Glutamate decarboxylase-1 is essential for efficient acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

Glutamate decarboxylase (GAD) is a Ca2+ -calmodulin-activated, cytosolic enzyme that produces γ-aminobutyrate (GABA) as the committed step of the GABA shunt. This pathway bypasses the 2-oxoglutarate to succinate reactions of the tricarboxylic acid (TCA) cycle. GABA also accumulates during many plant stresses. We tested the hypothesis that AtGAD1 (At5G17330) facilitates Arabidopsis acclimation to Pi deprivation. Quantitative RT-PCR and immunoblotting revealed that AtGAD1 transcript and protein expression is primarily root-specific, but inducible at lower levels in shoots of Pi-deprived (-Pi) plants. Pi deprivation reduced levels of the 2-oxoglutarate dehydrogenase (2-OGDH) cofactor thiamine diphosphate (ThDP) in shoots and roots by > 50%. Growth of -Pi atgad1 T-DNA mutants was significantly attenuated relative to wild-type plants. This was accompanied by: (i) an > 60% increase in shoot and root GABA levels of -Pi wild-type, but not atgad1 plants, and (ii) markedly elevated anthocyanin and reduced free and total Pi levels in leaves of -Pi atgad1 plants. Treatment with 10 mM GABA reversed the deleterious development of -Pi atgad1 plants. Our results indicate that AtGAD1 mediates GABA shunt upregulation during Pi deprivation. This bypass is hypothesized to circumvent ThDP-limited 2-OGDH activity to facilitate TCA cycle flux and respiration by -Pi Arabidopsis.

Improving Boron and Molybdenum Use Efficiencies in Contrasting Cultivars of Subirrigated Greenhouse-Grown Pot Chrysanthemums.

Fertilizer boron (B) and molybdenum (Mo) were provided to contrasting cultivars of subirrigated pot chrysanthemums at approximately 6-100% of current industry standards in an otherwise balanced nutrient solution during vegetative growth, and then all nutrients were removed during reproductive growth. Two experiments were conducted for each nutrient in a naturally lit greenhouse using a randomized complete block split-plot design. Boron (0.313-5.00 µmol L-1) or Mo (0.031-0.500 µmol L-1) was the main plot, and cultivar was the sub-plot. Petal quilling was observed with leaf-B of 11.3-19.4 mg kg-1 dry mass (DM), whereas Mo deficiency was not observed with leaf-Mo of 1.0-3.7 mg kg-1 DM. Optimized supplies resulted in leaf tissue levels of 48.8-72.5 mg B kg-1 DM and 1.9-4.8 mg Mo kg-1 DM. Boron uptake efficiency was more important than B utilization efficiency in sustaining plant/inflorescence growth with decreasing B supply, whereas Mo uptake and utilization efficiencies appeared to have similar importance in sustaining plant/inflorescence growth with decreasing Mo supply. This research contributes to the development of a sustainable low-input nutrient delivery strategy for floricultural operations, wherein nutrient supply is interrupted during reproductive growth and optimized during vegetative growth.

γ-Aminobutyrate Improves the Postharvest Marketability of Horticultural Commodities: Advances and Prospects.

Postharvest deterioration can result in qualitative and quantitative changes in the marketability of horticultural commodities, as well as considerable economic loss to the industry. Low temperature and controlled atmosphere conditions (low O2 and elevated CO2) are extensively employed to prolong the postharvest life of these commodities. Nevertheless, they may suffer from chilling injury and other physiological disorders, as well as excessive water loss and bacterial/fungal decay. Research on the postharvest physiological, biochemical, and molecular responses of horticultural commodities indicates that low temperature/controlled atmosphere storage is associated with the promotion of γ-aminobutyrate (GABA) pathway activity, with or without the accumulation of GABA, delaying senescence, preserving quality and ameliorating chilling injury. Regardless of whether apple fruits are stored under low temperature/controlled atmosphere conditions or room temperature, elevated endogenous GABA or exogenous GABA maintains their quality by stimulating the activity of the GABA shunt (glutamate GABA succinic semialdehyde succinate) and the synthesis of malate, and delaying fruit ripening. This outcome is associated with changes in the genetic and biochemical regulation of key GABA pathway reactions. Flux estimates suggest that the GABA pool is derived primarily from glutamate, rather than polyamines, and that succinic semialdehyde is converted mainly to succinate, rather than γ-hydroxybutyrate. Exogenous GABA is a promising strategy for promoting the level of endogenous GABA and the activity of the GABA shunt in both intact and fresh-cut commodities, which increases carbon flux through respiratory pathways, restores or partially restores redox and energy levels, and improves postharvest marketability. The precise mechanisms whereby GABA interacts with other signaling molecules such as Ca2+, H2O2, polyamines, salicylic acid, nitric oxide and melatonin, or with phytohormones such as ethylene, abscisic acid and auxin remain unknown. The occurrence of the aluminum-activated malate transporter and the glutamate/aspartate/GABA exchanger in the tonoplast, respectively, offers prospects for reducing transpirational water in cut flowers and immature green fruit, and for altering the development, flavor and biotic resistance of apple fruits.

