Target of rapamycin signaling couples energy to oxygen sensing to modulate hypoxic gene expression in Arabidopsis.

Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs). Under hypoxia, PCOs are inactive and the ERF-VIIs activate transcription of the HRGs required for surviving hypoxia. However, if the plant exposed to hypoxia has limited sugar reserves, the activity of ERF-VIIs is severely dampened. This suggests that oxygen sensing by PCO/ERF-VII is fine-tuned by another sensing pathway, related to sugar or energy availability. Here, we show that oxygen sensing by PCO/ERF-VII is controlled by the energy sensor target of rapamycin (TOR). Inhibition of TOR by genetic or pharmacological approaches leads to a much lower induction of HRGs. We show that two serine residues at the C terminus of RAP2.12, a major ERF-VII, are phosphorylated by TOR and are needed for TOR-dependent activation of transcriptional activity of RAP2.12. Our results demonstrate that oxygen and energy sensing converge in plants to ensure an appropriate transcription of genes, which is essential for surviving hypoxia. When carbohydrate metabolism is inefficient in producing ATP because of hypoxia, the lower ATP content reduces TOR activity, thus attenuating the efficiency of induction of HRGs by the ERF-VIIs. This homeostatic control of the hypoxia-response is required for the plant to survive submergence.

Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes.

N-terminal cysteine oxidases (NCOs) use molecular oxygen to oxidise the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway. To expand the characterisation of the plant family of NCOs (plant cysteine oxidases [PCOs]), we performed a phylogenetic analysis across different taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups. A-type PCOs are conserved across all plant species and are generally unaffected at the messenger RNA level by oxygen availability. Instead, B-type PCOs appeared in spermatophytes to acquire transcriptional regulation in response to hypoxia. The inactivation of two A-type PCOs in Arabidopsis thaliana, PCO4 and PCO5, is sufficient to activate the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperms. Therefore, while it is possible to distinguish two clades within the PCO family, we conclude that they all contribute to restrain the anaerobic transcriptional programme in normoxic conditions and together generate a molecular switch to toggle the hypoxic response.

A Ratiometric Sensor Based on Plant N-Terminal Degrons Able to Report Oxygen Dynamics in Saccharomyces cerevisiae.

The ability to perceive oxygen levels is crucial to many organisms because it allows discerning environments compatible with aerobic or anaerobic metabolism, as well as enabling rapid switch between these two energy strategies. Organisms from different taxa dedicate distinct mechanisms to associate oxygen fluctuations with biological responses. Following from this observation, we speculated that orthogonal oxygen sensing devices can be created by transfer of essential modules from one species to another in which they are not conserved. We expressed plant cysteine oxidase (PCOs) enzymes in Saccharomyces cerevisiae, to confer oxygen-conditional degradability to a bioluminescent protein tagged with the Cys-exposing N-degron typical of plant ERF-VII factors. Co-translation of a second luciferase protein, not subjected to oxygen-dependent proteolysis, made the resulting Double Luciferase Oxygen Reporter (DLOR) ratiometric. We show that DLOR acts as a proxy for oxygen dynamics in yeast cultures. Moreover, since DLOR activity was enabled by the PCO sensors, we employed this device to disclose some of their properties, such as the dispensability of nitric oxide for N-terminal cysteine oxidation and the individual performance of Arabidopsis PCO isoforms in vivo. In the future, we propose the synthetic DLOR device as a convenient, eukaryotic cell-based tool to easily screen substrates and inhibitors of cysteine oxidase enzymes in vivo. Replacement of the luminescent proteins with fluorescent proteins will further turn our system into a visual reporter for oxygen dynamics in living cells.

Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation.

Agrobacterium tumefaciens infection of wounded plant tissues causes the formation of crown gall tumors. Upon infection, genes encoded on the A. tumefaciens tumor inducing plasmid are integrated in the plant genome to induce the biosynthesis of auxin and cytokinin, leading to uncontrolled cell division. Additional sequences present on the bacterial T-DNA encode for opine biosynthesis genes, which induce the production of opines that act as a unique carbon and nitrogen source for Agrobacterium. Crown galls therefore become a very strong sink for photosynthate. Here we found that the increased metabolic demand in crown galls causes an increase in oxygen consumption rate, which leads to a steep drop in the internal oxygen concentration. Consistent with this, plant hypoxia-responsive genes were found to be significantly upregulated in crown galls compared to uninfected stem tissue. Following this observation, we aimed at understanding whether the low-oxygen response pathway, mediated by group VII ethylene response factor (ERF-VII) transcription factors, plays a role in the development of crown galls. We found that quintuple knock-out mutants of all ERF-VII members, which are incapable of inducing the hypoxic response, show reduced crown gall symptoms. Conversely, mutant genotypes characterized by constitutively high levels of hypoxia-associated transcripts, displayed more severe crown gall symptoms. Based on these results, we concluded that uncontrolled cell proliferation of crown galls established hypoxic conditions, thereby requiring adequate anaerobic responses of the plant tissue to support tumor growth.

Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.

Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway.

In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants.