Activation of O(2) Uptake and NAD-Specific Isocitrate Dehydrogenase in Mitochondria Isolated from Cotyledons of Castor Bean by cis, trans-Abscisic Acid

The rate of O₂ uptake and the activity of NAD-specific isocitrate dehydrogenase (NAD-ICDH) of mitochondria isolated from castor bean cotyledons were increased by added cis, trans-abscisic acid (ABA) in an in vitro system, while the NADP-specific isocitrate dehydrogenase (NADP-ICDH) was not affected by cis, trans-ABA. Trans, trans-ABA showed only a slightly inhibitory effect on O₂ uptake. The V_max value for the isotherm of isocitrate by the enzyme was also increased by cis, trans-ABA. The isocitrate K_m value for the enzyme with cis, trans-ABA was calculated to be approximately 249.8 micromolar, while the S_0.5 for the enzyme without the ABA was 151.6 micromolar. The n value calculated from the slopes of Hill plots of the reaction velocity of NAD-ICDH against isocitrate concentration was 1.5 in the mitochondrial fraction in the absence of ABA, and cis, trans-ABA treatment decreased the value to 1.0. Cis, trans-ABA also partly overcame the inhibition of NAD-ICDH activity by ATP.     

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.     

Compartmentation and equilibration of abscisic Acid in isolated xanthium cells

The compartmentation of endogenous abscisic acid (ABA), applied (±)-[³H]ABA, and (±)-trans-ABA was measured in isolated mesophyll cells of the Chicago strain of Xanthium strumarium L. The release of ABA to the medium in the presence or absence of DMSO was used to determine the equilibration of ABA in the cells. It was found that a greater percentage of the (±)-[³H]ABA and the (±)-trans-ABA was released into the medium than of the endogenous ABA, indicating that applied ABA did not equilibrate with the endogenous material.     

Endogenous abscisic Acid in relation to bud growth in alternate bearing ;valencia' orange

An investigation was conducted into the relation of ABA (cis-trans-abscisic acid) in the dormant buds of alternate bearing `Valencia' orange (Citrus sinensis [L.] Osbeck) trees. ABA did not appear to be related to alternate bearing but t-ABA (2-transabscisic acid) did. There was 5- to 10-fold more t-ABA than ABA in the buds. There was more t-ABA in the buds of the “on” trees than in the buds of the “off” trees, and a drastic drop in t-ABA in both types of buds as spring growth approached. Bud dormancy and readiness for growth as related to t-ABA are discussed.     

Drought-induced increases in abscisic Acid levels in the root apex of sunflower

Abscisic acid (ABA) levels in 3-mm apical root segments of slowly droughted sunflower plants (Helianthus annuus L. cv Russian Giant) were analyzed as the methyl ester by selected ion monitoring gas chromatography-mass spectrometry using characteristic ions. An internal standard, hexadeuterated ABA (d6ABA) was used for quantitative analysis. Sunflower seedlings, grown in aeroponic chambers, were slowly droughted over a 7-day period. Drought stress increased ABA levels in the root tips at 24, 72, and 168 hour sample times. Control plants had 57 to 106 nanograms per gram ABA dry weight in the root tips (leaf water potential, −0.35 to −0.42 megapascals). The greatest increase in ABA, about 20-fold, was found after 72 hours of drought (leaf water potential, −1.34 to −1.47 megapascals). Levels of ABA also increased (about 7− to 54-fold) in 3-mm apical root segments which were excised and then allowed to dessicate for 1 hour at room temperature.     

Optically pure abscisic Acid analogs-tools for relating germination inhibition and gene expression in wheat embryos

We report an examination of the structural requirements of the abscisic acid (ABA) recognition response in wheat dormant seed embryos using optically pure isomers of ABA analogs. These compounds include permutations to the ABA structure with either an acetylene or a trans bond at C-4 C-5, and either a single or double bond at the C-2′ C-3′ double bond. (R)-ABA and the three isomers with the same configuration at C-1′ as natural ABA were found to be effective germination inhibitors. The biologically active ABA analogs exhibited differential effects on ABA-responsive gene expression. All the ABA analogs that inhibited germination induced two ABA-responsive genes, wheat group 3 lea and dhn (rab). However, (R)-ABA and (S)-dihydroABA were less effective in inducing the ABA-responsive gene Em within the time that embryonic germination was inhibited.     

