Trine Kjærsgaard:
Structure-function relationship of barley senescence-associated NAC transcription factors

Date: 30-09-2011    Supervisor: Karen Skriver

In this thesis, three studies focusing on the understanding of the role of NAC transcription factors (TFs) both globally and more particularly, in senescence, are presented. The NAC family constitutes a large family of TFs present in a wide range of land plants, and the family comprises more than 100 members in Arabidopsis thaliana (Arabidopsis). NAC proteins consist of two regions: N-terminal conserved DNA-binding domains (NAC domains), which ensure binding to target promoters, and divergent C-terminal transcriptional regulatory domains (TRDs), which confer transcriptional activity and contain sites for protein-protein interactions.

A study of the senescence-associated NAC TFs, HvNAC005 and HvNAC013, presents the fist structural analysis of NAC TRDs. Results show that they are largely intrinsically disordered in their TRDs. HvNAC013 has transactivational activity, which is conferred by a small conserved sequence motif, the LP motif. HvNAC013 furthermore interacted with barley Radical-Induced Cell Death1 (HvRCD1). This interaction was mediated between the disordered C-terminus of HvNAC013 and a conserved motif in HvRCD1. RCD1 also interacted with other TFs through their disordered domains, and may be a regulatory protein, which exploits the disorder in TFs for interaction. There is a large oxidative burst upon leaf senescence and reactive oxygen species (ROS) themselves can trigger senescence. Expression analyses indicated increased expression of both HvNAC013 and HvRCD1 upon stress treatment with ROS, providing a possible physiological link to the interaction.

A global study of Arabidopsis NAC family members revealed an interesting sub-grouping of NAC genes, relating structure and function. A meta-analysis revealed distinct temporal expression profiles of NAC genes upon stimuli with plant phytohormones. Furthermore, a large degree of intrinsic disorder in the NAC TRDs was identified. A study of 10 phylogenetically widely distributed NAC proteins showed that 9 out of 10 proteins are dependent on their C-terminal domains for transactivational activity. Furthermore, 9 out of 10 proteins bind a previously identified NAC DNA target sequence. The NAC family member ANAC019 was identified as a positive regulator of absiscic acid (ABA) signaling, conferring ABA hypersensitivity when ectopically expressed in Arabidopsis. In planta domain swap experiments to decipher the functional modularity of the NAC domains and the divergent C-terminal domains showed that the TRD could confer the ABA hypersensitivity. Altogether, the biochemical and functional specificity of NAC TFs is associated with both the NAC domains and the TRDs.