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Plant scientists have devoted considerable efforts to understanding the molecular bases of wood formation with the aim of increasing the diverse industrial uses of this important bioresource (pulp and paper, lumber, biofuels and others) by modifying the chemical composition of the wood cell walls (for reviews see Boerjan et al., 2003; Mellerowicz & Sundberg, 2008; Vanholme et al., 2008; Weng et al., 2008). Wood is a complex vascular tissue containing specialized cells types such as tracheary elements, fibres and tracheids derived from a secondary meristem called the vascular cambium (Plomion et al., 2001; Ye, 2002). These cells form thick secondary cell walls (SWs) typically composed of three main polymers: cellulose, hemicelluloses and lignin (Plomion et al., 2001; Boerjan et al., 2003; Mellerowicz & Sundberg, 2008). Formation and deposition of these components in SWs require fine temporal and spatial regulation. Transcriptional regulators are expected to precisely coordinate the expression of hundreds of genes participating in this process (Sivadon & Grima-Pettenati, 2004; Demura & Fukuda, 2007; Zhong & Ye, 2007).
Targeted transcriptome profiling experiments performed in Arabidopsis and in poplar (Hertzberg et al., 2001; Oh et al., 2003; Schrader et al., 2004; Ehlting et al., 2005; Kubo et al., 2005) have highlighted the stage-specific transcriptional regulation of lignin and cellulose biosynthetic genes during development and the importance of transcriptional regulators such as members of the R2R3 MYB, bHLH, bZIP, HD-ZIP and NAC-domain transcription factors families (NACs). Functional analyses of some of these transcription factors have provided insights into the complex network of transcriptional regulatory pathways involved in SW biosynthesis (reviewed in Baucher et al., 2007; Demura & Fukuda, 2007; Zhong & Ye, 2007; Zhong et al., 2008). Recently, a hierarchical network of transcription factors has been proposed to control SW formation in Arabidopsis. In this network SECONDARY WALL-ASSOCIATED NAC DOMAIN 1 protein (SND1/NST3) and its functional homologues (NST1 and NST2, vessel-specificVND6 and VND7) are master switches that turn on a subset of transcription factors (SND3, MYB46, MYB103 and KNAT7 (knotted1-like homeodomain protein) in different cell types, which, in turn, activate the SW biosynthetic pathways (Zhong et al., 2008). Based on functional analyses carried out in conifers, it was proposed that this pathway may be largely conserved across the entire plant kingdom (Bomal et al., 2008).
In addition to NACs, several MYB transcription factors were shown to be important regulators of SW formation. We have previously shown that the Eucalyptus gunnii EgMYB2 protein is able to bind to the promoters of the EgCCR (cinnamoyl coA reductase) and EgCAD2 (cinnamyl alcohol dehydrogenase) genes and activate their transcription (Goicoechea et al., 2005). Overexpression of EgMYB2 in tobacco was associated with significantly thicker xylem SWs as well as a modified lignin profile consistent with increased transcript abundance of most of the lignin biosynthesis genes. These data suggest that EgMYB2 is a positive regulator for both lignin biosynthesis and SW formation. Similarly, constitutive overexpression of AtMYB46, the putative EgMYB2 orthologue in Arabidopsis, has been shown to be associated with ectopic lignification, SW thickening and activation of lignin and other SW genes (Zhong et al., 2007; Ko et al., 2009). Interestingly, transient transcriptional activation analyses in Arabidopsis protoplasts indicated that AtMYB46 overexpression also activates other MYB factors, thereby underlining the complexity of the regulatory network involved (Ko et al., 2009). Other R2R3 MYB transcription factors were also shown to bind to AC elements and/or regulate the biosynthesis of phenylpropanoid and derived-products including lignins (Patzlaff et al., 2003). More recently, AtMYB58 and AtMYB63 were shown to function as specific transcriptional activators of lignin biosynthesis in Arabidopsis (Zhou et al., 2009).
Together, these reports clearly show that different transcription factors, including MYB proteins are involved in the temporal and spatial control of SW formation. However, the above factors appear to function as transcriptional activators. In the case of a tightly-regulated process such as SW formation it is possible that transcriptional repressors might also be required for precise control of gene expression. There is strong evidence that some MYB factors can act as repressors. For example, the overexpression of the snapdragon AmMYB308 and AmMYB330 genes in tobacco downregulated phenylpropanoid and lignin biosynthetic genes leading to reduction in lignin accumulation (Tamagnone et al., 1998). Similarly, AtMYB4, the proposed Arabidopsis orthologue of AmMYB308, has been shown to downregulate C4H gene expression and a knockout mutant showed increased amounts of sinapate esters, resulting in better tolerance to UV-B (Jin et al., 2000). In maize, it has been proposed that the R2R3 MYB genes ZmMYB31 and ZmMYB42 are negative regulators with complementary roles in lignin and phenylpropanoid metabolism (Fornaléet al., 2006). ZmMYB42 overexpression was shown to affect the cell wall structure, composition and degradability in Arabidopsis (Sonbol et al., 2009).
