ETD

• abiotic stress
• Agrobacterium tumefaciens
• Arabidopsis thaliana
• promoter activity
• Reporter lines
• transgene

Plants are continuously exposed to changing environmental conditions that can cause abiotic stress, reducing plant growth and productivity. This thesis aimed to validate a luciferase-based molecular tool to monitor nitrogen status and ABA-related stress signaling using two transcriptional reporter lines: pNRT2.4::LUC, based on a high-affinity nitrate transporter induced under nitrogen limitation, and pRD29B::LUC, a well-known marker of ABA-dependent drought responses. Promoter regions were selected by in silico analysis, and the reporter constructs were assembled, propagated in E. coli, verified, and introduced into A. tumefaciens for subsequent A. thaliana transformation with the floral dip method. For pRD29B::LUC, the 8 independent lines generated were screened in a 96-well plate using a plate luminometer to select the most robust ABA-responsive line, and this line was further validated by an ABA titration assay, which showed a clear dose-dependent induction and confirmed ABA-dependent regulation of the reporter. For pNRT2.4::LUC, the 8 independent lines generated were similarly screened to identify the best-performing line, and promoter specificity was then assessed by comparing control and low-nitrogen conditions: induction occurred only under nitrogen limitation, with a progressive increase in signal over time, while control remained at basal levels. These results are supported by NightShade in vivo imaging, which confirmed stronger activation in nitrogen-starvation seedlings over multiple days. Taken together, these results showed that both pRD29B::LUC and pNRT2.4::LUC can be used as robust transcriptional reporters for their respective pathways, providing quantitative readouts suitable for comparing how different treatments influence ABA signaling or nitrogen-responsive regulation.