Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Low-oxygen-induced root bending is altered by phytoglobin1 through mediation of ethylene response factors (ERFs) and auxin signaling.

MAIN CONCLUSION: phytoglobin1 positively regulates root bending in hypoxic Arabidopsis roots through regulation of ethylene response factors and auxin transport. Hypoxia-induced root bending is known to be mediated by the redundant activity of the group VII ethylene response factors (ERFVII) RAP2.12 and HRE2, causing changes in polar auxin transport (PAT). Here, we show that phytoglobin1 (Pgb1), implicated in hypoxic adaptation through scavenging of nitric oxide (NO), can alter root direction under low oxygen. Hypoxia-induced bending is exaggerated in roots over-expressing Pgb1 and attenuated in those where the gene is suppressed. These effects were attributed to Pgb1 repressing both RAP2.12 and HRE2. Expression, immunological and genetic data place Pgb1 upstream of RAP2.12 and HRE2 in the regulation of root bending in oxygen-limiting environments. The attenuation of slanting in Pgb1-suppressing roots was associated with depletion of auxin activity at the root tip because of depression in PAT, while exaggeration of root bending in Pgb1-over-expressing roots with the retention of auxin activity. Changes in PIN2 distribution patterns, suggestive of redirection of auxin movement during hypoxia, might contribute to the differential root bending responses of the transgenic lines. In the end, Pgb1, by regulating NO levels, controls the expression of 2 ERFVIIs which, in a cascade, modulate PAT and, therefore, root bending.

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.

The underestimated role of plant root nitric oxide emission under low-oxygen stress.

The biotic release of nitric oxide (NO), a greenhouse gas, into the atmosphere contributes to climate change. In plants, NO plays a significant role in metabolic and signaling processes. However, little attention has been paid to the plant-borne portion of global NO emissions. Owing to the growing significance of global flooding events caused by climate change, the extent of plant NO emissions has been assessed under low-oxygen conditions for the roots of intact plants. Each examined plant species (tomato, tobacco, and barley) exhibited NO emissions in a highly oxygen-dependent manner. The transfer of data obtained under laboratory conditions to the global area of farmland was used to estimate possible plant NO contribution to greenhouse gas budgets. Plant-derived and stress-induced NO emissions were estimated to account for the equivalent of 1 to 9% of global annual NO emissions from agricultural land. Because several stressors induce NO formation in plants, the actual impact may be even higher.

Iron toxicity increases oxidative stress and impairs mineral accumulation and leaf gas exchange in soybean plants during hypoxia.

Iron toxicity is a major challenge faced by plants in hypoxic soils; however, the consequences of such combined stress for soybean (Glycine max) remain to be determined. Here we assessed the physiological responses of soybean plants exposed to hypoxia and a high concentration of iron. Soil-grown plants cultivated in a greenhouse until the vegetative stage were transferred to a hydroponic system containing nutrient solution and subjected to two oxygen conditions (normoxia (6.2 mg L-1) and hypoxia (0.33 mg L-1)) and two iron concentrations (Fe-EDTA) (0.09 and 1.8 mM) for 72 h. During hypoxia, high concentrations of iron in the nutrient solution resulted in increased iron accumulation in roots and leaves. Under this condition, the concentrations of zinc, nitrogen, potassium, and calcium decreased in the roots, while the concentration of nitrogen and magnesium decreased in the leaves. Additionally, during hypoxia, the higher concentration of iron led to an increase in the activity of the antioxidant enzymes in roots and leaves, while decreased the levels of the photosynthetic pigments, leaf gas exchange, and plant growth. In conclusion, high iron concentration in the root medium results in a considerably more severe damage condition to soybean plants under hypoxia compared to plants grown under low iron availability.

An updated overview of the physiological and molecular responses of rice to anoxia.

