Temporal analysis of Arabidopsis genes activated by Eucalyptus grandis NAC transcription factors associated with xylem fibre and vessel development

Secondary cell wall (SCW) deposition in Arabidopsis is regulated among others by NAC transcription factors, where SND1 chiefly initiates xylem fibre differentiation while VND6 controls metaxylem vessel SCW development, especially programmed cell death and wall patterning. The translational relevance of Arabidopsis SCW regulation theory and the utility of characterized transcription factors as modular synthetic biology tools for improving commercial fibre crops is unclear. We investigated inter-lineage gene activation dynamics for potential fibre and vessel differentiation regulators from the widely grown hardwood Eucalyptus grandis (Myrtales). EgrNAC26, a VND6 homolog, and EgrNAC61, an SND1 homolog, were transiently expressed in Arabidopsis mesophyll protoplasts in parallel to determine early and late (i.e. 7 and 14 hours post-transfection) gene targets. Surprisingly, across the time series EgrNAC26 activated only a subset of SCW-related transcription factors and biosynthetic genes activated by EgrNAC61, specializing instead in targeting vessel-specific wall pit and programmed cell death markers. Promoters of EgrNAC26 and EgrNAC61 both induced reporter gene expression in vessels of young Arabidopsis plants, with EgrNAC61 also conferring xylem- and cork cambium-preferential expression in Populus. Our results demonstrate partial conservation, with notable exceptions, of SND1 and VND6 homologs in Eucalyptus and a first report of cork cambium expression for EgrNAC61.M.L. prepared the constructs, performed the protoplast RNA-seq analyses and yeast transactivation experiments, and co-wrote the manuscript with S.H. K.B., L.T. and S.H. conducted promoter::GUS analyses and drafted these sections of the manuscript. E.M. and A.M. edited the manuscript, co-supervised the study with S.H. and assisted with funding.A strategic (Eucalyptus Genomics Platform) grant from the Department of Science and Technology (DST) and the National Research Foundation of South Africa (NRF Bioinformatics and Functional Genomics Programme grant UID 97911). Seed funding was provided by the Genomics Research Institute (University of Pretoria). Finally, the work was supported by Sappi and Mondi through the Forest Molecular Genetics (FMG) Programme and matching funding from the Technology and Human Resources for Industry Programme (THRIP gr ant UID 96413).http://www.nature.com/srepam2018BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog