Beatrice Giuntoli

Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization

The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low—as during flooding—RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops.

HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana.

Plants often experience challenging hypoxic conditions imposed by soil waterlogging or complete flooding. In rice, Sub1A, a flooding-induced ethylene responsive factor (ERF) plays a crucial role in submergence tolerance. In this study, we examined two Arabidopsis Hypoxia Responsive ERF genes (HRE1 and HRE2), belonging to the same ERF group as Sub1A. Transgenic Arabidopsis plants, which over-expressed HRE1, showed an improved tolerance of anoxia, whereas a double-knockout mutant hre1hre2 was more susceptible than the wild type. HRE1 over-expressing plants showed an increased activity in the fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase together with increased ethanol production under hypoxia, but not in normoxia. Whole-genome microarray analyses suggested that an over-expression of HRE1, but not HRE2, increased the induction of most anaerobic genes under hypoxia. Real-time quantitative (q)PCR analyses confirmed a positive effect of HRE1 over-expression on several anaerobic genes, whereas the double-knockout mutant hre1hre2 showed a decreased expression in the same genes after 4 h of hypoxia. Single-knockout mutants did not show significant differences from the wild type. We found that the regulation of HRE1 and HRE2 by low oxygen relies on different mechanisms, since HRE1 requires protein synthesis to be induced while HRE2 does not. HRE2 is likely to be regulated post-transcriptionally by mRNA stabilization. We propose that HRE1 and HRE2 play a partially redundant role in low oxygen signalling in Arabidopsis thaliana, thus improving the tolerance of the plant to the stress by enhancing anaerobic gene expression and ethanolic fermentation.

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.

The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2.12, RAP2.2 and RAP2.3

The ethylene response factor VII (ERF-VII) transcription factor RELATED TO APETALA2.12 (RAP2.12) was previously identified as an activator of the ALCOHOL DEHYDROGENASE1 promoter::luciferase (ADH1-LUC) reporter gene. Here we show that overexpression of RAP2.12 and its homologues RAP2.2 and RAP2.3 sustains ABA-mediated activation of ADH1 and activates hypoxia marker genes under both anoxic and normoxic conditions. Inducible expression of all three RAP2s conferred tolerance to anoxia, oxidative and osmotic stresses, and enhanced the sensitivity to abscisic acid (ABA). Consistently, the rap2.12-2 rap2.3-1 double mutant showed hypersensitivity to both submergence and osmotic stress. These findings suggest that the three ERF-VII-type transcription factors play roles in tolerance to multiple stresses that sequentially occur during and after submergence in Arabidopsis. Oxygen-dependent degradation of RAP2.12 was previously shown to be mediated by the N-end rule pathway. During submergence the RAP2.12, RAP2.2 and RAP2.3 are stabilized and accumulates in the nucleus affecting the transcription of stress response genes. We conclude that the stabilized RAP2 transcription factors can prolong the ABA-mediated activation of a subset of osmotic responsive genes (e.g. ADH1). We also show that RAP2.12 protein level is affected by the REALLY INTERESTING GENE (RING) domain containing SEVEN IN ABSENTIA of Arabidopsis thaliana 2 (SINAT2). Silencing of SINAT1/2 genes leads to enhanced RAP2.12 abundance independently of the presence or absence of its N-terminal degron. Taken together, our results suggest that RAP2.12 and its homologues RAP2.2 and RAP2.3 act redundantly in multiple stress responses. Alternative protein degradation pathways may provide inputs to the RAP2 transcription factors for the distinct stresses.

A Trihelix DNA Binding Protein Counterbalances Hypoxia-Responsive Transcriptional Activation in Arabidopsis

DNA binding protein controls plant transcription when oxygen is at a premium - During hypoxia, the plant transcription factor HRA1 counterbalances the upregulation of anaerobic gene expression triggered by a stabilized plant ethylene responsive factor.

