Day and Night Fluctuations in GABA Biosynthesis Contribute to Drought Responses in Nicotiana tabacum L.

When synchronized with the light/dark cycle the circadian rhythm is termed a diurnal rhythm and this organizes an organism's daily life cycle in relation to the metabolic shifts during the day/night cycles. This is a complex task, particularly under stress conditions. Accurate maintenance of the diurnal rhythm becomes an issue under environmental extremes, such as drought due to the impairment of metabolism, redox balance, and structural integrity. In plants, the non-proteinogenic amino acid GABA accumulates to high levels in response to several stress factors but this is not always dependent on the activation of its biosynthesis. Here we propose a regulatory role to GABA during the diurnal rhythm in plants which is similar to its function in animals where it adjusts the circadian rhythm. Here we investigated whether GABA-biosynthesis was affected by drought stress during the diurnal cycle. For this, we took samples from leaves of N. tabacum plants subjected to PEG-mediated drought stress (-0.73 MPa) during the day and night cycle during a 24 hour period. Glutamate, GABA, and proline contents, along with GDH, GAD enzyme activities and transcript profiles were analyzed. Overall, we conclude that the oscillations in GABA biosynthesis during day and night cycle have an impact on drought stress responses which needs to be elucidated by further analysis.

N-acyl homoserine lactone-mediated modulation of plant growth and defense against Pseudoperonospora cubensis in cucumber.

N-acyl-homoserine lactones (AHLs), a well-described group of quorum sensing molecules, may modulate plant defense responses and plant growth. However, there is limited knowledge regarding the defense responses of non-model crops to AHLs and the mechanism of action responsible for the modulation of defense responses against microbial pathogens. In the present study, long-chain N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) was shown to have a distinct potential to prime cucumber for enhanced defense responses against the biotrophic oomycete pathogen Pseudoperonospora cubensis and the hemibiotrophic bacterium Pseudomonas syringae pv. lachrymans. We provide evidence that AHL-mediated enhanced defense against downy mildew disease is based on cell wall reinforcement by lignin and callose deposition, the activation of defense-related enzymes (peroxidase, ß-1,3-glucanase, phenylalanine ammonia-lyase), and the accumulation of reactive oxygen species (hydrogen peroxide, superoxide) and phenolic compounds. Quantitative analysis of salicylic acid and jasmonic acid, and transcriptional analysis of several of genes associated with these phytohormones, revealed that defense priming with oxo-C14-HSL is commonly regulated by the salicylic acid signaling pathway. We also show that treatment with short- (N-hexanoyl-l-homoserine lactone) and medium-chain (N-3-oxo-decanoyl-l-homoserine lactone) AHLs promoted primary root elongation and modified root architecture, respectively, resulting in enhanced plant growth.

The involvement of gamma-aminobutyric acid shunt in the endoplasmic reticulum stress response of Arabidopsis thaliana.

The endoplasmic reticulum (ER) is the main site of secretory protein production and folding and its homeostasis under environmental stress is vital for the maintenance of the protein secretory pathway. The loss of homeostasis and accumulation of unfolded proteins in the ER is referred to as ER stress. Although, γ-aminobutyric acid (GABA) is an important regulator of stress response in plants, its roles during ER stress remains unclear. This study investigated the involvement of GABA in the ER stress response of plants. For this, changes in GABA metabolism under ER stress was analysed in Arabidopsis thaliana, then to study the response of the ER-folding machinery, plants were treated with exogenous GABA under ER stress. The antibiotic tunicamycin, which inhibits N-glycosylation was used to specifically induce ER stress. This stress up-regulated the expression of five glutamate decarboxylase (GAD) genes except GAD2 and GABA content of A. thaliana plants increased with an increasing concentration of tunicamycin (0.1 µg ml-1 and 0.25 µg ml-1). Moreover, expressions of genes involved in the conversion of GABA to succinate was also induced, while genes involved in transport across plasma and mitochondrial membrane showed no response to ER stress. The exogenous treatment of plants with 1-and 5-mM GABA increased plant performance under ER stress but 0.1 mM proved ineffective. Plants treated with GABA under ER stress had decreased expression of ER stress marker genes such as BIP1, BIP3 or CNX, but the expression of genes related to ER stress perception or ER-associated protein degradation showed no changes with respect to GABA treatments.

Ozone triggers different defence mechanisms against powdery mildew (Blumeria graminis DC. Speer f. sp. tritici) in susceptible and resistant wheat genotypes.

