An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

8[prime]-Methylene Abscisic Acid (An Effective and Persistent Analog of Abscisic Acid)

We report here the synthesis and biological activity of a new persistent abscisic acid (ABA) analog, 8[prime]-methylene ABA. This ABA analog has one additional carbon atom attached through a double bond to the 8[prime]-carbon of the ABA molecule. (+)-8[prime]-Methylene ABA is more active than the natural hormone (+)-ABA in inhibiting germination of cress seed and excised wheat embryos, in reducing growth of suspension-cultured corn cells, and in reducing transpiration in wheat seedlings. The (+)-8[prime]-methylene analog is slightly weaker than (+)-ABA in increasing expression of ABA-inducible genes in transgenic tobacco, but is equally active in stimulating a transient elevation of the pH of the medium of corn cell cultures. In corn cells, both (+)-ABA and (+)-8[prime]-methylene ABA are oxidized at the 8[prime] position. ABA is oxidized to phaseic acid and (+)-8[prime]-methylene ABA is converted more slowly to two isomeric epoxides. The alteration in the ABA structure causes the analog to be metabolized more slowly than ABA, resulting in longer-lasting and more effective biological activity relative to ABA.     

The Physiological Role of Abscisic Acid in Eliciting Turion Morphogenesis

The exogenous application of hormones has led to their implication in a number of processes within the plant. However, proof of their function in vivo depends on quantitative data demonstrating that the exogenous concentration used to elicit a response leads to tissue hormone levels within the physiological range. Such proof is often lacking in many investigations. We are using abscisic acid (ABA)-induced turion formation in Spirodela polyrrhiza L. to investigate the mechanism by which a hormone can trigger a morphogenic switch. In this paper, we demonstrate that the exogenous concentration of ABA used to induce turions leads to tissue concentrations of ABA within the physiological range, as quantified by both enzyme-linked immunosorbent assay and high-performance liquid chromatography/gas chromatography-electron capture detection analysis. These results are consistent with ABA having a physiological role in turion formation, and they provide an estimate of the changes in endogenous ABA concentration required if environmental effectors of turion formation (e.g. nitrate deficiency, cold) act via an increased level of ABA. In addition, we show that the (+)- and (-)-enantiomers of ABA are equally effective in inducing turions. Moreover, comparison of the ABA; levels attained after treatment with (+)-, (-)-, and ([plus or minus])-ABA and their effect on turion induction and comparison of the effectiveness of ABA on turion induction under different pH regimes suggest that ABA most likely interacts with a plasmalemma-located receptor system to induce turion formation.     

Biological activity of optically pure C-1 altered abscisic acid analogs in Brassica napus microspore embryos

We have examined the effects of stereochemically pure analogs of abscisic acid (ABA) on three responses in Brassica napus microspore embryos. The analogs used include modifications to natural (S-) (+)-ABA (= N-ABA) at the C-1 and C-1 positions. At the C-1 position, the carboxylic acid function was replaced with an alcohol, aldehyde, or methyl ester functional group, and at the chiral C-1 position both enantiomers were prepared. The rationale for choosing these particular analogs was that they had previously shown some potential as slow release forms of ABA (Gusta LV, Ewan B, Reaney MJT, Abrams SR (1992) Can J Bot. 70:1550–1555). The responsiveness of microspore-derived embryos of B. napus to these analogs was investigated. Three types of responses were evaluated: (1) the inhibition of precocious germination; (2) induction of oleosin gene expression; and (3) induction of napin gene expression. All of the structural analogs of ABA tested were effective in the three assays, regardless of functional group substitution or stereochemistry. However, the three assays showed differential sensitivity to the various analogs. The U-forms of abscisyl alcohol and abscisyl aldehyde were very effective in inhibiting precocious germination (greater than natural ABA). Oleosin mRNA accumulation responded most effectively to U-abscisyl alcohol, while the N-abscisyl aldehyde and ABA methyl ester were the most effective at inducing napin mRNA accumulation. This work highlights the distinct differences in activity which result from using stereochemically pure analogs. In addition, surprisingly potent responses are reported in one or more of the assays for abscisyl aldehyde and abscisyl alcohol.Abbreviations ABA abscisic acid - LEA late embryogenic abundant - HPLC high performance liquid chromatography - MOPS 4-morpholinepropanesulfonic acid - SDS sodium dodecyl sulfate     

