Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana.

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

ERFVII-controlled hypoxia responses are in part facilitated by MEDIATOR SUBUNIT 25 in Arabidopsis thaliana.

Flooding impairs plant growth through oxygen deprivation, which activates plant survival and acclimation responses. Transcriptional responses to low oxygen are generally associated with the activation of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors. However, the exact mechanisms and molecular components by which ERFVII factors initiate gene expression are not fully elucidated. Here, we show that the ERFVII factors RELATED TO APETALA 2.2 (RAP2.2) and RAP2.12 cooperate with the Mediator complex subunit AtMED25 to coordinate gene expression under hypoxia in Arabidopsis thaliana. Respective med25 knock-out mutants display reduced low-oxygen stress tolerance. AtMED25 physically associates with a distinct set of hypoxia core genes and its loss partially impairs transcription under hypoxia due to decreased RNA polymerase II recruitment. Association of AtMED25 with target genes requires the presence of ERFVII transcription factors. Next to ERFVII protein stabilisation, also the composition of the Mediator complex including AtMED25 is potentially affected by hypoxia stress as shown by protein-complex pulldown assays. The dynamic response of the Mediator complex to hypoxia is furthermore supported by the fact that two subunits, AtMED8 and AtMED16, are not involved in the establishment of hypoxia tolerance, whilst both act in coordination with AtMED25 under other environmental conditions. We furthermore show that AtMED25 function under hypoxia is independent of ethylene signalling. Finally, functional conservation at the molecular level was found for the MED25-ERFVII module between A. thaliana and the monocot species Oryza sativa, pointing to a potentially universal role of MED25 in coordinating ERFVII-dependent transcript responses to hypoxia in plants.

An apical hypoxic niche sets the pace of shoot meristem activity.

Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere1; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism2. Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation3. Here we demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves. We show that hypoxia localized to the shoot meristem inhibits the proteolysis of an N-degron-pathway4,5 substrate known as LITTLE ZIPPER 2 (ZPR2)-which evolved to control the activity of the class-III homeodomain-leucine zipper transcription factors6-8-and thereby regulates the activity of shoot meristems. Our results reveal oxygen as a diffusible signal that is involved in the control of stem-cell activity in plants grown under aerobic conditions, which suggests that the spatially distinct distribution of oxygen affects plant development. In molecular terms, this signal is translated into transcriptional regulation by the N-degron pathway, thereby linking the control of metabolic activity to the regulation of development in plants.

The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype.

BACKGROUND: Plant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens. RESULTS: Here, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000. CONCLUSION: We demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

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.

Molecular oxygen as a signaling component in plant development.

While traditionally hypoxia has been studied as a detrimental component of flooding stress, the last decade has flourished with studies reporting the involvement of molecular oxygen availability in plant developmental processes. Moreover, proliferating and undifferentiated cells from different plant tissues were found to reside in endogenously generated hypoxic niches. Thus, stress-associated acute hypoxia may be distinguished from constitutively generated chronic hypoxia. The Cys/Arg branch of the N-degron pathway assumes a central role in integrating oxygen levels resulting in proteolysis of transcriptional regulators that control different aspects of plant growth and development. As a target of this pathway, group VII of the Ethylene Response Factor (ERF-VII) family has emerged as a hub for the integration of oxygen dynamics in root development and during seedling establishment. Additionally, vegetative shoot meristem activity and reproductive transition were recently associated with oxygen availability via two novel substrates of the N-degron pathways: VERNALISATION 2 (VRN2) and LITTLE ZIPPER 2 (ZPR2). Together, these observations support roles for molecular oxygen as a signalling molecule in plant development, as well as in essential metabolic reactions. Here, we review recent findings regarding oxygen-regulated development, and discuss outstanding questions that spring from these discoveries.

Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in Arabidopsis.

