Antag0onistic histone modifiers coordinate flooding stress tolerance and memory in plants

Flooding is an increasing problem around the world. In addition to having devastating effects on people's homes and businesses, it severely impacts livestock and crop survival, which can ultimately affect farmer's livelihoods and food production. Many of the key crops we grow (wheat, rice, potatoes) are particularly susceptible to floods, which cause plant death by limiting oxygen availability. If we want to develop new improved crop varieties that have a better chance at withstanding floods, then we first need to understand how plants sense and respond to flooding stress at the molecular and genetic level. Lab based experiments on model plant species, such as Arabidopsis (a common weed), can help to identify the underlying biological principles that control flooding (and other) stress responses, which can then be the focus of more targeted breeding and biotechnology approaches in crops.

An emerging phenomenon in plant stress biology is the concept that plants can sense and "remember" previous stresses by chemically modifying genes (referred to as epigenetics). We recently showed that a conserved plant protein called VRN2, which helps to establish this type of environmental epigenetic memory by switching off gene expression, increases in response to flooding stress. Therefore, we predicted it might be involved in promoting a memory of floods. We have now shown that Arabidopsis plants that were previous exposed to a flood have better survival rates than plants that did not receive this prior stress, and that this is dependent on VRN2. We have also identified a second protein, called REF6, that targets flood-associated genes. Interestingly, REF6 is also an epigenetic regulator, but works in the opposite way to VRN2 by activating gene expression. We propose that these antagonistic memory proteins are part of a previously uncharacterised network of flood-responsive regulators that is important or controlling short- and long-term gene expression changes that promote flooding resilience and provide plants with a positive memory of stress. With this grant, we will explore this concept in more detail, with particular focus on characterising the REF6 component.

To achieve this, we will use a range of molecular, genetic, biochemistry and physiological experiments in Arabidopsis to answer the following questions: (1) What are flood-responsive gene targets of REF6? (2) How is REF6 targeted to these genes during floods? (3) What are the shared and distinct gene targets of REF6 and VRN2? (4) How does the combined activity of these proteins coordinate global gene expression to promote flooding stress tolerance and memory? By answering these questions, our work will reveal how flooding stress signals are directly converted into epigenetic changes, providing fundamental new and detailed insight into how plants can sense, respond, and adapt to stresses in their environment. Whilst Arabidopsis is not a crop, it is hoped that the results of this project will provide plant breeders and biotechnologists with new information that could help them to develop improved varieties of important crops that are better able to withstand floods, and which will help to improve global food security.

Flooding causes oxygen (O2) deprivation (hypoxia) that can lead to plant death. Plants have therefore evolved mechanisms for regulating the transcriptional response to flooding based on O2 sensing. ERFVII transcription factors are O2-labile proteins that accumulate under hypoxia to redirect transcription and promote flood survival. VRN2, part of the polycomb repressive complex 2 (PRC2) that represses gene expression via H3K27 trimethylation, is also regulated by O2, and we have shown it is important for establishing an epigenetic memory of flooding stress. We have now identified a third regulatory protein - the H3K27 demethylase REF6 - that physically interacts with ERFVIIs and modulates hypoxic gene expression. Our work suggests that stress-responsive changes in histone methylation through antagonistic enzyme activities is important for coordinating the transcriptional response to flooding stress. We propose that REF6 and VRN2-PRC2 act alongside ERFVIIs to orchestrate gene expression changes that promote immediate tolerance and long-term stress memory. Here we will explore this further by characterising the role of REF6 in mediating flooding responses. We will use stress assays and omics approaches to define its function and stress-responsive global gene targets. We will explore the mechanistic basis of its interaction with ERFVIIs and how this controls its recruitment to hypoxia response genes during floods. We will explore the crosstalk between REF6 and VRN2-PRC2 by investigating their shared and distinct targets, and we will also investigate how early flooding signals feed into the control of REF6 activity. In doing so, this work will provide new mechanistic insight into how this emerging O2-responsive regulatory network integrates flooding stress-signals to coordinate short- and long-term transcriptional responses, establishing a molecular framework that could be targeted for manipulating flooding resilience in diverse crop species of agronomic significance.