Response of Cultured Maize Cells to (+)-Abscisic Acid, (-)-Abscisic Acid,and Their Metabolites

The metabolism and effects of (+)-S- and (-)-R-abscisic acid (ABA) and some metabolites were studied in maize (Zea mays L. cv Black Mexican Sweet) suspension-cultured cells. Time-course studies of metabolite formation were performed in both cells and medium via analytical high-performance liquid chromatography. Metabolites were isolated and identified using physical and chemical methods. At 10 [mu]M concentration and 28[deg] C, (+)-ABA was metabolized within 24 h, yielding natural (-)-phaseic acid [(-)-PA] as the major product. The unnatural enantiomer (-)-ABA was less than 50% metabolized within 24 h and gave primarily (-)-7[prime]-hydroxyABA [(-)-7[prime]-HOABA], together with (+)-PA and ABA glucose ester. The distribution of metabolites in cells and medium was different, reflecting different sites of metabolism and membrane permeabilities of conjugated and nonconjugated metabolites. The results imply that (+)-ABA was oxidized to (-)-PA inside the cell, whereas (-)-ABA was converted to (-)-7[prime]-HOABA at the cell surface. Growth of maize cells was inhibited by both (+)- and (-)-ABA, with only weak contributions from their metabolites. The concentration of (+)-ABA that caused a 50% inhibition of growth of maize cells was approximately 1 [mu]M, whereas that for its metabolite (-)-PA was approximately 50 [mu]M. (-)-ABA was less active than (+)-ABA, with 50% growth inhibition observed at about 10 [mu]M. (-)-7[prime]-HOABA was only weakly active, with 50% inhibition caused by approximately 500 [mu]M. Time-course studies of medium pH indicated that (+)-ABA caused a transient pH increase (+0.3 units) at 6 h after addition that was not observed in controls or in samples treated with (-)-PA. The effect of (-)-ABA on medium Ph was marginal. No racemization at C-1[prime] of (+)-ABA, (-)-ABA, or metabolites was observed during the studies.     

Abscisic acid regulation of heterophylly in Marsilea quadrifolia L.: effects of R-(-) and S-(+) isomers

The plant hormone abscisic acid (ABA) induces a developmental switch in the aquatic fern Marsilea quadrifolia, causing the formation of aerial type characteristics, including the elongation of petioles and roots, a change in leaf morphology, the expansion of leaf surface area, and the shortening of the internodes. A number of ABA-responsive heterophylly (ABRH) genes are induced early during the transition. Using optically pure isomers of ABA, it was found that both the natural S-(+)-ABA and the unnatural R-(-)-ABA are capable of inducing a heterophyllous switch and regulating ABRH gene expression. When dose responses are compared, the unnatural ABA gives stronger morphogenic effects than the natural ABA at the same concentration, it is effective at lower concentrations, and its optimal concentration is also lower compared with the natural ABA. Deuterium-labelled ABA enantiomers were used to trace the fate of applied ABA and to distinguish the applied compound and its metabolites from the endogenous counterparts. In tissues, the supplied (+)-ABA was metabolized principally to dihydrophaseic acid, while the supplied (-)-ABA was converted at a slower rate to 7'-hydroxy abscisic acid. Treatment with either enantiomer resulted in increased biosynthesis of ABA, as reflected in the accumulation of endogenous dihydrophaseic acid. Taken together, these results suggest two distinct mechanisms of action for (-)-ABA: either (-)-ABA is intrinsically active, or its activity is due to the stimulation of ABA biosynthesis.     

