Characterization of regulatory roles of DPBF4 and SNF4 in sugar and stress signaling in Arabidopsis

In higher plants, the regulation of metabolic and developmetal processes is tightly connected to a complex crosstalk between hormone signaling pathways. Many responses controlled by osmotic stress (salinity, drought and cold) stimuli are synergictically induced by the stress hormone abscisic acid and by glucose and sucrose. The ancient pathway of glucose repression, which is found in all organisms, also plays a central role in the regulation of transcription and growth responses in plants, though this pathway negatively controls the activity of genes involved in photosynthesis. Glucose and sucrose essentially signal through stimulation of ABA synthesis, whereas ABA signaling requires elements of the ethylene signaling pathway. Within this web of signaling functions, the closely related SnRK1 and SnRK2 families of protein kinases play a distinguished role. Whereas SnRK1 enzymes are key cellular glucose and AMP sensors, which monitor the cells� energy homeostasis, their relatives in the SnRK2 kinase family participate in the regulation cellular responses to osmotic stress stimuli by estabilishing connections between early signaling events, such as generation of secondary messengers, and direct regulation of the activity of transcription factors by phosphorylation. In this work a study of the function and regulation of an Arabidopsis bZIP transcription factor, DPBF4, is described. DPBF4 was identified as a G-box binding factor, which recognizes glucose -responsive regulatory sequences in the promoter of the aspartate kinase gene AK1/HSD1 implicated in the control of biosynthesis of essential amino acids. DPBF4 is a member of the ABI5/DPBF/AREB/ABF bZIP family, which include closely related transcription factors. Analysis of dpbf4 knockout mutations suggests fuctional redundancy of Arabidopsis DPBF homologs. DPBF4 is a substrate of the SnRK2 protein kinase OST1 (OPEN STOMATA). Characterization of OST1 shows that this kinase is capable of autophosphorylation within its activating T-loop and recognizes common substrates with members of the SnRK1 kinase family. Mapping of critical amino acid residues by site-specific mutagenesis provides an insight into details of regulation of SnRK2 kinase activity. OST1 is probably required for stimulation of dimerization and DNA-binding of DPBF4, which was found in chromatin-associated protein complexes. Chromatin crosslinking experiments show that DPBF4 indeed occupies specific cis-regulatory elements in the promoter of the model AK1/HSD1 aspartokinase gene. DPBF4 turned out to be also a substrate of SnRK1 kinases, whose activation is controled by their SNF4 gamma subunits. Characterization of knockout mutations demonstrates that SNF4 is essential for male gametogenesis. SNF4 is a nuclear kinase subunit and its nuclear import appears to be light-regulated. The work described in this thesis offers a wide repertoire of molecular, genetic and biochemical tools for dissection of regulatory elements of glucose and stress signaling pathway, in particular for identification of partners and regulators of DPBF-like bZIP proteins and protein kinases of SnRK1 and SnRK2 families.