Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-010-9728-y) contains supplementary material, which is available to authorized users.

No abstract

We discuss the origin of 4-aminobutyrate (GABA) from glutamate and polyamines, and its subsequent catabolism to succinic semialdehyde and either succinate or 4-hydroxybutyrate. Promiscuous activities of GABA transaminase, glyoxylate/succinic semialdehyde reductases, and aldehyde dehydrogenase 10As appear to be important determinants of cross-talk among metabolic pathways during stress. Imposition of abiotic stress, as well as genetic or chemical disruption of glutamate decarboxylase, GABA transaminase, and tricarboxylic acid cycle reactions, results in non-cyclic carbon flux in the tricarboxylic acid cycle, demonstrating that stress-induced GABA metabolism is strongly linked with respiration. Metabolic generation of 4-hydroxybutyrate is probably linked to the stimulation of succinic semialdehyde reductase activity by an increasing NADPH/NADP+ ratio. We discuss the potential signaling role of GABA in various processes, including pollen tube guidance, interaction with fungal, bacterial, and invertebrate pests, and stomatal functioning, and argue that further research on short-term responses to stress is required to determine whether or not GABA functions by binding to or regulating the activity of GABA receptor molecules. Finally, we describe how emerging information about the metabolic and signaling roles of GABA is being used to improve plant defenses against biotic and abiotic stresses, and benefit human health.

4-Aminobutyrate (GABA) accumulates in apple fruit during controlled atmosphere storage. A potential source of GABA is the polyamine putrescine, which can be oxidized via copper-containing amine oxidase (CuAO), resulting in the production 4-aminobutanal/Δ(1)-pyrroline, with the consumption of O2 and release of H2O2 and ammonia. Five putative CuAO genes (MdAO genes) were cloned from apple (Malus domestica Borkh. cv. Empire) fruit, and the deduced amino acid sequences found to contain the active sites typically conserved in CuAOs. Genes encoding two of these enzymes, MdAO1 and MdAO2, were highly expressed in apple fruit and selected for further analysis. Amino acid sequence analysis predicted the presence of a C-terminal peroxisomal targeting signal 1 tripeptide in MdAO1 and an N-terminal signal peptide and N-glycosylation site in MdAO2. Transient expression of green fluorescent fusion proteins in Arabidopsis protoplasts or onion epidermal cells revealed a peroxisomal localization for MdAO1 and an extracellular localization for MdAO2. The enzymatic activities of purified recombinant MdAO1 and MdAO2 were measured continuously as H2O2 production using a coupled reaction. MdAO1 did not use monoamines or polyamines and displayed high catalytic efficiency for 1,3-diaminopropane, putrescine and cadaverine, whereas MdAO2 exclusively utilized aliphatic and aromatic monoamines, including 2-phenylethylamine and tyramine. Together, these results indicate that MdAO1 may contribute to GABA production via putrescine oxidation in the peroxisome of apple fruit under controlled atmosphere conditions. MdAO2 seems to be involved in deamination of 2-phenylethylamine, which is a step in the biosynthesis of 2-phenylethanol, a contributor to fruit flavor and flower fragrance.

4-Aminobutyrate accumulates in plants under abiotic stress. Here, targeted quantitative profiling of metabolites and transcripts was conducted to monitor glutamate- and polyamine-derived 4-aminobutyrate production and its subsequent catabolism to succinate or 4-hydroxybutyrate in apple (Malus x domestica Borkh.) fruit stored at 0 °C with 2.5 kPa O2 and 0.03 or 5 kPa CO2 for 16 weeks. Low-temperature-induced protein hydrolysis appeared to be responsible for the enhanced availability of amino acids during early storage, and the resulting higher glutamate level stimulated 4-aminobutyrate levels more than polyamines. Elevated CO2 increased the levels of polyamines, as well as succinate and 4-hydroxybutyrate, during early storage, and 4-aminobutyrate and 4-hydroxybutyrate over the longer term. Expression of all of the genes likely involved in 4-aminobutyrate metabolism from glutamate/polyamines to succinate/4-hydroxybutyrate was induced in a co-ordinated manner. CO2-regulated expression of apple GLUTAMATE DECARBOXYLASE 2, AMINE OXIDASE 1, ALDEHYDE DEHYDROGENASE 10A8 and POLYAMINE OXIDASE 2 was evident with longer term storage. Evidence suggested that respiratory activities were restricted by the elevated CO2/O2 environment, and that decreasing NAD+ availability and increasing NADPH and NADPH/NADP+, respectively, played key roles in the regulation of succinate and 4-hydroxybutyate accumulation. Together, these findings suggest that both transcriptional and biochemical mechanisms are associated with 4-aminobutyrate and 4-hydroxybutyrate metabolism in apple fruit stored under multiple abiotic stresses.

BackgroundThe ubiquitous, non-proteinaceous amino acid GABA (γ-aminobutyrate) accumulates in plants subjected to abiotic stresses such as chilling, O2 deficiency and elevated CO2. Recent evidence indicates that controlled atmosphere storage causes the accumulation of GABA in apple (Malus x domestica Borkh.) fruit, and now there is increasing interest in the biochemical mechanisms responsible for this phenomenon. Here, we investigated whether this phenomenon could be mediated via Ca2+/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity.ResultsGAD activity in cell-free extracts of apple fruit was stimulated by Ca2+/CaM at physiological pH, but not at the acidic pH optimum. Based on bioinformatics analysis of the apple genome, three apple GAD genes were identified and their expression determined in various apple organs, including fruit. Like recombinant Arabidopsis GAD1, the activity and spectral properties of recombinant MdGAD1 and MdGAD2 were regulated by Ca2+/CaM at physiological pH and both enzymes possessed a highly conserved CaM-binding domain that was autoinhibitory. In contrast, the activity and spectral properties of recombinant MdGAD3 were not affected by Ca2+/CaM and they were much less sensitive to pH than MdGAD1, MdGAD2 and Arabidopsis GAD1; furthermore, the C-terminal region neither bound CaM nor functioned as an autoinhibitory domain.ConclusionsPlant GADs typically differ from microbial and animal GAD enzymes in possessing a C-terminal 30–50 amino acid residue CaM-binding domain. To date, rice GAD2 is the only exception to this generalization; notably, the C-terminal region of this enzyme still functions as an autoinhibitory domain. In the present study, apple fruit were found to contain two CaM-dependent GADs, as well as a novel CaM-independent GAD that does not possess a C-terminal autoinhibitory domain.

Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

Jasmonates are plant signaling molecules that play key roles in protection against certain pathogens and against insects by switching on the expression of genes encoding defense proteins including enzymes involved in the biosynthesis of toxic secondary metabolites. In Catharanthus roseus, the ethylene response factor (ERF) transcription factor ORCA3 controls the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. Its promoter contains an autonomous jasmonate-responsive element (JRE). Here we describe the jasmonate-responsive activity of the JRE from the ORCA3 promoter in Arabidopsis thaliana. We found that it interacts in vitro and in vivo with the basic helix-loop-helix transcription factor AtMYC2. Analysis of JRE-mediated reporter gene expression in an atmyc2-1 mutant background showed that the activity was strictly dependent on AtMYC2.