Supervisors info:
Κοσμάς Χαραλαμπίδης, Αναπληρωτής Καθηγητής Μοριακής Βιολογίας Ανάπτυξης Φυτών, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ)
Summary:
In plants, several specific transcription factor binding sites and their respective transcriptional factors (TFs) regulate the expression of genes involved in various developmental processes and/or biotic and abiotic stress responses. Two of these elements are the SMRE and SNBE binding sites, that are located at multiple sites within the Arabidopsis thaliana VPNB1 promoter. The VPNB1 gene is expressed exclusively in the plant vascular tissue and seems to be involved in the biogenesis and differentiation of the vascular cambium, the phloem, and the xylem cells. The final step during xylem differentiation comprises the deposition of secondary cell wall materials. The latter involves a highly complex network of transcriptional factors, which bind among other sites, to the SMRE and SNBE promoter elements. During this study, a promoter deletion analysis of the VPNB1 gene was carried out. In this context, several promoter::GUS transgenic lines, harboring various fragments of the Arabidopsis thaliana VPNB1 promoter, were analyzed both quantitatively and qualitatively by using colorimetric GUS-staining and fluorometry, respectively. VISUAL (Vascular cell Induction culture Using Arabidopsis Leaves) was also performed on selected transgenic lines, to investigate the responsiveness of the VPNB1 promoter in the differentiation of mesophyll cells into tracheary elements. The quantitative results showed a statistically significant reduction of promoter activity, in both 3 and 8 DAS (Days After Sowing) seedlings as well as reproductive tissues (flowers), by the sequential deletion of the SNBE located in -1422, the SMRE at position -1247, the SNBE at position -1146 and SNBE at position -1041. Furthermore, the qualitative GUS-staining assays confirmed that the deletion of the SMRE and the two SNBEs located at position -1247, -1146 and -1041, respectively, affected the GUS staining pattern. Finally, the VISUAL experiments showed that the tissue cultures responded to the induction, as strong GUS staining was evident in all the transgenic lines used. Taken together, the results indicate that the SNBE and SMRE transcription factor binding sites located within the VPNB1 regulatory sequences act cooperatively in promoter induction and consequently in VPNB1 gene expression during vascular development and differentiation. VISUAL, on the other hand, confirmed that the SNBE and SMRE promoter elements are essential for the differentiation of mesophyll cells into tracheary elements.
Keywords:
promoter deletion analysis, Arabidopsis thaliana, vascular development, GUS assay, VISUAL