Hormonal characterization of a nonrooting naphthalene-acetic Acid tolerant tobacco mutant by an immunoenzymic method

The comparative analysis of plant hormones was undertaken on a 1-naphthaleneacetic acid tolerant mutant and normal tobacco (Nicotiana tabacum cv Xanthi) plantlets. The mutant plantlet was scrubby and impaired in its root morphogenesis. Degeneration of the root meristem was studied on tissue sections; it appeared very fast (as early as the 3rd or 4th day after sowing), after which the root was further transformed into a callus. Indoleacetic acid (IAA), abscisic acid (ABA), and the isopentenyladenine (iP)- and trans-zeatin(Z)-type cytokinin levels were measured in terminal buds and root tips 13 days after sowing, by enzyme linked immunosorbent assay of high performance liquid chromatography fractions. Some differences appeared between the apical buds of the two genotypes, but the mutant tobacco differed from the wild type mainly by the presence of higher levels of IAA, ABA, and iP + isopentenyladenosine (iPA) in its small root. Thus, the IAA, ABA, and iP + iPA contents were increased by a factor of 15, 7, and 24 times, respectively, in mutant root compared to wild-type tobacco roots. Previous work has shown that the mutation impairs membrane polarization effects induced by auxin at the cell level. The present results would favor the hypothesis that the mutation has also affected the control of growth regulator accumulation in tissues.     

Carrier-Mediated Uptake of Abscisic Acid by Suspension-Cultured Amaranthus tricolor Cells

Abscisic acid (ABA) uptake by Amaranthus tricolor cell suspensions was found to include both a nonsaturable component and a saturable part with K_m of 3.74 ± 0.43 micromolar and an apparent V_max of 1.5 ± 0.12 nanomoles per gram per minute. These kinetic parameters as well as the uptake by intact cells at 0°C or by frozen and thawed cells, are consistent with operation of a saturable carrier. This carrier-mediated ABA uptake was partially energized by ΔpH: it increased as the external pH was lowered to pH 4.0; it decreased after the lowering of the ΔpH by the proton ionophore carbonylcyanide-m-chlorophenylhydrazone or after the altering of metabolically maintained pH gradient by metabolic inhibitors (KCN, oligomycin). The carrier is specific for ABA among the plant growth regulators tested, is unaffected by (RS)-trans-ABA and was inhibited by (S)-ABA, (R)-ABA, and also by the ABA analog LAB 173711.     

Abscisic (ABA)-Aldehyde Is a Precursor to, and 1',4'-trans-ABA-Diol a Catabolite of, ABA in Apple

Previous ¹⁸O labeling studies of abscisic acid (ABA) have shown that apple (Malus domestica Borkh. cv Granny Smith) fruits synthesize a majority of [¹⁸O]ABA with the label incorporated in the 1′-hydroxyl position and unlabeled in the carboxyl group (JAD Zeevaart, TG Heath, DA Gage [1989] Plant Physiol 91: 1594-1601). It was proposed that exchange of ¹⁸O in the side chain with the medium occurred at an aldehyde intermediate stage of ABA biosynthesis. We have isolated ABA-aldehyde and 1′-4′-trans-ABA-diol (ABA-trans-diol) from ¹⁸O-labeled apple fruit tissue and measured the extent and position of ¹⁸O incorporation by tandem mass spectrometry. ¹⁸O-Labeling patterns of ABA-aldehyde, ABA-trans-diol, and ABA indicate that ABA-aldehyde is a precursor to, and ABA-trans-diol a catabolite of, ABA. Exchange of ¹⁸O in the carbonyl of ABA-aldehyde can be the cause of loss of ¹⁸O from the side chain of [¹⁸O]ABA. Results of feeding experiments with deuterated substrates provide further support for the precursor-product relationship of ABA-aldehyde → ABA → ABA-trans-diol. The ABA-aldehyde and ABA-trans-diol contents of fruits and leaves were low, approximately 1 and 0.02 nanograms per gram fresh weight for ABA-aldehyde and ABA-trans-diol, respectively, while ABA levels in fruits ranged from 10 to 200 nanograms per gram fresh weight. ABA biosynthesis was about 10-fold lower in fruits than in leaves. In fruits, the majority of ABA was conjugated to β-d-glucopyranosyl abscisate, whereas in leaves ABA was mainly hydroxylated to phaseic acid. Parallel pathways for ABA and trans-ABA biosynthesis and conjugation in fruits and leaves are proposed.     

The Uptake of (+)-S- and (-)-R-Abscisic Acid by Suspension Culture Cells of Hopbush (Dodonaea viscosa)

