Ethylene-Regulated Glutamate Dehydrogenase Fine-Tunes Metabolism during Anoxia-Reoxygenation

Abstract

Ethylene is an essential hormone in plants that is involved in low-oxygen and reoxygenation responses. As a key transcription factor in ethylene signaling, ETHYLENE INSENSITIVE3 (EIN3) activates targets that trigger various responses. However, most of these targets are still poorly characterized. Through analyses of our microarray data and the published Arabidopsis (Arabidopsis thaliana) EIN3 chromatin immunoprecipitation sequencing data set, we inferred the putative targets of EIN3 during anoxia-reoxygenation. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further studies for its role in tricarboxylic acid cycle replenishment. We demonstrated that both GDH1 and GDH2 are induced during anoxia and reoxygenation and that this induction is mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the level of GDH during anoxia-reoxygenation decreased in the ethylene-insensitive mutants ein2-5 and ein3eil1 Global metabolite analysis indicated that the deamination activity of GDH might regenerate 2-oxoglutarate, which is a cosubstrate that facilitates the breakdown of alanine by alanine aminotransferase when reoxygenation occurs. Moreover, ineffective tricarboxylic acid cycle replenishment, disturbed carbohydrate metabolism, reduced phytosterol biosynthesis, and delayed energy regeneration were found in gdh1gdh2 and ethylene mutants during reoxygenation. Taken together, these data illustrate the essential role of EIN3-regulated GDH activity in metabolic adjustment during anoxia-reoxygenation.

Figure 1.

EIN3 stabilization during A/R. 35S::EIN3-GFP seedlings (7-d-old) in the ein3eil1 mutant background (35S::EIN3GFP-OE/dm) were used to follow EIN3 levels by immunoblot detection of EIN3-GFP under A/R treatment. Anoxia treatment was performed in the dark. Reoxygenation was carried out either in the dark (A) or light (B). Nor, Plants grown under aerated conditions (normoxia); Re0, after 4 h of anoxia (beginning of reoxygenation); Re1, Re3, and Re6, 1, 3, and 6 h of reoxygenation, respectively. Treatment with AgNO₃ (20 μm) or 1-methylcyclopropene (1-MCP) was used as a negative control to inhibit ethylene signal transduction. The quantified signal values were normalized to time point Nor and are shown below the gels.

Figure 2.

Analyses of EIN3 direct targets and A/R-regulated genes. Venn diagrams and heat maps were used to demonstrate the EIN3-regulated targets in response to A/R. A, The gene lists of Targets_ET-R and Targets_N-ET-R, which represent EIN3 targets under ethylene regulation and EIN3 targets not under ethylene regulation, respectively, were extracted from Chang et al. (2013). The clustering of these EIN3 targets was defined according to their responses to ethylene treatment. Some of the EIN3 targets were regulated by ethylene alone, and some were not (Chang et al., 2013). The gene list of 4h-AR_C (> 2FC), which represents genes with 2-fold changes in response to 4 h of A/R (P < 0.05), was extracted from the microarray data presented by Tsai et al. (2014). B, Representative genes from each group were selected to present as a heat map by applying them to previous A/R array data. Nor, Plants grown under aerated conditions (normoxia); Re0, after 4 h of anoxia (beginning of reoxygenation); Re0.5, Re1, Re3, and Re6, after 0.5, 1, 3, and 6 h of reoxygenation, respectively. The scales at right are presented as log₂ values. C, Class I and class II genes were used to perform Venn diagram analysis with the core hypoxia-responsive genes that were reported by Mustroph et al. (2009).

Figure 3.

Ethylene participates in the regulation of GDH genes during A/R. Quantitative real-time PCR was used to verify the effects of ethylene signaling on the GDHs. A, Transcript levels of GDH1, GDH2, and GDH3 in 7-d-old wild-type, ein2-5, and ein3eil1 seedlings in response to A/R in the dark. B, Transcript levels were determined in plants under A/R in light. C, Transcript levels in 7-d-old wild-type seedlings treated with AgNO₃ or 1-MCP before and after anoxia treatment in the dark. Nor, Plants grown under aerated conditions (normoxia); Re0, after 4 h of anoxia (beginning of reoxygenation); Re0.5, Re1, Re3, and Re6, after 0.5, 1, 3, and 6 h of reoxygenation, respectively. The results are mean values of at least four biological replicates with se. Asterisks indicate P < 0.05 by Student’s t test.

Figure 4.

