RETINOBLASTOMA-RELATED 1 roles in Nicotiana tabacum glandular trichomes development

Details

Supervisors

Hachez, Charles ; Peeters, Manon

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

Nicotiana tabacum L. (Tobacco) is a model plant belonging to the Solanaceae family. Tobacco harbors epidermal outgrowths on its aerial parts known as "trichomes". These structures play a significant role in plant-environment interactions, particularly through the production and secretion of bioactive compounds called "secondary metabolites". Due to their unique properties, these compounds are of increasing interest in the agricultural, pharmaceutical, and biotechnological sectors. It is therefore essential to enhance our understanding of the developmental processes of glandular trichomes for the optimization of their industrial applications and the deepening of research in related fields. The present study investigates the roles of RETINOBLASTOMA-RELATED 1 (RBR1) in regulating the development of long-stalked glandular trichomes in Nicotiana tabacum. RBR1 has been previously identified as a pivotal cell cycle regulator in various plant species; however, its specific functions in tobacco glandular trichomes remain to be elucidated. The present thesis centers on the targeted silencing of RBR1 in trichome cells through the utilization of artificial microRNA (AmiRNA) technology, with the aim of elucidating its implications in cell cycle regulation, endoreduplication, and trichome branching. AmiRNA constructs designed to target RBR1 were synthesized and integrated with specific promoters (pMYBML3, pCYCD3, and pAGL66), thereby enabling spatiotemporal silencing in developing trichomes. Transgenic plants exhibited an increase in the number of branched glandular trichomes, with 6.7% observed in certain mutant lines versus 1.3% in wild types (WT). Furthermore, nuclear area measurements revealed that the first cell of glandular trichomes stalks is bigger than in other epidermal cells (other trichome cells, pavement and stomata guard cells) of WT plants, suggesting that endoreduplication naturally occurs in these cells. This observation was accurate in WT but even more in mutant lines. These findings suggest an increase of endoreduplication amount in these cells, which is consistent with RBR1's known role as a negative regulator of this process in Arabidopsis thaliana. However, enhanced cell division was also observed, with more branched trichomes and glandular trichomes comprising twice as many cells in mutant lines as in WT, suggesting a dual role for RBR1 in balancing endoreduplication and mitosis. However, challenges such as inconsistent plant growth and undetectable AmiRNA expression in plant tissues were encountered. Consequently, future projects should optimize silencing approaches, including refining AmiRNA-based approaches to optimize gene silencing and exploring alternative strategies, such as inducible expression systems. Additionally, improving the plant screening process is crucial to save precious research time.