Functional genomics of NAC transcription factor SND2 regulating secondary cell wall biosynthesis in Arabidopsis and Eucalyptus

Abstract

Wood formation is heavily exploited for the manufacturing of pulp, paper, sustainable biomaterials and, potentially, biofuels. Eucalyptus is a favourable fast-growing, short rotation plantation crop grown over millions of hectares globally for its superior fiber properties. Understanding the molecular biology of secondary cell wall (SCW) formation in trees, and in particular how it is transcriptionally and epigenetically regulated, lays the foundation for enhanced woody trait improvement strategies in tree biotechnology. Transcriptional networks regulating SCW biosynthesis have been discovered in the herbaceous model plant Arabidopsis thaliana, in which NAC domain transcription factors (TFs) play a prominent role. The functions of many NAC domain TFs remain to be resolved, and their regulatory roles and evolution in Eucalyptus is unknown. Functional genomics studies of Eucalyptus TFs are currently challenged by a lack of established high-throughput genomics techniques commonly applied to model organisms. In this study, we aimed to better understand NAC family evolution and the epigenetic regulation of xylogenesis in E. grandis, and characterize the role of NAC domain TF SND2 in transcriptional regulation of SCW biosynthesis in A. thaliana and E. grandis. Comparative genomics and bioinformatics analyses of 189 curated gene models of the E. grandis NAC family, one of the largest described to date, revealed extensive tandem duplication in stress response-associated subfamilies, while SCW-associated subfamilies were generally conserved among angiosperms. Novel candidates for wood and tension wood formation as well as cold-stress tolerance were identified from transcriptional profiling in E. globulus and E. grandis. We identified the phenotypic effects and putative targets of the NAC domain TF SND2 in A. thaliana using microarray, microscopy and cell wall chemistry analyses. Moderate SND2 overexpression upregulated genes involved in cellulose, xylan, mannan, signaling and lignin polymerization processes and affected mannose, rhamnose and lignin components of stem cell walls, while strong overexpression resulted in reduced interfascicular fiber SCW deposition. SND2 overexpression in Eucalyptus somatic xylem sectors increased cross-sectional fiber cell area. We optimized a chromatin immunoprecipitation sequencing (ChIP-seq) approach and applied it to developing secondary xylem of mature E. grandis trees to identify the targets of the E. grandis ortholog of SND2, EgrNAC170. In validating the approach, we addressed the regulatory role of the epigenetic mark trimethylated lysine 4 of histone H3 (H3K4me3) in this tissue, showing a strong association with expressed loci, occupation of regions close to transcriptional start sites and tight correlation with transcript abundance, especially that of broadly expressed genes but also genes associated with SCW formation. A pilot study of EgrNAC170 targets was performed using the high-throughput ChIP-seq approach, identifying over 3,000 putative targets in E. grandis developing secondary xylem, but showing evidence that further ChIP-seq data are required for reliable target identification. The results of this thesis contribute to science an understanding of the unique evolution of NAC proteins in Eucalyptus, knowledge of the function of SND2/EgrNAC170 as possible candidates for tree biotechnology, the first genomic profile of a histone modification in developing wood and a high-throughput ChIP-seq protocol for the study of native protein-DNA interactions in developing xylem.