplast transformation, Dr. A.M. Lloyd (University of Texas at Austin) for kindly providing MYB75pro::GUS seeds, the Arabidopsis Biological Resource Center for pap1-D seeds, RIKEN (Yokohama, Japan) for myb75-1 seeds, and Dr. Mathias Schuetz (University of British Columbia) for critical reviewing of the manuscript. We also thank Dr. Joachim Uhrig (Botanical Institute, Universität Köln) for the kind gift of pBatTL vectors used for BiFC assays.

References

  • Bernhardt C, Lee MM, Gonzalez A, Zhang F, Lloyd A, Schiefelbein J. (2003) The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root. Development 130: 6431–6439 [PubMed]
  • Bloom AJ, Chapin FS, III, Mooney HA. (1985) Resource limitation in plants-an economic analogy. Annu Rev Ecol Syst 16: 363–392
  • Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C. (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12: 2383–2394 [PMC free article] [PubMed]
  • Brown DM, Zeef LA, Ellis J, Goodacre R, Turner SR. (2005) Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. Plant Cell 17: 2281–2295 [PMC free article] [PubMed]
  • Brown DM, Zhang Z, Stephens E, Dupree P, Turner SR. (2009) Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis. Plant J 57: 732–746 [PubMed]
  • Coleman HD, Park JY, Nair R, Chapple C, Mansfield SD. (2008) RNAi-mediated suppression of p-coumaroyl-CoA 3′-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism. Proc Natl Acad Sci USA 105: 4501–4506 [PMC free article] [PubMed]
  • Davies KM, Schwinn KE. (2003) Transcriptional regulation of secondary metabolism. Funct Plant Biol 30: 913–925
  • Do CT, Pollet B, Thévenin J, Sibout R, Denoue D, Barrière Y, Lapierre C, Jouanin L. (2007) Both caffeoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta 226: 1117–1129 [PubMed]
  • Douglas CJ, Ehlting J. (2005) Arabidopsis thaliana full genome longmer microarrays: a powerful gene discovery tool for agriculture and forestry. Transgenic Res 14: 551–561 [PubMed]
  • Dubos C, Willment J, Huggins D, Grant GH, Campbell MM. (2005) Kanamycin reveals the role played by glutamate receptors in shaping plant resource allocation. Plant J 43: 348–355 [PubMed]
  • Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS. (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45: 616–629 [PubMed]
  • Ehlting J, Mattheus N, Aeschliman DS, Li E, Hamberger B, Cullis IF, Zhuang J, Kaneda M, Mansfield SD, Samuels L, et al. (2005) Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation. Plant J 42: 618–640 [PubMed]
  • Gonzalez A, Zhao M, Leavitt JM, Lloyd AM. (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53: 814–827 [PubMed]
  • Gutierrez L, Mauriat M, Pelloux J, Bellini C, Van Wuytswinkel O. (2008) Towards a systematic validation of references in real-time rt-PCR. Plant Cell 20: 1734–1735 [PMC free article] [PubMed]
  • Joshi CP, Mansfield SD. (2007) The cellulose paradox—simple molecule, complex biosynthesis. Curr Opin Plant Biol 10: 220–226 [PubMed]
  • Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T. (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19: 1855–1860 [PMC free article] [PubMed]
  • Kuromori T, Hirayama T, Kiyosue Y, Takabe H, Mizukado S, Sakurai T, Akiyama K, Kamiya A, Ito T, Shinozaki K. (2004) A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis. Plant J 37: 897–905 [PubMed]
  • Lea US, Slimestad R, Smedvig P, Lillo C. (2007) Nitrogen deficiency enhances expression of specific MYB and bHLH transcription factors and accumulation of end products in the flavonoid pathway. Planta 225: 1245–1253 [PubMed]
  • Li E. (2009) Identification and characterization of regulatory genes associated with secondary wall formation in Populus and Arabidopsis thaliana. PhD thesis. University of British Columbia, Vancouver, Canada
  • Malamy JE, Benfey PN. (1997) Analysis of SCARECROW expression using a rapid system for assessing transgene expression in Arabidopsis roots. Plant J 12: 957–963 [PubMed]
  • Meyer K, Shirley AM, Cusumano JC, Bell-Lelong DA, Chapple C. (1998) Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis. Proc Natl Acad Sci USA 95: 6619–6623 [PMC free article] [PubMed]
  • Mir Derikvand M, Sierra JB, Ruel K, Pollet B, Do CT, Thévenin J, Buffard D, Jouanin L, Lapierre C. (2008) Redirection of the phenylpropanoid pathway to feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1. Planta 227: 943–956 [PubMed]
  • Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R, Ortet P, Creff A, Somerville S, Rolland N, et al. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc Natl Acad Sci USA 102: 11934–11939 [PMC free article] [PubMed]
  • Morcuende R, Bari R, Gibon Y, Zheng W, Pant BD, Bläsing O, Usadel B, Czechowski T, Udvardi MK, Stitt M, et al. (2007) Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus. Plant Cell Environ 30: 85–112 [PubMed]