The uptake of (+)-S- and (−)-R-abscisic acid (ABA) by suspension culture cells of hopbush (Dodonaea viscosa L. Jacqu.) was followed over a range of temperatures, pH values, and time intervals. The natural (+)-S-ABA was taken up about five times faster than the unnatural (−)-R-ABA. Each 10°C rise in temperature from 1 to 31°C increased the rate of uptake (Q₁₀) of (+)-S-ABA about 2.2-fold, whereas that of the (−)-R increased with a Q₁₀ of 1.4. (+)-ABA was taken into the cells by a saturable carrier, but (−)-ABA and both enantiomers of 2-trans-ABA were not; they appeared to enter by passive diffusion. The uptake of (+)-ABA was linear over the first 8 hours but concentrations within the cells decreased after 2 hours to remain constant after 4 hours as rapid metabolism was induced. Electron microscopy of thin sections of the cells, combined with a stereological analysis of their shape, showed that the vacuoles comprised 80% of the cell volume and the cytoplasm plus nucleus comprised 20%. There were no photosynthetically active plastids in the cells. Concentrations of the endogenous ABA in the cytoplasm (pH 7.32) and vacuoles (pH 5.88) were calculated by applying the Henderson-Hasselbalch equation (ABA pK_a 4.7) so that, provided no active metabolic redistribution occurred, the concentration in the cytoplasm was 7.9 micromolar and that in the vacuole was 0.3 micromolar. In vivo pH was measured by ³¹P nuclear magnetic resonance spectroscopy.     

Kinetics of maize leaf elongation IV. Effects of (+)- and (-)-abscisic acid

Abscisic acid (ABA) is involved in many of the responses of plants to environmental stress. This study focuses on the inhibitory effect of ABA on leaf expansion. In addition, the effects of (+)-ABA, the natural form of ABA, were compared to the effects of (-)-ABA. Leaf elongation rates (LER) were measured for the 3rd leaf of maize plants. ABA concentrations were measured by RIA for total ABA and an ELISA specific for (+)-ABA. ABA was added to the hydroponic solution and changes in the LER were measured over time. ABA could inhibit LER within 30 min ad reached steady-state LER within 4 h. Internal ABA concentrations in the growing zone of the leaf also reached steady-state concentrations after 4 h. This effect of ABA was reversible, because LER was fully restored upon removal of externally applied ABA, and internal concentrations of ABA in the growing zone returned to normal levels, whereas ABA concentrations remained elevated in mature tissue. Thus, steady-state LER was highly correlated with the steady-state internal ABA concentration of the growing zone. ABA inhibited leaf expansion by increasing the apparent cell wall yield threshold; no other growth parameters were affected. The (-)-enantiomer of ABA had much less effect on LER than (+)-ABA when compared upon an external concentration basis. Internal ABA concentrations rationalized the response, showing that (-)-ABA accumulation was very low, most likely due to low uptake rates. From this analysis, it was determined that LER was equally sensitive to internal concentrations of (+)- or (-)-ABA.     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.     

Endogenous gibberellins and inhibitors in caryopses of rye

Gibberellins A₈, A₁₆, A₂₄, and abscisic acid were identified by GC-MS of derivatized extracts from immature fruits of Secale cereale. Mature caryopses contained ABA and trans-ABA in a ratio 1:1 as well as 4′-dihydrophaseic acid. During milk ripeness a neutral GA conjugate was detected. Free GA, afforded by enzymatic hydrolysis of the conjugate, was chromatographically identified as GA₁₆     

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 Specificity of the Carrier-Mediated Uptake of ABA by Root Segments of Phaseolus coccineus L.

Earlier work has established that the saturable component ofuptake of RS-[2¹⁴C]ABA by bean (Phaseolus coccineus L. cv. Prizewinner)root segments can be attributed to the action of a carrier.We now show that the carrier-mediated uptake is unaffected byRS-2-trans-ABA and lunularic acid and the unnatural R-ABA alsoappears to be ineffective. The specificity for S-ABA requiresthe halving of the K_m value for ABA determined previously (2.6mmol m_-3 for RS-; 1.3 mmol m_-3 for S-ABA). The RS-1', 4'-cis-dioland RS-1'-deoxy ABA reduce the uptake of RS-[2-₁₄C]ABA aboutas strongly as does unlabelled ABA, the K¹ for 1'-deoxy ABAwas similar to the K_m for ABA. The K₁ for RS-1', 4'-trans-diolwas 15.7 mmol m_-3. Consideration of the stereochemistry of thesecompounds suggests that the face of the ring of ABA away fromthe 1'-hydroxyl group interacts with the carrier site. Labelled material diffused out of undamaged root surfaces whichhad absorbed RS-[³H]ABA through an apical cut, suggesting thatABA is present in the apoplast. A simplified hypothesis is presented that can account for polartransport of ABA based on a gradient of a carrier in a tissuebut where the carrier is distributed uniformly on the apicaland basal ends of each cell. Key words: Uptake carrier, Abscisic acid, 1', 4'-Diol, Lunularic acid, Phaseolus coccineus, Polar-transport, Deoxyabscisic acid     

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [2¹⁴-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.Abbreviations ABA abscisic acid - 3,3-DGA 3,3-dimethyl-glutaric acid - DPA dihydrophaseic acid - PA phaseic acid - GCMS gas chromatography-mass spectrometry     

