Synthesis and movement of abscisic Acid in water-stressed cotton leaves

Synthesis and movement of abscisic acid (ABA) into the apoplast of water-stressed cotton (Gossypium hirsutum L.) leaves were examined using pressure dehydration techniques. The exudates of leaves dehydrated in a pressure chamber contained ABA. The level of ABA in the exudates was insensitive to the leaf water potential when dehydration occurred over a 3-hour period. When leaves were rapidly dehydrated in the pressure chamber and held at a balance pressure coincident with the point of zero turgor, ABA accumulated in the leaf tissue and then in the apoplast, but only after 2 to 3 hours of zero turgor. Slow dehydration of leaves by equilibration over varying mannitol concentrations resulted in some accumulation of ABA prior to the point of zero turgor, but ABA accumulated in the tissue and apoplast most rapidly after the onset of zero turgor.     

Conversion of xanthoxin to abscisic Acid by cell-free preparations from bean leaves

Cell-free extracts from the leaves of Phaseolus vulgaris L. convert xanthoxin to abscisic acid. The enzyme activity in dialyzed or acetone-precipitated extracts shows a strong dependence on either NAD or NADP. The enzyme activity appears to be cytosolic with no significant activity observed in chloroplasts. The activity was observed in extracts from roots of Phaseolus vulgaris, and also in extracts prepared from the leaves of Pisum sativum L., Zea mays L., Cucurbita maxima Duchesne, and Vigna radiata L. Neither water stress nor cycloheximide appear to significantly affect the level of enzyme activity in leaves. No intermediates between xanthoxin and abscisic acid were detected.     

Changes in free amino acids and osmotic adjustment in leaves of water-stressed bean

Bean ( Phaseolus vulgaris L. cv. Topcrop) plants were raised in a growth chamber in small pots or flower boxes and kept at full water regime until the full development of primary leaves (14–16 days). Both potted and flower box-grown plants were subjected to a gradually increased water stress of about 60–70 kPa day⁻¹ leaf water potential (stressed plants) or full water regime (control). The water potential, osmotic potential and turgor pressure in freshly detached primary leaves and osmotic potential at zero turgor were calculated using pressure/volume curves. Changes in free amino acids and amides were also measured in parallel trials. Water relation parameters documented that in the stressed leaves there was moderate osmotic adjustment, which was more evident in the potted plants. If considered 0% ionised, the accumulation of free amino acids and amides (μmol g⁻¹ H₂O) accounts for a van't Hoff's value of about 10.2 kPa in the small pot-grown and 5.5 kPa in the box-grown plants. The values are twice as high if considered 100% ionised. Proline accumulation accounted for about 6.4% of the pool enlargement in the potted plants and 22.3% in the flower box plants.     

The relationship between stomatal resistance and abscisic-acid levels in leaves of water-stressed bean plants

Leaf water potentials of Phaseolus vulgaris L. plants exposed to a -3.0 bar root medium were reduced to between -7 and -9 bars within 25 min and remained constant for the next several hours. This treatment led to considerable variation between leaves in both abscisic-acid (ABA) content and R_s, although the two were well correlated after a 5-h treatment. There was an apparent 7-fold increase in leaf ABA levels necessary to initiate stomatal closure when plants were exposed to a -3.0 bar treatment, but when plants were exposed to a -5.0 bar stress R_s values increased prior to any detectable rise in ABA levels. To explain these seemingly contradictory results, we suggest that the rate of ABA synthesis in the leaf, rather than the total ABA content, determines the status of the stomatal aperture.Abbreviations ABA abscisic acid - PEG polyethylene glycol - R_s stomatal diffusion resistance of lower leaf surface - leaf water potential     

Influence of cadmium on water relations, stomatal resistance, and abscisic Acid content in expanding bean leaves

