Periodic Reporting for period 1 - StickyFT (Genetic engineering as an approach to enhance the lipid-binding properties of the Flowering locus T, the member of the Phosphatidylethanolamine-binding protein family)
Periodo di rendicontazione: 2023-11-06 al 2025-11-05
Rising global temperatures are already disrupting plant development, including the timing of flowering, which is essential for successful crop production. The IPCC predicts a temperature increase of at least 1.5°C by 2050, and many crop species have already begun flowering earlier, by roughly two days per decade over the last 50 years. Earlier flowering shortens the reproductive phase, reduces yield, and threatens future food security. Understanding how temperature affects flowering is therefore a key challenge for agriculture in a warming climate. This project focuses on a newly discovered temperature-sensitive mechanism involving FLOWERING LOCUS T (FT), a protein known as florigen that triggers flowering. My recent findings show that FT interacts with negatively charged lipid membranes in a temperature-dependent way: at cooler temperatures (around 16°C) FT is trapped on membranes, while at warmer temperatures (around 22°C) it is released and able to move to the shoot apex to activate flowering. This suggests that membrane binding may serve as an additional regulatory layer that links climate warming directly to flowering time. The overall goal of the project is to use genetic engineering to create modified versions of FT with weaker or stronger lipid-binding properties, and to test how these variants behave under different temperatures. By producing FT versions that range from “super-sticky” to “non-binding,” and by measuring their interactions with membranes, the project aims to reveal how temperature influences FT mobility and flowering regulation. This includes testing known FT mutants as well as generating new combinatorial variants designed to shift lipid-binding strength. The project will fill an important knowledge gap, as almost no research has examined how temperature alters protein–membrane interactions in the context of plant development. Preliminary results already show that FT affects membrane fluidity, while FT mutants change membrane rigidity—suggesting that FT is uniquely suited for studying this mechanism. By uncovering how temperature affects FT positioning and activity, the project will provide fundamental knowledge needed to understand and potentially stabilize flowering time in crops under climate change. This research will contribute to long-term strategies for improving plant resilience, supporting both agricultural adaptation and global food security in a changing climate.
The researcher successfully designed, constructed, cloned, and purified two sets of mutant FT proteins, all obtained in soluble form and suitable for downstream analyses. These variants were screened using qualitative lipid-binding approaches, including lipid–overlay and liposome–sedimentation assays. Most mutants behaved similarly to the wild-type FT, whereas the 3R variant showed reduced membrane affinity, identifying it as a key target for continued investigation.
Quantitative analyses were then performed to characterise FT–membrane interactions under different ambient temperatures. The results showed that wild-type FT binds more strongly at 25 °C than at 15 °C, suggesting that in vivo additional membrane components may stabilize FT association during cold conditions. The 3R mutant displayed slower dissociation kinetics, indicating altered membrane-interaction dynamics. To gain deeper structural and biophysical insight, the wild-type FT protein was examined using Cryo-EM, while selected wild-type and mutant proteins (including 3R and FTdC) were further analysed with surface plasmon resonance (SPR) and thermal stability profiling using the Prometheus Panta instrument. These analyses revealed that although thermal behaviour differed depending on storage and buffer conditions, all FT variants exhibited similar unfolding and refolding patterns once mixed with liposomes.
Overall, the work produced a detailed biochemical and biophysical characterisation of wild-type and mutant FT proteins, pinpointing residues important for membrane association and providing mechanistic insights into how FT contributes to temperature-dependent control of flowering.
Quantitative analyses were then performed to characterise FT–membrane interactions under different ambient temperatures. The results showed that wild-type FT binds more strongly at 25 °C than at 15 °C, suggesting that in vivo additional membrane components may stabilize FT association during cold conditions. The 3R mutant displayed slower dissociation kinetics, indicating altered membrane-interaction dynamics. To gain deeper structural and biophysical insight, the wild-type FT protein was examined using Cryo-EM, while selected wild-type and mutant proteins (including 3R and FTdC) were further analysed with surface plasmon resonance (SPR) and thermal stability profiling using the Prometheus Panta instrument. These analyses revealed that although thermal behaviour differed depending on storage and buffer conditions, all FT variants exhibited similar unfolding and refolding patterns once mixed with liposomes.
