Mitochondrial succinic-semialdehyde dehydrogenase of the gamma-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants

Abstract

The gamma-aminobutyrate (GABA) shunt is a metabolic pathway that bypasses two steps of the tricarboxylic acid cycle, and it is present in both prokaryotes and eukaryotes. In plants the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase and the mitochondrial enzymes GABA transaminase and succinic-semialdehyde dehydrogenase (SSADH). The activity of the GABA shunt in plants is rapidly enhanced in response to various biotic and abiotic stresses. However the physiological role of this pathway remains obscure. To elucidate its role in plants, we analyzed Arabidopsis T-DNA knockout mutants of SSADH, the ultimate enzyme of the pathway. Four alleles of the ssadh mutation were isolated, and these exhibited a similar phenotype. When exposed to white light (100 micromol of photons per m2 per s), they appear dwarfed with necrotic lesions. Detailed spectrum analysis revealed that UV-B has the most adverse effect on the mutant phenotype, whereas photosynthetic active range light has a very little effect. The ssadh mutants are also sensitive to heat, as they develop necrosis when submitted to such stress. Moreover, both UV and heat cause a rapid increase in the levels of hydrogen peroxide in the ssadh mutants, which is associated with enhanced cell death. Surprisingly, our study also shows that trichomes are hypersensitive to stresses in ssadh mutants. Our work establishes a role for the GABA shunt in preventing the accumulation of reactive oxygen intermediates and cell death, which appears to be essential for plant defense against environmental stress.

Fig. 1.

Schematic presentation of the GABA shunt metabolic pathway. The GABA shunt is composed of three enzymes (depicted in boldface type): glutamate decarboxylase (GAD; EC 4.1.1.15), GABA transaminase (GABA-T; EC 2.6.1.19), and succinic-semialdehyde dehydrogenase (SSADH; EC 1.2.1.16). TCA cycle, tricarboxylic acid cycle; SSA, succinic semialdehyde; SCS, succinyl-CoA synthetase; α-KGDH, α-ketoglutarate dehydrogenase; dashed lines, effectors; solid lines, substrates and products.

Fig. 2.

Genotypes and phenotypes of ssadh mutants. (A) Schematic presentation of the structure of the SSADH ORF (At1g79440). The 20 exons are represented by gray boxes (drawn to scale). The T-DNA location for each of the four ssadh knockouts is indicated, namely, ssadh-1 (Versailles collection, line CSV5), ssadh-2 (Salk collection, line 03223), ssadh-3 (Syngenta collection, line 1278_B12), and ssadh-4 (Syngenta collection, line 205_C07). (B) Expression analysis of SSADH mRNA in the ssadh-1 mutant and WT by RT-PCR. Total RNAs from the ssadh-1 mutant and WT were isolated from 3-week-old seedlings and used as templates for reverse transcription (RT). A 439-bp DNA fragment corresponding to a region of the SSADH mRNA was amplified with primers flanking the T-DNA integration site. Amplification of the corresponding region from the SSADH cDNA clone (SSADH cDNA cloned in pZL1 vector; GenBank accession no. AF117335) served as a positive control. -RT designates negative control experiments in which reverse transcriptase was omitted before the final PCR amplification step. Control primers were used to amplify a region of the Arabidopsis mRNA (after RT) encoding elongation factor 1-α (GenBank accession no. AY039583). MW, molecular weight markers, with lengths in bp. (C) Phenotype of the ssadh-1 mutant. After 3 weeks of in vitro growth, seedlings were transferred to soil and grown for a total of 3 months under high-fluence WL at 100μmol·m^-2·s^-1 before being photographed. (D) Phenotype of the four ssadh alleles. Seeds of the mutants and WT (Arabidopsis ecotype Wassilewskija) were grown in vitro for 3 weeks. Seedlings were transferred to soil and grown for an additional 4 weeks under high-fluence WL at 100 μmol·m^-2·s^-1 before being photographed.

Fig. 3.

