Investigation of the functional role of the tomato SlBBX16/17 MicroProteins and optimization of a method for silencing genes involved in the reproductive development by exogenous dsRNA

Climate change poses a major threat to global agricultural systems, as plants must rapidly adjust to increasingly fluctuating environmental conditions, such as extreme temperatures, drought, and variable light conditions. Understanding how plants perceive and respond to these environmental cues at the molecular level is essential for developing adaptive strategies in agriculture. In this context, the B-box (BBX) family of transcription factors has emerged as a key player in regulating plant development and responses to environmental stress. BBX proteins, which contain one or two B-box domains that mediate protein-protein interactions, often possess a CCT domain (CONSTANS, CO-like and TIMING OF CAB: TOC1) responsible for DNA binding and transcriptional regulation. This family plays essential roles in processes such as photomorphogenesis, flowering time control, and abiotic stress tolerance. This thesis explores the functional role of two microProteins (miPs) belonging to group V BBX, SlBBX16 and SlBBX17, in tomato (Solanum lycopersicum) reproductive development, alongside the potential application of exogenous double-stranded RNA (dsRNA) for gene silencing to regulate fruit growth. To investigate the role of SlBBX16, we generated transgenic tomato plants overexpressing SlBBX16 and analyzed their phenotypes and hormonal gene expression. Overexpression of SlBBX16 caused a delay in fruit growth, particularly between the early green and breaker stages, implying a role for SlBBX16 in fruit expansion. Furthermore, a previous PhD study demonstrated that SlBBX17 overexpression prolonged the flowering period and reduced fruit yield in MicroTom plants. In both SlBBX16 and SlBBX17 transgenic lines, changes in gibberellin metabolism were observed, confirming their potential regulatory role in tomato fruit development. To explore the molecular mechanisms underlying the regulatory functions of SlBBX16 and SlBBX17, yeast two-hybrid (Y2H) assays were performed to assess their interactions with key flowering regulators in tomato. Specifically, interactions with CONSTANS (SlCOL1), a flowering time regulator, and TOPLESS (SlTPL1), a co-repressor, were tested. In Arabidopsis, miP1a and miP1b the closest homologs of SlBBX16 and SlBBX17 have been shown to form a flowering repression complex together with CO and TPL, the Arabidopsis homologs of SlCOL1 and SlTPL1, respectively (Graeff et al., 2016). We also tested a mutant version of SlBBX16 (SlBBX16*) containing a PFVLF motif found in Arabidopsis, but absent in SlBBX16. Results showed that neither SlBBX16 nor SlBBX16* interacted with SlCOL1 or SlTPL1, suggesting that these proteins do not form a flowering inhibitor complex in tomato, as observed in Arabidopsis. These findings indicate that either additional factors are necessary for the interaction, or other CO-like family members in tomato may be involved in these pathways. To identify additional interacting partners of SlBBX16 and SlBBX17, we will employ Affinity Purification-Mass Spectrometry analysis (AP-MS). For this purpose, new genetic constructs were generated by fusing a 3xFLAG epitope to the C-terminal regions of SlBBX16 and SlBBX17. Transgenic MicroTom plants expressing these constructs were produced and confirmed through PCR screening and dot-blot assays to detect FLAG-tagged fusion protein expression. Western blot analysis further validated the expression of FLAG-tagged proteins in T1 plants, providing a foundation for subsequent AP-MS experiments. Another objective of this research was to suppress the expression of endogenous genes involved in ovary development by applying dsRNA to young flower buds. Specifically, we targeted key regulators of ovary growth, such as the tomato AUX/IAA9 (SlIAA9) and SlAGAMOUS-like 6 (SlAGL6), which are well-known inhibitors of ovary expansion before fertilization. To enhance the efficiency of gene silencing, we coupled the dsRNA with layered double hydroxide (LDH) nanoparticles, which significantly improved both the stability of the RNA and its delivery into the tomato flower buds. This work demonstrates that dsRNA can be injected into floral pedicels as a quick and effective way to temporarily silence endogenous genes, allowing to study the molecular events that control fruit set. Additionally, using nanovectors to deliver dsRNA can help maintain and prolong gene silencing. These findings could be useful for future applications, including the possibility of transiently silencing SlBBX16 and SlBBX17 genes by exogenous dsRNA, as well as other Group V BBXs, to further investigate their role in tomato reproductive development.