Thiol dioxygenases: from structures to functions.

Thiol oxidation to dioxygenated sulfinic acid is catalyzed by an enzyme family characterized by a cupin fold. These proteins act on free thiol-containing molecules to generate central metabolism precursors and signaling compounds in bacteria, fungi, and animal cells. In plants and animals, they also oxidize exposed N-cysteinyl residues, directing proteins to proteolysis. Enzyme kinetics, X-ray crystallography, and spectroscopy studies prompted the formulation and testing of hypotheses about the mechanism of action and the different substrate specificity of these enzymes. Concomitantly, the physiological role of thiol dioxygenation in prokaryotes and eukaryotes has been studied through genetic and physiological approaches. Further structural characterization is necessary to enable precise and safe manipulation of thiol dioxygenases (TDOs) for therapeutic, industrial, and agricultural applications.

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Unearthing the secrets of ERFVIIs: new insights into hypoxia signaling.

Group VII ethylene-responsive factor (ERFVII) transcription factors are crucial for the adaption of plants to conditions that limit oxygen availability. A recent study by Zubrycka et al. reveals new aspects of ERFVII stabilization through the PLANT CYSTEINE OXIDASE (PCO)-N degron pathway and non-autonomous regulation in response to different endogenous and exogenous cues.

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 synthetic switch based on orange carotenoid protein to control blue-green light responses in chloroplasts.

Synthetic biology approaches to engineer light-responsive systems are widely used, but their applications in plants are still limited due to the interference with endogenous photoreceptors and the intrinsic requirement of light for photosynthesis. Cyanobacteria possess a family of soluble carotenoid-associated proteins named orange carotenoid proteins (OCPs) that, when activated by blue-green light, undergo a reversible conformational change that enables the photoprotection mechanism that occurs on the phycobilisome. Exploiting this system, we developed a chloroplast-localized synthetic photoswitch based on a protein complementation assay where two nanoluciferase fragments were fused to separate polypeptides corresponding to the OCP2 domains. Since Arabidopsis (Arabidopsis thaliana) does not possess the prosthetic group needed for the assembly of the OCP2 complex, we first implemented the carotenoid biosynthetic pathway with a bacterial ß-carotene ketolase enzyme (crtW) to generate keto-carotenoid-producing plants. The photoswitch was tested and characterized in Arabidopsis protoplasts and stably transformed plants with experiments aimed to uncover its regulation by a range of light intensities, wavelengths, and its conversion dynamics. Finally, we applied the OCP-based photoswitch to control transcriptional responses in chloroplasts in response to green light illumination by fusing the two OCP fragments with the plastidial SIGMA FACTOR 2 and bacteriophage T4 anti-sigma factor AsiA. This pioneering study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses upon exposure to specific light spectra.

Exploiting the Gal4/UAS System as Plant Orthogonal Molecular Toolbox to Control Reporter Expression in Arabidopsis Protoplasts.

The ability of protein domains to fold independently from the rest of the polypeptide is the principle governing the generation of fusion proteins with customized functions. A clear example is the split transcription factor system based on the yeast GAL4 protein and its cognate UAS enhancer. The rare occurrence of the UAS element in the transcriptionally sensitive regions of the Arabidopsis genome makes this transcription factor an ideal orthogonal platform to control reporter induction. Moreover, heterodimeric transcriptional complexes can be generated by exploiting posttranslational modifications hampering or promoting the interaction between GAL4-fused transcriptional partners, whenever this leads to the reconstitution of a fully functional GAL4 factor.The assembly of multiple engineered proteins into a synthetic transcriptional complex requires preliminary testing, before its components can be stably introduced into the plant genome. Mesophyll protoplast transformation represents a fast and reliable technique to test and optimize synthetic regulatory modules. Remarkable properties are the possibility to transform different combinations of plasmids (co-transformation) and the physiological resemblance of these isolated cells with the original tissue.Here we describe an extensive protocol to produce and exploit Arabidopsis mesophyll protoplasts to investigate the transcriptional output of GAL4/UAS-based complexes that are sensitive to posttranslational protein modifications.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity.

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

The Contribution of Plant Dioxygenases to Hypoxia Signaling.

