Cytosolic calcium levels and stress induced y-amino butyrate synthesis in asparagus mesophyll cells

Numerous investigations have demonstrated large increases in y-amino butyrate (GABA) levels in response to a variety of stresses such as touch or cold shock (Wallace et ale 1984) Circumstantial evidence indicating a role of Ca2 + in these increases includes elevated Ca2+ levels in response to touch and cold shock (Knight et ale 1991), and the demonstration of a calmodulin binding domain on glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (Baum et al 1993) In the present study the possible role of Ca2+ and calmodulin in stimulation of GAD and subsequent GABA accumulation was examined using asparagus mesophyll cells. Images of cells loaded with the Ca2+ indicator Fluo-3 revealed a rapid and transient increase in cytosolic Ca2+ in response to cold shock. GABA levels increased by 106% within 15 min. of cold shock. This increase was inhibited 70% by the calmodulin antagonist W7, and 42% by the Ca2+ channel blocker La3+.. Artificial elevation of intracellular Ca2+ by the Ca2+ionophore A23187 resulted in an 61% increase in GABA levels. Stimulation of GABA synthesis by ABA resulted in an 83% increase in GABA levels which was inhibited 55% by W7. These results support the hypothesis that cold shock stimulates Ca2+ entry into the cytosol of the cells which results in Ca2+/calmodulin mediated activation of GAD and consequent GABA synthesis.

The production of 4-adminobutyrate in response to treatments reducing cytosolic pH and the regulation of intracellular pH

GABA (4-aminobutyrate) is synthesized through the decarboxylation of LGlu- (L-Glu-+ H+ ---> GABA + C02), and compared to many free amino acids is present in high concentrations in plant cells. GABA levels rise rapidly and dramatically in response to varied stress conditions including anaerobiosis. Recent papers suggest that GABA production and associated H+ consumption are parts of a metabolic pH-stat mechanism which ameliorates the intracellular pH decline associated with anaerobiosis or other treatments. To test this hypothesis GABA production and efflux have been measured in isolated Asparagus sprengeri cells in response to three treatments which potentially cause intracellular acidification. Acid loads were imposed using 60 min of (i) anaerobiosis, (ii) H+/LGlu- cotransport, and (iii) treatment with permeant weak acids (butyric, acetic and propionic). Both intra- and extracellular GABA concentrations increased more than 100% after anaerobiosis, almost 1000% after H+/L-Glu- cotransport (light or dark) and almost 5000/0 after addition of 5 mM butyric acid at pH 5.0. HPLC analysis of amino acids indicates that as GABA concentrations increased in response to butyric acid addition, glutamate concentrations decreased. Time-course studies demonstrated that added butyric acid stimulates GABA production by 2800/0 within 15 seconds. A fluorescent determination of cytosolic pH indicates that addition of butyric or other weak acids resulted in a rapid reduction in cytosolic pH of 0.6 pH units. The half time for the response to butyric acid addition is 2.1 seconds, indicating that the decline in cytosolic pH is rapid enough to account for the rapid stimulation of GABA production. The acid load in response to butyric acid addition was assayed by measurements of 14C-butyric acid uptake. Calculations indicate that GABA production accounted for 45% of the imposed acid load. The biological significance of GABA efflux is not yet understood. The results support the original hypothesis suggesting a role for GABA production in cellular pH regulation.

The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme by Hâ ½ and metabolites : kinetic evidence for and against a cytosolic pH-stat

Phosphoenolpyruvate carboxylase (PEPC) and malic enzyme activities in soluble protein extracts of Avena coleoptiles were investigated to determine whether their kinetics were consistent with a role in cytosol pH regulation. Malic enzyme activity was specific for NADP+ and Mn2+. Maximal labelled product formation from [14C]-substrates required the presence of all coenzymes, cofactors and substrates. Plots of rate versus malate concentration, and linear transformations there- 2 of, indicated typical Michaelis-Menten kinetics at non-saturating malate levels and substrate inhibition at higher malate levels. pH increases between 6.5 and 7.25 increased near-optimal activity, decreased the degree of substrate inhibition and the Kmapp(Mn2+) but did not affect the Vmax or Kmapp(malate). Transformed data of PEPC activity demonstrated non-linear plots indicative of non-Michaelian kinetics. pH increases between 7.0 and 7.6 increased the Vmax and decreased the Km app (Mg2+) but did not affect the Kmapp(PEP). Various carboxylic acids and phosphorylated sugars inhibited PEPC and malic enzyme activities, and these effects decreased with pH increases. Metabolite inhibited malic enzyme activity was non-competitive and resulted mainly from Mn2+ chelation. In contrast, metabolite inhibited PEPC activity was unique for each compound tested, being variously dependent on the PEP concentration and the pH employed. These results indicate that fluctuations in pH and metabolite levels affect PEPC and malic enzyme activities similarly and that 3 the in vitro properties of PEPC are consistent with its proposed role in a pH-stat, whereas the in vitro properties of the malic enzyme cannot be interpreted in terms of a role in pH regulation.

Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.