Ozone has been proposed as a convenient elicitor against pathogens since it is known to generate different reactive oxygen species (ROS) and induce nonspecific defence by altering gene expression. The mode of action and its interaction with other defence pathways are yet to be elucidated. Besides its negative effects on plants, ozone can be used for triggering defence against environmental stresses, including pathogens, when used at appropriate concentrations. Powdery mildew, caused by the obligate biotrophic fungus Blumera graminis f.sp. tritici (Bgt), is an important plant disease that reduces crop yield and quality. We hypothesised that ozone treatment may elicit defence against Bgt by inducing ROS signalling or other routes such as the salicylic acid (SA) or jasmonic acid (JA) pathways. We conducted experiments with Bgt-susceptible (cv. Pamukova) and resistant (cv. Tahirova) wheat (Triticum aestivum L,) cultivars and treated them with different ozone concentrations before Bgt inoculation. Stress response and defence-related features such as antioxidative enzyme activity; lipid peroxidation; H2O2 and Ca+2 levels; PR1, LOX, PAL and RBOH gene expression; and disease severity were assayed. Clear discrepancies between the responses of susceptible and resistant cultivars were found, suggesting that different defence routes were activated. Here, we showed that ozone treatment was effective for diminishing Bgt invasion in the susceptible cultivar in the short term, which was probably related to defence induced via the SA pathway. Moreover, the resistant cultivar Tahirova exhibited a different mode of action against the pathogen that was triggered by ozone treatment, plausibly related to the JA pathway.

Arabidopsis NATA1 Acetylates Putrescine and Decreases Defense-Related Hydrogen Peroxide Accumulation.

Biosynthesis of the polyamines putrescine, spermidine, and spermine is induced in response to pathogen infection of plants. Putrescine, which is produced from Arg, serves as a metabolic precursor for longer polyamines, including spermidine and spermine. Polyamine acetylation, which has important regulatory functions in mammalian cells, has been observed in several plant species. Here we show that Arabidopsis (Arabidopsis thaliana) N-ACETYLTRANSFERASE ACTIVITY1 (NATA1) catalyzes acetylation of putrescine to N-acetylputrescine and thereby competes with spermidine synthase for a common substrate. NATA1 expression is strongly induced by the plant defense signaling molecule jasmonic acid and coronatine, an effector molecule produced by DC3000, a Pseudomonas syringae strain that initiates a virulent infection in Arabidopsis ecotype Columbia-0. DC3000 growth is reduced in nata1 mutant Arabidopsis, suggesting a role for NATA1-mediated putrescine acetylation in suppressing antimicrobial defenses. During infection by P. syringae and other plant pathogens, polyamine oxidases use spermidine and spermine as substrates for the production of defense-related H2O2 Compared to wild-type Columbia-0 Arabidopsis, the response of nata1mutants to P. syringae infection includes reduced accumulation of acetylputrescine, greater abundance of nonacetylated polyamines, elevated H2O2 production by polyamine oxidases, and higher expression of genes related to pathogen defense. Together, these results are consistent with a model whereby P. syringae growth is improved in a targeted manner through coronatine-induced putrescine acetylation by NATA1.

Histone acetylation influences the transcriptional activation of POX in Beta vulgaris L. and Beta maritima L. under salt stress.

Acetylation of histone proteins is a type of chromatin modification which facilitates the activation of genes. Recent studies brought up the importance of this reversible and rapid process for the regulation of gene expression especially in plant defense against a variety of environmental stresses. Deciphering the exact mechanisms of chromatin modifications under abiotic stress conditions is important for improving crop plants' performance and yield. In a previous study we compared the salt stress responses of Beta vulgaris (sugar beet) and Beta maritima (wild beet). In accordance with those results we suggested that chromatin remodeling can be an active process in the regulation of genes related to salt stress tolerance of these plants. Therefore we performed ChIP assay in control and salt stressed (250 and 500 mM NaCl) plants and compared the enrichment of acetylation in the associated chromatin sites. We found that the transcriptional activation of one peroxidase (POX) encoding gene was associated with the elevated levels of acetylation in H3K9 and H3K27 sites. The acetylation patterns were remarkably different between two species in which the highest acetylation levels were found at H3K9 and H3K27 in wild beet and sugar beet respectively.

The impact of GABA in harpin-elicited biotic stress responses in Nicotiana tabaccum.

