Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress

Abstract

Timely perception of adverse environmental changes is critical for survival. Dynamic changes in gases are important cues for plants to sense environmental perturbations, such as submergence. In Arabidopsis thaliana, changes in oxygen and nitric oxide (NO) control the stability of ERFVII transcription factors. ERFVII proteolysis is regulated by the N-degron pathway and mediates adaptation to flooding-induced hypoxia. However, how plants detect and transduce early submergence signals remains elusive. Here we show that plants can rapidly detect submergence through passive ethylene entrapment and use this signal to pre-adapt to impending hypoxia. Ethylene can enhance ERFVII stability prior to hypoxia by increasing the NO-scavenger PHYTOGLOBIN1. This ethylene-mediated NO depletion and consequent ERFVII accumulation pre-adapts plants to survive subsequent hypoxia. Our results reveal the biological link between three gaseous signals for the regulation of flooding survival and identifies key regulatory targets for early stress perception that could be pivotal for developing flood-tolerant crops.

Fig. 1

Ethylene pre-treatment enhances hypoxia tolerance. a, b Arabidopsis (Col-0) seedling root tip a and adult rosette b survival after 4 h of air (white) or ~5μllˉ¹ ethylene (blue) followed by hypoxia and recovery (3 days for root tips, 7 days for rosettes). Values are relative to control (normoxia) plants (mean ± sem). Asterisks indicate significant differences between air and ethylene (p < 0.05, Generalized linear model, negative binomial error structure, n = 4-8 rows consisting of ~23 seedlings a, n = 30 plants b). c Arabidopsis (Col-0) rosette phenotypes after 4 h of pre-treatment (air/ ~5μllˉ¹ ethylene) followed by hypoxia and 7 days recovery. All experiments were replicated at least 3 times

Fig. 2

Ethylene-induced hypoxia tolerance is regulated by RAP-type ERFVIIs. a Seedling root tip survival of Col-0, Ler-0, rap2.2 rap2.12 (2 independent lines in Col-0 x Ler-0 background), a constitutively expressed stable version of RAP2.12 and N-degron pathway mutant prt6-1 after 4 h air or ~5μllˉ¹ ethylene followed by 4 h of hypoxia and 3 days recovery. Values are relative to control (normoxia) plants (mean ± sem). Statistically similar groups are indicated using the same letter (p < 0.05, 2-way ANOVA, Tukey’s HSD, n = 20-28 rows consisting of ~23 seedlings). b, c Representative root tip images showing promRAP2.12:RAP2.12-GUS staining and confocal images of 35 S:RAP2.12-GFP intensity in root tips after 4 h of air or ~5μllˉ¹ ethylene. Cell walls were visualized using Calcofluor White stain c. Scale bar of b and c is 50 μm. All experiments were replicated at least 3 times

Fig. 3

Ethylene impairs NO levels leading to ERFVII stability and enhanced hypoxia survival. a, b Representative confocal images visualizing a and quantifying b NO using fluorescent probe DAF-FM diacetate, in Col-0 seedling root tips after 4 h of air or ~5μllˉ¹ ethylene (scale bar= 50 μm). (Letters indicate significant differences (1-way ANOVA, Tukey’s HSD, n = 3–4). c, d Representative confocal images visualizing c and quantifying d 35 S:RAP2.12-GFP intensity in seedling root tips after indicated pre-treatments and subsequent hypoxia (4 h). Cell walls were visualized using Calcofluor White stain (scale bar= 50μm). (Letters indicate significant differences (p < 0.05, 2-way ANOVA, Tukey’s HSD, n = 5-7). e RAP2.3 protein levels in 35 S:MC-RAP2.3-HA seedlings (Col-0 background) after indicated treatments. f Seedling root tip survival of Col-0, rap2.2 rap2.12 line A mutants and an over-expressed stable version of RAP2.12 after indicated pre-treatments followed by hypoxia (4 h) and 3 days recovery. Values are relative to control (normoxia) plants. Letters indicate significant differences (p < 0.05, 2-way ANOVA, Tukey’s HSD, n = 12 rows consisting of ~23 seedlings). All data shown are mean ± sem. All experiments were replicated at least 3 times, except for c, d and f (2 times)

Fig. 4

Ethylene mediates NO levels, ERFVII stability and hypoxia survival through PHYTOGLOBIN1. a Relative transcript abundance of PGB1 in root tips of Col-0, pgb1-1 and 35 S:PGB1 after 4 h air or ~5μllˉ¹ ethylene followed by (4 h) hypoxia. Values are relative to Col-0 air treated samples. Letters indicate significant differences (p < 0.05, 2-way ANOVA, n = 3 replicates of ~200 root tips each). b PGB1 protein levels in Col-0 , pgb1-1 and 35 S:PGB1 root tips after 4 h air or ~5μllˉ¹ ethylene followed by (4 h) hypoxia. c, d Representative confocal images visualizing c and quantifying d NO using fluorescent probe DAF-FM diacetate in Col-0, pgb1-1 and 35 S:PGB1 seedling root tips after 4 h air or ~5μllˉ¹ ethylene (scale bar= 50μm). Letters indicate significant differences (p < 0.05, 2-way ANOVA, Tukey’s HSD, n = 5). e RAP2.3 and PGB1 protein levels in 35 S:MC-RAP2.3-HA (in Col-0, pgb1-1 and 35 S:PGB1 backgrounds) seedling root tips after indicated pre-treatments and subsequent hypoxia (4 h). f Seedling root tip survival of Col-0, pgb1-1 and 35 S:PGB1 after indicated pre-treatments followed by 4 h hypoxia and 3 days recovery. Values are relative to control (normoxia) plants. Letters indicate significant differences (p < 0.05, 2-way ANOVA, Tukey’s HSD, n = 12 rows of ~23 seedlings). All data shown are mean ± sem. All experiments were replicated at least 2 times

Fig. 5

Proposed mechanism of ethylene-induced hypoxia tolerance upon submergence. I Upon submergence ethylene (C₂H₄) accumulates within minutes in plant tissues due to restricted gas diffusion. II+III Ethylene perception leads to EIN2 and EIN3EIL1 dependent signalling and enhanced production of NO-scavenger PHYTOGLOBIN1 (PGB1) within 1 h of ethylene signalling. IV+V Within 4 h, these enhanced PGB1 levels lead to NO depletion, in turn limiting PRT6 N-degron pathway targeted proteolysis of RAP-type group VII Ethylene Response Factor transcription factors (ERVIIs). V+VII Stabilized ERFVIIs translocate to the nucleus where they induce enhanced hypoxia gene expression only when O₂ deprivation occurs. This amplified hypoxia response increases hypoxia tolerance of Arabidopsis root and shoot apical meristems (created with BioRender.com)