Dioxygenases catalyze the incorporation of one or two oxygen atoms into target organic substrates. Besides their metabolic role, these enzymes are involved in plant signaling pathways as this reaction is in several instances required for hormone metabolism, to control proteostasis and regulate chromatin accessibility. For these reasons, alteration of dioxygenase expression or activity can affect plant growth, development, and adaptation to abiotic and biotic stresses. Moreover, the requirement of co-substrates and co-factors, such as oxygen, 2-oxoglutarate, and iron (Fe2+), invests dioxygenases with a potential role as cellular sensors for these molecules. For example, inhibition of cysteine deoxygenation under hypoxia elicits adaptive responses to cope with oxygen shortage. However, biochemical and molecular evidence regarding the role of other dioxygenases under low oxygen stresses is still limited, and thus further investigation is needed to identify additional sensing roles for oxygen or other co-substrates and co-factors. Here, we summarize the main signaling roles of dioxygenases in plants and discuss how they control plant growth, development and metabolism, with a focus on the adaptive responses to low oxygen conditions.

Similar and Yet Different: Oxygen Sensing in Animals and Plants.

The ability to perceive oxygen levels and adapt metabolism on the basis of its availability is vital for most eukaryotic cells. Here, we retrace the key steps that led to the identification of oxygen-sensing mechanisms in animals and plants and compare the essential features of the two strategies.

Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.

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.

Zinc Excess Induces a Hypoxia-Like Response by Inhibiting Cysteine Oxidases in Poplar Roots.

Poplar (Populus spp.) is a tree species considered for the remediation of soil contaminated by metals, including zinc (Zn). To improve poplar's capacity for Zn assimilation and compartmentalization, it is necessary to understand the physiological and biochemical mechanisms that enable these features as well as their regulation at the molecular level. We observed that the molecular response of poplar roots to Zn excess overlapped with that activated by hypoxia. Therefore, we tested the effect of Zn excess on hypoxia-sensing components and investigated the consequence of root hypoxia on poplar fitness and Zn accumulation capacity. Our results suggest that high intracellular Zn concentrations mimic iron deficiency and inhibit the activity of the oxygen sensors Plant Cysteine Oxidases, leading to the stabilization and activation of ERF-VII transcription factors, which are key regulators of the molecular response to hypoxia. Remarkably, excess Zn and waterlogging similarly decreased poplar growth and development. Simultaneous excess Zn and waterlogging did not exacerbate these parameters, although Zn uptake was limited. This study unveils the contribution of the oxygen-sensing machinery to the Zn excess response in poplar, which may be exploited to improve Zn tolerance and increase Zn accumulation capacity in plants.

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.

A Synthetic Oxygen Sensor for Plants Based on Animal Hypoxia Signaling.

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.

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.

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.

APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs.

Transcription factors belonging to the APETALA2/Ethylene Responsive Factor (AP2/ERF) family are conservatively widespread in the plant kingdom. These regulatory proteins are involved in the control of primary and secondary metabolism, growth and developmental programs, as well as responses to environmental stimuli. Due to their plasticity and to the specificity of individual members of this family, AP2/ERF transcription factors represent valuable targets for genetic engineering and breeding of crops. In this review, we integrate the evidence collected from functional and structural studies to describe their different mechanisms of action and the regulatory pathways that affect their activity.

New role for an old rule: N-end rule-mediated degradation of ethylene responsive factor proteins governs low oxygen response in plants(F).

The N-end rule pathway regulates protein degradation, which depends on exposed N-terminal sequences in prokaryotes and eukaryotes. In plants, conserved and specific enzymes stimulate selective proteolysis. Although a number of developmental and growth phenotypes have been reported for mutants in the N-end rule, its function has remained unrelated to specific physiological pathways. The first report of the direct involvement of the N-end rule in stress responses focused on hypoxic signaling and how the oxygen-dependent oxidation of cystein promotes the N-end rule-mediated degradation of ethylene responsive factor (ERF)-VII proteins, the master regulators of anaerobic responses. It has been suggested that plants have evolved specific mechanisms to tune ERF-VII availability in the nucleus. In this review, we speculate that ERF-VII proteins are reversibly protected from degradation via membrane sequestration. The oxidative response in plants subjected to anoxic conditions suggests that reactive oxygen and nitrogen species (reactive oxygen species and reactive nitrogen species) may interact or interfere with the N-end rule pathway-mediated response to hypoxia.

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.

Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.