Oxygen Sensing via the Ethylene Response Transcription Factor RAP2.12 Affects Plant Metabolism and Performance under Both Normoxia and Hypoxia

Abstract

Subgroup-VII-ethylene-response-factor (ERF-VII) transcription factors are involved in the regulation of hypoxic gene expression and regulated by proteasome-mediated proteolysis via the oxygen-dependent branch of the N-end-rule pathway. While research into ERF-VII mainly focused on their role to regulate anoxic gene expression, little is known on the impact of this oxygen-sensing system in regulating plant metabolism and growth. By comparing Arabidopsis (Arabidopsis thaliana) plants overexpressing N-end-rule-sensitive and insensitive forms of the ERF-VII-factor RAP2.12, we provide evidence that oxygen-dependent RAP2.12 stability regulates central metabolic processes to sustain growth, development, and anoxic resistance of plants. (1) Under normoxia, overexpression of N-end-rule-insensitive Δ13RAP2.12 led to increased activities of fermentative enzymes and increased accumulation of fermentation products, which were accompanied by decreased adenylate energy states and starch levels, and impaired plant growth and development, indicating a role of oxygen-regulated RAP2.12 degradation to prevent aerobic fermentation. (2) In Δ13RAP2.12-overexpressing plants, decreased carbohydrate reserves also led to a decrease in anoxic resistance, which was prevented by external Suc supply. (3) Overexpression of Δ13RAP2.12 led to decreased respiration rates, changes in the levels of tricarboxylic acid cycle intermediates, and accumulation of a large number of amino acids, including Ala and γ-amino butyric acid, indicating a role of oxygen-regulated RAP2.12 abundance in controlling the flux-modus of the tricarboxylic acid cycle. (4) The increase in amino acids was accompanied by increased levels of immune-regulatory metabolites. These results show that oxygen-sensing, mediating RAP2.12 degradation is indispensable to optimize metabolic performance, plant growth, and development under both normoxic and hypoxic conditions.

Figure 1.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to decreased growth and delayed development under normoxic conditions. A, Rosette size; B, fresh weight; C, development of 35S::Δ13RAP2.12 plants in comparison to 35S::RAP2.12 and the wild type (WT). Plants were grown for 4 weeks (A) or for the times indicated in the respective figures (B and C). Results in B and C are means ± se (n = 5–6 biological replicates for fresh weight determination or 18 for development analysis) with significant differences indicated by different letters (according to one-way ANOVA for fresh weight determination and two-way ANOVA for development analysis, P < 0.05). Developmental stages were determined according to the BBCH-scale (Hess et al., 1997). FW, Fresh weight.

Figure 2.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to constitutive expression of anoxic marker genes. After 16 h incubation in normoxia (A and C) or hypoxia (B and D) in the absence of external sugars in the dark, 5-week-old wild-type, RAP2.12, and Δ13RAP2.12-overexpressing plants were sampled to analyze the mRNA expression levels of ADH1, PDC1, HB1, SUS4, and RAP2.12 (A and B) as well as AlaAT1 and AlaAT2 (C and D) in whole rosettes. Results are means ± se (n = 8–10 biological replicates) normalized to the normoxic wild type. Values of the different genotypes that significantly differ from each other are indicated by different letters (according to one-way ANOVA test, P < 0.05). WT, Wild type.

Figure 3.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to a constitutive activation of fermentative enzymes. After 16 h incubation in normoxia or hypoxia in the absence of external sugars in the dark, 5-week-old wild-type, RAP2.12, and Δ13RAP2.12 overexpressing plants were sampled to analyze activities of A, ADH; B, PDC; C, LDH; and D, AlaAT in whole rosettes. Results are means ± se (n = 8–10 biological replicates for activity assay of ADH and 6 for PDC, LDH, and AlaAT). Values of the different genotypes that significantly differ from each other are indicated by different letters (according to one-way ANOVA test, P < 0.05). Growth of plants and rosette stage analyzed was similar as in Fig. 2. WT = wild type.

Figure 4.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to increased ethanol production and decreased respiration rates under normoxic conditions. A, To analyze ethanol levels, seedlings were grown in liquid culture containing 1/2 MS medium and 30 mm Suc for 1 week under long-day conditions. After 10 h into the photoperiod, the medium was exchanged to 1/2 MS containing 90 mm Suc and the seedlings were subjected to normoxia or anoxia for further 16 h in the dark, before ethanol levels were measured. B, To analyze respiration rates, whole rosettes of 5-week-old plants were sampled at the end of the night and leaf material representing the whole rosette was introduced into the measuring chamber of an oxygen electrode to analyze oxygen consumption rates in normoxic or hypoxic conditions in the absence of external Suc in the dark. Results are means ± se (n = 25–35 different sets of plants for ethanol assay and seven biological replicates for respiration rates). Values of the different genotypes that significantly differ from each other are indicated by different letters (according to one-way ANOVA test, P < 0.05). WT, Wild type.

