Periodic Reporting for period 1 - FD Net (Deciphering different layers of regulation of FLOWERING LOCUS D, a bZIP transcription factor that promotes flowering of Arabidopsis)
Berichtszeitraum: 2021-09-01 bis 2023-08-31
Floral transition is a crucial step in the plant life cycle, and is controlled by multiple endogenous and environmental cues such as photoperiod, temperature, hormones and sugars. The timing of floral transition affects the reproductive success of the plant by influencing several downstream traits such as plant architecture, yield and effective seed dispersal. Flowering plants dominate terrestrial ecosystems and are a major source of food for human society. Thus, understanding how plants regulate flowering is essential to secure food production, as the detrimental effects of global warming on agriculture are already visible. Arabidopsis has been widely used as a model system to decipher the genetic pathways controlling floral transition, especially in response to photoperiod. Under long-day conditions (LD), transcription of FLOWERING LOCUS T (FT) is induced in leaf phloem companion cells and the FT protein is transported to the shoot apical meristem to initiate floral development. In the meristem, FT-mediated activation of flowering-time and floral-meristem identity genes requires the interaction of FT with the basic leucine zipper (bZIP) transcription factor FD.
Recently, a growing body of evidence supports the idea that FD performs other functions in plant development in addition to its role in floral transition. The overall objective of the Action was to decipher different layers of regulation of FD, which would reveal different modes of action of FD during plant development. For this, the FD phosphorylation status, its protein interactome, and its relationship with two group A bZIP transcription factors were investigated. In conclusion, the results obtained during this Action have advanced our knowledge of how photoperiod regulates different developmental processes through an intricate network composed of FT, FD and several group A bZIP transcription factors.
Recently, a growing body of evidence supports the idea that FD performs other functions in plant development in addition to its role in floral transition. The overall objective of the Action was to decipher different layers of regulation of FD, which would reveal different modes of action of FD during plant development. For this, the FD phosphorylation status, its protein interactome, and its relationship with two group A bZIP transcription factors were investigated. In conclusion, the results obtained during this Action have advanced our knowledge of how photoperiod regulates different developmental processes through an intricate network composed of FT, FD and several group A bZIP transcription factors.
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.
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.
Floral transition is a crucial step in the plant life cycle, and the timing of floral transition affects the reproductive success of the plant. Flowers are one of the most important evolutionary innovations that have contributed to the diversification of angiosperms. Flowering plants nowadays dominate terrestrial ecosystems and are a major food source for human society. Many important crop species flower in response to changes in photoperiod and understanding how plants respond to photoperiod is essential to secure food production, as the detrimental effects of global warming on agriculture are already visible.
Two decades ago, the identification of the FT–FD pathway was a major advance in understanding how photoperiod regulates flowering, and this knowledge was later shown to be conserved among angiosperms. Within this context, I expect that the findings of this project will be readily translated to other species. The work developed during this Action aimed to characterise further FD, a core transcription factor of the plant response to photoperiod at the meristem. For the first time in vivo, the phosphorylation of the FD SAP motif was identified, and that this post-translational modification is important to maintain a uniform distribution of the FD protein in the nucleus. The identification that phosphorylation of the FD SAP motif affects its solubility opens up novel ways to understand how this pathway is finely regulated at cellular level. Additionally, the functional characterisation of the relationship between FD with AREB3 and bZIP13 revealed how FD regulates different phases of plant development. We have shown that AREB3 is responsible to perform an FD-like function by mediating FT signalling at the shoot apical meristem partially redundant with FD. I have demonstrated that bZIP13, a previously uncharacterised transcription factor, directly or indirectly represses cell proliferation and that FD is responsible to shape bZIP13 function in response to photoperiod.
The results generated during this Action are of broad interest because they advance our understanding of how photoperiod regulates different developmental processes through a complex network composed of FT and several group A bZIP transcription factors.
Two decades ago, the identification of the FT–FD pathway was a major advance in understanding how photoperiod regulates flowering, and this knowledge was later shown to be conserved among angiosperms. Within this context, I expect that the findings of this project will be readily translated to other species. The work developed during this Action aimed to characterise further FD, a core transcription factor of the plant response to photoperiod at the meristem. For the first time in vivo, the phosphorylation of the FD SAP motif was identified, and that this post-translational modification is important to maintain a uniform distribution of the FD protein in the nucleus. The identification that phosphorylation of the FD SAP motif affects its solubility opens up novel ways to understand how this pathway is finely regulated at cellular level. Additionally, the functional characterisation of the relationship between FD with AREB3 and bZIP13 revealed how FD regulates different phases of plant development. We have shown that AREB3 is responsible to perform an FD-like function by mediating FT signalling at the shoot apical meristem partially redundant with FD. I have demonstrated that bZIP13, a previously uncharacterised transcription factor, directly or indirectly represses cell proliferation and that FD is responsible to shape bZIP13 function in response to photoperiod.
The results generated during this Action are of broad interest because they advance our understanding of how photoperiod regulates different developmental processes through a complex network composed of FT and several group A bZIP transcription factors.