Harpin is a bacterial elicitor protein that was first isolated from Erwinia amylovora. Infiltration of this elicitor into the leaves of plants activates systemic acquired resistance against a variety of plant pathogens via the salicyclic acid defense pathway. The non-protein amino acid, neurotransmission inhibitor molecule of mammals-GABA- is found in all organisms and is known to be an important component of stress responses in plants. We hypothesized a possible interaction between harpin-induced defense responses and GABA shunt. Therefore, we conducted experiments on harpin-infiltrated tobacco and analyzed the components of GABA shunt in relation to growth, photosynthesis and H2O2 levels. RGR, RWC and photosynthetic efficiency were all affected in harpin-infiltrated tobacco leaves, but the rate of decline was more remarkable on RGR. H2O2 levels showed significant difference on 7 days after harpin infiltration when the necrotic lesions were also visible. GABA accumulation was increased and glutamate levels were decreased parallel to the differences in GDH and GAD enzyme activities, especially on days 5 and 7 of harpin infiltration. Transcript abundance of GDH and GAD encoding genes were differentially regulated in harpin-infiltrated leaves as compared to that of control and mock groups. In the present study, for the first time we showed a relationship between harpin-elicited responses and GABA in tobacco that was not mediated by H2O2 accumulation. Harpin infiltration significantly induced the first components of the GABA shunt such as GDH, GAD, glutamate and GABA in tobacco.

Interspecific diversity in root antioxidative enzyme activities reflect root turnover strategies and preferred habitats in wetland graminoids.

Antioxidant enzymes protect cells against oxidative stress and are associated with stress tolerance and longevity. In animals, variation in their activities has been shown to relate to species ecology, but in plants, comparative studies with wild species are rare. We investigated activities of five antioxidant enzymes - ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase (POX), and superoxide dismutase (SOD) - in roots of four perennial graminoid wetland species over a growing season to find out whether differences in root turnover or habitat preferences would be associated with variation in seasonal patterns of antioxidant enzyme activities. The investigated species differ in their root turnover strategies (fine roots senesce in the fall or fine roots survive the winter) and habitat preferences (nutrient-poor vs. productive wetlands). Roots were collected both in the field and from garden-grown plants. Antioxidant enzyme activities were higher and lipid peroxidation rates lower in species with annual root systems, and for species of the nutrient-poor wetland, compared with perennial roots and species of productive wetlands, respectively. There was variation in the activities of individual antioxidant enzymes, but discriminant analyses with all enzymes revealed a clear picture, indicating consistent associations of antioxidant enzyme activities with the type of root turnover strategy and with the preferred habitat. We conclude that antioxidant enzyme activities in plant roots are associated with the species' ecological strategies and can be used as traits for the characterization of the species' position along plant economics spectrum.

Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants.

Plants accumulate high levels of Gamma amino butyric acid (GABA) in response to different environmental stresses and GABA metabolism has different functions such as osmotic and pH regulation, bypass of tricarboxylic acid cycle, and C:N balance. The cytoplasmic male sterile (CMS) II mutant of Nicotiana sylvestris has a deletion in the mitochondrial gene nad7 which encodes the NAD7 subunit of complex I which causes increased leaf respiration, impaired photosynthesis, slower growth and increased amino acid levels. In this study we aimed to elucidate the role of GABA and GABA metabolism in different genotypes of the same plant system under salt stress (100mM NaCl) in short (24h) and long (7, 14 and 21 days) terms. We have investigated the differences in leaf fresh and dry weights, relative water content, photosynthetic efficiency (F(v)/F(m)), glutamate dehydrogenase (GDH, EC 1.4.1.4) and glutamate decarboxylase (GAD, EC 4.1.1.15) enzyme activities, GABA content and GAD gene expression profiles. GDH activity showed variations in CMSII and wild type (WT) plants in the first 24h. GAD gene expression profiles were in good agreement with the GAD enzyme activity levels in CMSII and WT plants after 24h. In long-term salinity, GAD activities increased in WT but, decreased in CMSII. GABA accumulation in WT and CMSII plants in short and long term was induced by salt stress. Variations in GDH and GAD activities in relation to GABA levels were discussed and GABA metabolism has been proposed to be involved in better performance of CMSII plants under long term salinity.

Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris.

Mitochondrial electron transport pathways exert effects on carbon-nitrogen (C/N) relationships. To examine whether mitochondria-N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered 'nitrate-sensing' phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.