Eucalyptus trees represent one of the main sources of wood worldwide and are widely used in industrial plantations. In order to learn more about wood formation in this species our group has initiated a programme to identify and characterize regulator genes involved in this process. We identified an R2R3 E. gunnii MYB, EgMYB1 preferentially expressed in E. gunnii xylem (Legay et al., 2007). EgMYB1 recombinant protein was shown to specifically bind the MYB-binding sites present in the cis-regulatory regions of the E. gunnii CCR and CAD promoters. In addition, in vivo transient coexpression in tobacco leaves with either EgCCR::GUS or EgCAD2::GUS reporter constructs showed that EgMYB1 can repress both EgCAD2 and EgCCR promoter activities suggesting that it could act as a negative regulator of lignin biosynthesis gene expression in planta (Legay et al., 2007).
Eucalyptus is still difficult to transform and so we decided to investigate the role of EgMYB1 in planta in two model systems: Arabidopsis thaliana, an herbaceous annual and Populus tremula × Populus alba, a woody perennial. Our findings (this paper) in both model species are consistent with a role of EgMYB1 in transcriptional repression of lignin biosynthesis and SW formation.
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Wood is the most abundant biomass on earth. It is mainly composed of SWs whose biosynthesis requires the coordinated transcriptional regulation of hundreds of genes to synthesize the main SW polymers, that is, cellulose, xylan and lignins. Although the SW biosynthetic pathway has been well characterized biochemically and genetically, our current knowledge of the signals and transcriptional regulators that are responsible for controlling the SW biosynthetic program is still limited. In previous studies, we have functionally characterized the promoters of two key lignin genes (EgCCR and EgCAD2) from eucalyptus (Lacombe et al., 2000; Lauvergeat et al., 2002) and provided functional evidence showing that the coordinated vascular expression of these two genes is mediated through MYB transcription factors (Rahantamalala et al., 2010).
We have also shown that EgMYB1, an R2R3 MYB transcription factor is preferentially expressed in secondary xylem from stems and roots of eucalyptus and binds specifically to the MBSIIG sites located in the promoters of the EgCAD2 and EgCCR lignin biosynthetic genes. The EgMYB1 protein sequence harbours an active repressor motif in the regulatory domain and is able to repress the activity of both EgCCR and EgCAD2 promoters in vivo, as shown by transient assays performed in tobacco leaves (Legay et al., 2007). These data led us to hypothesize that EgMYB1 acts as a transcriptional repressor of lignin biosynthesis genes. In order to test this hypothesis and to further investigate the role of EgMYB1 we compared the effects of EgMYB1 overexpression in both Arabidopsis and poplar. The data reported here support the hypothesis that EgMYB1 acts as a negative regulator of SW formation in vascular tissue, the first example to our knowledge of such a repressor in highly lignified woody tissues.
EgMYB1 overexpression in both Arabidopsis and poplar affected growth and development by reducing stem height and diameter, leaf size and altering leaf shape. It also reduced lignin content by a modest but consistent percentage of total SW weight. Similar modifications in growth and development have been observed in different plant species downregulated for lignin biosynthetic genes. For example, PAL down-regulated tobacco plants had reduced height, cup-shaped leaves and decreased lignin content (Elkind et al., 1990). Similarly, CCR downregulated plants were shorter, had smaller leaves with altered morphology and contained less acid-insoluble lignin (Piquemal et al., 1998; Goujon et al., 2003; Leple et al., 2007). These observations suggest that changes in lignin content/phenylpropanoid metabolism can often have an important effect on plant growth and development.
Histochemical analyses indicated that stem vascular tissue SWs were significantly thinner in both Arabidopsis and poplar EgMYB1-overexpressing plants. In agreement with these observations, quantitative RT-PCR analysis in both species indicated that EgMYB1 overexpression downregulated three different SW CesA genes (CesA4/IRX5, CesA7/IRX3, CesA8/IRX1: Taylor et al., 2004) and two glycosyltransferases (IRX8: Pena et al., 2007; IRX7/FRA8: Zhong et al., 2005) associated with xylan biosynthesis, in addition to a number of lignin biosynthetic genes. The negative impact on overall SW thickening could explain the apparent discrepancy between the moderate decrease in Klason lignin (11% relative to cell wall weight) and the marked effect on lignin staining visualized by histochemistry (particularly dramatic in Arabidopsis interfascicular zones). Together, these results could suggest that EgMYB1 functions as a negative regulator of the SW developmental program and not just as a negative regulator of lignification.
Interestingly, we recently reported (Goicoechea et al., 2005) that overexpression of the transcriptional activator EgMYB2 in tobacco plants had the opposite effect (i.e. thicker SW and slight increase of lignin content). Similar observations have been made for AtMYB46, the closest Arabidopsis orthologue of EgMYB2. Whereas dominant repression of AtMYB46 caused a drastic reduction of SW thickening in fibres and vessels, its overexpression led to an activation of the biosynthetic pathways of lignin as well as those of cellulose and xylan (Zhong et al., 2007; Ko et al., 2009). Thus AtMYB46 (and most likely EgMYB2) is considered as a key master switch activating the SW developmental programme through coordinated regulation of the biosynthetic pathways of all three major SW components (Zhong et al., 2007; Ko et al., 2009). The fact that EgMYB1 overexpression resulted in the downregulation of SW genes in both Arabidopsis and poplar suggests that it could also play a central role in SW formation in higher plants, at least in dicots. Interestingly, the dominant repression form used for AtMYB46 by Zhong et al. (2007) corresponding to a fusion between AtMYB46 and the EAR domain is structurally quite similar to EgMYB1 (R2R3-MYB containing an EAR domain).
