Objective 1: We have shown that VRN2 is regulated through the PRT6 N-degron pathway, via the successive actions of PCO, ATE and PRT6 enzymes on its conserved N-terminal cysteine. Spatiotemporal analysis revealed that VRN2 protein is restricted to root and shoot meristems, as well as lateral root primordia. However, in response to reduced oxygen (hypoxia), reduced NO, or long-term cold, degradation of VRN2 is inhibited and it accumulates outside of meristems. This suggests that the N-degron pathway has a dual function, restricting VRN2 to specific tissues, whilst permitting its enhanced abundance in response to environmental change. We also explored the subcellular dynamics of its regulation, and investigated how oxygen availability and cold-exposure are able to converge on the N-terminus of VRN2 to regulate its stability. Moreover, we have shown that other signals also trigger VRN2 stabilisation, including pathogen-derived immune-response elicitors. Many of the findings linked to objective 1 are published in Gibbs et al 2018 (Nature Communications) and Barreto et al (2022) Current Biology, with others still the focus of ongoing research.
Objective 2: VRN2 was original identified as a regulator of vernalization, the process by which long-term cold exposure triggers flowering in spring. VRN2 also has other known developmental functions and we investigated how control of VRN2 stability impacts its functions during development. We showed that restriction of VRN2 to shoot meristems controls photoperiod-dependent flowering, whilst in roots it negatively regulates root system architecture. Ectopic stabilisation of VRN2 outside of meristems is insufficient to trigger vernalization, since it requires other cold-induced factors to coordinate this response. Our work reveals that post-translational restriction of VRN2 to meristems is important for controlling its functions in growth, whilst environment-triggered accumulation outside of meristems allows it to adopt a different set of context-specific roles. Work from Objective 2 is published in Labandera et al (2021) New Phytologist.
Objective 3: We carried out a combined omic analysis (RNAseq and ChIPseq) in a range of different genetic backgrounds and identified a number of VRN2 targets, which are linked to cell-expansion mediated growth. This led us to discover that VRN2-PRC2 is required for repressing PIF signalling, which helps to prevent ectopic gene expression and appropriately coordinate growth. This work is nearing completion and will be a major output of the GasPlaNt project (Osborne et al., in prep). In parallel, we also discovered that VRN2 contributes to the capacity for plants to encode a memory of flooding stress, and identified diverse VRN2-regulated memory genes that contribute to this. This novel role for VRN2 in regulating flooding stress responses is being investigated in collaboration with Dr Sjon Hartman (Freiburg), and will also be a key output of GasPlaNt when published (Maric et al., in prep).
Objectives 4 and 5: How did VRN2 evolve as a target of this pathway and is the same mechanism conserved in monocots such as barley? We have shown that the conserved VRN2 N-degron is functional across broad flowering plant taxa and propose that co-option to this pathway may have permitted neofunctionalisation of PRC2 in angiosperms, perhaps providing increased plasticity and flexibility in the capacity for environmental signals to regulate the epigenome. Part of this work is published in Gibbs et al (2018) Nature Communications.
Many of the findings linked to GasPlaNt were disseminated by the PI and associated project personnel at a range of international meetings. Several key papers have been published (as outlined above) and it is expected that several further core outputs from the phase of GasPlaNt will be published in the next 12 months and beyond.