Waterlogging or flooding in agricultural soil constructs a complex abiotic stress-web in crop plants throughout the lowland agricultural system. In rice, a staple grain crop in the world, submergence creates a long-term and recurrent problem for crops withstanding and their succeeding productivity. Therefore, to restore a satisfactory yield instead of a failure of crop in such submerged areas, the analysis of plants' responses is important. Such analysis will facilitate research about the entity components of plants responses to anoxia or submergence. For example, the development of cellular and molecular cascade in gene expression of ROS signaling and its subsequent responses may lead to either tolerance or susceptibility in plants. Interplay of plant growth regulators [e.g., ethylene (ET), abscisic acid (ABA), gibberellic acid (GA) etc.] is the well-recognized residues in the coordination of signaling, its transmission through cellular network, and finally, modulation of gene expression are the keys to such tolerance. Nucleotide elements that are specifically sensitive to ethylene have been rescued from land-races of aus-type group of rice (Oryza sativa) and those are considered as the prime determinants for tolerance against anoxia. In this comprehensive text, we tried to accommodate and revise the fundamental and pivotal mechanisms of submergence stress at different angles of physiological and cellular responses of plants. These have also been reviewed for modern state of art techniques deciphering the molecular rejoinders like microRNA, protein-protein interaction, feedback regulation of expression, sugar sensing, amplification of elicitor's responses and others. Finally, strategies including physiological selection, metabolic engineering, marker assisted selection, genetical manipulation, interspecific hybridization are involved in developing stress resilience and plants' architecture to support sustainable agriculture under lowland systems.

Hypometabolic Responses to Chronic Hypoxia: A Potential Role for Membrane Lipids.

Metabolic suppression is an essential strategy to cope with chronic hypoxia. This review examines the physiological processes used to survive in low oxygen environments. It proposes a novel mechanism-the remodeling of membrane lipids-to suppress ATP use and production. Temperature (homeoviscous adaptation), diet (natural doping in migrant birds) and body mass (membrane pacemaker of metabolism) have an impact on the lipid composition of membranes, which, in turn, modulates metabolic capacity. Vertebrate champions of hypoxia tolerance show extensive changes in membrane lipids upon in vivo exposure to low oxygen. These changes and those observed in hibernating mammals can promote the downregulation of ion pumps (major ATP consumers), ion channels, mitochondrial respiration capacity (state 3, proton leak, cytochrome c oxidase), and energy metabolism (ß-oxidation and glycolysis). A common membrane signal regulating the joint inhibition of ion pumps and channels could be an exquisite way to preserve the balance between ATP supply and demand in hypometabolic states. Membrane remodeling together with more traditional mechanisms could work in concert to cause metabolic suppression.

Regulation of the Central Carbon Metabolism in Apple Fruit Exposed to Postharvest Low-Oxygen Stress.

After harvest, fruit remain metabolically active and continue to ripen. The main goal of postharvest storage is to slow down the metabolic activity of the detached fruit. In many cases, this is accomplished by storing fruit at low temperature in combination with low oxygen (O2) and high carbon dioxide (CO2) partial pressures. However, altering the normal atmospheric conditions is not without any risk and can induce low-O2 stress. This review focuses on the central carbon metabolism of apple fruit during postharvest storage, both under normal O2 conditions and under low-O2 stress conditions. While the current review is focused on apple fruit, most research on the central carbon metabolism, low-O2 stress, and O2 sensing has been done on a range of different model plants (e.g., Arabidopsis, potato, rice, and maize) using various plant organs (e.g., seedlings, tubers, roots, and leaves). This review pulls together this information from the various sources into a coherent overview to facilitate the research on the central carbon metabolism in apple fruit exposed to postharvest low-O2 stress.

Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit.

The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions.

Introduction to the Special Issue: Electrons, water and rice fields: plant response and adaptation to flooding and submergence stress.

Flooding and submergence impose widespread and unpredictable environmental stresses on plants and depress the yield of most food crops. The problem is increasing, as is the need for greater food production from an expanding human population. The incompatibility of these opposing trends creates an urgent need to improve crop resilience to flooding in its multifarious forms. This Special Issue brings together research findings from diverse plant species to address the challenge of enhancing adaptation to flooding in major crops and learning from tactics of wetland plants. Here we provide an overview of the articles, with attempts to summarize how recent research results are being used to produce varieties of crop plants with greater flooding tolerance, notably in rice. The progress is considerable and based firmly on molecular and physiological research findings. The article also sets out how next-generation improvements in crop tolerance are likely to be achieved and highlights some of the new research that is guiding the development of improved varieties. The potential for non-model species from the indigenous riparian flora to uncover and explain novel adaptive mechanisms of flooding tolerance that may be introduced into crop species is also explored. The article begins by considering how, despite the essential role of water in sustaining plant life, floodwater can threaten its existence unless appropriate adaptations are present. Central to resolving the contradiction is the distinction between the essential role of cellular water as the source of electrons and protons used to build and operate the plant after combining with CO2 and O2 and the damaging role of extracellular water that, in excess, interferes with the union of these gases with photosynthetic or respiratory electrons and protons.