| Potassium (K+) is one of the most abundant cations in plant cells, and participates in many physiological processes in plants. Potassium deficiency is one of major abiotic stresses that leads to the reduction of crop yield and quality. To adapt to K+-deficiency, plants have evolved a complex signaling pathways that help them to survive under K+-deficiency. Previous reports showed that regulation of K+ transporters at transcriptional level or post-translational level are two major mechanisms in plants response to low-K+ stress. HAK5 is one of the most important K+ transporters in Arabidopsis, which mediates high-affinity K+ transport in roots. The transcript of HAK5 is remarkably induced by K+-deficiency in order to enhance high-affinity K+ uptake. However, the transcriptional regulation of HAK5 is still unclear. The focus of this dissertation work is to characterize the role of transcription factor ARF2 (Auxin Responsive Factor 2) in K+ uptake regulation, and analyze ARF2-mediated HAK5 transcriptional regulation in Arabidopsis response to low-K+ stress.Using reverse genetic approach, we found that the mutant of transcription factor ARF2 showed low-K+-tolerant phenotype. ARF2 strongly expresses in both seedling roots and shoots. The primary root lengths of arf2 were longer than that of wild-type plants, when they directly germinated on low-K+ (0.01 mM) medium. ARF2-overexpressing lines were sensitive to low-K+ stress, whose primary root lengths were shorter than wild-type. The complementation lines could restore the phenotype of arf2 to wild-type level. The results of K+ content measurement and 6Rb+ uptake assays indicated that arf2 had higher K+ uptake capacity and accumulated more K+ in plants under low-K+ conditons.As a transcription factor, ARF2 can bind to auxin-responsive elements (AuxRE) within gene promoter, and repress the gene expression. The qRT-PCR and GUS/LUC assays showed that ARF2 repressed HAK5 expression. EMSA and ChIP results showed that ARF2 can directly bind to HAK5 promoter in vitro and in vivo. Phenotype test indicated that the low-K+-tolerant phenotype of ar/2 was entirely abolished in arf2 hak5 double mutant, whose primary root length was as short as hak5 single mutant under low-K+ conditions. It is suggested that HAK5 is genetically epistatic to ARF2. In addition, we found that the root hair lengths of arf2 mutant and HAK5-overexpressing lines were longer than that of wild-type only under low-K+ condition. Contrarily, hak5 mutant and arf2 hak5 double mutant exhibited extremely short root hairs on low-K+ medium.ChIP-qPCR results showed that the interaction between ARF2 protein and HAK5 promoter was severely impaired under K+-deficient conditions, which was regulated by phosphorylation. It’s observed that low-K+ treatment can trigger a rapid phosphorylation of ARF2, which released the repression on HAK5 transcription. The S689 of ARF2 can be phosphorylated after low-K+ treatment, suggesting its crucial role in ARF2 response to low-K+ stress. Mutation of S689D abolished ARF2 DNA-binding activity to HAK5 promoter, and removed the repression of HAK5 transcription. Moreover, ARF2 was degraded after low-K+ treatment, which was dependent on 26S proteasome-mediated pathway,In summary, the results presented in this dessertation reveal that ARF2 plays important roles in Arabidopsis response to external K+ supply and regulates HAK5 transcription accordingly. Under K+ sufficient conditions, ARF2 binds to HAK5 promoter and represses HAK5. When plants are subjected to low-K+ stress, ARF2 is rapidly phosphorylated and loses DNA-binding activity. ARF2 is removed from HAK5 promoter and relieves the repression on HAK5 transcription. Finally, the elevated HAK5 expression enhances HAK5-mediated high-affinity K+ uptake, which can help plants to survive under K+ deficient conditions. |
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