During the Action, several rounds of protocol optimisation were performed to efficiently isolate nuclei from meristem-enriched samples, as access to the inner tissues of the meristem is a critical step in efficiently purifying cells expressing FD. Another major bottleneck faced was the long harvesting times required to obtain sufficient material, which led me to test protocols to isolate nuclei from frozen tissues. However, the original strategy of isolating nuclei containing Venus and GFP fluorescent markers proved inappropriate.
To characterise the phosphorylation status of FD in vivo, protein immunoprecipitation followed by mass spectrometry (IP-MS) was employed. Phosphorylation of the FD SAP motif was the only phosphorylated site identified in vivo. Although FD SAP motif phosphorylation has been proposed and implicated in the photoperiodic control of flowering for decades, this is the first in vivo data that demonstrate its phosphorylation. Confocal microscopy and high sensitivity western blotting were used to analyse FD protein localisation and stability. The phosphorylation of the FD SAP motif does not affect the stability of the protein, but rather influences its solubility in the nucleus.
The characterisation of the FD protein interactome was performed using the complementary IP-MS and yeast two-hybrid (Y2H) approaches. These strategies identified FD interaction with several 14-3-3 proteins and group A bZIP transcription factors, including ABA-RESPONSIVE ELEMENT BINDING PROTEIN 3 (AREB3) and bZIP13. Although 14-3-3 proteins have been proposed to be components of the Florigen Activation Complex, this work is the first evidence in vivo for their participation in this complex. In a collaboration with the University of Milan, we recently demonstrated that AREB3 colocalises with FD to the SAM and regulates floral transition by relaying FT signals partially redundantly with FD. AREB3 was originally assigned to the ABA-related clade of group A bZIP TFs, and our results suggest an extensive interaction between ABA responses and flowering-time regulation by group A bZIP TFs. Additionally, the relationship of bZIP13, a previously uncharacterised group A bZIP transcription factor, and FD revealed that bZIP13 heteromeric complexes with FD shape bZIP13 function in response to photoperiod.
The data generated by “FD Net” was disseminated by scientific publications and participation in local and international scientific meetings. One of the main outputs of this Action was a joint publication with the University of Milan in the Open Access journal PLoS Genetics (Martignago & Falavigna et al. 2023). This collaboration was established during the Flowering Symposium held in Milan, Italy. Additionally, I attended international scientific conferences such as the Workshop on Molecular Mechanisms Controlling Flowering held in Alicante, Spain, and the 33rd International Conference on Arabidopsis Research held in Chiba, Japan. In-house seminars were also organised with the participation of the local academic community to present and discuss preliminary results, and improved protocols and methods.
