Effect of abscisic Acid on the linoleic Acid metabolism in developing maize embryos

Partially purified protein extracts from maize (Zea mays L.) embryos, whether treated or not with abscisic acid (ABA), were incubated with linoleic acid (LA) and 1-[¹⁴C]LA. The resulting LA metabolites were monitored by high performance liquid chromatography with a radioactivity detector and identified by gas chromatography-mass spectrometry. α- and γ-ketol metabolites arising from 9-lipoxygenase activity were the more abundant compounds detected in the incubates, although the corresponding metabolites produced by 13-lipoxygenase were also present in the samples. In addition, a group of stereoisomers originating from two isomeric trihydroxy acids (9, 12, 13-trihydroxy-10-octadecenoic and 9, 10, 13-trihydroxy-11-octadecenoic acids) are described. Important variations in the relative proportions of the LA metabolites were observed depending on the embryo developmental stage and on ABA treatment. Two new ABA-induced compounds have been detected. These compounds are present in embryos at all developmental stages, being more abundant in old (60 days) embryos. Furthermore, ABA induction of these compounds is maximum at very young developmental stages, decreasing as maturation progresses. A tentative structure for these compounds (10-oxo-9, 13-dihydroxy-11-octadecenoic acid and 12-oxo-9, 13-dihydroxy-10-octadecenoic acid) is also provided. This study revealed an early stage in maize embryogenesis characterized by a higher relative sensitivity to ABA. The physiological importance of ABA on LA metabolism is discussed.     

Effect of abscisic acid on the cell cycle in the growing maize root

The mechanism by which the rate of cell proliferation is regulated in different regions of the root apical meristem is unknown. The cell populations comprising the root cap and meristem cycle at different rates, proliferation being particularly slow in the quiescent centre. In an attempt to detect the control points in the cell cycle of the root apical meristem of Zea mays L. (cv. LG 11), quiescent-centre cells were stimulated to synthesise DNA and to enter mitosis either by decapping or by immersing intact roots in an aqueous 3,3-dimethyl-glutaric acid buffer solution. From microdensitometric and flow-cytometric data, we conclude that, upon immersion, the G₂ phase of the cell cycle of intact roots was shortened. However, when 50 M abscisic acid (ABA) was added to the immersion buffer, parameters of the cell cycle were restored to those characteristic of intact roots held in a moist atmosphere. On the other hand, decapping of primary roots preferentially shortened the G₁ phase of the cell cycle in the quiescent centre. When supplied to decapped roots, ABA reversed this effect. Therefore, in our model, applied ABA retarded the completion of the cell cycle and acted upon the exit from either the G₁ or the G₂ phase. Immersion of roots in buffer alone seems to trigger cells to more rapid cycling and may do so by depleting the root of some ABA-like factor.Abbreviations ABA cis-abscisic acid - DGA 3,3-dimethyl-glutaric acid - DAPI 4,6-diamidino-2-phenylindole - LI labelling index We thank Pierre Zaech of the Ludwig Institute, Epalinges, Switzerland, for expert assistance in flow cytometry and Dr. Jean-Marcel Ribaut of our Institute for providing data on exodiffusion and metabolism of ABA.     

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     

Increased endogenous abscisic Acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials

Roots of maize (Zea mays L.) seedlings continue to grow at low water potentials that cause complete inhibition of shoot growth. In this study, we have investigated the role of abscisic acid (ABA) in this differential growth sensitivity by manipulating endogenous ABA levels as an alternative to external applications of the hormone. An inhibitor of carotenoid biosynthesis (fluridone) and a mutant deficient in carotenoid biosynthesis (vp 5) were used to reduce the endogenous ABA content in the growing zones of the primary root and shoot at low water potentials. Experiments were performed on 30 to 60 hour old seedlings that were transplanted into vermiculite which had been preadjusted to water potentials of approximately −1.6 megapascals (roots) or −0.3 megapascals (shoots). Growth occurred in the dark at near-saturation humidity. Results of experiments using the inhibitor and mutant approaches were very similar. Reduced ABA content by either method was associated with inhibition of root elongation and promotion of shoot elongation at low water potentials, compared to untreated and wild-type seedlings at the same water potential. Elongation rates and ABA contents at high water potential were little affected. The inhibition of shoot elongation at low water potential was completely prevented in fluridone-treated seedlings during the first five hours after transplanting. The results indicate that ABA accumulation plays direct roles in both the maintenance of primary root elongation and the inhibition of shoot elongation at low water potentials.     

