Identification of Norway Spruce MYB-bHLH-WDR Transcription Factor Complex Members Linked to Regulation of the Flavonoid Pathway

Abstract

Transcription factors (TFs) forming MYB-bHLH-WDR complexes are known to regulate the biosynthesis of specialized metabolites in angiosperms through an intricate network. These specialized metabolites participate in a wide range of biological processes including plant growth, development, reproduction as well as in plant immunity. Studying the regulation of their biosynthesis is thus essential. While MYB (TFs) have been previously shown to control specialized metabolism (SM) in gymnosperms, the identity of their partners, in particular bHLH or WDR members, has not yet been revealed. To gain knowledge about MYB-bHLH-WDR transcription factor complexes in gymnosperms and their regulation of SW, we identified two bHLH homologs of AtTT8, six homologs of the MYB transcription factor AtTT2 and one WDR ortholog of AtTTG1 in Norway spruce. We investigated the expression levels of these genes in diverse tissues and upon treatments with various stimuli including methyl-salicylate, methyl-jasmonate, wounding or fungal inoculation. In addition, we also identified protein-protein interactions among different homologs of MYB, bHLH and WDR. Finally, we generated transgenic spruce cell lines overexpressing four of the Norway spruce AtTT2 homologs and observed differential regulation of genes in the flavonoid pathway and flavonoid contents.

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective

“…However, although R2R3MYB and bHLH genes do regulate flavonoid biosynthesis in liverworts, and there are conserved WDR sequences in the genome (Bowman et al, 2017), there is no answer yet on whether the MBW complex exists in bryophytes. A flavonoidrelated MBW complex has been characterized in the gymnosperm Norway spruce (Picea abies) (Nemesio-Gorriz et al, 2017), supporting an origin for the MBW complex in the plant lineage prior to the last common ancestor of gymnosperms and angiosperms, around 350-300 MYA. However, although the conserved amino acid motif ([D/E]Lx2[R/K]x3Lx6Lx3R) identified as necessary for R2R3MYB proteins to bind the bHLH partners (Zimmermann et al, 2004) is present in the S. moellendorffii sequence SmXP002978781, it is lacking in bryophyte R2R3MYBs studied to date.…”

Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L.

“…Accumulating evidence demonstrates that TT8 is a central component of the well‐conserved complex that controls flavonoid accumulation in various crops (Escaray et al , ; Li et al , ; Li et al , ; Lim et al , ; Nemesio‐Gorriz et al , ; Schaart et al , ). Taking into account the close phylogenetic relationship between Arabidopsis and Brassica , TT8 gene homologs could play comparable roles in Brassica species.…”

“…In A. thaliana, a ternary complex (known as the MBW complex) comprising three TF regulators, namely AtMYB123/TT2 (R2R3-MYB), TT8 (basic helix-loop-helix, bHLH) and TTG1 (WD40 protein), plays a key role in activating PA-specific genes in seed coat development (Xu et al, 2014(Xu et al, , 2015. Accumulating evidence demonstrates that TT8 is a central component of the well-conserved complex that controls flavonoid accumulation in various crops (Escaray et al, 2017;Li et al, 2016;Lim et al, 2017;Nemesio-Gorriz et al, 2017;Schaart et al, 2013). Taking into account the close phylogenetic relationship between Arabidopsis and Brassica, TT8 gene homologs could play comparable roles in Brassica species.…”

Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants

“…Although the MBW complex is often regarded as universal for anthocyanin regulation, it is actually an open question as to what aspects of the system are conserved across the major land plant groups (Albert et al ., ; Liu et al ., ; Xu et al ., ; Lloyd et al ., ). The recent finding of a conserved MBW complex able to activate flavonoid biosynthesis in a gymnosperm species (Nemesio‐Gorriz et al ., ) suggests an ancient origin in the plant lineage, as angiosperms and gymnosperms are thought to have diverged c . 300 Ma (Lu et al ., ).…”

“…Although the MBW complex is often regarded as universal for anthocyanin regulation, it is actually an open question as to what aspects of the system are conserved across the major land plant groups (Albert et al, 2014;Liu et al, 2015;Xu et al, 2015;Lloyd et al, 2017). The recent finding of a conserved MBW complex able to activate flavonoid biosynthesis in a gymnosperm species (Nemesio-Gorriz et al, 2017) suggests an ancient origin in the plant lineage, as angiosperms and gymnosperms are thought to have diverged c. 300 Ma (Lu et al, 2014). However, genomes of the basal plant groups liverworts (Bowman et al, 2017) and mosses (Rensing et al, 2008) contain markedly fewer MYB and bHLH genes than those of angiosperms and gymnosperms, suggesting a more limited range of regulatory activities.…”