Mutants of GABA transaminase (POP2) suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh) mutants in Arabidopsis.
BACKGROUND
The gamma-aminubutyrate (GABA) shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-T; POP2) and succinic semialdehyde dehydrogenase (SSADH). We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs) and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported.


RESULTS
To elucidate the role of succinic semialdehyde (SSA), gamma-hydroxybutyrate (GHB) and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants.


CONCLUSIONS
We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.
Ludewig
Frank
F
Botanical Institute, University of Cologne, Cologne, Germany.
Hüser
Anke
A
Fromm
Hillel
H
Beauclair
Linda
L
Bouché
Nicolas
N
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000033:anatomy & histology / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004843:Epistasis, Genetic; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism; D016415:Sequence Alignment; D012978:Sodium Oxybate / Q000378:metabolism; D050644:Succinate-Semialdehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D029681:Arabidopsis Proteins; D017382:Reactive Oxygen Species; D005680:gamma-Aminobutyric Acid; D012978:Sodium Oxybate; D050644:Succinate-Semialdehyde Dehydrogenase; D000637:Transaminases; C489779:POP2 protein, Arabidopsis