Hypersensitivity of the ssadh-1 mutant to light and heat stress. (A) Necrosis formation on leaves of ssadh-1 plants exposed to heat and light. WT and ssadh-1 plants were grown under low-fluence WL at 10 μmol·m^-2·s^-1 for 6 weeks (WL-low) or for 4 weeks under low-fluence WL followed by 2 weeks under high-fluence WL at 90 μmol·m^-2·s^-1 (WL-low + WL-high). For heat stress treatment, plants were grown for 2 weeks under low-fluence WL (10 μmol·m^-2·s^-1) at 21°C and 65% relative humidity and for 2 more weeks were heated at 37°C daily for 5 hr in the dark (WL-low + HS). Between 5 and 25 plants (6- to 10-leaf-rosette stage) of each line were measured in each treatment. TB, leaves stained with trypan blue. Red arrows show necrotic lesions. (B) Spectrum analysis of ssadh-1 sensitivity to light. WT and ssadh-1 plants were grown under low-fluence WL at 10 μmol·m^-2·s^-1 for 4 weeks followed by exposure to photosynthetically active radiation (PAR; 400–700 nm) alone at 30 μmol·m^-2·s^-1 (PAR-low) or 70 μmol·m^-2·s^-1 (PAR-high). Wherever indicated, PAR was supplemented with UV-A irradiation at 4.5 μmol·m^-2·s^-1 (UVA-low) or 11.7 μmol·m^-2·s^-1 (UVA-high) or UV-B irradiation at 0.65 μmol·m^-2·s^-1 (UVB-low) or 3.6 μmol·m^-2·s^-1 (UVB-high). Seedlings were visualized after 24 hr, 48 hr, and 7 days of irradiation.

Fig. 6.

Cell death and H₂O₂ accumulation in ssadh-1 trichomes. Plants were grown at 10 μmol·m^-2·s^-1 WL for 4 weeks and then subjected to low-fluence PAR at 30 μmol·m^-2·s^-1 (PAR-low) or transferred to high-fluence PAR at 70 μmol·m^-2·s^-1 supplemented by UV-B at 3.6 μmol·m^-2·s^-1 (PAR-high UVB-high). After 2, 24, or 48 hr, plants were treated with TB or DAB, as indicated. Numbers indicate the percentage of representative trichomes shown in pictures. Values represent a mean of 20 trichomes per time point.

Fig. 4.

H₂O₂ accumulation detected in situ by DAB staining. (A) Plants grown at 60 μmol·m^-2·s^-1 WL for 3 weeks. (B) Plants grown at 10 μmol·m^-2·s^-1 WL for 2 weeks and subjected to heat treatments (HS), as described in the legend of Fig. 3A. Numbers indicate the percentages of leaves showing DAB staining similar to that presented in the pictures (total number of leaves observed was 72 for WT - HS, 70 for WT + HS, 96 for ssadh-1 - HS, and 102 for ssadh-1 + HS). (C) Quantitative analysis of DAB staining of leaves from ssadh-1 plants exposed to UV-B. Plants were grown under low-fluence WL at 10 μmol·m^-2·s^-1 for 4 weeks followed by exposure at 70 μmol·m^-2·s^-1 of PAR supplemented with UV-B irradiation at 3.6 μmol·m^-2·s^-1 for 2 or 24 hr. Leaves (four to seven) from three plants were pictured, and the DAB-stained area was determined as a percentage of total leaf area. Error bars represent SD.

Fig. 5.

H₂O₂ content in the ssadh-1 mutant exposed to various light conditions. H₂O₂ was quantified in ssadh-1 and WT shoots as described in Materials and Methods. Plants were grown in vitro for 4 weeks in complete darkness (Dark) or under low-fluence WL at 10 μmol·m^-2·s^-1 for 4 weeks (WL-low) or for 2 weeks under low-fluence WL followed by 2 weeks under high-fluence WL at 90 μmol·m^-2·s^-1 (WL-low + WL-high). Measurements were performed on bulks of 5–10 individual seedlings. For each light condition, data represent an average of six measurements performed in two independent experiments. Error bars represent SD.