The NAC domain transcription factor family is found only in plants and is involved in important developmental and defense processes. A detailed understanding of the evolutionary relationships within the NAC family would facilitate the development of hypotheses about the function of specific NAC domain proteins. To this end, we conducted several bioinformatic analyses of all Arabidopsis, poplar, and rice NAC domain transcription factors. A phylogenetic analysis of these proteins assembled into 52 clades. Seventeen clades contained sequences from all three species and are predicted to include proteins with conserved functions in plants. Members of the remaining clades are predicted to fall into broad functional groups based on their genome of origin and that of the other proteins with which they clustered (if any). To identify relatively short stretches of conserved amino acids that might activate transcription, convey functional specificity or affect dimerization, the MEME and MAST programs were used to identify conserved motifs outside of the larger NAC domain. In total, 129 conserved motifs were identified as conserved within individual clades. Seventy-seven of these motifs were present in at least one other sequence in another clade, while 23 of them were present in multiple members of other clades. Proteins that share motifs are predicted to have related functions. Secondary wall thickening is a hallmark of cells involved in support or water transfer, and is usually seen in vascular cells. Understanding the molecular switches that control secondary wall deposition in plants is important for basic plant biology and understanding the development of vascular tissues. PtNAC068 is a Populus trichocarpa NAC domain protein that was predicted to regulate vascular development on the basis of its similarity to vascular related NAC domain proteins in Arabidopsis and the presence of its transcription in poplar vascular tissue in EST libraries. Analyses of PtNAC068 over-expression, knockdown, and dominant repression lines of transgenic poplar plants were used to elucidate the role of PtNAC068 in vascular development. PtNAC068 over-expression mutants displayed ectopic lignin deposition in leaf epidermal cells and primary leaf vein pith cells, as well as increased lignin deposition in and around phloem fiber bundles and their associated cells. Dominant repression PtNAC068 mutants displayed a reduction in lignin deposition associated with phloem fiber bundles in primary leaf veins and stems, and a loss of sclereids associated with phloem fiber bundles in stems. Thus we conclude that PtNAC068 is a positive regulator of secondary wall development in phloem fibers and associated cells.