Plant response to environmental stimuli involves integration of multiple signals. Upon low-oxygen stress, plants initiate a set of adaptive responses to circumvent an energy crisis. Here, we reveal how these stress responses are induced by combining (i) energy-dependent changes in the composition of the acyl-CoA pool and (ii) the cellular oxygen concentration. A hypoxia-induced decline of cellular ATP levels reduces LONG-CHAIN ACYL-COA SYNTHETASE activity, which leads to a shift in the composition of the acyl-CoA pool. Subsequently, we show that different acyl-CoAs induce unique molecular responses. Altogether, our data disclose a role for acyl-CoAs acting in a cellular signaling pathway in plants. Upon hypoxia, high oleoyl-CoA levels provide the initial trigger to release the transcription factor RAP2.12 from its interaction partner ACYL-COA BINDING PROTEIN at the plasma membrane. Subsequently, according to the N-end rule for proteasomal degradation, oxygen concentration-dependent stabilization of the subgroup VII ETHYLENE-RESPONSE FACTOR transcription factor RAP2.12 determines the level of hypoxia-specific gene expression. This research unveils a specific mechanism activating low-oxygen stress responses only when a decrease in the oxygen concentration coincides with a drop in energy.

Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport.

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli.

Mass spectrometry-based plant metabolomics: Metabolite responses to abiotic stress.

Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include analysis of a wide range of chemical species with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. This complexitiy brings huge challenges to the analytical technologies employed in current plant metabolomics programs, and powerful analytical tools are required for the separation and characterization of this extremely high compound diversity present in biological sample matrices. The use of mass spectrometry (MS)-based analytical platforms to profile stress-responsive metabolites that allow some plants to adapt to adverse environmental conditions is fundamental in current plant biotechnology research programs for the understanding and development of stress-tolerant plants. In this review, we describe recent applications of metabolomics and emphasize its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress (or a combination of them). Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:620-649, 2016.

Oxygen Sensing via the Ethylene Response Transcription Factor RAP2.12 Affects Plant Metabolism and Performance under Both Normoxia and Hypoxia.

Subgroup-VII-ethylene-response-factor (ERF-VII) transcription factors are involved in the regulation of hypoxic gene expression and regulated by proteasome-mediated proteolysis via the oxygen-dependent branch of the N-end-rule pathway. While research into ERF-VII mainly focused on their role to regulate anoxic gene expression, little is known on the impact of this oxygen-sensing system in regulating plant metabolism and growth. By comparing Arabidopsis (Arabidopsis thaliana) plants overexpressing N-end-rule-sensitive and insensitive forms of the ERF-VII-factor RAP2.12, we provide evidence that oxygen-dependent RAP2.12 stability regulates central metabolic processes to sustain growth, development, and anoxic resistance of plants. (1) Under normoxia, overexpression of N-end-rule-insensitive Δ13RAP2.12 led to increased activities of fermentative enzymes and increased accumulation of fermentation products, which were accompanied by decreased adenylate energy states and starch levels, and impaired plant growth and development, indicating a role of oxygen-regulated RAP2.12 degradation to prevent aerobic fermentation. (2) In Δ13RAP2.12-overexpressing plants, decreased carbohydrate reserves also led to a decrease in anoxic resistance, which was prevented by external Suc supply. (3) Overexpression of Δ13RAP2.12 led to decreased respiration rates, changes in the levels of tricarboxylic acid cycle intermediates, and accumulation of a large number of amino acids, including Ala and γ-amino butyric acid, indicating a role of oxygen-regulated RAP2.12 abundance in controlling the flux-modus of the tricarboxylic acid cycle. (4) The increase in amino acids was accompanied by increased levels of immune-regulatory metabolites. These results show that oxygen-sensing, mediating RAP2.12 degradation is indispensable to optimize metabolic performance, plant growth, and development under both normoxic and hypoxic conditions.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution.

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [(13)C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [(13)C]glutamate and [(15)N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the (13)C/(15)N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis.

Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops.

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.

Nighttime sugar starvation orchestrates gibberellin biosynthesis and plant growth in Arabidopsis.

A plant's eventual size depends on the integration of its genetic program with environmental cues, which vary on a daily basis. Both efficient carbon metabolism and the plant hormone gibberellin are required to guarantee optimal plant growth. Yet, little is known about the interplay between carbon metabolism and gibberellins that modulates plant growth. Here, we show that sugar starvation in Arabidopsis thaliana arising from inefficient starch metabolism at night strongly reduces the expression of ent-kaurene synthase, a key regulatory enzyme for gibberellin synthesis, the following day. Our results demonstrate that plants integrate the efficiency of photosynthesis over a period of days, which is transduced into a daily rate of gibberellin biosynthesis. This enables a plant to grow to a size that is compatible with its environment.

