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 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

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

A Multi-OMICs approach sheds light on the higher yield phenotype and enhanced abiotic stress tolerance in tobacco lines expressing the carrot lycopenelycopene β−cyclase1\beta-cyclase1 gene

International audienceRecently, we published a set of tobacco lines expressing the Daucus carota (carrot) DcLCYB1 gene with accelerated development, increased carotenoid content, photosynthetic efficiency, and yield. Because of this development, DcLCYB1 expression might be of general interest in crop species as a strategy to accelerate development and increase biomass production under field conditions. However, to follow this path, a better understanding of the molecular basis of this phenotype is essential. Here, we combine OMICs (RNAseq, proteomics, and metabolomics) approaches to advance our understanding of the broader effect of LCYB expression on the tobacco transcriptome and metabolism. Upon DcLCYB1 expression, the tobacco transcriptome (~2,000 genes), proteome (~700 proteins), and metabolome (26 metabolites) showed a high number of changes in the genes involved in metabolic processes related to cell wall, lipids, glycolysis, and secondary metabolism. Gene and protein networks revealed clusters of interacting genes and proteins mainly involved in ribosome and RNA metabolism and translation. In addition, abiotic stress-related genes and proteins were mainly upregulated in the transgenic lines. This was well in line with an enhanced stress (high light, salt, and H$_2$ O$_2$ ) tolerance response in all the transgenic lines compared with the wild type. Altogether, our results show an extended and coordinated response beyond the chloroplast (nucleus and cytosol) at the transcriptome, proteome, and metabolome levels, supporting enhanced plant growth under normal and stress conditions. This final evidence completes the set of benefits conferred by the expression of the DcLCYB1 gene, making it a very promising bioengineering tool to generate super crops

Interaction of 29,39-cAMP with Rbp47b plays a role in Stress Granule Formation

29,39-cAMP is an intriguing small molecule that is conserved among different kingdoms. 29,39-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 29,39-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 29,39- cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 29,39-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 29,39-cAMP. Furthermore, SG formation was promoted in 29,39-cAMP-treated Arabidopsis seedlings, and interactions between 29,39-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 29,39-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.We acknowledge the Knut and Alice Wallenberg Foundation for supporting the work of E.G.-B

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 production1. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion2, 3, 4. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses5, 6, 7, 8, 9, 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 plants10, 11, 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

The nuclear factor HRA1 attenuates the expression of hypoxia-responsive genes.

<p>(A) Subcellular localization of the HRA1:GFP protein in root cells. Nuclei were visualized by DAPI staining. Scale bar, 30 µm. (B) Differential gene expression in <i>OE-HRA1</i> and wild type seedlings under air or hypoxia (2 h), compared with <i>35S:HA:RAP2.12</i> transgenics. (C) Venn diagram describing the overlap between genes with opposing regulation by HRA1 or RAP2.12 and 49 genes induced across cell types by hypoxia in wild type seedlings <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001950#pbio.1001950-Mustroph1" target="_blank">[13]</a>. (D) ADH enzyme activity is affected by altered levels of HRA1 in plants at the seedling stage (mean ± s.d., one-way ANOVA, <i>p</i><0.05, <i>n</i> = 3). Hypoxia, 3 h.</p

Model summarizing how the balanced action of RAP2.12 and HRA1 tunes transcription of hypoxia target genes.

<p>In plant cells, hypoxia promotes the relocalization of RAP2.12 to the nucleus, which triggers the expression of <i>HRA1</i> and other RAP2.12 target genes. Once synthesized, the encoded HRA1 protein generates two negative loops of feedback regulation, one acting on RAP2.12 and another on HRA1 itself. The first one dampens RAP2.12 activity, thereby limiting the anaerobic gene expression after its initial burst. The negative self-regulation is, instead, supposed to contribute to the subsequent down-regulation of HRA1 and makes a later wave of anaerobic gene expression possible under prolonged hypoxia.</p

A negative feedback loop acting on HRA1 determines the extent of anaerobic gene expression over the time of stress.

<p>(A) Modulation of <i>HRA1</i> promoter activity, as visualized through the firefly luciferase reporter, by co-transfection of protoplasts with a stabilized RAP2.12_14–358, alone (blue bars) or in combination with a HRA1 effector construct (yellow bars). Data are mean ± s.d. (<i>n</i> = 4). (B) Steady-state levels, in Arabidopsis seedlings, of the full-length <i>HRA1</i> mRNA (<i>HRA1_Endo</i>), measured with specific <i>HRA1 3′-UTR</i> primers, and of full-length (in the wild type), truncated (in <i>hra1-1</i> and <i>-2</i>), and overexpressed transcripts (in <i>OE-HRA1-#1</i> and <i>-#2</i>) (<i>HRA1_Tot</i>), measured with primers specific for <i>HRA1</i> coding sequence; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001950#pbio.1001950.s004" target="_blank">Figure S4B</a> for the position of primers used for <i>HRA1_Tot</i> and <i>HRA1_Endo</i> mRNA abundance measurement. Hypoxia, 2 h. Data are mean ± s.d. (<i>n</i> = 3 ). The absence of expression is indicated by grey rectangles (masked). Numeric expression values are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001950#pbio.1001950.s021" target="_blank">Table S5</a>. (C) Abundance of <i>HRA1</i> and hypoxia marker gene mRNAs over prolonged hypoxia stress in <i>OE-HRA1</i> and <i>hra1</i> seedlings. Data are mean ± s.d. (<i>n</i> = 4).</p

HRA1 contributes to plant submergence survival.

<p>(A) Effect of <i>HRA1</i> misexpression on rosette growth in air, or after recovery from 72 h submergence in darkness. Scale bar, 2 cm. (B) Percentage of plants surviving flooding-induced hypoxia (<i>n</i> = 5), dry weight of rosette plants kept under control growth conditions (<i>n</i> = 6), and dry weight of rosettes after postsubmergence recovery (<i>n</i> = 6). Data are mean ± s.d.; *<i>p</i><0.05, significant differences from the wild type after one-way ANOVA. (C) HRA1 regulates target gene transcripts in an age-dependent manner in leaves of plants treated with complete submergence. Transcripts were measured before submergence (“control conditions”), after 4 h submergence in darkness (“submergence”), and after 1 h de-submergence in the light (“reoxygenation”). Relative transcript values were calculated using old leaves of the wild type under control conditions as the reference sample. Data are mean ± s.d. (<i>n</i> = 3); letters indicate statistically significant differences between genotypes after one-way ANOVA (<i>p</i><0.05) performed independently on each leaf type. (D) Western blot analysis of ADH and PDC protein accumulation in leaves at different developmental stages from control and submerged (4 h) plants. The full-size images of the hybridized membranes can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001950#pbio.1001950.s010" target="_blank">Figure S10</a>. (E) Stability of the translational fusion RAP2.12:RrLuc protein (RrLuc, <i>Renilla reniformis</i> luciferase) in Arabidopsis mesophyll protoplasts upon transfection with increasing amounts of <i>35S:HRA1</i>. RAP2.12:RrLuc abundance was evaluated from the RrLuc relative activity, measured through a dual luciferase assay. Data are mean ± s.d. (<i>n</i> = 4), and the asterisks indicate statistically significant differences (<i>p</i><0.05) from protoplasts expressing RAP2.12:RrLuc alone, after one-way ANOVA.</p