Effects of Ultraviolet-B Radiation Stress on the Abscisic Acid Status of Rumex patientia Leaves

To determine if increased abscisic acid (ABA) levels are associated with ultraviolet-B (UV-B) radiation (288–320 nm) stress, Rumex patientia L. plants were exposed to visible light with high or low (control) UV-B irradiance and then assayed for ABA by gas chromatography. Though leaf growth was inhibited by the UV-B irradiation, no differences in levels of free ABA were found between leaves from the two treatments after 1, 3 or 5 days of exposure. Unlike the situation in most environmental stresses, increased ABA levels are not associated with UV-B radiation stress. Further, photolysis or isomerization are not likely explanations for the absence of ABA increase. Calculations indicate that if ABA in the leaf were fully exposed to solar UV-B radiation only about 6.7% would be photolyzed by existing daily radiation at 40°N latitude, and 11.3% by radiation resulting from a 40% decrease in atmospheric ozone. The quantum yield for isomerization of cistrans-ABA by radiation in the 288–312 nm waveband was determined. Existing daily solar UV-B irradiation is capable of isomerizing ABA in solution to an equilibrium mixture of 50% cisjrans and 50%irans, trans-ABA., However, because of radiation attenuation in the leaf, it is estimated that only a small fraction of the cistrans-A.BA. would be isomerized.     

Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes

Using root- and cell-pressure probes, the effects of the stress hormone abscisic acid (ABA) on the water-transport properties of maize roots (Zea mays L.) were examined in order to work out dose and time responses for root hydraulic conductivity. Abscisic acid applied at concentrations of 100–1,000 nM increased the hydraulic conductivity of excised maize roots both at the organ (root Lp_r: factor of 3–4) and the root cell level (cell Lp: factor of 7–27). Effects on the root cortical cells were more pronounced than at the organ level. From the results it was concluded that ABA acts at the plasmalemma, presumably by an interaction with water channels. Abscisic acid therefore facilitated the cell-to-cell component of transport of water across the root cylinder. Effects on cell Lp were transient and highly specific for the undissociated (+)-cis-trans-ABA. The stress hormone ABA facilitates water uptake into roots as soils start drying, especially under non-transpiring conditions, when the apoplastic path of water transport is largely excluded. Received: 26 February 2000 / Accepted: 17 August 2000     

2-Trans-Abscisic Acid Biosynthesis and Metabolism of ABA-Aldehyde and Xanthoxin in Wild Type and the aba Mutant of Arabidopsis thaliana

Abscisic acid (ABA) and 2-trans-ABA (t-ABA) biosynthesis werestudied in wild type Landsberg erecta and the three allelicaba mutants of Arabidopsis thaliana (L.) Heynh., which are impairedin epoxy-carotenoid biosynthesis. Labelling experiments with¹⁸O₂and mass spectrometric analysis of [¹⁸O]ABA and its catabolitesABA-glucose ester (ABA-GE) and phaseic acid (PA), and t- ABAand t-ABA-GE, showed that t-ABA biosynthesis was less affectedthan ABA biosynthesis by mutations at the ABA locus. The aba-4allele caused the most severe impairment of ABA biosynthesiscompared with the other two mutant alleles aba-1 and aba-3,yet aba-4 plants synthesized as much t-ABA as wild type Landsbergerecta plants. Feeding experiments with RS- [²H₆]ABA-aldehydeisomers and unlabelled xanthoxin isomers suggest that t-xanthoxinand t-ABA-aldehyde are precursors to ABA and t-ABA in Arabidopsis Key words: ABA-alcohol, ABA-aldehyde, ABA-glucose ester, ¹⁸O₂ labelling, phaseic acid     

The Effects of Mechanical Impedance to Growth on the Levels of ABA and IAA in Root Tips of Zea mays L.

Extraction and analytical methods have been refined and newones devised to allow precise determinations by GC-EC of thelevels of abscisic acid (ABA) and indol-3ylacetic acid (IAA)in samples of maize root tips as small as 1.0 g fr. wt. Seminalroots of 5-d-old maize seedlings grown in normal (bulk density1200 kg m^–3) and compacted (bulk density 1600 kg m^–3)sand/garden loam mixtures have been examined. Seminal rootsfrom compacted soil had an average length of about 40% of thatof control roots and were much thicker. The ABA levels in 10mm tips of impeded roots (c. 25–35 ng g^–1 fr.wt.)did not differ significantly from those of normal root tipson both a fresh and dry weight basis. The levels in 0–1mm tips were approximately double those in the remaining 1–10mm zones. IAA levels were increased by about 3 times in impededroots (176.3 as compared with 52.4 ng g^–1 fr.wt) and itis concluded that this response is likely to be the main causeof the morphological and growth changes brought about by soilcompaction.