  • Muller R, Morant M, Jarmer H, Nilsson L, Nielsen TH. (2007) Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol 143: 156–171 [PMC free article] [PubMed]
  • Murashige T, Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497
  • Neff MM, Chory J. (1998) Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development. Plant Physiol 118: 27–35 [PMC free article] [PubMed]
  • Pauly M, Keegstra K. (2008) Cell-wall carbohydrates and their modification as a resource for biofuels. Plant J 54: 559–568 [PubMed]
  • Pena MJ, Zhong R, Zhou GK, Richardson EA, O’Neill MA, Darvill AG, York WS, Ye ZH. (2007) Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell 19: 549–563 [PMC free article] [PubMed]
  • Persson S, Caffall KH, Freshour G, Hilley MT, Bauer S, Poindexter P, Hahn MG, Mohnen D, Somerville C. (2007) The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity. Plant Cell 19: 237–255 [PMC free article] [PubMed]
  • Persson S, Wei H, Milne J, Page GP, Somerville CR. (2005) Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets. Proc Natl Acad Sci USA 102: 8633–8638 [PMC free article] [PubMed]
  • Pourtau N, Jennings R, Pelzer E, Pallas J, Wingler A. (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta 224: 556–568 [PubMed]
  • Preston J, Wheeler J, Heazlewood J, Li SF, Parish RW. (2004) AtMYB32 is required for normal pollen development in Arabidopsis thaliana. Plant J 40: 979–995 [PubMed]
  • Robinson AR, Mansfield SD. (2009) Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling. Plant J 58: 706–714 [PubMed]
  • Rowan DD, Cao M, Lin-Wang K, Cooney JM, Jensen DJ, Austin PT, Hunt MB, Norling C, Hellens RP, Schaffer RJ, et al. (2009) Environmental regulation of leaf colour in red 35S:PAP1 Arabidopsis thaliana. New Phytol 182: 102–115 [PubMed]
  • Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU. (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37: 501–506 [PubMed]
  • Sibout R, Eudes A, Mouille G, Pollet B, Lapierre C, Jouanin L, Séguin A. (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17: 2059–2076 [PMC free article] [PubMed]
  • Smith AM, Stitt M. (2007) Coordination of carbon supply and plant growth. Plant Cell Environ 30: 1126–1149 [PubMed]
  • Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, et al. (2004) Toward a systems approach to understanding plant cell walls. Science 306: 2206–2211 [PubMed]
  • Storey J. (2002) A direct approach to false discovery rates. J R Stat Soc Series B Stat Methodol 64: 479–498
  • Taylor NG, Gardiner JC, Whiteman R, Turner SR. (2004) Cellulose synthesis in the Arabidopsis secondary cell wall. Cellulose 11: 329–338
  • Teng S, Keurentjes J, Bentsink L, Koornneef M, Smeekens S. (2005) Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene. Plant Physiol 139: 1840–1852 [PMC free article] [PubMed]
  • Tohge T, Matsui K, Ohme-Takagi M, Yamazaki M, Saito K. (2005a) Enhanced radical scavenging activity of genetically modified Arabidopsis seeds. Biotechnol Lett 27: 297–303 [PubMed]
  • Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara M, Inoue E, Takahashi H, Goodenowe DB, Kitayama M, et al. (2005b) Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J 42: 218–235 [PubMed]
  • Vanderauwera S, Zimmermann P, Rombauts S, Vandenabeele S, Langebartels C, Gruissem W, Inzé D, Van Breusegem F. (2005) Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis. Plant Physiol 139: 806–821 [PMC free article] [PubMed]
  • Wang S, Chang Y, Guo J, Chen JG. (2007) Arabidopsis Ovate Family Protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J 50: 858–872 [PubMed]
  • Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15. [PMC free article] [PubMed]
  • Zhong R, Iii WH, Negrel J, Ye ZH. (1998) Dual methylation pathways in lignin biosynthesis. Plant Cell 10: 2033–2046 [PMC free article] [PubMed]
  • Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH. (2008) A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20: 2763–2782 [PMC free article] [PubMed]
  • Zhong R, Peña MJ, Zhou GK, Nairn CJ, Wood-Jones A, Richardson EA, Morrison WH, III, Darvill AG, York WS, Ye ZH. (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, Ye ZH. (2007) Regulation of cell wall biosynthesis. Curr Opin Plant Biol 10: 564–572 [PubMed]
  • Zhong R, Ye ZH. (2009) Transcriptional regulation of lignin biosynthesis. Plant Signal Behav 4: 1028–1034 [PMC free article] [PubMed]
  • Zhou J, Lee C, Zhong R, Ye ZH. (2009) MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21: 248–266 [PMC free article] [PubMed]
  • Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF. (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40: 22–34 [PubMed]

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