Figure 5.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to severe changes in metabolite profiles. Metabolite levels from whole rosettes of 5-week-old wild-type, RAP2.12, and Δ13RAP2.12-overexpressing plants sampled after 16 h incubation in normoxia or hypoxia in the absence of sugars in the dark are visualized using the following VANTED diagrams: A, Carbohydrate metabolism and pentose P-pathway; B, glycolysis, fermentation, Ser, and shikimate pathways; and C, tricarboxylic acid cycle, Asp, and Glu metabolism. Results are means ± se (n = 8–10 biological replicates) and are expressed as arbitrary values; the scaling of the y axis was therefore omitted. Values of the different genotypes that significantly differ from each other within one oxygen treatment are indicated by different letters (according to one-way ANOVA test, P < 0.05). Metabolite ratios are shown in boxes with gray background color. In Supplemental Table S1, the complete metabolite profile is shown together with a statistical analysis of the data. Growth of plants and rosette stage analyzed was similar as in Fig. 2. WT, Wild type.

Figure 5.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to severe changes in metabolite profiles. Metabolite levels from whole rosettes of 5-week-old wild-type, RAP2.12, and Δ13RAP2.12-overexpressing plants sampled after 16 h incubation in normoxia or hypoxia in the absence of sugars in the dark are visualized using the following VANTED diagrams: A, Carbohydrate metabolism and pentose P-pathway; B, glycolysis, fermentation, Ser, and shikimate pathways; and C, tricarboxylic acid cycle, Asp, and Glu metabolism. Results are means ± se (n = 8–10 biological replicates) and are expressed as arbitrary values; the scaling of the y axis was therefore omitted. Values of the different genotypes that significantly differ from each other within one oxygen treatment are indicated by different letters (according to one-way ANOVA test, P < 0.05). Metabolite ratios are shown in boxes with gray background color. In Supplemental Table S1, the complete metabolite profile is shown together with a statistical analysis of the data. Growth of plants and rosette stage analyzed was similar as in Fig. 2. WT, Wild type.

Figure 6.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to a decrease in the adenylate energy state under normoxic conditions. After 16 h incubation in normoxia or hypoxia in the absence of sugars in the dark, 5-week-old wild-type, RAP2.12, and Δ13RAP2.12-overexpressing plants were sampled to analyze the levels of A, ATP; B, ADP; C, ATP/ADP; D, NADH; E, NAD⁺; F, NADH/NAD⁺; G, NADPH; H, NADP⁺; and I, NADPH/NADP⁺ in whole rosettes. Results are means ± se (n = 9–10 biological replicates). Values of the different genotypes that significantly differ from each other are indicated by different letters (according to one-way ANOVA test, P < 0.05). Growth of plants and rosette stage analyzed was similar as in Fig. 2. WT, Wild type.

Figure 7.

Overexpression of the N-end-rule-insensitive Δ13RAP2.12 transcription factor under the control of the 35S promoter leads to decreased anoxic survival rates, which can be prevented by external feeding of 30 mm Suc. A, Seedlings of 1-week-old wild-type, RAP2.12, and Δ13RAP2.12-overexpressing plants grown on MS medium with (+) or without (−) 30 mm Suc were exposed to anoxic conditions for 6.5 and 8 h, respectively, before survival rates were measured 1 week later. B, To put the data into context, starch levels were measured in the shoots of the seedlings at the time point of transfer to anoxic conditions (2 h into the photoperiod). Results are means ± se (n = 10 different sets of plants for survival rates or 5–6 for starch levels). Values of the different genotypes that significantly differ from each other are indicated by different letters (according to one-way ANOVA test, P < 0.05). WT, Wild type.

Figure 8.

Summary model of RAP2.12 regulation of plant metabolism based on the results of this work. When oxygen falls to concentrations below 10% (v/v), the transcription factor RAP2.12 is stabilized and moves to the nucleus to activate the expression of specific genes (Kosmacz et al., 2015). First, mRNA levels and enzyme activities of AlaAT are increased, resulting in increased levels of Ala and γ-amino butyrate and associated changes in the flux modus of the TCA cycle, including an inhibition of succinate dehydrogenase, which finally leads to an inhibition of respiration. The precise mechanism of RAP2.12 regulation of AlaAT expression has not been clarified yet. Second, mRNA levels and enzyme activities of ADH, PDC, and LDH are increased, resulting in elevated rates of ethanol and lactate fermentation. In Arabidopsis plants overexpressing a deregulated (constitutively stabilized) RAP2.12, these metabolic changes are also observed under normoxic conditions, leading to decreased growth and anoxic stress resistance. Green = promoting effects; red = inhibiting effects; dotted arrow = precise mechanism is not known yet.