Differential growth responses to sodium salts involve different abscisic acid metabolism and transport in Prosopis strombulifera

In this work, the response of the halophytic shrub Prosopis strombulifera to lowering an osmotic potential (Ψ_o) to ?1.0, ?1.9, and ?2.6 MPa generated by NaCl, Na₂SO₄, and the iso-osmotic combination of them was studied at 6, 12, and 24 h after reaching such values in the growing media. By analyzing the content of abscisic acid (ABA) and related metabolites and transpiration rates, we observed that ABA content varied depending on type of salt, salt concentration, organ analyzed, and age of a plant. ABA content in leaves was much higher than in roots, presumably because of rapid biosynthesis and transport from roots. Leaves of Na₂SO₄-treated plants had the highest ABA content at Ψ_o ?2.6 MPa (24 h) associated with sulfate toxicity symptoms. Significant content of ABA-glucose ester (ABA-GE) was found in both the roots and leaves, whereas only low content of phaseic acid (PA) and dihydrophaseic acid (DPA). The roots showed high ABA-GE accumulation in all treatments. The highest content of free ABA was correlated with ABA-GE glucosidase activity. The results show that ABA-GE and free ABA work together to create a specific stress signal.     

Impact of Irrigation Strategies on Abscisic Acid and its Catabolites Profiles in Leaves and Berries of Baco noir Grapes

To understand the relationship among soil and plant water status, plant physiology, and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA, 8′-hydroxy-ABA, neophaseic acid, and abscisic acid glucose ester (ABA-GE)] in leaves and berries from wine grape cultivar Baco noir (Folle blanche × Vitis riparia) were analyzed. The experiment was conducted during the growing seasons 2006 and 2007 in an irrigation trial set up in a commercial vineyard located in Niagara-on-the-Lake, ON, Canada. ABA and its metabolites were quantified using liquid chromatography with ion trap combined with electrospray ionization-mass spectrometry. The hormonal profile indicated a direct relationship between the amount of ABA and climatic factors. The ABA varied between 582 and 4,026 ng g^?1 dry matter (DM), DPA between 417 and 562 ng g^?1, and ABA-GE between 337 and 2,764 ng g^?1 DM. At many sampling times PA in the leaves was undetectable, and its highest concentration (260 ng g^?1 DM) was at beginning of July 2007. ABA followed different catabolic pathways depending on the plant water status. ABA was likely catabolized by conjugation to form ABA-GE in treatments at higher water deficit levels, whereas in treatments with high water status, the oxidation pathway leading to DPA or PA was likely preferred. The ABA and ABA-GE concentrations in the berries at harvest showed high correlation with soil and plant water status.     

Use of Persistent Analogs of Abscisic Acid as Palliatives against Salt-stress Induced Damage in Citrus Plants

The effectiveness of several abscisic acid (ABA) analogs as palliatives against salt stress in intact citrus plants has been tested in this work. The effect of ABA, 8′-methylene ABA, 8′-acetylene ABA, ABA methyl ester, 8′-methylene ABA methyl ester, and 8′-acetylene ABA methyl ester on citrus responses to salt stress was studied on 2-year-old grafted plants. Leaf abscission, chloride accumulation, ethylene production, and net photosynthetic rate were the parameters used to characterize the performance of plants under stress. Data indicate that 8′-methylene ABA was the most effective compound in delaying the deleterious effects of high salinity on citrus plants. Its regular application reduced leaf chloride concentration, ethylene production, and leaf abscission. Furthermore, it delayed the depletion of CO₂ assimilation under these adverse conditions. Abscisic acid and 8′-acetylene ABA also reduced salt-stress induced injuries in citrus, although to a lower extent. Neither ABA methyl ester nor its 8′-C modified analogs showed biological activity in these assays.     

Metabolism of R,S-[2-14C]abscisic acid by non-stressed and water-stressed detached leaves of wheat (Triticum aestivum L.)

