Cross-Kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differ-entially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.Instituto de Biotecnologia y Biologia Molecula

Cross-Kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differ-entially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.Instituto de Biotecnologia y Biologia Molecula

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

Cross-Kingdom Comparison of Transcriptomic Adjustments to Low-Oxygen Stress Highlights Conserved and Plant-Specific Responses1[W][OA]

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance

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

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

<i>HRA</i>1 is a low oxygen-inducible gene from Arabidopsis.

<p>(A) <i>HRA1</i> mRNA steady state levels in hypoxia-treated seedlings, in comparison with other abiotic stress treatments. Data are mean ± s.d. (<i>n</i> = 3). (B) Visualization of <i>HRA1</i> promoter activity by GUS-reporter staining. Nucleotide positions in the schematic are relative to <i>HRA1</i> transcription start site. Scale bar, 2 mm. (C) Transcript accumulation of <i>HRA1</i> and the hypoxic marker <i>ADH1</i> in seedlings, over an initial and more prolonged time course of sublethal hypoxia (upper and lower left diagrams). Data are mean ± s.d. (<i>n</i> = 3).</p

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

HRA1 modulates transcription of anaerobic genes through protein–protein interaction with RAP2.12.

<p>(A) HRA1 binding site on the upstream region of the <i>HRA1</i> gene. The number next to the peak indicates the peak summit. ChIP-seq peak area for <i>HRA1</i> was subsequently divided into six regions for confirmation of DNA binding using ChIP-qPCR (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001950#pbio.1001950.s020" target="_blank">Table S4</a> for primer sequences). TSS, transcription start site. (B) Yeast-two-hybrid assay between an HRA1 C-terminal fragment (HRA1_194–431) and the five Arabidopsis ERF-VII proteins. AD, activation domain; DBD, DNA binding domain; UAS, upstream activating sequence. SC-LW, control medium −Leu −Trp; SC-LWH+3AT, selective medium −Leu −Trp −His +3AT; LacZ, β-galactosidase assay. (C) Bimolecular fluorescence complementation between HRA1 and RAP2.12 in Arabidopsis mesophyll protoplasts. DAPI staining indicates the position of the nucleus. Scale bar, 20 µm. (D) Transcriptional activation of the <i>PDC1</i> promoter, visualized through a firefly <i>luciferase</i> (<i>PpLuc</i>) reporter fusion, by RAP2.12 alone (blue bars) and in combination with HRA1 (yellow bars) or its C-terminal fragment HRA1_194–431 (orange bars). Data are mean ± s.d. (<i>n</i> = 4). (E) Dual luciferase assay showing that HRA1 repression of RAP2.12 is independent of HRA1 binding to DNA. A heterologous promoter made up of four repeats of the yeast <i>GAL4</i> upstream activating sequence (“<i>GAL4 UAS</i>”) was introduced into plant protoplasts and could only be recognized by chimeric GAL4DBD (GAL4 DNA binding domain)-containing TFs, in this case by RAP2.12-GAL4DBD <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001950#pbio.1001950-Licausi1" target="_blank">[7]</a>. GFP was used as a negative control, as a RAP2.12 noninteracting protein. Data are mean ± s.d. (<i>n</i> = 3); *<i>p</i><0.05, statistically significant difference from the positive interaction produced by RAP2.12-GAL4DBD.</p