Regulation of adaptive responses to flood-induced hypoxia in Marchantia polymorpha

Similar to animals, plants need oxygen to respire and thrive in their habitat. When exposed to light, plants release oxygen from water molecules through the green tissues that perform photosynthesis. However, conditions that limit gas diffusion, such as flooding, cause a dramatic reduction in oxygen availability. This leads to a stressful situation that diminishes crop productivity, when not directly causing death of the plants. In the UK, flood events such as those of Summer 2007 that hit the North Eastern England and the West Midlands, and of winter 2013/14 that affected Yorkshire and Somerset have been estimated to cause an economic damage to agriculture of over £84 million. In some cases, costs have exceeded £200,000 per farm. Economies of developing countries are even more sensitive to such events (Shresta et al. 2018). Considering this, flooding stress play a major more, along environmental factors, to limit agriculture yield worldwide and restricts the possibility to feed the human population, especially when considering its rapid growth, estimated to reach 9 bn within the next 30 years.
Together with the University of Nottingham, my team characterized the mechanism by which plants measure the oxygen concentration available in the environment and activate adaptive response in time to cope with this stress. Using Arabidopsis thaliana as a model plant species, we discovered an enzyme conserved with animals and fungi that controls the abundance of regulatory protein that mediate the molecular response to low oxygen. Moreover, we and colleagues from Australia have provided evidence that the cellular energy powerplants, the mitochondria, also participate to signal the need to activate an adaptive response via regulatory protein called ANACs. These are normally bound to cellular membranes from which they are rapidly released in case of stress, to enter cell nuclei and induce the synthesis of proteins required to face the stress.
Land plants derive from aquatic ancestors that colonized emerged lands about 500 M years ago. This big change of ambient feature drove plant evolution towards that enabled to exploit the opportunities and overcome the limitations of nonaquatic environments. These innovations have led land plant to development specialized features that ensure their success and fitness in environments characterized by strong fluctuations in water and oxygen availability. Liverworts, such as Marchantia polymorpha, thrive in humid habitats and yet we found them extremely sensitive to prolonged submergence, while Arabidopsis colonized a range of environments. These species largely differ under several aspects of their physiology, and thus we expect them to use alternative strategies to cope with low oxygen. We believe that the identification of differences and similarities in this process across plant species with such divergent morphology and ecology holds untapped potential to the discovery of useful and yet unknown pathways to response to oxygen fluctuations and activate response that protect from oxidative stress and allow metabolic adjustments to cope with hypoxia.
In summary, we will study the response to hypoxia in the two species (Marchantia and Arabidopsis) by means of state-of-the-art technologies and applying the most recent molecular analytic techniques. By doing so, we envisage to identify and characterize the molecular mechanisms involved in oxygen perception and oxidative stress signalling in plants. We will compare the mechanisms set into place in Marchantia and Arabidopsis, and exploit the strategy to induce or repress each mechanism in both model species, to identify those essential or more effective to protect plants from flooding stress. Our final aim is the identification of key elements or pathways that can be effectively transferred to crop species to improve their submergence tolerance.

Despite the strides accomplished in the recent years towards the understanding of oxygen sensing in angiosperms, its evolution and the extent of its conservation across land plants remains an enigma. The present proposal aims at filling this knowledge gap by merging transcript and metabolic profiling of Marchantia polymorpha with the data previously collected on angiosperms. These datasets will lay the foundations to challenge experimentally the hypothesis of a general response to oxidative stress as main constituent of low oxygen condition in bryophytes, and investigate the role of retrograde mitochondrial signalling in the activation of the response. To achieve this goal, I propose to:
1. Reconstruct regulatory networks that enable metabolic adjustments and oxidative stress defense in Marchantia. We will define the role of transcriptionally upregulated transcription factors but also identify those involved in initiating the signalling cascade, guided by the knowledge gathered on angiosperms and by an unbiased RNA-seq approach.
2. Compare two hypoxia-related regulatory pathways (the Cys branch of the N-degron pathway and the membrane-associated NAC pathway) in Marchantia and Arabidopsis to identify shared elements that indicate common origin and conservation or divergence in land plants. These conserved components will be studied in detail to identify the essential features that enable effective signalling.
3. Investigate the relevance of adaptive metabolic adjustments to hypoxia in parallel in Marchantia and Arabidopsis, with a double strategy based on gene inactivation (via T-DNA insertion of CRISPR) and inducible gene activation.