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/−/−; +/+/−; +/+/+) or incapable (−/−/−) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.     

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.     

Effect of inhibition of abscisic Acid accumulation on the spatial distribution of elongation in the primary root and mesocotyl of maize at low water potentials

Previous work showed that accumulation of endogenous abscisic acid (ABA) acts both to maintain primary root growth and inhibit shoot growth in maize seedlings at low water potentials (ψ_w) (IN Saab, RE Sharp, J Pritchard, GS Voetberg [1990] Plant Physiol 93: 1329-1336). In this study, we have characterized the growth responses of the primary root and mesocotyl of maize (Zea mays L. cv FR27 × FRMo 17) to manipulation of ABA levels at low ψ_w with a high degree of spatial resolution to provide the basis for studies of the mechanism(s) of ABA action. In seedlings growing at low ψ_w and treated with fluridone to inhibit carotenoid (and ABA) biosynthesis, ABA levels were decreased in all locations of the root and mesocotyl growing zones compared with untreated seedlings growing at the same ψ_w. In the root, low ψ_w (−1.6 megapascals) caused a shortening of the growing zone, as reported previously. The fluridone treatment was associated with severe inhibition of root elongation rate, which resulted from further shortening of the growing zone. In the mesocotyl, low ψ_w (−0.3 megapascal) also resulted in a shortened growing zone. In contrast with the primary root, however, fluridone treatment prevented most of the inhibition of elongation and the shortening of the growing zone. Final cell length measurements indicated that the responses of both root and mesocotyl elongation to ABA manipulation at low ψ_w involve large effects on cell expansion. Measurements of the relative changes in root and shoot water contents and dry weights after transplanting to a ψ_w of −0.3 megapascal showed that the maintenance of shoot elongation in fluridone-treated seedlings was not attributable to increased water or seed-reserve availability resulting from inhibition of root growth. The results suggest a developmental gradient in tissue responsiveness to endogenous ABA in both the root and mesocotyl growing zones. In the root, the capacity for ABA to protect cell expansion at low ψ_w appears to decrease with increasing distance from the apex. In the mesocotyl, in contrast, the accumulation of ABA at low ψ_w appears to become increasingly inhibitory to expansion as cells are displaced away from the meristematic region.     

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. ¹⁸O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.     

Stress induction of abscisic acid in maize roots

Moderate water stresses in the range 0 to −0.6 MPa applied with PEG 6000 to excised roots of Zea mays L. var. LG 11 induced increases of up to four-fold in the amount of abscisic acid (ABA) determined in the tissue after a 12 h period of xylem exudation. The ABA concentration of xylem exudate collected after a 2 h water stress also increased by up to four-fold. Salt stresses, induced with NaCl solutions, resulted in similar increases in the ABA concentrations. ABA concentrations in both root tissue and xylem exudate were highest 4 h after removal of the stress and then declined over a subsequent 8 h period. These results are interpreted in support of the concept that root-produced ABA may have a role in the fine control of the plant's water balance.     

Lactic Acid efflux as a mechanism of hypoxic acclimation of maize root tips to anoxia

Hypoxic pretreatment (3 kPa oxygen) of maize (Zea mays L.) root tips improved their survival time in a subsequent anoxic incubation from 10 h to more than 3 d, provided that glucose was added to the medium to sustain metabolism. The glycolytic flux (lactate + ethanol) was the same in both pretreated and untreated root tips during the 1st h after transfer to anoxia. It was only after 2 h that it declined sharply in untreated tips, but was sustained in pretreated ones. Right after the transition from normoxia to anoxia of untreated root tips, the only fermentative product detected was lactic acid, which accumulated in a 7:1 proportion after 30 min in tissue and medium, respectively. It took 10 min before ethanol could be detected and 20 min for it to be produced at its maximum rate at the expense of lactate production, which slowed down. In contrast, in hypoxically pretreated root tips, ethanol was produced at a maximum rate right after the transfer to anoxia. Concurrently, low amounts of lactic acid were produced that accumulated in a 1:1 proportion after 30 min in tissue and medium, respectively. This large efflux of lactic acid could account for the higher cytoplasmic pH values always found in pretreated tissues. The presence of cycloheximide during pretreatment abolished this difference, suggesting that the greater efficiency of lactate efflux was linked to protein synthesis. The role of lactate in cytosolic pH regulation and in sensitivity to anoxia is discussed.     