Metabolism of R,S-[2-¹⁴C]abscisic acid (ABA) was studied in detached leaves of six wheat (Triticum aestivum) cultivars, using non-stressed leaves or leaves water stressed by desiccation to 90% of their original fresh weight. The rate constant of ABA metabolism was similar in nonstressed leaves of all cultivars. Water stress resulted in significantly lower rate constants in two cultivars which accumulated high levels of ABA when stressed, the constants decreasing by a factor of about 1.5. Rate constants for the remainder of the cultivars were not significantly different from those for the non-stressed controls. It was calculated that if decreased metabolism was the mechanism for the accumulation of ABA following water stress the rate constants of metabolism would have to be reduced by a factor of between 25 and 70. The results therefore support the hypothesis that enhanced synthesis rather than reduced degradation is the main process by which ABA levels are elevated following experimentally induced water stress. There were differences between the six cultivars in the products of ABA metabolism. Over the time period studied, oxidation to phaseic acid and dihydrophaseic acid as well as to other unidentified metabolites appeared to be the predominant pathway of ABA metabolism, rather than conjugation to ABA glucose ester and other more polar compounds.Abbreviations ABA abscisic acid - ABAGE abscisic-acid glucose ester - DPA dihydrophaseic acid - PA phaseic acid     

A simple system for determining the phloem mobility of compounds using excised pods of lupin (Lupinus albus L.)

Lupin pods were isolated from the plant and the pedicels placed in solutions of either radiolabeled sucrose, glucose, abscisic acid, or gibberellin A₉ (GA₉). Phloem exudate was collected from the cut stylar ends of the pods and both quantitative and qualitative data were obtained on the activity in the sap. Fed sucrose, abscisic acid, and GA₉ were found in phloem exudate, but labeled glucose was immobile as the monosaccharide and activity in exudate appeared in sucrose.Abbreviations GA gibberellic acid - ABA abscisic acid - TLC thin layer chromatography     

Anaerobiosis and Release from Dormancy in Apple Embryos: Leaching of (+/-) [C]Abscisic Acid and its Metabolites under Aerobic and Anaerobic Conditions

An anaerobic treatment released Pyrus malus L. cv Golden Delicious embryos from their primary dormancy. It also suppressed the inhibitory effect induced by exogenous abscisic acid (ABA) on after-ripened embryos. For the study of ABA metabolism, a two-step culture method was developed. Embryos in primary dormancy were cultivated aerobically in the presence of [¹⁴C]ABA (first culture). Some were directly analyzed to evaluate metabolism of absorbed ABA. The remaining embryos were cultivated on moist cotton without ABA, either in aerobic or anaerobic conditions (second culture). The amounts of ABA and its metabolites were measured both in the embryos and the water-leachates. After the second culture, the embryos showed a spectacular decrease in ABA content, with no difference between anaerobic and aerobic cultures. The amount of ABA glucose ester increased slightly in aerobiosis but diminished markedly in anaerobiosis. Radioactivity of the butanol fraction, which corresponded to polar conjugates, decreased considerably in anaerobiosis, whereas it increased in aerobiosis.

Analysis of the water-leachates indicated that, compared to aerobic conditions, anaerobiosis increased total leaching of radioactive materials (× 4.2) as well as leaching of ABA (× 1.4). In addition, anaerobiosis induced leaching of conjugates, such as ABA glucose ester and butanol-soluble metabolites. We concluded that the anaerobic treatment affects mainly membrane permeability.

     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.     

The effect of dormancy on glucose uptake in gladiolus cormels

The uptake of glucose and of 3-O-methyl-d-glucose by cormel slices of Gladiolus X gandavensis Van Houtte was studied in relation to cormel dormancy. Uptake was higher in nondormant cormels. Incubation of nondormant cormels with abscisic acid (ABA) reduced their uptake capacity. Treatment of dormant cormels with 6-benzyladenine (BA) did not affect their uptake rate. ABA and BA promoted O₂ uptake, indicating that differences in uptake are not related to differences in energy supply.     