γ-Aminobutyrate (GABA) Regulated Plant Defense: Mechanisms and Opportunities.

Global climate change and associated adverse abiotic and biotic stress conditions affect plant growth and development, and agricultural sustainability in general. Abiotic and biotic stresses reduce respiration and associated energy generation in mitochondria, resulting in the elevated production of reactive oxygen species (ROS), which are employed to transmit cellular signaling information in response to the changing conditions. Excessive ROS accumulation can contribute to cell damage and death. Production of the non-protein amino acid γ-aminobutyrate (GABA) is also stimulated, resulting in partial restoration of respiratory processes and energy production. Accumulated GABA can bind directly to the aluminum-activated malate transporter and the guard cell outward rectifying K+ channel, thereby improving drought and hypoxia tolerance, respectively. Genetic manipulation of GABA metabolism and receptors, respectively, reveal positive relationships between GABA levels and abiotic/biotic stress tolerance, and between malate efflux from the root and heavy metal tolerance. The application of exogenous GABA is associated with lower ROS levels, enhanced membrane stability, changes in the levels of non-enzymatic and enzymatic antioxidants, and crosstalk among phytohormones. Exogenous GABA may be an effective and sustainable tolerance strategy against multiple stresses under field conditions.

Does the GABA Shunt Regulate Cytosolic GABA?

The GABA shunt has long been known as a metabolic pathway that produces GABA in, and removes GABA from, the cytosol. There is no consensus regarding its function. The hypothesis presented here is that the GABA shunt regulates cytosolic GABA levels and GABA signaling.

Spermine Is a Potent Plant Defense Activator Against Gray Mold Disease on Solanum lycopersicum, Phaseolus vulgaris, and Arabidopsis thaliana.

Polyamines (PAs) are ubiquitous aliphatic amines that play important roles in growth, development, and environmental stress responses in plants. In this study, we report that exogenous application of spermine (Spm) is effective in the induction of resistance to gray mold disease, which is caused by the necrotrophic fungal pathogen Botrytis cinerea, on tomato (Solanum lycopersicum), bean (Phaseolus vulgaris), and Arabidopsis thaliana. High throughput transcriptome analysis revealed a priming role for the Spm molecule in the genus Arabidopsis, resulting in strong upregulation of several important defense-associated genes, particularly those involved in systemic-acquired resistance. Microscopic analysis confirmed that Spm application potentiates endogenous defense responses in tomato leaves through the generation of reactive oxygen species and the hypersensitive response, which effectively contained B. cinerea growth within the inoculated area. Moreover, co-application of Spm and salicylic acid resulted in a synergistic effect against the pathogen, leading to higher levels of resistance than those induced by separate applications of the two compounds. The Spm plus salicylic acid treatment also reduced infection in systemic nontreated leaves of tomato plants. Our findings suggest that Spm, particularly when applied in combination with salicylic acid, functions as a potent plant defense activator that leads to effective local and systemic resistance against B. cinerea.

Spermine Differentially Refines Plant Defense Responses Against Biotic and Abiotic Stresses.

Roles of the major polyamines (mPA), putrescine, spermidine, and spermine (Spm), in various developmental and physiological processes in plants have been well documented. Recently, there has been increasing focus on the link between mPA metabolism and defense response during plant-stress interactions. Empirical evidence is available for a unique role of Spm, distinct from the other mPA, in eliciting an effective defense response to (a)biotic stresses. Our understanding of the precise molecular mechanism(s) by which Spm modulates these defense mechanisms is limited. Further analysis of recent studies indicates that plant Spm functions differently during biotic and abiotic interactions in the regulation of oxidative homeostasis and phytohormone signaling. Here, we summarize and integrate current knowledge about Spm-mediated modulation of plant defense responses to (a)biotic stresses, highlighting the importance of Spm as a potent plant defense activator with broad-spectrum protective effects. A model is proposed to explain how Spm refines defense mechanisms to tailor an optimal resistance response.

Targeted quantitative profiling of metabolites and gene transcripts associated with 4-aminobutyrate (GABA) in apple fruit stored under multiple abiotic stresses.

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.

Metabolic Alterations in Postharvest Pear Fruit As Influenced by 1-Methylcyclopropene and Controlled Atmosphere Storage.