Some aspects of the control of root growth and georeaction: the involvement of indoleacetic Acid and abscisic Acid

Apical segments of roots of Zea mays L. cv. Orla and cv. Anjou show a strong georeaction during 7 hours geostimulation. This is abolished by detipping the segments and restored by replacing the tips upon the apical cut surfaces. After exodiffusion of endogenous indoleacetic acid (IAA) the retipped segments showed a significantly lower geocurvature. Application of low concentrations of IAA to the basal cut surface of root segments from which endogenous IAA had not been allowed to exodiffuse increased the geocurvature of retipped Orla segments but decreased geocurvature of Anjou segments. At appropriate concentration basally applied IAA restored the georeaction capacity of root segments from which the endogenous auxin had exodiffused. The implications of the interaction between exogenous and endogenous IAA in the control of root georeaction are discussed with special reference to the normal role of endogenous IAA in the regulation of root georeaction and the variation in endogenous IAA content of roots of different cultivars of maize. The probability that the normal control of root growth and georeaction involves concomitant actions in the elongation zone of IAA moving preferentially in the acropetal direction and basipetally transported growth inhibitors (such as abscisic acid) produced in the cap cells is stressed.     

Characterization of a maize root proteinase.

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.     

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.     

The effect of abscisic acid on cell growth, cell division and DNA synthesis in the maize root meristem

Cis -abscisic acid (ABA), when applied to maize ( Zea mays L. cv. LG 11) roots, decreases the rates of cell growth and cell division in the meristem. It also decreases the rate at which nuclei become labelled with [³H]-thymidine and enter mitosis. Removing the root cap accelerates the entry of nuclei into the DNA synthetic phase of the mitotic cycle and enhances the rate of cell proliferation in the quescent centre. ABA diminishes these effects, but does not suppress them. Thus, ABA cannot wholely substitute for the presence of a cap. One of the primary effects of applied ABA is to retard cell enlargement which may in turn affect the rate of cell division; natural endogenous ABA may act similarly. ABA might in this way assist in maintaining the quiescent centre in intact roots, but cannot be the sole agent involved.     

Phytotropin-induced root phototropism in maize

Phytotropins, even those not absorbing in the visible region of the spectrum, can induce a phototropic response in maize ( Zea mays L. cv. PX-75) roots when illuminated unilaterally with white light. The most active phytotropin, 2-(1-pyrenoyl) benzoic acid (PBA) can elicit a full response at 10 μ M , while the other active molecules, 2-carboxyphenyl-3-phenylpropane-1,3-dione (CPD), 2-carboxyphenyl-3-phenyl-1,2-pyrazole (CPP), 1-N-naphthylphthalamic acid (NPA) and erythrosin elicit a full response at 100 μ M . The less active phytotropins BBA and fluorescein give a reduced response. It is suggested that the observed effect cannot be explained solely on the basis of auxin transport inhibition. There is a photoreceptor in the extension zone of the root, which may be associated in some way with the receptor for NPA. The results are consistent with the proposal that the phototropic process may form part of the root gravitropic response mechanism.     

Indoleacetic Acid and abscisic Acid antagonism: I. On the phytochrome-mediated attachment of mung bean root tips on glass

The phytochrome-mediated attachment of mung bean (Phaseolus vulgaris L., var. Oklahoma 612) root tips on glass is quickly affected by indoleactic acid and abscisic acid at concentrations of 10 nm or less. Indoleacetic acid induces detachment, whereas abscisic acid induces attachment. Both plant regulators rapidly antagonize the action of the other. None of several cytokinins, gibberellins, and ethylene tested over a wide range in concentration had any effect on either attachment or detachment of root tips. It is postulated that phytochrome could control the endogenous levels of indoleacetic acid and abscisic acid and perhaps other hormones under certain circumstances, that this action is the first process initiated by phytochrome, and that indoleacetic acid and abscisic acid act on the plasmalemma to bring about opposing changes in the surface electric charges of plant cells.     

