ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana

Abstract

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.

Fig. 1. ERFVIIs as determinants of plant hypoxia tolerance.

a Schematic of the currently accepted PCO N-degron pathway. The changing N-terminus of ERFVIIs is shown in relation to enzyme activities. MetAP, METHIONINE AMINOPEPTIDASE; PCO, PLANT CYSTEINE OXIDASE; ATE, ARGINYL TRANSFERASE; PRT6, PROTEOLYSIS6. The conversion of amino-terminal Cys to Cys-sulfinate (SO₂^-) is shown. Residues indicated by three letter code. b Root tip survival of wild-type (Col-0) and mutant seedlings subjected to 4 h hypoxia and 3 days recovery. MA-ERFVIIs indicates individual promERFVII:C²A-ERFVII transgene in Col-0 background. Data are presented as mean ± SD (n = 9) significant differences denoted with letters for one-way ANOVA (p < 0.05). c Germination under different levels of ambient oxygen for wild-type (Col-0) and mutant seeds. Data are presented as mean ± SD (n = 3). d Images of four-week-old wild-type (Col-0) or erfVII plants grown in sandy-clay loam soil in control conditions or upon waterlogging treatment for 7 days. Scale bar is 1 cm. e Weights of rosettes and roots of four-week-old wild-type (Col-0) and erfVII mutant with or without waterlogging treatment of the duration of 7 days. Data are presented as mean ± SD (n = 8), t-test, ***p-value < 0.0002 and ****p-value < 0.0001, ‘ns’ not significant.

Fig. 2. ERFVII influence on root structure in soil.

a Representative 3D rendered X-ray computed micro-tomography front view images of four-week-old wild-type (Col-0) and erfVII mutant roots grown in sandy-clay loam soil under control conditions or waterlogging treatment conditions for 7 days. Scale bar is 15 mm. b Root total volume (mm³), root region of interest (ROI) surface area (mm²) and maximum root depth calculated from X-ray computed micro-tomography images of wild type (Col-0) and erfVII mutant roots with or without waterlogging treatment, as described in (a). Data are presented as mean ± SD (n = 4). For µCT imaging, four replicates were imaged, data were analysed by two-way ANOVA and different letters indicate significant differences (p-value < 0.05). Scale bar is 15 mm.

Fig. 3. Influence of ERFVIIs on the seedling transcriptome.

a Proportional Venn diagram representation of gene sets for Col-0 (wild-type) vs. prt6 erfVII and prt6 vs. prt6 erfVII. In each case numbers of total differentially regulated genes (up or down) are shown. b Top 10 most significant GO terms associated with transcripts differentially regulated in prt6 vs. prt6 erfVII, Fisher’s one-tailed test (p < 0.05). c Proportional Venn diagram representation showing overlapping gene sets between genes differentially expressed between prt6 vs prt6 erfVII and hypoxia vs normoxia. Total number of differentially regulated genes are shown next to comparisons, in set identities within the intersecting set. d Differential expression of a set of core 49 hypoxia upregulated genes in transcriptome comparisons, sorted by fold difference in prt6 vs prt6 erfVII.

Fig. 4. Modulation of RAP2.3 stability by genetic and environmental factors.

a In situ immunolocalization of RAP2.3^3xHA in control or bortezomib (50 μM) treated roots of 4-day-old seedlings, scale bars 20 μm. b Western blot analysis of RAP2.3^3xHA abundance in control or bortezomib treated WT or prt6-1 mutant seedlings. c Time-course Western blot analysis of RAP2.3^3xHA abundance in WT seedlings in response to submergence or hypoxia treatment. d Time-course of expression of ADH1 in WT seedlings in response to submergence or hypoxia treatment. Data are presented as mean ± SD (n = 3). f Western blot analysis of RAP2.3^3xHA abundance in WT seedlings in response to constant contact or transfer to media with no, low or high supplemented Fe²⁺. e Western blot analysis of RAP2.3^3xHA abundance in WT seedlings following transfer from hypoxia (60 min) to normoxia. g Western blot analysis showing relative abundance of RAP2.3^3xHA in response to bortezomib (BZ), cPTIO (NO scavenger), hypoxia or submergence. h Western blot analysis showing relative abundance of RAP2.3^3xHA in low altitude accession Col-0 in comparison to high altitude Sha at either 21% or 15% ambient oxygen. BZ treatment for light-grown seedlings. Ponceau or CBB staining of Western blots is shown.

Fig. 5. Analysis of the chemical structure at the amino-termini of RAP2.3 and RAP2.12 in a plant extract.

MS/MS spectra of the Nt tryptic peptides of (a) RAP2.3 ([M + 2H]2 + = m/z 856.4) and (b) RAP2.12 ([M + 2H]2 + = m/z 823.9) following expression of RAP2.3^3xHA and RAP2.12^3xHA proteins, respectively, in a wheat germ extract showing the presence of Cys-sulfonic acid (C^3ox) and arginylation at the amino-terminus (R-) in each case.

Fig. 6. Model for plant oxygen sensing through the PCO N-degron pathway based on data presented in this work.

ERFVII transcription is shown at the top of the model. Subsequent enzymatic steps on the protein are shown, either oxygen-independent (MetAP) or oxygen dependent. Activation of nuclear gene expression by stabilised ERFVIIs in the absence of oxygen, through the Hypoxia Responsive Promoter Element (HRPE) is indicated. “?” indicates an unknown component required for the addition of a third oxygen to form Cys sulfonate.