Low oxygen response mechanisms in green organisms

Low oxygen stress often occurs during the life of green organisms, mostly due to the environmental conditions affecting oxygen availability. Both plants and algae respond to low oxygen by resetting their metabolism. The shift from mitochondrial respiration to fermentation is the hallmark of anaerobic metabolism in most organisms. This involves a modified carbohydrate metabolism coupled with glycolysis and fermentation. For a coordinated response to low oxygen, plants exploit various molecular mechanisms to sense when oxygen is either absent or in limited amounts. In Arabidopsis thaliana, a direct oxygen sensing system has recently been discovered, where a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the fate of the protein under normoxia/hypoxia. In Oryza sativa, this same group of ERFs drives physiological and anatomical modifications that vary in relation to the genotype studied. The microalga Chlamydomonas reinhardtii responses to low oxygen seem to have evolved independently of higher plants, posing questions on how the fermentative metabolism is modulated. In this review, we summarize the most recent findings related to these topics, highlighting promising developments for the future.

Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana

Plant adaptation to hypoxic conditions is mediated by the transcriptional activation of genes involved in the metabolic reprogramming of plant cells to cope with reduced oxygen availability. Recent studies indicated that members of the group VII of the Ethylene Responsive Transcription Factor (ERFs) family act as positive regulators of this molecular response. In the current study, the five ERF-VII transcription factors of Arabidopsis thaliana were compared to infer a hierarchy in their role with respect to the anaerobic response. When the activity of each transcription factor was tested on a set of hypoxia-responsive promoters, RAP2.2, RAP2.3 and RAP2.12 appeared to be the most powerful activators. RAP2.12 was further dissected in transactivation assays in Arabidopsis protoplasts to identify responsible regions for transcriptional activation. An ultimate C-terminal motif was identified as sufficient to drive gene transcription. Finally, using realtime RT-PCR in single and double mutants for the corresponding genes, we confirmed that RAP2.2 and RAP2.12 exert major control upon the anaerobic response.

Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana

The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions.

Group VII Ethylene Response Factors in Arabidopsis: Regulation and Physiological Roles

The role of ERF-VII TFs in higher plants is to coordinate their signature response to oxygen deficiency, but additional layers of modulation of ERF-VII activity enrich their regulatory range.

Functional Balancing of the Hypoxia Regulators RAP2.12 and HRA1 Takes Place in vivo in Arabidopsis thaliana Plants

Plants are known to respond to variations in cellular oxygen availability and distribution by quickly adapting the transcription rate of a number of genes, generally associated to improved energy usage pathways, oxygen homeostasis and protection from harmful products of anaerobic metabolism. In terrestrial plants, such coordinated gene expression program is promoted by a conserved subfamily of ethylene responsive transcription factors called ERF-VII, which act as master activators of hypoxic gene transcription. Their abundance is directly regulated by oxygen through a mechanism of targeted proteolysis present under aerobic conditions, which is triggered by ERF-VII protein oxidation. Beside this, in Arabidopsis thaliana, the activity of the ERF-VII factor RAP2.12 has been shown to be restrained and made transient by the hypoxia-inducible transcription factor HRA1. This feedback mechanism has been proposed to modulate ERF-VII activity in the plant under fluctuating hypoxia, thereby enhancing the flexibility of the response. So far, functional balancing between RAP2.12 and HRA1 has been assessed in isolated leaf protoplasts, resulting in an inverse relationship between HRA1 amount and activation of RAP2.12 target promoters. In the present work, we showed that HRA1 is effective in balancing RAP2.12 activity in whole arabidopsis plants. Examination of a segregating population, generated from RAP2.12 and HRA1 over-expressing plants, led to the first quantitative proof that, over a range of either transgene expression levels, HRA1 counteracts the phenotypic and transcriptional effects of RAP2.12. This report supports the occurrence of fine-tuned regulation of the hypoxic response under physiological growth conditions.