AtMYB46 was shown to be a direct target of a SW NAC protein, SND1, a master switch which turns on downstream direct target genes SND3, MYB46, MYB103 and KNAT7 in different cell types, which in turn activate the SW biosynthetic pathway. Whether EgMYB1 could be part of a similar SND1-mediated transcriptional network regulating SW synthesis is a testable hypothesis as potential eucalyptus SND1 orthologues were recently identified (Rengel et al., 2009). The very recent release of the Eucalyptus grandis genome will provide access to EgMYB1 promoter sequences and should help elucidate the position of EgMYB1 in a putative regulatory network. Identifying direct targets of EgMYB1 is a tangible goal which could take advantage of experimental systems such as the steroid-receptor based inducible systems described by Zhong et al. (2008) in combination with deep sequencing (Ko et al., 2009) and may help uncover other transcription factors involved in SW formation.
As two important MYB regulators playing apparently opposite roles in SW biogenesis and lignin biosynthesis are both preferentially expressed in eucalyptus xylem, it is possible that dynamic competition between EgMYB1 and EgMYB2 for the same promoters will allow the formation of either a repressing or an activating regulatory complex, thereby providing a sophisticated mechanism for the spatial and temporal control of lignified SW formation. Consistent with this idea, we have recently shown that the EgCAD2 promoter has a complex organization with its cis-elements arranged in two similar modules (containing MYB sites) suggesting that redundancy and mechanisms of cooperation or competition between cis-elements and trans-acting factors might be involved in the regulation of promoter activity (Rahantamalala et al., 2010). In particular, EMSAs with recombinant EgMYB2 and transactivation assays clearly demonstrated that the two MYB sites of the EgCAD2 promoter cooperated for binding and activation and it is possible that one of these sites could bind a repressor MYB factor (Rahantamalala et al., 2010).
The activity of transcription factors can be regulated at the transcriptional or post-transcriptional level, and may involve combinatorial action with other transcription factors. It is known that bHLH proteins are capable of interacting with MYBs (Zimmermann et al., 2004) and the fact that the EgMYB1 sequence contains a conserved motif for bHLH interaction suggests that MYB–bHLH interaction might be necessary for the control of SW synthesis in xylem. Indirect evidence in support of this hypothesis include the preferential accumulation of several bHLH transcripts in eucalyptus xylem, and the close association of putative bHLH binding sites sequences and MYB binding sites in the promoters of many poplar lignin biosynthetic genes (Legay et al., 2007; Table S3).
A number of reports in different species have shown that modifying the expression of a single lignin biosynthesis gene affects the expression of many other genes in various unanticipated pathways, thereby revealing previously unsuspected and uncharacterized interactions between monolignol and other metabolic pathways (Rohde et al., 2004; Sibout et al., 2005; Dauwe et al., 2007; Leple et al., 2007). As EgMYB1 overexpression in poplar also downregulated a number of lignin biosynthetic genes, we decided to analyse the poplar expression profiles in order to investigate the potential interactions between lignin and other metabolic pathways. In agreement with results obtained on CCR-/CAD-downregulated tobacco (Dauwe et al., 2007) transcript accumulation in EgMYB1-overexpressing poplar was altered for genes associated with phenylpropanoid-, starch- and hemicellulose-metabolisms, stress metabolism and light-regulated genes. Of interest was the downregulation of the gene encoding prephenate dehydrogenase/chorismate mutase, involved in the synthesis of phenylalanine and modulation of carbon flux into the phenylpropanoid pathway. However, further work is necessary to determine whether this gene was downregulated because it is a direct target of EgMYB1 or because of indirect feedback mechanisms resulting from the general repression of lignin biosynthesis, as observed in CCR downregulated tobacco (Dauwe et al., 2007).
In conclusion, this study shows that EgMYB1 overexpression leads to consistent molecular phenotypes in both a woody perennial and a herbaceous annual species and provides evidence for the biological role of EgMYB1 as a negative regulator of lignification and SW formation. Based on these new findings, as well as on previous studies, we propose that the combinatorial control of gene expression through the action of positive regulators (such as EgMYB2, Goicoechea et al., 2005) and negative regulators (such as EgMYB1) could provide the necessary flexibility to ensure tight temporal and spatial regulation of SW biosynthesis in vascular tissues. The identification of a repressor of SW development opens new avenues to dissect the complex networks of transcriptional regulators involved in SW biosynthesis, thereby contributing to a better understanding of SW formation in plants, and in particular in woody species of major economic importance.