Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Identification and functional evaluation of accessible chromatin associated with wood formation in Eucalyptus grandis.

Accessible chromatin changes dynamically during development and harbours functional regulatory regions which are poorly understood in the context of wood development. We explored the importance of accessible chromatin in Eucalyptus grandis in immature xylem generally, and MYB transcription factor-mediated transcriptional programmes specifically. We identified biologically reproducible DNase I Hypersensitive Sites (DHSs) and assessed their functional significance in immature xylem through their associations with gene expression, epigenomic data and DNA sequence conservation. We identified in vitro DNA binding sites for six secondary cell wall-associated Eucalyptus MYB (EgrMYB) transcription factors using DAP-seq, reconstructed protein-DNA networks of predicted targets based on binding sites within or outside DHSs and assessed biological enrichment of these networks with published datasets. 25 319 identified immature xylem DHSs were associated with increased transcription and significantly enriched for various epigenetic signatures (H3K4me3, H3K27me3, RNA pol II), conserved noncoding sequences and depleted single nucleotide variants. Predicted networks built from EgrMYB binding sites located in accessible chromatin were significantly enriched for systems biology datasets relevant to wood formation, whereas those occurring in inaccessible chromatin were not. Our study demonstrates that DHSs in E. grandis immature xylem, most of which are intergenic, are of functional significance to gene regulation in this tissue.