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During angiosperm reproduction, pollen grains form a tube that navigates through female tissues to the micropyle, delivering sperm to the egg; the signals that mediate this process are poorly understood. Here, we describe a role for gamma-amino butyric acid (GABA) in pollen tube growth and guidance. In vitro, GABA stimulates pollen tube growth, although vast excesses are inhibitory. The Arabidopsis POP2 gene encodes a transaminase that degrades GABA and contributes to the formation of a gradient leading up to the micropyle. pop2 flowers accumulate GABA, and the growth of many pop2 pollen tubes is arrested, consistent with their in vitro GABA hypersensitivity. Some pop2 tubes continue to grow toward ovules, yet they are misguided, presumably because they target ectopic GABA on the ovule surface. Interestingly, wild-type tubes exhibit normal growth and guidance in pop2 pistils, perhaps by degrading excess GABA and sharpening the gradient leading to the micropyle.
The γ-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.
To elucidate the role of succinic semialdehyde (SSA), γ-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.
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.
Copyright: 2008 Ludewig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
GABA synthesis (Figure 1A) from glutamate is controlled by the cytosolic glutamate decarboxylase (GAD), a Ca2+/calmodulin regulated enzyme in plants [10]. GABA is catabolized in mitochondria through the GABA-shunt [2], a metabolic pathway that bypasses two successive steps of the tricarboxylic acid cycle catalyzed by α-ketoglutarate dehydrogenase and succinyl CoA ligase. The enzymes involved in GABA catabolism are GABA transaminase (GABA-T) which converts GABA to succinic semialdehyde (SSA), and succinic semialdehyde dehydrogenase (SSADH) which oxidizes SSA to succinate coupled to NADH production. Hence, GABA is a metabolite en route from glutamate to the tricarboxylic acid cycle which provides succinate and NADH to the respiratory machinery. The production of succinate via the GABA-shunt seems to be of primary importance when the TCA cycle does not provide enough succinate. In fact, transgenic plants exhibiting decreased expression of the succinyl CoA ligase present a mild phenotype and slightly reduced rates of respiration; in these plants, GAD activities are increased and the production rate of succinate derived from the GABA-shunt is elevated, thus compensating the deficiency of succinate production by the TCA cycle [9]. SSA can also be converted into γ-hydroxybutyric acid (GHB) by a succinic semialdehyde reductase present in animals and recently discovered in plants [11].
In humans, SSADH deficiency, known as GHB aciduria, is a genetically inherited disease causing non-specific neurological disorders due to the accumulation of GHB and GABA in the brain [12]. GHB was shown to accumulate in mice deficient for SSADH [13] as well as in Arabidopsis ssadh knockout mutants [14]. GABA and GHB are playing crucial roles as neurotransmitters by binding to receptors in the mammalian brain. In plants, evidence is lacking to assign a function as signaling molecules to them because plant receptors binding GABA or GHB are still to be identified. Genes homologous to ionotropic glutamate receptors of animals are present in the genome of Arabidopsis [15], [16] and encode proteins that likely mediate sodium or calcium entry into cells [17]. Very recently, the characterization of a short-root mutant of Oryza sativa that has defects in a glutamate receptor gene (GLR3.1) provided genetic and molecular evidence for an important function of GLR3.1 in seedling development [18]. Whether GABA could possibly interact with glutamate receptors in plants remains an open question [19]. Another putative GABA receptor could be the high affinity GABA transporter (AtGAT1) localized in the plasma membrane of Arabidopsis and expressed under conditions of elevated GABA content like mechanical wounding or senescence [20].
A GABA concentration gradient is essential for the growth and guidance of pollen tubes and suggests that this amino acid is involved in intercellular signaling [21]. The pop2 (pollen-pistil incompatibility2) mutant is unable to produce a functional GABA-T enzyme, and, as a consequence, this leads to growth inhibition and misguidance of pop2 pollen tubes in pop2 pistils because the GABA gradient guiding pollen tubes is disturbed. GABA binding sites (i.e. putative receptors) were detected on protoplast membranes of both pollen and somatic cells using a fluorescent probe [22]. The main question raised by these studies is whether GABA itself serves as a signaling molecule in plants. Recent findings point toward a role of GABA as a signal between plants and pathogenic bacteria since GABA can modulate quorum sensing in Agrobacterium tumefaciens [23], [24]. A role for GABA in mediating responses to volatiles produced during wounding or pathogen attacks was proposed since some of these volatiles induce GABA accumulation, and pop2 alleles (her1) were isolated in mutagenesis screens to select Arabidopsis mutants with altered responses to such volatiles [25].
We have previously shown that compromising the function of the GABA-shunt causes enhanced accumulation of reactive oxygen intermediates (ROIs) and cell death in response to light and heat stress [26]. We described the phenotype of ssadh mutants impaired in the last step of the GABA-shunt. In brief, when grown under standard light conditions, the four isolated ssadh homozygous mutants (ssadh-1 to ssadh-4) are dwarfs and present necrotic lesions and bleached spots on leaves, reduced leaf area, lower chlorophyll content, and fewer flowers compared to wild type plants. In addition, ssadh mutants are more sensitive to at least two types of environmental stresses. The development of ssadh mutants exposed to UV-B light or heat stress is significantly retarded and associated with the appearance of necrotic lesions. H2O2 contents are increased in ssadh plants exposed to stress, as shown by 3,3-diaminobenzidine (DAB) staining and direct H2O2 quantification [26]. Thus, the phenotype of ssadh plants grown in standard conditions is probably associated with the UV-B light contained in white light. To explain the accumulation of H2O2 in ssadh mutants and its severe growth retardation, we mentioned two possible hypotheses [26]. First, the lack of supply of essential metabolites to the TCA cycle (particularly succinate and NADH), and secondly, the accumulation of a toxic compound (SSA or GHB, or both) due to the block in the metabolism of SSA. Here we combined two genetic approaches to suppress the severe phenotype of ssadh mutants, with physiological and metabolic investigations of single mutants, double mutants, and second-site suppressors to elucidate the cause of the ssadh phenotype and the role of SSA, GABA and GHB in the accumulation of ROIs.
To dissect the phenotypic effects of ssadh mutations, we first crossed ssadh mutants with pop2 mutants. Such pop2 ssadh double mutants are expected to lack the potential toxic compounds SSA, GHB (Figure 1A), or metabolites derived from their catabolism. However, pop2 ssadh double mutants would not be able to supply NADH and succinate to the TCA cycle via the GABA shunt, similar to ssadh mutants. Several mutations in the GABA-T (POP2) gene encoding the enzyme degrading GABA into SSA (Figure 1A), were described (pop2-1, pop2-2 and pop2-3 mutants; [21]). By comparing the GABA-T (At3g22200) genomic sequence of Arabidopsis with T-DNA flanking genomic sequences deposited in the databases we identified two additional pop2 alleles designated pop2-4 and pop2-5 (Figure 1B). The genomic DNAs of the mutants were characterized by PCR (Materials and Methods) and the junctions between the T-DNAs and the gene were sequenced. Plants homozygous for the pop2-4 or pop2-5 mutations were phenotypically similar to other pop2 alleles [21]. Homozygous plants grew as wild type but were partially sterile with no or very small siliques (Figure 1C).
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