Functional characterization of the Arabidopsis heat shock factor A4A, identified by a novel genetic screen

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Plants, as sessile organisms are constantly subjected to external stimuli, which determine their growth, development and survival. The environmental conditions frequently fluctuate beyond optimal parameters, becoming a source of stress for the plants. To cope with the unfavourable environment, plants have developed several strategies, which involve different biochemical and physiological adjustments. Mechanisms of stress acclimation and tolerance are controlled by complex signalling pathways that regulate gene expression. Our knowledge of stress perception is limited, downstream components of the signalling cascade are activated by specific receptors that are mostly unknown. The signal is transmitted by secondary messenger molecules such as phospholipids, cyclic nucleotides, hormones, Ca2+ and reactive oxygen species (ROS) and activates enzymes involved in post-translational modifications, i. g. protein phosphorylation by CIPKs, CDPKs and MAP kinases. Phosphorylation regulates the activity of several stress response molecules, including transcription factors. In general, mechanisms of stress tolerance are based on the maintenance of homeostasis, ROS detoxification and direct protection of cellular components. substantial amount of information on abiotic stress signalling pathways has been obtained using Arabidopsis thaliana as a model plant. Publication of the genome sequence of Arabidopsis, together with development of genetic tools has been essential to dissect molecular mechanisms behind stress responses. Particularly, genetic screens have proven to be very useful tools to identify stress-related genes. The aim of this project was to identify new components of plant stress tolerance using a novel genetic approach. For this purpose, an Arabidopsis random cDNA library under the control of an estradiol induced promoter (Papdi, 2008) was expressed in Arabidopsis cell suspension, and 1.2 million transformed cells were screened for enhanced salt tolerance. Four cell suspension derived microcolonies were identified with salt tolerant growth that was dependent of estradiol. One of the colonies carried the full-length cDNA of the heat shock transcription factor HSFA4A, this gene was chosen for further functional characterization. The conditional salt-tolerance phenotype was confirmed by re-transforming the estradiol-induced ER8-HSFA4A into Arabidopsis cell suspension. Thereafter, ectopic expression of HSFA4A in Arabidopsis plants provided tolerance to salt, paraquat, H2O2, anoxia, CdCl2 and mannitol, all these stressors converge in oxidative stress. During salt stress, HSFA4A overexpression resulted in lower lipid peroxidation rate and reduced H2O2 content indicating a decreased effect of the salt-induced oxidative damage. These results suggested that HSFA4A contributes to Arabidopsis oxidative stress tolerance. Corresponding with public available transcript profiling, gene expression analysis showed that HSFA4A expression can be induced by several stress conditions. After H2O2 treatment, rapid induction of HSFA4A was detected and high levels of HSFA4A mRNA was sustained for several days. To find target genes of HSFA4A, a transcript profiling was performed, comparing HSFA4A overexpressing plants with wild type. A set of stress response genes was identified as transcriptional targets of HSFA4A: ZAT6, ZAT12, HSP17.6A, ATL31, CRK13, WRKY30 and CTP1. Characterization of hsfa4a T-DNA insertion mutant revealed that the inactivation of HSFA4A causes salt hypersensitive growth, higher lipid peroxidation and increased H2O2 accumulation during salt stress. However, genetic complementation of hsfa4a could restore the wild type phenotype. Salt-treated hsfa4a plants showed reduced levels of ZAT6, ZAT12, WRKY30 and CTP1 comparing to wild type. A model previously proposed that in plants, ROS accumulation results in multimer formation of heat shock factors, which can be connected to their function. Performing yeast two-hybrid assay and Bimolecular Fluorescence Complementation (BIFC), it was demonstrated that HSFA4A is able to create homodimers, furthermore, during salt stress, nuclear accumulation of HSFA4A homodimers was observed. Multimer formation occurs between cysteine residues of two or more HSFA4A proteins. Substitution of 3 highly conserved cysteine residues to alanine greatly reduced HSFA4A homodimerization in yeast cells and in Arabidopsis protoplasts. Using yeast two-hybrid assay and BIFC in tobacco leaves, was showed that HSFA4A interacts with two mitogen-activated kinases MPK3 and MPK6. With in-gel and in vitro kinase assays, was demonstrated that MPK3 and MPK6 phosphorylate HSFA4A,and phosphorylation sites were identified by mass spectrometry. Site-directed mutagenesis followed by an in vitro kinase assay revealed Ser309 as the major phosphorylation site. Trans-activation of pHSP17.6A-LUC reporter construct by the Ser309Ala mutant HSFA4A was significantly reduced compared to the wild type HSFA4A.

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