Different relative humidity conditions combined with chloride and sulfate salinity treatments modify abscisic acid and salicylic acid levels in the halophyte Prosopis strombulifera

It has been shown that abscisic acid (ABA) and salicylic acid (SA) act as endogenous signal molecules responsible for inducing abiotic stress tolerance in plants. However, our knowledge on the role of both phytohormones in response to environmental conditions in halophytic plants is still limited. In this study endogenous ABA and SA levels, growth parameters and chlorophylls content were determined in leaves and roots of the halophyte Prosopis strombulifera cultivated under increasing NaCl and Na₂SO₄ concentrations, at 30 and 70 % relative humidity (RH) conditions. Endogenous ABA and SA content differed depending on the salt type and concentration, RH, plant age and the organ analyzed. Under low RH conditions P. strombulifera growth was strongly inhibited and chlorophyll a and b content were decreased. In leaves of Na₂SO₄-treated plants at 30 % RH, high ABA levels were correlated with protection against dehydration and ion toxicity. Instead, high SA levels were correlated with the damaging effect of sulfate anion and low RH on plant growth. NaCl-treated plants growth was also inhibited at 30 % RH although levels of both hormones were not significantly increased. Taken together, the salt toxic effects on growth parameters and photosynthetic pigments were accentuated by low RH conditions and these responses were reflected on ABA and SA content.     

Identification of Abscisic Acid in Tulipa gesneriana L. by Gas-Liquid Chromatography with Electron Capture and Combined Gas-Liquid Chromatography and Mass Spectrometry

A major growth inhibitory substance of tulip bulbs (Tulipa gesneriana L. cv Paul Richter) has been unequivocally shown to be abscisic acid (ABA). The ABA methyl ester of the free ether-soluble acid fractions of tulip organs had the identical retention time on gas-liquid chromatography with electron capture detector as authentic ABA methyl ester. In addition, the mass spectra were the same. On a unit dry matter basis, the basalplate and floral shoot contained 3.6 and 2.6 times more ABA than the fleshy scales, respectively.     

Convergent Induction of Osmotic Stress-Responses : Abscisic Acid, Cytokinin, and the Effects of NaCl

In Mesembryanthemum crystallinum, salt stress induces the accumulation of proline and a specific isoform of the enzyme phosphoenolpyruvate carboxylase (PEPCase) prior to the switch from C₃ to Crassulacean acid metabolism (CAM). To determine whether plant growth regulators initiate or imitate these responses, we have compared the effects elicited by NaCl, abscisic acid (ABA), and cytokinins using PEPCase and proline levels as diagnostic tools. Exogenously applied ABA is a poor substitute for NaCl in inducing proline and CAM-specific PEPCase accumulation. Even though ABA levels increase 8- to 10-fold in leaves during salt stress, inhibition of ABA accumulation does not affect these salt-induced responses. In contrast, the addition of cytokinins (6-benzylaminopurine, zeatin, 2-isopentyladenine) mimic salt by greatly increasing proline and PEPCase amounts. Endogenous zeatin levels remain unchanged during salt stress. We conclude: (a) The salt-induced accumulation of proline and PEPCase is coincident with, but is not attributable to, the rise in ABA or zeatin concentration. (b) For the first time, cytokinins and NaCl are implicated as independent initiators of a sensing pathway that signals leaves to alter PEPCase gene expression. (c) During stress, the sensing of osmotic imbalances leading to ABA, proline, and CAM-specific PEPCase accumulation may be mediated directly by NaCl.     

Abscisic Acid metabolism by source and sink tissues of sugar beet

The fate of exogenously applied, labeled abscisic acid (±)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.

Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10⁻⁶ molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues.

     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

Abscisic alcohol is an intermediate in abscisic Acid biosynthesis in a shunt pathway from abscisic aldehyde

It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples [1988] Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate trans-ABA and trans-ABA glucose ester and that this accumulation is due to trans-ABA biosynthesis. ¹⁸O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [¹⁸O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [¹⁸O]ABA with two ¹⁸O atoms in the carboxyl group. We further show, by feeding experiments with [²H₆]ABA-alcohol and ¹⁸O₂, that this doubly-carboxyl-labeled ABA is synthesized from [¹⁸O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [²H₆]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Likewise, carbon monoxide inhibits the synthesis of doubly-carboxyl-labeled ABA in ¹⁸O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant.     