This study assessed the impact of 1-methylcyclopropene (1-MCP) and controlled atmosphere (CA) on the metabolism of targeted amino acids, organic acids, and antioxidants in stored 'AC Harrow Crisp' pears and their relationships to storage disorders. Pears were treated with 0 or 300 nL L-1 1-MCP and stored at 0 °C under ambient air or CA. Spectrophotometric assays demonstrated that glutathione levels fluctuated with storage and were most preserved by 1-MCP under ambient air. HPLC analysis revealed that ascorbate concentrations declined with storage and were little affected by 1-MCP and CA. Citrate, lactate, and fumarate accumulated with storage but were differentially affected by 1-MCP. Aspartate and glutamate concentrations were greater with 1-MCP; γ-aminobutyrate accumulated in disordered fruit. Principal component analysis demonstrated that alterations in citrate and fumarate were, respectively, correlated with internal breakdown and senescent scald. γ-Aminobutyrate and alanine were associated with internal cavities. All disorders were associated with antioxidant depletion.

Corrigendum: Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

This corrects the article DOI: 10.1038/srep35115.

Plant Glyoxylate/Succinic Semialdehyde Reductases: Comparative Biochemical Properties, Function during Chilling Stress, and Subcellular Localization.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2) are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh.) and rice (Oryza sativa L.) GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress.

Ancient Plant Glyoxylate/Succinic Semialdehyde Reductases: GLYR1s Are Cytosolic, Whereas GLYR2s Are Localized to Both Mitochondria and Plastids.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2) are considered to be involved in detoxifying harmful aldehydes, thereby preserving plant health during exposure to various abiotic stresses. Phylogenetic analysis revealed that the two GLYR isoforms appeared in the plant lineage prior to the divergence of the Chlorophyta and Streptophyta, which occurred approximately 750 million years ago. Green fluorescent protein fusions of apple (Malus x domestica Borkh.), rice (Oryza sativa L.) and Arabidopsis thaliana [L.] Heynh GLYRs were transiently expressed in tobacco (Nicotiana tabaccum L.) suspension cells or Arabidopsis protoplasts, as well in methoxyfenozide-induced, stably transformed Arabidopsis seedlings. The localization of apple GLYR1 confirmed that this isoform is cytosolic, whereas apple, rice and Arabidopsis GLYR2s were localized to both mitochondria and plastids. These findings highlight the potential involvement of GLYRs within distinct compartments of the plant cell.

Subcellular compartmentation of 4-aminobutyrate (GABA) metabolism in arabidopsis: An update.

This addendum discusses the compartmentation of γ-aminobutyrate (GABA) metabolism, highlighting recent progress with Arabidopsis thaliana and raising new questions about the roles of mitochondria, plastids and peroxisomes in abiotic stress tolerance.

Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

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 ß-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.

Plant GABA: Not Just a Metabolite.

γ-Aminobutyric acid (GABA) accumulates rapidly when plants are exposed to stress. Whether GABA accumulation represents the regulation of metabolism in response to stress or an adaptive response to mitigate stress is unknown. Genetic manipulation of GABA levels has revealed that GABA accumulation functions in defense against drought and insect herbivory.

Oxidative metabolism is associated with physiological disorders in fruits stored under multiple environmental stresses.

In combination with low temperature, controlled atmosphere storage and 1-methylcyclopropene (ethylene antagonist) application are used to delay senescence of many fruits and vegetables. Controlled atmosphere consists of low O2 and elevated CO2. When sub-optimal partial pressures are used, these practices represent multiple abiotic stresses that can promote the development of physiological disorders in pome fruit, including flesh browning and cavities, although there is some evidence for genetic differences in susceptibility. In the absence of surface disorders, fruit with flesh injuries are not easily distinguished from asymptomatic fruit until these are consumed. Oxidative stress metabolites tend to accumulate (e.g., γ-aminobutyrate) or rapidly decline (e.g., ascorbate and glutathione) in vegetative tissues exposed to hypoxic and/or elevated CO2 environments. Moreover, these phenomena can be associated with altered energy and redox status. Biochemical investigations of Arabidopsis and tomato plants with genetically-altered levels of enzymes associated with the γ-aminobutyrate shunt and the ascorbate-glutathione pathway indicate that these metabolic processes are functionally related and critical for dampening the oxidative burst in vegetative and fruit tissues, respectively. Here, we hypothesize that γ-aminobutyrate accumulation, as well energy and antioxidant depletion are associated with the development of physiological injury in pome fruit under multiple environmental stresses. An improved understanding of this relationship could assist in maintaining the quality of stored fruit.

NAD(+)-aminoaldehyde dehydrogenase candidates for 4-aminobutyrate (GABA) and ß-alanine production during terminal oxidation of polyamines in apple fruit.