Overall, the work produced a detailed biochemical and biophysical characterisation of wild-type and mutant FT proteins, pinpointing residues important for membrane association and providing mechanistic insights into how FT contributes to temperature-dependent control of flowering.
Key Scientific Results
The project achieved substantial scientific progress, beginning with the successful generation of two complete sets of FT mutant proteins. All variants were obtained in soluble form at satisfactory concentrations, stored under low-temperature conditions, and prepared to a high standard for downstream biochemical and biophysical analyses. These proteins formed the basis for a series of assays that revealed important insights into how florigen interacts with lipid membranes and how these interactions respond to temperature changes.
Main Results
1. The first-wave FT mutants (E84K, P94L, R119H, G171E) and second-wave mutants (VSR, 3R, FTdC, 3RdC) were successfully constructed, purified, and stabilised for further experimentation.
2. Temperature-dependent membrane behaviour was observed: wild-type FT binds more strongly at 25 °C than at 15 °C, suggesting that in vivo additional membrane-associated factors may stabilise FT under cold conditions.
3. The 3R mutant displayed slower dissociation from membranes, indicating altered interaction dynamics and identifying this variant as especially promising for deeper mechanistic and structural studies.
Together, these findings provide new evidence for how FT responds to environmental cues at the molecular level and lay the groundwork for exploring temperature-dependent flowering regulation.
Potential Impacts and Needs for Further Uptake
The assembled FT mutant library now serves as a powerful resource for dissecting the structural features that govern FT–membrane interactions. The temperature sensitivity observed opens a path toward understanding how environmental conditions modulate florigen activity, with potential applications in developing climate-resilient crops. The unique behaviour of the 3R mutant further highlights specific residues that merit targeted investigation.
Key needs to support further uptake
1. Continued quantitative biophysical studies to refine our understanding of how individual mutations alter membrane-binding dynamics.
2. High-resolution structural analysis (e.g. Cryo-EM, NMR) to map FT–membrane interfaces at atomic detail.
3. In vivo validation in model plants to connect biochemical behaviour with flowering phenotypes and assess translational potential.
The project achieved substantial scientific progress, beginning with the successful generation of two complete sets of FT mutant proteins. All variants were obtained in soluble form at satisfactory concentrations, stored under low-temperature conditions, and prepared to a high standard for downstream biochemical and biophysical analyses. These proteins formed the basis for a series of assays that revealed important insights into how florigen interacts with lipid membranes and how these interactions respond to temperature changes.
Main Results
1. The first-wave FT mutants (E84K, P94L, R119H, G171E) and second-wave mutants (VSR, 3R, FTdC, 3RdC) were successfully constructed, purified, and stabilised for further experimentation.
2. Temperature-dependent membrane behaviour was observed: wild-type FT binds more strongly at 25 °C than at 15 °C, suggesting that in vivo additional membrane-associated factors may stabilise FT under cold conditions.
3. The 3R mutant displayed slower dissociation from membranes, indicating altered interaction dynamics and identifying this variant as especially promising for deeper mechanistic and structural studies.
Together, these findings provide new evidence for how FT responds to environmental cues at the molecular level and lay the groundwork for exploring temperature-dependent flowering regulation.
Potential Impacts and Needs for Further Uptake
The assembled FT mutant library now serves as a powerful resource for dissecting the structural features that govern FT–membrane interactions. The temperature sensitivity observed opens a path toward understanding how environmental conditions modulate florigen activity, with potential applications in developing climate-resilient crops. The unique behaviour of the 3R mutant further highlights specific residues that merit targeted investigation.
Key needs to support further uptake
1. Continued quantitative biophysical studies to refine our understanding of how individual mutations alter membrane-binding dynamics.
2. High-resolution structural analysis (e.g. Cryo-EM, NMR) to map FT–membrane interfaces at atomic detail.
3. In vivo validation in model plants to connect biochemical behaviour with flowering phenotypes and assess translational potential.