Ten day old bush bean plants (Phaseolus vulgaris L. cv Contender) were used to analyze the effects of 3 micromolar Cd on the time courses of expansion growth, dry weight, leaf water relations, stomatal resistance, and abscisic acid (ABA) levels in roots and leaves. Control and Cd-treated plants were grown for 144 hours in nutrient solution. Samples were taken at 24 hour intervals. At the 96 and 144 hour harvests, additional measurements were made on excised leaves which were allowed to dry for 2 hours. From the 48 hour harvest, Cd-treated plants showed lower leaf relative water contents and higher stomatal resistances than controls. At the same time, root and leaf expansion growth, but not dry weight, was significantly reduced. The turgor potentials of leaves from Cd-treated plants were nonsignificantly higher than those of control leaves. A significant increase (almost 400%) of the leaf ABA concentration was detected after 120 hours exposure to Cd. But Cd was found to inhibit ABA accumulation during drying of excised leaves. It is concluded that Cd-induced decrease of expansion growth is not due to turgor decrease. The possible mechanisms of Cd-induced stomatal closure are discussed.     

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.     

Levels of short-chain fatty acids and of abscisic acid in water-stressed and non-stressed leaves and their effects on stomata in epidermal strips and excised leaves

Straight-chain saturated fatty acids (C6-C11) and abscisic acid (ABA) accumulate in the leaves of Phaseolus vulgaris L. and Hordeum vulgare L. under water stress. ABA and certain of the fatty acids, particularly decanoic and undecanoic acid, can inhibit stomatal opening and cause stomatal closure in epidermal strips of Commelina communis L. depending on the incubating medium used. 10^-4 M (±)-ABA inhibits opening in media containing either high or relatively low concentrations of KCl but causes closure only in the latter medium. The fatty acids (at 10^-4 M) prevent opening in both media while significant closure of open stomata was caused only by undecanoic acid in both media and, additionally, by decanoic acid in the low-KCl medium. 10^-4 M formic acid also caused stomatal closure and prevented opening to significant extents in the low-KCl medium (it was not tested in the high-KCl medium). The efficacy of undecanoic acid in causing 50% inhibition of opening is about three orders of magnitude lower than that of ABA. At a concentration of 10^-3 M, nonanoic, decanoic and particularly undecanoic acid and all-trans-farnesol cause increased cell leakage in Beta vulgaris L. root tissue. Undecanoic acid (10^-4 M) also causes some loss of guard cell integrity in C. communis within 1.5 h of treatment. ABA (10^-4 M) reduces transpiration rates in barley and C. communis leaves when applied via the transpiration stream but decanoic and undecanoic acids did not have this effect. Transpiration was not affected when ABA or the fatty acids were applied to the leaf surfaces.Abbreviations ABA abscisic acid - RWC relative water content - SCFA short-chain fatty acids Deceased May 1977     

The effects of abscisic Acid on growth and nucleic Acid synthesis in excised embryonic bean axes

Abscisic acid (ABA) is an effective inhibitor of cell elongation in excised embryonic bean axes whether added prior to or after the initiation of cell elongation. Zeatin partially reverses this growth inhibition. ABA inhibits ³²P incorporation into ribosomal RNA, transfer RNA, and DNA but not into the tenaciously bound fraction of elongating axes in a manner resembling 5-fluorouracil, a compound which does not inhibit axis growth. The methylated albumin on kie-selguhr elution profiles of nucleic acids obtained from axes treated with either ABA, 5-fluorouracil, or a combination of the two are similar, and zeatin treatment has little apparent effect on these results. Our results suggest that the inhibition of growth in the axes by ABA is not due to its inhibition of DNA synthesis.     

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.     