Inhibitors of abscisic acid 8'-hydroxylase

Structural analogues of the phytohormone (+)-abscisic acid (ABA) have been synthesized and tested as inhibitors of the catabolic enzyme (+)-ABA 8'-hydroxylase. Assays employed microsomes from suspension-cultured corn cells. Four of the analogues [(+)-8'-acetylene-ABA, (+)-9'-propargyl-ABA, (-)-9'-propargyl-ABA, and (+)-9'-allyl-ABA] proved to be suicide substrates of ABA 8'-hydroxylase. For each suicide substrate, inactivation required NADPH, increased with time, and was blocked by addition of the natural substrate, (+)-ABA. The most effective suicide substrate was (+)-9'-propargyl-ABA (K(I) = 0.27 microM). Several analogues were competitive inhibitors of ABA 8'-hydroxylase, of which the most effective was (+)-8'-propargyl-ABA (K(i) = 1.1 microM). Enzymes in the microsomal extracts also hydroxylated (-)-ABA at the 7'-position at a low rate. This activity was not inhibited by the suicide substrates, showing that the 7'-hydroxylation of (-)-ABA was catalyzed by a different enzyme from that which catalyzed 8'-hydroxylation of (+)-ABA. Based on the results described, a simple model for the positioning of substrates in the active site of ABA 8'-hydroxylase is proposed. In a representative physiological assay, inhibition of Arabidopsis thaliana seed germination, (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA exhibited substantially stronger hormonal activity than (+)-ABA itself.     

Abscisic acid does not evoke calcium influx in murine primary microglia and immortalised murine microglial BV-2 and N9 cells

Brain microglia are resident macrophage-like cells representing the first and main form of active immune response during brain injury. Microglia-mediated inflammatory events in the brain are known to be associated with chronic degenerative diseases such as Multiple Sclerosis, Parkinson’s, or Alzheimer’s disease. Therefore, identification of mechanisms activating microglia is not only important in the understanding of microglia-mediated brain pathologies, but may also lead to the development of new anti-inflammatory drugs for the treatment of chronic neurodegenerative diseases. Recently, abscisic acid (ABA), a phytohormone regulating important physiological functions in higher plants, has been proposed to activate murine microglial cell line N9 through increased intracellular calcium. In the present study, we determined the response to ABA and its analogues from murine primary microglia and immortalized murine microglial cell line BV-2 and N9 cells. A Fura-2-acetoxymethyl ester (Fura-2AM)-based ratiometric calcium imaging and measurement technique was used to determine the intracellular calcium changes in these cells when treated with (−)-ABA, (+)-ABA, (−)-trans-ABA and (+)-trans-ABA. Both primary microglia and microglial cell lines (BV-2 and N9 cells) showed significant increase in intracellular calcium ([Ca²⁺]i) in response to treatment with ATP and ionomycine. However, ABAs failed to evoke dose- and time-dependent [Ca²⁺]i changes in mouse primary microglia, BV-2 and N9 cells. Together, these surprising findings demonstrate that, contrary to that reported in N9 cells [3], ABAs do not evoke intracellular calcium changes in primary microglia and microglial cell lines. The broad conclusion that ABA evokes [Ca²⁺]i in microglia requires more evidence and further careful examination.     

The monoclonal antibody MAC252 does not react with the (-) enantiomer of abscisic acid

An RIA procedure has been developed for ABA quantification using MAC62, a monoclonal antibody raised against (+)-cis, trans -ABA. This widely used method now relies on MAC252, a recloned version of the exhausted MAC62. Recently, it has been suggested that MAC252 was not able to discriminate between the (+) and (-) enantiomers of ABA. As this can be misleading when interpreting RIA results, it has been carefully examined here whether MAC252 reacts with (-)-ABA. MAC252 exhibited negligible cross-reactivity with (-)-ABA, which was confirmed with commercial mixtures of ABA isomers. It is concluded that the RIA protocol can continue to be used with MAC252 as it was with MAC62.     

Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds.

Yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds exhibit prolonged coat-imposed dormancy following their dispersal from the parent plant. Analyses were undertaken using S-(+)-[(3)H] abscisic acid (ABA) to monitor the capacity of embryos to metabolize ABA following their isolation from seeds subjected to various dormancy-breaking and control treatments. Radiolabelled phaseic acid (PA) and dihydrophaseic acid (DPA) were detected in embryos and, to a greater extent in the surrounding media, by 48 h regardless of whether the embryos had been excised from seed previously subjected to only a 3 d soak or to a full dormancy-breaking treatment. Of the two enantiomers of ABA, only the natural S-(+)-ABA effectively inhibited germination of isolated embryos. A metabolism-resistant synthetic ABA analogue S-[8',8',8',9',9',9']-hexadeuteroabscisic acid, S-(+)-d6-ABA, consistently slowed the germination rate of excised embryos to a greater extent than that caused by natural S-(+)-ABA. The deuterium-labelled ring methyl groups of the analogue made it more resistant to oxidation by yellow-cedar embryos and thus rendered the analogue more persistent and possessing greater activity. With increasing time of exposure to moist chilling, yellow-cedar embryos became increasingly insensitive to both ABA and to the analogue. Subjecting seed to chemical treatments (GA(3) in combination with 1-propanol) prior to moist chilling strongly enhanced the germinability of whole seeds. This treatment also had a relatively greater impact on ABA metabolism than did moist chilling alone, as indicated by a greater capacity of S-(+)-d6-ABA to inhibit the germination of embryos as compared to S-(+)-ABA. Moist chilling was most critical for reduced ABA sensitivity of embryos. A change in the embryo's ability to metabolize ABA and reduced embryo sensitivity to ABA are two factors associated with dormancy termination of whole seeds of yellow cedar; a change in only one of these factors is insufficient to elicit high germinability.     

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.     

Alterations in Water Status,Endogenous Abscisic Acid Content,and Expression of rab18 Gene during the Development of Freezing Tolerance in Arabidopsis thaliana

Treatments as diverse as exposure to low temperature (LT), exogenous abscisic acid (ABA), or drought resulted in a 4 to 5[deg]C increase in freezing tolerance of the annual herbaceous plant Arabidopsis thaliana. To correlate the increase in freezing tolerance with the physiological changes that occur in response to these treatments, we studied the alterations in water status, endogenous ABA levels, and accumulation of rab18 (V. Lang and E.T. Palva [1992] Plant Mol Biol 20: 951-962) mRNA. Exposure to LT and exogenous ABA caused only a minor decline in total water potential ([psi]w), in contrast to a dramatic decrease in [psi]w during drought stress. Similarly, the endogenous ABA levels were only slightly and transiently increased in LT-treated plants in contrast to a massive increase in ABA levels in drought-stressed plants. The expression of the ABA-responsive rab18 gene was low during the LT treatment but could be induced to high levels by exogenous ABA and drought stress. Taken together, these results suggest that the moderate increases in freezing tolerance of A. thaliana might be achieved by different mechanisms. However, ABA-deficient and ABA-insensitive mutants of A. thaliana have impaired freezing tolerance, suggesting that ABA is, at least indirectly, required for the development of full freezing tolerance.     

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     

Oxidation of 8'-hydroxy abscisic acid in Black Mexican Sweet maize cell suspension cultures

In a biotransformation study to prepare deuterium labelled phaseic acid (PA) from deuterated abscisic acid (ABA), the product contained fewer deuterium atoms than expected. Thus, spectroscopic data of isolated deuterated PA prepared from biotransformation of (+)-5,8',8',8'-d4-ABA in maize (Zea mays L. cv. Black Mexican Sweet) cell suspension cultures showed 83% deuterium incorporation at the 8'-exo position. Also, metabolism studies of (+)-4,5-d2-ABA in maize resulted in the isolation of deuterium labelled ABA derivatives, namely PA, dihydrophaseic acid (DPA), 4'-O-beta-D-glucopyranosylDPA, 8'-hydroxyPA, 8'-hydroxyDPA and 8'-oxoDPA, as deduced from spectroscopic methods. These combined results suggested the presence of an aldehyde intermediate which is either: (a) reduced to 8'-hydroxyABA and cyclized to PA, or (b) is hydrated and cyclized to 8'-hydroxyPA or (c) is further oxidized to the acid and cyclized to 8'-oxoPA. The chemical synthesis of this intermediate, as well as its biotransformation in maize cell cultures is presented.