Periodic Reporting for period 2 - SynOxyS (Synthetic Oxygen-related molecular Systems in plants)
Reporting period: 2022-10-01 to 2024-03-31
Aerobic organisms developed systems to measure cellular oxygen levels and activate adaptive responses in case of hypoxia. This condition can be caused by the environment or associated with developmental programmes. Recent reports have revealed that complex eukaryotes, such as higher plants and metazoans, converged in their oxygen sensing strategies towards the recruitment of dioxygenase enzymes to regulate the activity of specific transcription factors. Despite the functional comparability of the mechanisms, their molecular components are different. These similarities and differences between animal and plant hypoxia machineries can now be exploited to acquire better understanding of the dynamics of oxygen sensing in the two kingdoms and more precise manipulation of these mechanisms for applicative purposes. For example this can be used to improve crops' ability to cope with hypoxic conditions induced by the surrounding environment, as it happens during flooding. Moreover, this can be used to re-engineer the metabolic and structural organisation that plant cells establish in response to oxygen gradients and fluctuations.
With SynOxyS, we apply a novel approach that merges the synthetic biology framework, molecular physiology, and developmental biology to transfer features of the metazoan oxygen sensing and delivery systems to plants. This strategy is instrumental to investigate for the first time whole oxygen machineries and characterize their performance in the context of plant cells.
The proposed project expands in three distinct although interlinked directions that explore (1) the exploitation of 2-oxoglutarate (OG)-dependent dioxygenase to drive selective proteolysis in plants, (2) the investigation of endogenous or heterologous control of chromatin accessibility by 2-OG dependent dioxygenases and (3) the engineering of a chimeric delivery system to alter oxygen provision or perception specifically in shoot apical meristems. The design and optimization of these synthetic oxygen machineries, and their comparison with endogenous ones, will pinpoint the features that enable efficient control of hypoxic responses in plants and will foster precise control of adaptive responses that ameliorate hypoxia tolerance.
With SynOxyS, we apply a novel approach that merges the synthetic biology framework, molecular physiology, and developmental biology to transfer features of the metazoan oxygen sensing and delivery systems to plants. This strategy is instrumental to investigate for the first time whole oxygen machineries and characterize their performance in the context of plant cells.
The proposed project expands in three distinct although interlinked directions that explore (1) the exploitation of 2-oxoglutarate (OG)-dependent dioxygenase to drive selective proteolysis in plants, (2) the investigation of endogenous or heterologous control of chromatin accessibility by 2-OG dependent dioxygenases and (3) the engineering of a chimeric delivery system to alter oxygen provision or perception specifically in shoot apical meristems. The design and optimization of these synthetic oxygen machineries, and their comparison with endogenous ones, will pinpoint the features that enable efficient control of hypoxic responses in plants and will foster precise control of adaptive responses that ameliorate hypoxia tolerance.
We designed a system to control plant gene expression in response to hypoxia independently from the endogenous oxygen sensing system. To this end, we decided to mimic the mechanisms in place in metazoan cells, where hydroxylation of specific prolyl residues in the HIF-α transcription factor induces its degradation through the proteasome. We therefore introduced a sufficient number of DNA constructs in plant cells to establish this pathway. This part of the project has required several optimisation steps to identify the optimal sequences that enable oxygen-dependent proteolysis. As a result, we generated stable Arabidopsis lines able to control the stability of reporter proteins in an oxygen-dependent manner. We then moved on to apply this property to transcriptional regulators. We decided to restore oxygen responsiveness to mutated transcription factors that lost this ability in Arabidopsis plants. We are currently characterising these plants, and preliminary results indicate that modulation of gene expression in response to hypoxia is possible in our transgenic system, although with different dynamics from those measured in wild type plants.
In parallel, we are currently studying the contribution of dioxygenases not belonging to the thiol-dioxygenase family to the regulation of oxygen sensing in plant cells. By using a combination of overexpression and knock-out mutants we identified potential candidates that participate to adaptive responses to oxygen gradients in plants. We are testing their potential to plant growth and plant regeneration in vitro.
In parallel, we are currently studying the contribution of dioxygenases not belonging to the thiol-dioxygenase family to the regulation of oxygen sensing in plant cells. By using a combination of overexpression and knock-out mutants we identified potential candidates that participate to adaptive responses to oxygen gradients in plants. We are testing their potential to plant growth and plant regeneration in vitro.
The current work has allowed us to better understand the mechanisms of oxygen sensing pathway in plants and the dynamics with which responses to hypoxia are regulated. For example, we demonstrated the role of non-hypoxia inducible enzymes in repressing the low oxygen response in plants, and also their participation to developmental programmes under aerobic and anaerobic (flooding) conditions.
In parallel, this study has allowed us to study the evolution and diversification of oxygen sensing plants in plants and in animals. By carrying out this research we were also able to explore the potential to engineer oxygen sensing pathways for drug discovery and breeding purposes.
Our expectation for the remaining part of the project is to comparatively characterise the mechanisms of oxygen sensing in plant and animal cells, and provide models for studying their dynamics. We hope to be able to define the contribution of oxygen sensing pathways to developmental programmes in wild plants and crops, and guide their breeding towards enhanced tolerance to flash-flood events.
In parallel, this study has allowed us to study the evolution and diversification of oxygen sensing plants in plants and in animals. By carrying out this research we were also able to explore the potential to engineer oxygen sensing pathways for drug discovery and breeding purposes.
Our expectation for the remaining part of the project is to comparatively characterise the mechanisms of oxygen sensing in plant and animal cells, and provide models for studying their dynamics. We hope to be able to define the contribution of oxygen sensing pathways to developmental programmes in wild plants and crops, and guide their breeding towards enhanced tolerance to flash-flood events.