Indoleacetic Acid biosynthesis in Avena coleoptile tips and excised bean shoots

Avena coleoptiles did not elongate when incubated with tryptophan under sterile conditions. Indole, anthranilic acid, and tryptamine promoted elongation. Under the same conditions, the tissue converted tryptophan-¹⁴C to IAA-¹⁴C. More IAA-¹⁴C was produced from indole-¹⁴C than from tryptophan-¹⁴C; however, the free tryptophan content of the tissue was also greatly increased by the indole treatment. Tryptophan-¹⁴C was readily taken up by the tissue but was mainly incorporated into protein and did not increase the free tryptophan level. When bean shoots were labeled with tryptophan-¹⁴C or indole-¹⁴C, the label incorporation into IAA-¹⁴C was very nearly the same. In this tissue the free tryptophan level in the tryptophan-¹⁴C and indole-¹⁴C treatments was also about equal. These results suggest that failure of exogenously supplied tryptophan to promote the elongation of Avena coleoptiles is a result of its predominant incorporation into protein and consequent unavailability for conversion to IAA.     

Dynamic pathway allocation in early terpenoid biosynthesis of stress-induced lima bean leaves

Two independent pathways contribute in higher plants to the formation of isopenteny1 diphosphate (IDP), the central building block of isoprenoids. In general, the cytosolic mevalonate pathway (MVA) provides the precursors for sesquiterpenes and sterols, whereas the plastidial methylerythritol pathway (MEP) furnishes the monoterpene-, diterpene- and carotenoids. Administration of deuterium labeled 1-deoxy-d-xylulose and mevalolactone to lima beans (Phaseolus lunatus), followed by gas chromatographic separation and mass spectrometric analysis of de novo produced volatiles revealed that the strict separation of both pathways does not exist. This could be confirmed by blocking the pathways individually with cerivastatin((R)) (MVA) and fosmidomycin (MEP), respectively. Isotopic ratio mass spectrometry (IRMS) at natural abundance levels demonstrated independently and without the need for labeled precursors a dynamic allocation of the MVA- or the MEP-pathway in the biosynthesis of the nerolidol-derived homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT). Insect-feeding upregulated predominantly the MVA-pathway, while the fungal elicitor alamethicin stimulated the biosynthesis of DMNT via the MEP-pathway.     

A water potential threshold for the increase of abscisic Acid in leaves

A relationship between abscisic acid concentration and leaf water status is reported. Water potentials were measured in leaves of Ambrosia artemisiifolia L. and Ambrosia trifida L. throughout a period of dehydration of intact plants. Tissues from the same leaves were analyzed for abscisic acid. For both species, abscisic acid began to increase in a critical water potential range (−10 to −12 atmospheres). These data suggest a threshold water potential that stimulates abscisic acid synthesis. The data support the hypothesis that a small change in water potential could affect stomatal resistance to water loss by means of a very sensitive chemical feedback control mechanism.     

Effect of paclobutrazol on water stress-induced abscisic Acid in apple seedling leaves

Abscisic acid (ABA) was quantitated by enzyme-linked immunosorbent assay (ELISA) in water-stressed leaves from control apple seedlings, and also from apple seedlings treated for 28 days with paclobutrazol ([2RS, 3RS]-1-[4-chlorophenyl]-4,4-dimethyl-2-[1,2,4-triazol-1-yl] pentan-3-ol). The ELISA quantitative estimates were also validated by gas chromatography-electron capture detector and lettuce seed germination inhibition bioassay. Paclobutrazol treatment reduced endogenous ABA levels by about one-third, and prevented the marked accumulation of water-stress-induced ABA that occurred in untreated seedlings. The presence of ABA in the apple leaf extracts was confirmed by gas chromatography-mass spectrometry.     

Regulation of key enzymes of sucrose biosynthesis in soybean leaves : effect of dark and light conditions and role of gibberellins and abscisic Acid

