Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana.

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9 +/- 1.7 and 1.7 +/- 0.3 mM at pH 4, respectively. The simultaneous transport of [1-14C]GABA and [2,3-3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-14C]GABA transport was inhibited by proline and quaternary ammonium compounds.

LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen.

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons.

The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.

The Synthesis of [gamma]-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH.

[gamma]-Aminobutyric acid (GABA) synthesis (L-glutamic acid + H+ -> GABA + CO2) is rapidly stimulated by a variety of stress conditions including hypoxia. Recent literature suggests that GABA production and concomitant H+ consumption ameliorates the cytosolic acidification associated with hypoxia or other stresses. This proposal was investigated using isolated asparagus (Asparagus sprengeri Regel) mesophyll cells. Cell acidification was promoted using hypoxia, H+/L-glutamic acid symport, and addition of butyrate or other permeant weak acids. Sixty minutes of all three treatments stimulated the levels of both intracellular and extracellular GABA by values ranging from 100 to 1800%. At an external pH of 5.0, addition of 5 mM butyrate stimulated an increase in overall GABA level from 3.86 (0.56 [plus or minus] SE) to 20.4 (2.16 [plus or minus] SE) nmol of GABA/106 cell. Butyrate stimulated GABA levels by 200 to 300% within 15 s, and extracellular GABA was observed after 10 min. The acid load due to butyrate addition was assayed by measuring [14C]butyrate uptake. After 45 s of butyrate treatment, H+-consuming GABA production accounted for 45% of the imposed acid load. The cytosolic location of a fluorescent pH probe was confirmed using fluorescent microscopy. Spectrofluorimetry indicated that butyrate addition reduced cytosolic pH by 0.60 units with a half-time of approximately 2 s. The proposal that GABA synthesis ameliorates cytosolic acidification is supported by the data. The possible roles of H+ and Ca2+ in stimulating GABA synthesis are discussed.