Enzyme activity of GDH during A/R. An in vitro enzyme activity assay was used to measure GDH activity in 7-d-old wild-type, ein2-5, ein3eil1, and gdh1gdh2 seedlings during A/R in the dark. The x axis shows the treatment time course, and the zero point was set as the beginning of reoxygenation. The time points −4, −3.5, and −3 represent normoxia, 30 min, and 1 h of anoxia treatment, respectively. The time points 0.5, 1, 3, and 6 represent 0.5, 1, 3, and 6 h of reoxygenation, respectively. The results are mean values of at least four biological replicates with se. Asterisks indicate P < 0.05 by Student’s t test.

Figure 5.

Changes of metabolites in the wild type and gdh1gdh2 in response to A/R. GC-TOF-MS was applied to investigate the metabolite profiles during anoxia and reoxygenation. Black lines with diamonds correspond to the wild type, and dotted lines with circles correspond to the gdh1gdh2 mutant. The x axis shows the treatment time course. The time point −4 corresponds to normoxia, and the zero point is set as the beginning of reoxygenation. The time points 1, 3, and 6 represent 1, 3, and 6 h of reoxygenation, respectively. The level of metabolites is expressed as relative fold change calculated by normalizing values of the wild type (normoxia). The results are mean values of at least four biological replicates with se. Asterisks indicate P < 0.05 by Student’s t test. ADH, Alcohol dehydrogenase; GABAT, GABA aminotransferase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; SSADH, succinic semialdehyde dehydrogenase; TCA, tricarboxylic acid.

Figure 6.

Energy-related metabolites in response to A/R treatment. Nor, Seven-day-old plants grown under aerated conditions (normoxia); Re0, after 4 h of anoxia (beginning of reoxygenation); Re1 and Re6, after 1 and 6 h of reoxygenation, respectively. Black bars correspond to the wild type, dark green bars correspond to ein2-5, light green bars correspond to ein3eil1, and magenta bars correspond to gdh1gdh2. The level of metabolites is expressed as fold change that was calculated by normalizing values of the wild type (Nor). The results are mean values of at least four biological replicates with se. Asterisks indicate P < 0.05 by Student’s t test.

Figure 7.

Changes of carbohydrates, α-tocopherol, and phytosterols in wild-type and gdh1gdh2 plants in response to A/R. GC-TOF-MS was applied to investigate carbohydrates, α-tocopherol, and phytosterols during A/R. Black lines with diamonds correspond to the wild type, and dotted lines with circles correspond to the gdh1gdh2 mutant. The x axis shows the treatment time course. The time point −4 corresponds to normoxia, and the zero point is set as the beginning of reoxygenation. The time points 1, 3, and 6 represent 1, 3, and 6 h of reoxygenation, respectively. The level of metabolites is expressed as relative fold change that is calculated by normalizing values of the wild type (normoxia). The results are mean values of at least four biological replicates with se. Asterisks indicate P < 0.05 by Student’s t test. F-6-P, Fru-6-P; G-6-P, Glc-6-P. Campesterol, stigmasterol, and sitosterol are common sterols in plants.

Figure 8.

Phenotypes of the gdh1gdh2 mutant in response to oxygen deprivation. A, Five-week-old Arabidopsis plants were submerged in water for 36 h and reaerated for 7 d. B and C, Quantification of damage was performed based on the number of wilted leaves (B), and survival rate was determined by observing growth (growing corresponds to survival) after submergence (C). D, Seven-day-old seedlings were treated with 9 h of anoxia and reaerated for 2 d with the anaerobic chamber. E, Quantification of damage was measured based on the number of wilted leaves. The results are mean values of at least four biological replicates with se. Asterisks indicate P < 0.05 by Student’s t test.

Figure 9.

Model of tricarboxylic acid (TCA) cycle replenishment in response to reoxygenation. To rapidly recover energy generation, tricarboxylic acid cycle replenishment is proposed to be essential during reoxygenation. Two metabolic routes for tricarboxylic acid cycle replenishment are suggested in this model. One was performed by Phosphoenolpyruvate carboxylase (PEPC), which converts Phosphoenolpyruvate (PEP) into OAA (A), and the other one was performed by GDH, which converts Glu into 2OG (B). In addition, PYRUVATE PHOSPHATE DIKINASE (PPDK), an enzyme that converts pyruvate into PEP during glycogenesis, might play a role in providing PEP at the recovery stage. Transcripts of PPDK and GDH are regulated by ethylene signaling during A/R. Increased transcripts of GDH lead to the enhanced enzymatic reaction in the direction of deamination to regenerate 2OG. 2OG can further react with Ala and facilitate Ala breakdown by the enzyme AlaAT and produce Glu and pyruvate. Pyruvate further reenters the tricarboxylic acid cycle, and Glu can be recycled through the enzyme GDH again. PEPCK, PEP carboxylase kinase.