Coordinated transcriptional regulation of two key genes in the lignin branch pathway - CAD and CCR - is mediated through MYB- binding sites

Abstract

BackgroundCinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the final steps in the biosynthesis of monolignols, the monomeric units of the phenolic lignin polymers which confer rigidity, imperviousness and resistance to biodegradation to cell walls. We have previously shown that the Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in vascular tissues suggesting that monolignol production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes. Although consensus motifs for MYB transcription factors occur in most gene promoters of the whole phenylpropanoid pathway, functional evidence for their contribution to promoter activity has only been demonstrated for a few of them. Here, in the lignin-specific branch, we studied the functional role of MYB elements as well as other cis-elements identified in the regulatory regions of EgCAD2 and EgCCR promoters, in the transcriptional activity of these gene promoters.ResultsBy using promoter deletion analysis and in vivo footprinting, we identified an 80 bp regulatory region in the Eucalyptus gunnii EgCAD2 promoter that contains two MYB elements, each arranged in a distinct module with newly identified cis-elements. A directed mutagenesis approach was used to introduce block mutations in all putative cis-elements of the EgCAD2 promoter and in those of the 50 bp regulatory region previously delineated in the EgCCR promoter. We showed that the conserved MYB elements in EgCAD2 and EgCCR promoters are crucial both for the formation of DNA-protein complexes in EMSA experiments and for the transcriptional activation of EgCAD2 and EgCCR promoters in vascular tissues in planta. In addition, a new regulatory cis-element that modulates the balance between two DNA-protein complexes in vitro was found to be important for EgCAD2 expression in the cambial zone.ConclusionsOur assignment of functional roles to the identified cis-elements clearly demonstrates the importance of MYB cis-elements in the transcriptional regulation of two genes of the lignin-specific pathway and support the hypothesis that MYB elements serve as a common means for the coordinated regulation of genes in the entire lignin biosynthetic pathway.

Identification of a cis-acting regulatory motif recognized by MYB46, a master transcriptional regulator of secondary wall biosynthesis

“…Moreover, M46RE is complementary to a class of CA-rich regulatory elements named ACI and ACII (i.e., AC element), which are known to mediate transcriptional activation of lignin biosynthetic genes (Hatton et al 1995;Raes et al 2003). For example, PtMYB4 and Eg-MYB2 bind these elements to activate AC elementdependent transcription of lignin biosynthesis genes (Patzlaff et al 2003;Rahantamalala et al 2010). It appears that M46RE serve as a cis-acting element responsible for MYB46-mediated regulation of not only lignin biosynthesis but also cellulose and xylan biosynthesis (Tables 1, 2).…”

Transcriptional Regulation of the Lignin Biosynthetic Pathway Revisited: New Players and Insights

“…The Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in xylem tissues in different plants suggesting that the control of lignin gene expression is conserved between annual herbaceous and woody perennials and that the lignin production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes (Baghdady et al, 2006;Feuillet et al, 1995;Lacombe et al, 2000;Lauvergeat et al, 2002). Using a combination of promoter deletion/mutation analysis, electrophoretic mobility shift assay (EMSA) and/or in vivo footprinting, conserved AC elements in EgCAD2 and EgCCR promoters were shown to be crucial both for the formation of DNA-protein complexes and for the transcriptional activation of EgCAD2 and EgCCR in vascular tissues (Lacombe et al, 2000;Rahantamalala et al, 2010). Given the importance of AC elements for specific vascular expression, an in silico bioinformatic analysis of the promoters of all the lignin biosynthetic genes in Arabidopsis was undertaken (Raes et al, 2003).…”

Biotechnological improvement of lignocellulosic feedstock for enhanced biofuel productivity and processing

“…Several xylem-specific promoters have been isolated, characterized and utilized successfully to drive transgene expression in xylem tissue (Coleman et al 2006(Coleman et al , 2009Hu et al 1999;Li et al 2003;Osakabe et al 2009;Rahantamalala et al 2010;Shi et al 2010;Winzell et al 2010). However, the reported observations were made with different promoters driving different transgenes in different hosts, so it is difficult to quantitatively interpret the effectiveness of any given utility promoters.…”