Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in Arabidopsis

Abstract

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.

Keywords: ACBP; ERFVII; acyl-CoA; integrative signaling; low-oxygen stress.

Fig. 1.

ACBP is an important element of the low-oxygen stress response pathway in plants. (A) Multiple sequence alignment of class II ACBP ankyrin domain compared with ACBP1 and ACBP2 ankyrin domains in A. thaliana (27). (B) Multiple sequence alignment of ERFVII DNA-binding domain compared with RAP2.12 and RAP2.2 in A. thaliana (26). Sequences of homologous proteins were obtained from Phytozome v12.0 and aligned with Clustal X. (C) Eleven-day-old seedlings of wild type and acbp1 after 9 h of anoxia and 3-d recovery. (Scale bar: 1.0 cm.) (D) Survival scores for wild type and acbp1 after 9-h anoxia and 3-d recovery. Data are mean values ± SD; *P < 0.05, n = 4 (15 seedlings per replicate). (E) Effect of 2 h of hypoxia treatment (1% O₂) on the localization of GFP-tagged ACBP1 in epidermal cells. Representative pictures are shown. (Scale bar: 10 µm.)

Fig. 2.

Application of C18:1-CoA induces RAP2.12 relocalization into the nucleus. (A) Representative Western blot showing in vitro ACBP1:RAP2.12 complex stability after treatment with C18:1-CoA or C16:0-CoA. Pluronic F68 treatment served as control. (B) Quantification of ACBP1-to-RAP2.12-ratio as shown in A. Data are mean values ± SD *P < 0.05, n = 8. (C) Percentage of epidermal cells with nuclear localization of RAP2.12-GFP after treatment with different acyl-CoAs. Data are mean values ± SD; *P < 0.05. (D) Localization of RAP2.12-GFP in detached leaves incubated with 0.1% C18:1-CoA, C18:0-CoA or C16:0-CoA dissolved in 0.01% pluronic F68 under normoxic conditions for 3 h. Treatment with pluronic F68 only served as negative control. DAPI staining was used to identify nuclei. Arrows indicate nuclei with GFP signal. (Scale bar: 10 µm.)

Fig. 3.

Changing oleoyl-CoA levels induces low-oxygen responsive gene expression. (A) Number of significantly differentially expressed genes in leaves after 1.5-h treatment with different acyl-CoAs as determined by RNA-Seq (FDR-adjusted P value <0.05). (B) Number of GO classes in which differentially expressed genes are significantly overrepresented under acyl-CoA treatments as shown in A. (C) qPCR analysis of differential expression of hypoxia-responsive genes after acyl-CoA treatment in air (reference: F68 only). Data are presented as mean values *P < 0.05, n = 5. (D) C18:1-CoA levels increase upon hypoxia in wild type. Data are mean values ± SD; *P < 0.05, n = 3. (E) C16:0-CoA levels decrease upon hypoxia in wild type. Data are mean values ± SD; *P < 0.05, n = 3. (F) C18:1-CoA levels are increased in lacs4lacs9 double mutants grown in air. Data are mean values ± SD; *P < 0.05, n = 3. (G) C16:0-CoA levels are lowered in lacs4lacs9 double mutants. Data are mean values ± SD; *P < 0.05, n = 3. (H) Expression data for hypoxia-responsive genes comparing wild type and lacs4-1 lacs9-2 in air or hypoxia (2 h 1% O₂; mean values, *P < 0.05, n = 4).

Fig. 4.

Decreased tolerance of lacs4 lacs9 knockout lines to anoxia and submergence. (A) Eleven-day-old seedlings of wild type and lacs4-1 lacs9-2 after 9 h of anoxia and 3-d recovery. (Scale bar: 1.0 cm.) (B) Survival scores for wild type and lacs4-1 lacs9-2 after 9-h anoxia and 3-d recovery. Data are mean values ± SD; *P < 0.05, n = 4 (15 seedlings per replicate). (C) Phenotype of wild type and lacs4 lacs9 mutant grown in air (control), or after 3- or 4-d submergence-induced hypoxic treatment. (Scale bar: 2 cm.) Photographs were taken 4 d after the submergence treatment. (D) Absolute dry weight of wild-type and lacs4-1 lacs9-2 plants grown in air. Data represent mean ± SD (three replicate experiments with every 12 plants per genotype). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (E) Absolute fresh weight of wild-type and lacs4-1 lacs9-2 plants grown in air. Data represent mean ± SD (three replicate experiments with every 12 plants per genotype). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (F) Relative fresh weight of wild-type and lacs4-1 lacs9-2 plants grown in air, or after 3 or 4 d of submergence followed by 4 d of recovery. Data represent mean ± SD (three replicate experiments with every 12 plants per genotype). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (G) Percentage of plants that survived the 3 or 4 d of flooding-induced hypoxia, respectively (mean values ± SD, three replicate experiments with every 12 plants per genotype). *P < 0.05 according to one-way ANOVA.

Fig. 5.

Decreasing the cellular ATP level constitutes limiting conditions for LACS activity and induces the expression of low-oxygen responsive genes. (A) ATP levels under hypoxia (mean ± SD, *P < 0.05, n = 5). (B) Concentration of ADP in wild-type seedlings grown under long-day conditions and exposed to hypoxia. Data shown are given in nanomoles per gram fresh weight and represent the mean ± SD of independent replicates (n = 5). (C) ATP-to-ADP-ratio under hypoxia (mean ± SD, *P < 0.05, n = 5). (D) In vitro LACS activity depends on ATP concentration (mean ± SD, *P < 0.05, n = 5). The gray area marks the ATP-concentration range usually determined in plant cells. (E) Differential expression of hypoxia-responsive genes after 3 h of 1 mM DMTU and/or 50 µM antimycin-A (AA) treatment under aerobic conditions (reference: mock-treated control). Data are presented as mean ± SD, *P < 0.05, n = 5. (F) ATP levels after 3 h of 50 µM AA treatment (mean ± SD, *P < 0.05, n = 5). (G) Concentration of ADP in wild-type seedlings exposed to 3 h of 50 µM AA treatment. Data represent mean ± SD (n = 5). Asterisk indicates significant differences after one-way ANOVA (P < 0.05). (H) ATP-to-ADP-ratio after 3 h of 50 µM AA treatment (mean ± SD, *P < 0.05, n = 5). (I) Oxygen consumption rate in wild-type leaves upon 3 h of 50 µM AA treatment. Data represent mean ± SD (n = 7). Asterisk indicates significant difference after Student’s t test (P < 0.05).

Fig. 6.

Triggering low-oxygen responses in plants integrates the cellular energy and oxygen status via modulation of oleoyl-CoA levels. Oxygen limitation reduces cellular ATP levels, which results in increased C18:1-CoA levels. Dissociation of ERFVII protein (as shown here for RAP2.12) bound to ACBP1 at the plasma membrane is promoted by C18:1-CoA. Free ERFVII protein is stable under low-oxygen conditions and relocalizes into the nucleus to activate hypoxic responses.