Periodic Reporting for period 4 - GasPlaNt (Gas sensing in plants:Oxygen- and nitric oxide-regulated chromatin modification via a targeted protein degradation mechanism)
Reporting period: 2021-09-01 to 2023-06-30
As the global population continues to increase in size, and weather patterns become more unpredictable due to climate change, there is pressing need to produce more food (i.e. grow more crops) and less land. Understanding the basic mechanisms governing the capacity for plants to adapt and survive in diverse environments, including stressful situations, has the potential to identify new genetic and biochemical targets that could be manipulated in agronomically relevant species to enhance crop productivity and food security.
Plants have to maintain developmental flexibility to ensure survival in dynamic environments. As such they have evolved mechanisms to sense and respond to their environment. One way in which they can do this is through epigenetic control of their gene expression, which controls cell identity, developmental transitions, and environmental memory. Although epigenetic responses to environmental change have been known for many years, molecular mechanisms that connect the perception of environmental signals to these changes are less well known. It was previously shown that the N-degron pathway, a conserved system for targeted proteolysis, regulates responses to oxygen and nitric oxide availability through controlling the stability of transcription factors called the ERFVIIs. We showed that the angiosperm specific PRC2 component VERNALIZATION2 is also regulated by this degradation pathway. The PRC2 is a widely conserved holoenzyme that methylates histones in chromatin to trigger gene silencing. The discovery of VRN2 as a target of the N-degron pathway led to the hypothesis that VRN2 might directly sense and transduce oxygen/NO availability into chromatin changes to coordinate epigenetic responses to to the environment. The GasPlaNt project sought to fully characterise the mechanisms regulating VRN2 stability within the context of environmental change, to connect this regulation to its known functions, and to identify new gene targets and processes regulated by VRN2. Ultimately, GasPlaNt aimed to establish a framework for environmental triggered epigenetic changes that drive growth and development in plants. The project was largely successful in achieving these aims through characterising, at the biochemical level, the post-translational regulation of VRN2, linking this to known and new functions, and ultimately providing a platform for further exploration of environment-responsive control of PRC2 activity and its roles in regulating diverse plant responses of agronomic significance.
Plants have to maintain developmental flexibility to ensure survival in dynamic environments. As such they have evolved mechanisms to sense and respond to their environment. One way in which they can do this is through epigenetic control of their gene expression, which controls cell identity, developmental transitions, and environmental memory. Although epigenetic responses to environmental change have been known for many years, molecular mechanisms that connect the perception of environmental signals to these changes are less well known. It was previously shown that the N-degron pathway, a conserved system for targeted proteolysis, regulates responses to oxygen and nitric oxide availability through controlling the stability of transcription factors called the ERFVIIs. We showed that the angiosperm specific PRC2 component VERNALIZATION2 is also regulated by this degradation pathway. The PRC2 is a widely conserved holoenzyme that methylates histones in chromatin to trigger gene silencing. The discovery of VRN2 as a target of the N-degron pathway led to the hypothesis that VRN2 might directly sense and transduce oxygen/NO availability into chromatin changes to coordinate epigenetic responses to to the environment. The GasPlaNt project sought to fully characterise the mechanisms regulating VRN2 stability within the context of environmental change, to connect this regulation to its known functions, and to identify new gene targets and processes regulated by VRN2. Ultimately, GasPlaNt aimed to establish a framework for environmental triggered epigenetic changes that drive growth and development in plants. The project was largely successful in achieving these aims through characterising, at the biochemical level, the post-translational regulation of VRN2, linking this to known and new functions, and ultimately providing a platform for further exploration of environment-responsive control of PRC2 activity and its roles in regulating diverse plant responses of agronomic significance.
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.
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.
The GasPlaNt project has identified a new mechanism operating on the angiosperm PRC2 machinery that connects the spatiotemporal abundance of a key subunit of the complex to diverse environmental inputs. We have also shown how this post-translational control of VRN2-PRC2 regulates its activity at a genome wide scale, which has identified a range of developmental and stress-responsive processes that are controlled by this signal responsive holoenzyme. Our work has provided new insight into how plants are able to sense environmental signals and directly transduce them into epigenetic changes to coordinate growth and environmental responses, and has thus achieved progress beyond the state of the art as originally envisioned when the work program was first developed. Whilst most of this fundamental work was conducted in the model plant Arabidopsis, the identified mechanisms could be targeted in crops to develop varieties that are better equipped for accommodating different environmental scenarios in the future.