The stability and nuclear localization of the transcription factor RAP2.12 are dynamically regulated by oxygen concentration.

Plants often experience low oxygen conditions as the consequence of reduced oxygen availability in their environment or due to a high activity of respiratory metabolism. Recently, an oxygen sensing pathway was described in Arabidopsis thaliana which involves the migration of an ERF transcription factor (RAP2.12) from the plasma membrane to the nucleus upon hypoxia. Moreover, RAP2.12 protein level is controlled through an oxygen-dependent branch of the N-end rule pathway for proteasomal degradation. Inside the nucleus, RAP2.12 induces the expression of genes involved in the adaptation to reduced oxygen availability. In the present study, we describe the oxygen concentration and time-resolved characterization of RAP2.12 activity. A reduction of the oxygen availability to half the concentration in normal air is sufficient to trigger RAP2.12 relocalization into the nucleus, while nuclear accumulation correlates with the first induction of the molecular response to hypoxia. Nuclear presence of RAP2.12 may not only depend on relocalization of existing protein, but involves de novo synthesis of the transcription factor as well. After re-oxygenation of the tissue, degradation of RAP2.12 in the nucleus was observed within 3 h, concomitant with reduction in hypoxia responsive gene transcripts to normoxic levels.

Diurnal changes of polysome loading track sucrose content in the rosette of wild-type arabidopsis and the starchless pgm mutant.

Growth is driven by newly fixed carbon in the light, but at night it depends on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night, and low CO2 in Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) and in the starchless phosphoglucomutase (pgm) mutant. In Col-0, polysome loading was 60% to 70% in the light, 40% to 45% for much of the night, and less than 20% in an extended night, while in pgm, it fell to less than 25% early in the night. Quantification of ribosomal RNA species using quantitative reverse transcription-polymerase chain reaction revealed that polysome loading remained high for much of the night in the cytosol, was strongly light dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared with Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from (13)CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modeling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in the previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon.

Potassium (K+) gradients serve as a mobile energy source in plant vascular tissues.

The essential mineral nutrient potassium (K(+)) is the most important inorganic cation for plants and is recognized as a limiting factor for crop yield and quality. Nonetheless, it is only partially understood how K(+) contributes to plant productivity. K(+) is used as a major active solute to maintain turgor and to drive irreversible and reversible changes in cell volume. K(+) also plays an important role in numerous metabolic processes, for example, by serving as an essential cofactor of enzymes. Here, we provide evidence for an additional, previously unrecognized role of K(+) in plant growth. By combining diverse experimental approaches with computational cell simulation, we show that K(+) circulating in the phloem serves as a decentralized energy storage that can be used to overcome local energy limitations. Posttranslational modification of the phloem-expressed Arabidopsis K(+) channel AKT2 taps this "potassium battery," which then efficiently assists the plasma membrane H(+)-ATPase in energizing the transmembrane phloem (re)loading processes.

Introducing the halophyte Salicornia europaea to investigate combined impact of salt and tidal submergence conditions.

Tolerance mechanisms to single abiotic stress events are being investigated in different plant species, but how plants deal with multiple stress factors occurring simultaneously is still poorly understood. Here, we introduce Salicornia europaea as a species with an extraordinary tolerance level to both flooding and high salt concentrations. Plants exposed to 0.5MNaCl (mimicking sea water concentrations) grew larger than plants not exposed to salt. Adding more salt reduced growth, but concentrations up to 2.5MNaCl were not lethal. Regular tidal flooding with salt water (0.5MNaCl) did not affect growth or chlorophyll fluorescence, whereas continuous flooding stopped growth while plants survived. Quantitative polymerase chain reaction (qPCR) analysis of plants exposed to 1% oxygen in air revealed induction of selected hypoxia responsive genes, but these genes were not induced during tidal flooding, suggesting that S. europaea did not experience hypoxic stress. Indeed, plants were able to transport oxygen into waterlogged soil. Interestingly, sequential exposure to salt and hypoxic air changed the expression of several but not all genes as compared to their expression upon hypoxia only, demonstrating the potential to use S . europaea to investigate signalling-crosstalk between tolerance reactions to multiple environmental perturbations.