Charlotte O Shea:
NAC transcription factors in stress responses and senescence: importance of intrinsic disorder and DNA binding

Date: 12-08-2014    Supervisor: Karen Skriver

Plant-specific NAM/ATAF/CUC (NAC) transcription factors have recently received considerable attention due to their significant roles in plant development and stress signalling. This interest has resulted in a number of physiological, genetic and cell biological studies of their functions. Some of these studies have also revealed emerging gene regulatory networks and protein-protein interaction networks. However, structural studies relating structure to function are lagging behind. Structure-function analysis of the NAC transcription factors has therefore been the main focus of this PhD thesis.

A systematic analysis has been performed of protein intrinsic disorder (ID), referring to the lack of a fixed tertiary structure, in NAC transcription factors. The transcription regulatory domains (TRDs) from six phylogenetically representative Arabidopsis thaliana NAC transcription factors have a similarly low degree of average structure but different patterns of disorder/order and molecular recognition features (MoRFs). For example, senescence-associated ANAC046 has a simple pattern with just a single MoRF. Analysis in yeast and thermodynamic characterisation suggested that the 11-residue C-terminal MoRF is a functional hotspot for both transcriptional activity and interaction with the cellular hub protein Radical Induced Cell Death1 (RCD1). Specific amino acid residues essential for the interaction were identified. These studies and structural analysis suggested that RCD1-ANAC046 complex formation does not involve significant folding-upon-binding but fuzziness or an extended ANAC046 region. The ANAC046 regulatory domain functions as an entropic chain with a bait for interactions with for example RCD1. RCD1 interacts with transcription factors from several different families, and the large stress-associated RCD1-transcription factor network represents a much-needed model for translation of in vitro obtained ID analysis, as presented here, to the in vivo organismal level.

A systematic analysis was performed of the DNA-binding-site landscape and regulatory network of NAC transcription factors in Arabidopsis as a prerequisite for a system-wide understanding of NAC transcription factors. This PhD thesis contributes to the work through the production and purification of NAC domain recombinant proteins. More importantly, the work presented here has created a platform for future verification of predicted NAC target genes identified by the systematic binding-site analysis. This platform uses tools such as knock-out phenotypes and over-expression available for the model plant Arabidopsis.

The final contribution to the NAC transcription factor field presented in this thesis is an overall summary of the contribution of these transcription factors to abiotic stress responses. Here the focus is on NAC transcription factors in abiotic stress responses and senescence, and on physiologically relevant interactions with DNA and other proteins.