Mode of action of abscisic Acid in barley aleurone layers : induction of new proteins by abscisic Acid

As part of a continuing effort to elucidate the mode of action of abscisic acid (ABA) in barley (Hordeum vulgare L. cv Himalaya) aleurone layers, we have investigated the induction of several polypeptides by ABA in this tissue. There were nine ABA-induced polypeptides as observed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and considerably more (at least 16 spots) on a two-dimensional gel. These proteins started to show enhanced synthesis 2 to 4 hours after ABA treatment, and their synthesis continued for at least 48 hours. In vitro translation using total RNA isolated from ABA-treated aleurone layers indicated that translatable mRNA levels of these proteins essentially paralleled the levels of in vivo synthesized proteins. The most abundant of the ABA-induced proteins was a 29 kilodalton polypeptide which was also synthesized in tissue incubated without ABA. In vivo synthesis of this protein declined as ABA concentration was decreased, with 1 nanomolar ABA approaching control level. Cell fractionation experiments located the 29 kilodalton major ABA-induced protein in 1,000g and 13,000g pellets; most other induced proteins were in the 80,000g supernatant. The 29 kilodalton protein appeared to be sensitive to degradation by sulfhydryl type proteases. As expected, the induction of these proteins by ABA was suppressed by gibberellic acid. Phaseic acid, the first stable metabolite of ABA, suppressed the gibberellic acid-enhanced α-amylase synthesis but was unable to induce the ABA-induced proteins. None of the ABA-induced proteins were secreted into the incubation medium. A 36 kilodalton ABA-induced protein showed cross-reactivity with antibody against a barley lectin specific for glucosamine, galactosamine, and mannosamine.     

Abscission: role of abscisic Acid

The effect of abscisic acid on cotton (Gossypium hirsutum L. cv. Acala 4-42) and bean (Phaseolus vulgaris L. cv. Red Kidney) explants was 2-fold. It increased ethylene production from the explants, which was found to account for some of its ability to accelerate abscission. Absci is acid also increased the activity of cellulase. Increased synthesis of cellulase was not du to an increase in aging of the explants but rather was an effect of abscisic acid on the processes that lead to cellulase synthesis or activity.     

Involvement of abscisic Acid in potato cold acclimation

Upon exposure to 2°C day/night (D/N), leaves of Solanum commersonii (Sc) began acclimating on the 4th day from a −5°C (killing temperature) hardy level to −12°C by the 15th day. Leaves of S. tuberosum L. (St) cv `Red Pontiac' typically failed to acclimate and were always killed at −3°C. Leaves of control (20/15°C, D/N) and treated plants (2°C, D/N) of St showed similar levels of free abscisic acid (ABA) during a 15-day sampling period. In treated Sc plants, however, free ABA contents increased 3-fold on the 4th day and then declined to their initial level thereafter. The increase was not observed in leaves of Sc control plants.

Treated St plants showed a slightly higher content of leaf soluble protein than controls. In Sc, leaves of controls maintained relatively constant soluble proteins, but leaves of treated plants showed a distinct increase. This significant increase was initiated on the 4th day, peaked on the 5th day, and remained at a high level throughout the 15-day sampling period.

Exogenously applied ABA induced frost hardiness in leaves of Sc plants whether plants were grown under a 20°C or 2°C temperature regime. When cycloheximide was added to the medium of stem-cultured plants at the beginning of 2°C acclimation, or at the beginning of the ABA treatment in the 20°C regime, it completely inhibited the development of frost hardiness. However, when cycloheximide was added to plants on the 5th day during 2°C acclimation, the induction of frost hardiness was not inhibited. The role of ABA in triggering protein synthesis needed to induce frost hardiness is discussed.