Figure 1.
The GABA shunt metabolic pathway and the pop2 mutants.
(A) Schematic presentation of the GABA shunt metabolic pathway. The GABA shunt is composed of three enzymes (in bold). The cytosolic glutamate decarboxylase (GAD) catalyses the irreversible decarboxylation of glutamate to produce GABA. GABA is transported in the mitochondria to be converted into succinic semialdehyde (SSA) by a GABA transaminase (GABA-T/POP2). SSA is then oxidized by a succinic semialdehyde dehydrogenase (SSADH) to form succinate, which enters the tricarboxylic acid (TCA) cycle. In animals and possibly in plants, SSA can be converted into γ-hydroxybutyric acid (GHB) by a SSA reductase (SSR) and GHB into SSA via another enzyme, the GHB dehydrogenase (GHBDH). α-KG; α-ketoglutarate. (B) Characterization of pop2-4 and pop2-5 T-DNA mutants. Gene structure of the GABA-T/POP2 (At3g22200) ORF and T-DNA insertions in both pop2-4 (SAIL_1230_C03) and pop2-5 (GABI_157D10) mutants. Exons are represented by boxes (drawn to scale). LB designs the left T-DNA borders. Junctions between the T-DNAs and the gene were sequenced. (C) Phenotype of the pop2-4 and pop2-5 mutants compared to WT (Col) plants. Seeds were sown on soil and grown for a total of 45 days in the greenhouse before being photographed.
Figure 2.
pop2-4 is epistatic to ssadh-3.
(A) Phenotype of six weeks old F2 plants resulting from a cross between pop2-4 and ssadh-3 homozygous plants. Genotypes are indicated, namely wild type (plant #25), ssadh-3/ssadh-3 (plant #10), ssadh-3/ssadh-3 pop2-4/POP2 (plant #22) and ssadh-3/ssadh-3 pop2-4/pop2-4 double mutants (plants #28 to 44). (B) PCR analysis of F2 plants shown in (A). DNAs from F2 plants were subjected to a PCR amplification using SSADH or GABA-T/POP2 gene specific oligos (Materials and Methods) and the T-DNA left border oligo corresponding to SAIL lines (LB3). MW; DNA molecular weight. (C) Expression analysis by RT-PCR of GABA-T/POP2 and SSADH mRNAs in F2 plants shown in (A). Total RNAs from F2 plants were extracted and used as templates for reverse transcription (Materials and Methods). (D) Average number of seeds for WT (Col-0), pop2-4, ssadh-3 and the corresponding double mutants. Seeds were counted for a total of 4 plants per genotype.
Figure 3.
GABA-T nucleotide and amino acid sequences of pop2-6 and pop2-7 EMS mutants.
(A) Position and nature of the point mutations (in bold) in the new pop2 alleles. Both pop2-6 and pop2-7 have a G→A transition resulting in the amino acid substitution G271E and G312D, respectively. The codons that have been changed in the mutants are underlined. (B) Comparison of the GABA-T amino acid sequences of Arabidopsis pop2-6 and pop2-7 and of Sus scrofa (GenBank accession 1OHV_A). ‘*’ indicates residues that are identical, ‘:’ homologous and ‘.’ similar . The active site residues of the pig enzyme are in bold, the pyridoxal phosphate recognition site is underlined, the attachment site is in italics, and the mutated residues are underlined, in bold and italics.
Figure 4.
H2O2 accumulation in pop2 and ssadh mutants detected by DAB staining.
In situ detection of H2O2 by using 3,3-diaminobenzidine (DAB) staining on pop2-4, ssadh-3, wild type and pop2-4 ssadh-3 plants grown at 150 µmol m−2 s−1 white light (WL) for three weeks (day/night cycle, 16/8 hours; temperature day/night, 20/15°C). Numbers indicate the percentages of leaves showing DAB staining similar to that presented in the pictures (total number of leaves observed was 12 for wild type, 17 for ssadh-3, 11 for pop2-4, and 15 for pop2-4 ssadh-3).
Figure 5.
Peroxide accumulation measured by high temperature thermoluminescence emission (HTL) in pop2 and ssadh mutants.
High temperature thermoluminescence emission measured in arbitrary units (a.u.) on rosette leaves fixed on aluminum foils using the custom-made apparatus and software described earlier [29]. Wild type (blue), pop2-4 (green), ssadh-3 (red) and pop2-4 ssadh-3 (black) plants were grown in vitro for three weeks under standard conditions (day/night cycle, 16/8 hours; light intensity, 100–150 µmol m−2 s−1; temperature day/night, 20/15 °C). The experiment was repeated 10 times and one representative signal is shown. Thermoluminescence heating rate; 0.1 °C s−1.
Figure 6.
Phenotypes of ssadh, pop2, pop2 ssadh and wild type plants grown on media containing SSA or GHB.
(A) Phenotype of the ssadh-3 mutants and Col-0 wild type plants grown in vitro for 14 days under standard conditions (day/night cycle, 16/8 hours; light intensity, 100–150 µmol m−2 s−1; temperature day/night, 20/15°C) with various amount of succinic semialdehyde (SSA) added to the medium. Photographs of Petri dishes and corresponding magnified pictures are shown. The experiment was repeated three times. (B) Phenotype of Col-0 wild type (WT), pop2-5 and pop2-5 ssadh-2 plants grown on ½ MS for 18 days under standard conditions (day/night cycle, 16/8 hours; light intensity, 100–150 µmol m−2 s−1; temperature day/night, 20/15°C) supplemented with the indicated concentrations of succinic semialdehyde (SSA) or γ-hydroxbutyrate (GHB).
Figure 7.
GHB and GABA contents of pop2, pop2 ssadh and wild type plants grown on either SSA or GHB.
(A) and (C) Relative amounts (highest value is 100%) of γ-hydroxbutyrate (A; GHB) and γ-aminobutyrate (C; GABA) in leaves of Col wild type (WT), pop2-5 and pop2-5 ssadh-2 mutant plants grown on the indicated concentrations of succinic semialdehyde (SSA). Data (relative arbitrary units normalized to plant weight and the abundance of the internal standard ribitol) were sampled using a GC/MS system, n = 4–8 (WT), n = 2–8 (pop2) and n = 2–8 (pop2 ssadh)±SE for GHB and n = 4–10 (WT), n = 2–10 (pop2) and n = 1–8 (pop2 ssadh)±SE for GABA. (B) and (D) Relative amounts (highest value is 100%) of GHB (B) and GABA (D) in leaves of Col wild type (WT), pop2-5 and pop2-5 ssadh-2 mutant plants grown on the indicated concentrations of GHB. Data (relative arbitrary units normalized to plant weight and the abundance of the internal standard ribitol) were sampled using a GC/MS system, n = 4–8 (WT), n = 4–8 (pop2) and n = 1–8 (pop2 ssadh)±SE for GHB and n = 4–10 (WT), n = 4–10 (pop2) and n = 2–8 (pop2 ssadh)±SE for GABA.