Binding of the transcription factors ZmDBB4 and ZmHSFTF28 to plasma membrane aquaporin promoters

Maize (Zea mays L.) is one of the world most important cereal crops, playing a crucial role in food security. Consequently, an in-depth study of this species is essential, particularly with regard to its responses to environmental stresses. Understanding the molecular mechanisms underlying these responses could help to improve the resilience of maize crops to the impacts of climate change. Aquaporins (AQP) are small channel proteins that facilitate membrane diffusion of molecules such as H2O, CO2, and other small solutes. Due to their crucial role in various physiological processes, the regulation of AQPs is a complex process meticulously orchestrated by the plant. In this research project, particular attention was paid to the transcriptional regulation of AQPs. In a pan-genomic association study, the transcription factors (TF) ZmDBB4, responsive to light stress, and ZmHSFTF28, responsive to heat stress, were statistically associated with variations in the expression of the AQP genes ZmPIP1;3 and ZmPIP1;6, respectively. Transactivation assays on maize leaf protoplasts then demonstrated that ZmDBB4 and ZmHSFTF28 increased the activity of the promoters of ZmPIP1;3 and ZmPIP1;6, respectively. The aim of this study was to investigate the interaction between the TFs, ZmDBB4 and ZmHSFTF28, and the ZmPIP1;3 and ZmPIP1;6 promoters. The TFs were expressed in fusion with a HaloTag in Escherichia coli BL21 to perform DNA affinity purification polymerase chain reaction (DAP-PCR). TFs fused with 6-histidines were also produced and purified for electrophoretic mobility shift assay (EMSA) to validate DAP-PCR results. We were able to optimize the DAP-PCR parameters and obtained preliminary results suggesting the binding of ZmDBB4 and ZmHSFTF28 to both ZmPIP1;3 and ZmPIP1;6 promoters. However, repetition of the experiments is necessary to confirm these observations. EMSA performed with the TF ZmDBB4 revealed binding to the ZmPIP1;6 promoter, although a migration problem was encountered, necessitating repetition of the experiment.