The last step of polyamine catabolism involves the oxidation of 3-aminopropanal or 4-aminobutanal via aminoaldehyde dehydrogenase. In this study, two apple (Malus x domestica) AMADH genes were selected (MdAMADH1 and MdAMADH2) as candidates for encoding 4-aminobutanal dehydrogenase activity. Maximal activity and catalytic efficiency were obtained with NAD(+) and 3-aminopropanal, followed by 4-aminobutanal, at pH 9.8. NAD(+) reduction was accompanied by the production of GABA and ß-alanine, respectively, when 4-aminobutanal and 3-aminopropanal were utilized as substrates. MdAMADH2 was peroxisomal and MdAMADH1 cytosolic. These findings shed light on the potential role of apple AMADHs in 4-aminobutyrate and ß-alanine production.

Apple fruit copper amine oxidase isoforms: peroxisomal MdAO1 prefers diamines as substrates, whereas extracellular MdAO2 exclusively utilizes monoamines.

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.

Impact of 1-methylcyclopropene and controlled atmosphere storage on polyamine and 4-aminobutyrate levels in "Empire" apple fruit.

1-Methylcyclopropene (1-MCP) delays ethylene-meditated ripening of apple (Malus domestica Borkh.) fruit during controlled atmosphere (CA) storage. Here, we tested the hypothesis that 1-MCP and CA storage enhances the levels of polyamines (PAs) and 4-aminobutyrate (GABA) in apple fruit. A 46-week experiment was conducted with "Empire" apple using a split-plot design with four treatment replicates and 3°C, 2.5 kPa O2, and 0.03 or 2.5 kPa CO2 with or without 1 µL L(-1) 1-MCP. Total PA levels were not elevated by the 1-MCP treatment. Examination of the individual PAs revealed that: (i) total putrescine levels tended to be lower with 1-MCP regardless of the CO2 level, and while this was mostly at the expense of free putrescine, large transient increases in soluble conjugated putrescine were also evident; (ii) total spermidine levels tended to be lower with 1-MCP, particularly at 2.5 kPa CO2, and this was mostly at the expense of soluble conjugated spermidine; (iii) total spermine levels at 2.5 kPa CO2 tended to be lower with 1-MCP, and this was mostly at the expense of both soluble and insoluble conjugated spermine; and (iv) total spermidine and spermine levels at 0.03 kPa were relatively unaffected, compared to 2.5 kPa CO2, but transient increases in free spermidine and spermine were evident. These findings might be due to changes in the conversion of putrescine into higher PAs and the interconversion of free and conjugated forms in apple fruit, rather than altered S-adenosylmethionine availability. Regardless of 1-MCP and CO2 treatments, the availability of glutamate showed a transient peak initially, probably due to protein degradation, and this was followed by a steady decline over the remainder of the storage period which coincided with linear accumulation of GABA. This pattern has been attributed to the stimulation of glutamate decarboxylase activity and inhibition of GABA catabolism, rather than a contribution of PAs to GABA production.

Effect of boron nutrition on American ginseng in field and in nutrient cultures.

Field and nutrient cultures of American ginseng (Panax quinquefolius L.) were used to establish foliar symptoms related to boron (B) concentration in leaves and soils, and to evaluate radish as a time-saving model system for B nutrition. Application of excess B, 8 kg/ha versus the recommended 1.5 kg/ha, to field plantings of 2-, 3-, and 4-yr-old American ginseng plants just prior to crop emergence caused, within 4 wk after crop emergence, leaf symptoms of chlorosis followed by necrosis starting at the tips and progressing along the margins. The B concentration in leaves of 2-4-yr-old plants receiving 1.5 kg/ha B was 30 µg/g dry mass compared to 460 µg/g dry mass where 8 kg/ha B was applied. Similarly, B concentration in soils receiving the lower B concentration was 1.8 µg/g dry mass and 2.2-2.8 µg/g dry mass where the higher B concentration was applied. Application of 8 kg/ha B reduced the dry yield of 3rd-yr roots by 20% from 2745 kg/ha to 2196 kg/ha and 4th-yr roots by 26% from 4130 kg/ha to 3071 kg/ha. Ginseng seedlings and radish were grown under greenhouse conditions in nutrient culture with four B concentrations ranging from 0 mg/L to 10 mg/L. At 5 mg/L and 10 mg/L ginseng and radish developed typical leaf B toxicity symptoms similar to those described above for field-grown plants. Increasing B in the nutrient solution from 0.5 mg/L to 10 mg/L decreased, in a linear fashion, the root and leaf dry mass of ginseng, but not radish. Given the many similarities of ginseng and radish to B utilization, radish might be used as a time-saving model system for the study of B, and other micronutrients, in the slow-growing perennial ginseng.