Nicolas Bouché, Aaron Fait, David Bouchez, Simon G. Møller, Hillel Fromm
The γ-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 μmol 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.
Light Spectrum Analysis. WT and ssadh mutant seedlings were germinated and grown under low-fluence white light (WL; 280–700 nm) for 4 weeks (short days) as described above followed by exposure to different irradiation conditions. Seedlings were irradiated for 7 days with Farnell 5-mm/T1¾ untainted clear-lens light-emitting diode rigs supplying either monochromatic blue light (458 nm, 11 μmol·m-2·s-1) or monochromatic red light (660 nm, 70 μmol·m-2·s-1). For UV irradiation, seedlings were exposed to low-fluence (30 μmol·m-2·s-1) or high-fluence (70 μmol·m-2·s-1) photosynthetically active radiation (PAR; 400–700 nm, including UV-Amax 0.45 μmol·m-2·s-1 and UV-Bmax 0.012 μmol·m-2·s-1) alone as a control, and supplemented with low- or high-fluence UV-A (320–400 nm; 4.5 μmol·m-2·s-1 or 11.7 μmol·m-2·s-1) or with low- or high-fluence UV-B (280–320 nm; 0.65 μmol·m-2·s-1 or 3.6 μmol·m-2·s-1) irradiation. UV-A and UV-B were supplied by Philips TL20W/09N and TL20W/01RS fluorescent tubes, respectively. All fluences were measured with a StellarNet EPP2000 fiber optic spectrometer (Tampa, FL).