The osmoadaptive response of the wine yeast Saccharomyces cerevisiae K1-V1116 during icewine fermentation

The adapted metabolic response of commercial wine yeast under prolonged exposure to concentrated solutes present in Icewine juice is not fully understood. Presently, there is no information regarding the transcriptomic changes in gene expression associated with the adaptive stress response ofwine yeast during Icewine fermentation compared to table wine fermentation. To understand how and why wine yeast respond differently at the genomic level and ultimately at the metabolic level during Icewine fermentation, the focus ofthis project was to identify and compare these differences in the wine yeast Saccharomyces cerevisiae KI-Vll16 using cDNA microarray technology during the first five days of fermentation. Significant differences in yeast gene expression patterns between fermentation conditions were correlated to differences in nutrient utilization and metabolite production. Sugar consumption, nitrogen usage and metabolite levels were measured using enzyme assays and HPLC. Also, a small subset of differentially expressed genes was verified using Northern analysis. The high osmotic stress experienced by wine yeast throughout Icewine fermentation elicited changes in cell growth and metabolism correlating to several fermentation difficulties, including reduced biomass accumulation and fermentation rate. Genes associated with carbohydrate and nitrogen transport and metabolism were expressed at lower levels in Icewine juice fermenting cells compared to dilute juice fermenting cells. Osmotic stress, not nutrient availability during Icewine fermentation appears to impede sugar and nitrogen utilization. Previous studies have established that glycerol and acetic acid production are increased in yeast during Icewine fermentation. A gene encoding for a glycerollW symporter (STL1) was found to be highly expressed up to 25-fold in the i Icewine juice condition using microarray and Northern analysis. Active glycerol transport by yeast under hyperosmotic conditions to increase cytosolic glycerol concentration may contribute to reduced cell growth observed in the Icewine juice condition. Additionally, genes encoding for two acetyl CoA synthetase isoforms (ACSl and ACS2) were found to be highly expressed, 19- and II-fold respectively, in dilute juice fermenting cells relative to the Icewine juice condition. Therefore, decreased conversion of acetate to acetyl-CoA may contribute to increased acetic acid production during Icewine fermentation. These results further help to explain the response of wine yeast as they adapt to Icewine juice fermentation. ii

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.

Hyperosmotic Stress and the Impact on Metabolite Formation and Redox Balance in Saccharomyces cerevisiae and Saccharomyces bayanus strains

Wine produced using an appassimento-type process represents a new and exciting innovation for the Ontario wine industry. This process involves drying grapes that have already been picked from the vine, which increases the sugar content due to dehydration and induces a variety of changes both within and on the surface of the grapes. Increasing sugar contents in musts subject wine yeast to conditions of high osmolarity during alcoholic fermentations. Under these conditions, yeast growth can be inhibited, target alcohol levels may not be attained and metabolic by-products of the hyperosmotic stress response, including glycerol and acetic acid, may impact wine composition. The further metabolism of acetic acid to acetylCoA by yeast facilitates the synthesis of ethyl acetate, a volatile compound that can also impact wine quality if present in sufficiently high concentrations. The first objective of this project was to understand the effect of yeast strain and sugar concentration on fermentation kinetics and metabolite formation, notably acetic acid and ethyl acetate, during fermentation in appassimento-type must. Our working hypotheses were that (1) the natural isolate Saccharomyces bayanus would produce less acetic acid and ethyl acetate compared to Saccharomyces cerevisiae strain EC-1118 fermenting the high and low sugar juices; (2) the wine produced using the appassimento process would contain higher levels of acetic acid and lower levels of ethyl acetate compared to table wine; (3) and the strains would be similar in the kinetic behavior of their fermentation performances in the high sugar must. This study determined that the S. bayanus strain produced significantly less acetic acid and ethyl acetate in the appassimento wine and table wine fermentations. Differences in acetic acid and ethyl acetate production were also observed within strains fermenting the two sugar conditions. Acetic acid production was higher in table wine fermented by S. bayanus as no acetic acid was produced in appassimento-style wine, and 1.4-times higher in appassimento wine fermented by EC-1118 over that found in table wine. Ethyl acetate production was 27.6-times higher in table wine fermented by S. bayanus, and 5.2-times higher by EC-1118, compared to that in appassimento wine. Sugar utilization and ethanol production were comparable between strains as no significant differences were determined. The second objective of this project was to bring a method in-house for measuring the concentration of pyridine nucleotides, NAD+, NADP+, NADH and NADPH, in yeast cytosolic extract. Development of this method is of applicative interest for our lab group as it will enable the redox balance of the NAD+/ NADH and NADP+/ NADPH systems to be assessed during high sugar fermentations to determine their respective roles as metabolic triggers for acetic acid production. Two methods were evaluated in this study including a UV-endpoint method using a set of enzymatic assay protocols outlined in Bergmeyer (1974) and a colorimetric enzyme cycling method developed by Sigma-Aldrich® using commercial kits. The former was determined to be limited by its low sensitivity following application to yeast extract and subsequent coenzyme analyses, while the latter was shown to exhibit greater sensitivity. The results obtained from the kits indicated high linearity, accuracy and precision of the analytical method for measuring NADH and NADPH, and that it was sensitive enough to measure the low coenzyme concentrations present in yeast extract samples. NADtotal and NADPtotal concentrations were determined to be above the lower limit of quantification and within the range of the respective calibration curves, making this method suitable for our research purposes.