An important part in the understanding of the regulation of carbon partitioning within the leaf is to investigate the endogenous variations of parameters related to carbon metabolism. This study of diurnal changes in the activities of sucrose-synthesizing enzymes and levels of nonstructural carbohydrates in intact leaves of field-grown soybean plants (Glycine max [L.]) showed pronounced diurnal fluctuations in sucrose phosphate synthase (SPS) activity. However, there was no distinct diurnal change in the activity of fructose-1,6-bisphosphatase (F1,6BPase). SPS activity in leaves from plants grown in controlled environments presented two peaks during the light period. In contrast to field-grown plants, F1,6BPase activity in leaves from growth chamber-grown plants manifested one peak during the first half of the light period. In plants grown under both conditions, sucrose and starch accumulation rates were highest during early hours of the light period. By the end of the dark period, most of the starch was depleted. A pattern of diurnal fluctuations of abscisic acid (ABA) levels in leaves was also observed under all growing conditions. Either imposition of water stress or exogenous applications of ABA inhibited F1,6BPase activity. However, SPS-extractable activity increased following water deficit but did not change in response to ABA treatment. Gibberellin application to intact soybean leaves increased levels of both starch and sucrose. Both gibberellic acid (10⁻⁶m) and gibberellins 4 and 7 (10⁻⁵m) increased the activity of SPS but had an inconsistent effect on F1,6BPase. Correlation studies between the activities of SPS and F1,6BPase suggest that these two enzymes are coordinated in their function, but the factors that regulate them may be distinct because they respond differently to certain environmental and physiological changes.     

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Effects of moisture stress and senescence on the synthesis of abscisic acid in the primary leaves of bean

The capacity for ABA synthesis during moisture stress of primary leaves of bean ( Phaseolus vulgaris cv. Kinghorn) was defined in terms of leaf age and associated changes in several physiological parameters. The leaves were fully expanded within 9 days after emergence. Fresh and dry weights per unit of leaf area fell during all 5 weeks of the study, from leaf expansion through advanced senescence. The most significant losses in weight occurred during the third and fourth weeks and coincided with a sharp drop in protein content that began immediately after full-leaf. Chlorophyll concentrations declined rapidly during leaf expansion and then more slowly through the end of the fifth week when the leaves were ready to abscise. The ratio of chlorophyll a to b rose steeply over the first 4 weeks of the study.
Although a rapid loss of protein provided the most definitive indication of the early stages of leaf senescence, a marked decline in the ability to synthesize ABA was more closely associated with the termination of rapid leaf growth. This relationship between leaf expansion and the capacity for ABA synthesis during moisture stress remained unchanged when ABA content was expressed on a per unit chlorophyll, protein or dry weight basis.
A water deficit between 5 and 10% of fresh weight, representing a drop in water potential of less than 150 kPa, was sufficient to initiate accumulation of ABA in young leaves. Slightly more intensive levels of stress were required to stimulate ABA synthesis in senescent leaves, but total accumulation was less than one-tenth of the amount recorded in the younger tissue.     

Effects of abscisic Acid on growth, RNA metabolism, and respiration in germinating bean axes

The effect of abscisic acid on growth, respiration, the ATP pool, and rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination has been measured in experiments where the duration of abscisic acid application and its concentration have been varied. At concentrations from 10(-7) to 10(-4)m, abscisic acid inhibits synthesis of RNA with maximal inhibition (80%) at 10(-5)m. RNA synthesis is inhibited by abscisic acid at all times examined (12, 18, and 24 hours), but the extent of inhibition is maximal at 18 hours. In 18-hour axes RNA synthesis is inhibited 42%, ATP pool size is reduced 3%, and O(2) consumption is decreased by 6% after 75 minutes of abscisic acid treatment. Inhibition of RNA synthesis is complete by 2 hours of treatment with abscisic acid, and recovery to near control levels occurs by the 3rd hour after removal from abscisic acid.     

Effect of cytokinins on the photosynthetic apparatus in water-stressed and rehydrated bean plants

Effects of the cytokinins 6-benzylaminopurine (BAP) and N-2-chloro-4-pyridyl-N′-phenylurea (4-PU-30) on the photochemical activity, oxygen flash yields, and thermoluminescence in bean plants under a water stress were studied. The cytokinins increased the photochemical (Hill reaction) activity and thermoluminescence "B"-band in control as well as in stressed and rehydrated plants, while the oxygen flash yields were affected only in the stressed and rehydrated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.