Validation of a radioimmunoassay for (+)-abscisic acid in extracts of apple and sweet-pepper tissue using high-pressure liquid chromatography and combined gas chromatography-mass spectrometry

A radioimmunoassay for (+)-abscisic acid (ABA) was developed and applied to the analysis of free ABA in extracts of apple (Malus pumila Mill.) and sweet pepper (Capsicum annuum L.) leaves at various stages during extract purification. Conjugates of ABA, were quantified after alkaline hydrolysis. The validity of the radioimmunoassay was tested by comparison of immunoassay estimates of ABA at different levels of extract purity with high-pressure liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. The antiserum, raised against (+)-ABA, was almost equally sensitive to (-)-ABA. Serum cross-reactivity with the methyl ester of ABA was 160% and with the glycosyl ester of ABA was 34%. Cross-reactivity with protein-ABA conjugates was very slight for C₄-conjugated keyholelimpet haemocyanin, but about 1000% for C₁-conjugated alkaline phosphatase. Other compounds tested showed extremely low or undetectable cross-reactivities. Further evidence for the specificity of the assay came from the agreement between the results using different assay methods for both apple and pepper extracts, and from the observation that the only zone of immunoreactivity on HPLC elution profiles corresponded with authentic (+)-ABA. The use of polyvinylpyrrolidone in the assay minimised interference by other substances in plant extracts. In pepper, free ABA levels increased rapidly during water stress and recovered to pre-stress levels within two days after rewatering. Levels of ABA conjugates were significantly lowr than free ABA in unstressed plants, and also increased rapidly with stress, although not to the same extent as free ABA, and did not recover as rapidly as did free ABA. In apple, levels of free ABA and of ABA conjugates both increased more than twofold over a two-week period of water stress. In contrast to pepper, however, immunoreactivity of the conjugate fraction was increased by hydrolysis, indicating that different ABA conjugates predominate in the two species.Abbreviations ABA abscisic acid - GC-MS combined gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography - Me-ABA methyl ester of ABA - PVP polyvinylpyrrolidone - RIA radioimmunoassay     

Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens

Plant hormone abscisic acid (ABA) is found in a wide range of land plants, from mosses to angiosperms. However, our knowledge concerning the function of ABA is limited to some angiosperm plant species. We have shown that the basal land plant Physcomitrella patens and the model plant Arabidopsis thaliana share a conserved abscisic acid (ABA) signaling pathway mediated through ABI1-related type 2C protein phosphatases (PP2Cs). Ectopic expression of Arabidopsis abi1-1, a dominant allele of ABI1 that functions as a negative regulator of ABA signaling, or targeted disruption of Physcomitrella ABI1-related gene (PpABI1A) resulted in altered ABA sensitivity and abiotic stress tolerance of Physcomitrella, as demonstrated by osmostress and freezing stress. Moreover, transgenic Physcomitrella overexpressing abi1-1 showed altered morphogenesis. These trangenic plants had longer stem lengths compared to the wild type, and continuous growth of archegonia (female organ) with few sporophytes under non-stress conditions. Our results suggest that PP2C-mediated ABA signaling is involved in both the abiotic stress responses and developmental regulation of Physcomitrella.Key words: ABA, ABI1, Physcomitrella patens, PP2C, signaling     

Changes of free and conjugated abscisic acid and phaseic acid in xylem sap of drought-stressed sunflower plants

Abscisic acid (ABA), conjugated abscisic acid, phaseic acid (PA), and conjugated phaseic acid were determined by enzyme-linked immunosorbent assay (ELISA) and gas chromatography (GC) in xylem sap of well-watered and drought-stressed sunflower plants. Conjugated ABA and conjugated PA were determined indirectly after chemical or enzymatic hydrolysis. Conjugated ABA was found to be the predominant ABA metabolite in xylem sap. In xylem sap from well-watered plants at least five, and in sap from drought-stressed plants at least six alkaline hydrolysable ABA conjugates were found. One of them corresponds chromatographically (HPLC) with abscisic acid glucose ester (ABAGE). Under drought conditions the concentrations of ABA, alkaline hydrolysable ABA conjugates, -glucosidase hydrolysable ABA conjugates, PA, and conjugated PA increased. After rewatering the drought-stressed plants, the ABA and the conjugated ABA content decreased. The possible function of the ABA conjugates in the xylem sap as a source of free ABA is discussed.